some properties of composites based on

Oxidation Communications 37, No 2, 405–415 (2014) Thermooxidative degradation of composite polymeric materials

Some ProPertieS of ComPoSiteS BaSed on tetrafluoroethylene-hexafluoroProPylene CoPolymer with white riCe huSk aSh and kinetiCS of itS thermooxidative degradation S. Genievaa, D. KiryaKovab, a. atanaSSovb*, L. vLaeva a

Department of Physical Chemistry, ‘Prof. Dr. Assen Zlatarov’ University, 1 Prof. Yakimov Street, 8010 Bourgas, Bulgaria b Department of Materials Science, ‘Prof. Dr. Assen Zlatarov’ University, 1 Prof. Yakimov Street, Bourgas, Bulgaria E-mail: [email protected]

aBStraCt Some properties of composites based on tetraluoroethylene-hexaluoropropylene copolymer illed with different quantities of white rice husk ash have been studied. With increase of iller content, the tensile strength and elongation of the composites sharply decreased while the Young modulus did not change signiicantly. The thermooxidative degradation kinetics of tetraluoroethylene-hexaluoropropylene copolymer and its composites was studied by non-isothermal differential thermal analysis at different heating rates. The values of apparent activation energy E, pre-exponential factor A in the Arrhenius equation, as well as the changes of entropy ∆S*, enthalpy ∆H* and free Gibbs energy ∆G* for the formation of the activated complex from the reagent were calculated. Keywords: tetraluoroethylene-hexaluoropropylene copolymer, white rice husk ash, composite materials, properties, thermooxidative degradation kinetics. aiMS anD BaCKGroUnD The luoropolymers such as polytetraluoroethylene (PTFE) and copolymers of tetraluoroethylene (TFE) with ethylene (E) and hexaluoropropylene (HFP) possess excellent antifriction, antiadhesion and electric properties. Because of their high thermal resistance, however, they are hard to process, they have low tensile strength and radiation resistance, and show small mass loss at higher processing temperatures1,2. These and some other disadvantages of the luoropolymers can be overcome by *

For correspondence.

405

synthesis of luorine containing copolymers with different comonomers to achieve properties similar to those of PTFE (Ref. 3). It is well known4 that the thermal degradation of PTFE and TFE–HFP copolymer gives predominantly the monomers TFE and HFP which can be used as initial materials for production of other perluorinated polymers. It is quite easier to manufacture products from the copolymers using the conventional methods of processing. The TFE copolymers and particularly TFE–HFP are relatively little studied as matrix for preparation of composite materials with various dispersed and ibrous secondary phases. The use of rice husk, waste product from rice processing, as iller in different polymer matrices has become one of the alternative methods for using this waste material, thus overcoming certain environmental problems5,6. The thermal treatment of raw rice husks in air occurs in three stages: drying (40–250°C), release of volatile organic substances (215–350°C) and burning of the carbon (350–690°C) (Refs 7–9). As a result, white rice husk ash (WRHA) is obtained containing 95 mass.% amorphous SiO2, or black rice husk ash (BRHA), containing 55 mass.% amorphous Sio2 and 45 mass.% carbon10–13 if the treatment is carried out in nitrogen atmosphere. Many authors14–16 studied the thermal characteristics of composite materials based on various polymers with rice husk ash. It was found that if BRHA is used as iller then the oxidative resistance is retained but the temperature of degradation is increased. The tensile strength and elongation at break signiicantly decrease with the increase of BRHA content while the elasticity modulus and density increase compared to the initial polymer. When WRHA is used, the oxidative stability of the polymer worsens and the composite begins decomposing at lower temperatures compared to that illed with BRHA and pure polymer17. However, to the authors knowledge, no detailed work has been carried out on the preparation and studies on thermooxidative degradation kinetics of composite materials based on TFE–HFP copolymer illed with rice husk ash, only few related to the mechanical, physical, thermal and tribological properties of the composite materials based on it with different ibres, minerals, graphite, etc.18,19 This has been done for materials of PTFE, TFE–E copolymer and polypropylene containing rice husk ash17,20. Studies on the thermal stability of TFE–HFP copolymer and composites based on it illed with various amounts of BRHA (Ref. 21) showed that the iller strongly affects the rheological characteristics and the thermal stability. The temperature of degradation decreases with the increase of BRHA content. The higher iller content, 10 mass.% BRHA, affects the mechanism of thermooxidative degradation of the copolymer and the kinetic parameters calculated for this composite have values close to these observed for the initial TFE–HFP copolymer. As a result from the higher content of amorphous SiO2 in the iller WRHA, it could be expected that the composites in this basis would decompose at even lower temperatures. The aim of the present work is to determine some physicomechanical properties of composites based on TFE–HFP copolymer and study the kinetics of thermooxida-

406

tive degradation of the copolymer and its composites illed with different quantity of WRHA. EXPERIMENTAL Materials and measurements. Samples of TFE–HFP copolymer (Du Pont, USA) with melt index 3.9 g/10 min (300°C, and loading 98 N, melting point 254°C) have been used in the experiments. WRHA obtained by thermal degradation of raw rice husks in air medium was used as iller of TFE–HFP copolymer. According to the data from X-ray analysis, the iller was amorphous22. After pyrolysis of raw rice husks in air the hard residue obtained (WRHA) was formed of almost pure amorphous and porous SiO2 (95.8 mass.%) with true density 2.2 g cm–3 and surface area 228 m2 g–1. As impurities, the sample contained about 4 mass.% oxides of alkaline, alkaline earth metals and Al2o3. After vigorous grounding in an agate mortar, the mean particle size of the sample from WRHA was about 60 μm. The compositions were prepared on plastograph ‘Brabender’ (Germany) by adding different quantities of the iller (5–10 mass.%) at 300°C. They were pressed on a laboratory press PHI (England) between aluminium foil under the following conditions: sample thickness about 1 mm, temperature 300ºС, time to melting 5 min, pressure 12 МРа and cooling rate 60ºС min–1. The melt low indices of the initial copolymer and the materials obtained from it were determined by MFI (g 10 min–1) method on an apparatus MFI 3350 (Prodemat, France) at temperature of 300ºС and load 98N (ASTM D 1238-65). The thermal stability was calculated by the weight loss (%) after 3 h at 300ºС. The shrinkage of the composites was determined by the reduction of the linear dimensions of the samples after heat treatment at 200ºС for 5 h compared to the initial dimensions. The tensile properties were measured on a dynamometer INSTRON 4203 (England) at speed of 100 mm min–1 and room temperature (ASTM D 638-89). The thermogravimetrical measurements were carried out under non-isothermal conditions on a derivatograph system F. Paulik–I. Paulik–L. Erdey (which records T, TG, DTG and DTA curves simultaneously) in air low at a rate of 25 cm3 min–1. Samples of 50 mg mass were used for the experiments carried out at heating rates from 6, 10, 13 and 15°C min–1 up to 700°C. The samples were loaded without pressing into an open platinum crucible. α-Alumina calcined up to 1100°C was used as a standard reference material. Theoretical approach and calculation procedures. TG/DTG technique is very useful for the determination of degradation temperature, degradation steps for solid substances. The kinetics of thermooxidative degradation reactions is described with various equations taking into account the special features of their mechanisms. The basic equations for treatment of the experimental data are described in details17,20. Different authors suggested different methods and calculation procedures23–26 to solve the conversion and temperature integral and ind the kinetic parameters of the process. 407

Coats and redfern23 suggested a calculation procedure which is successfully used in studies on the kinetics of dehydration, degradation or combustion of various solid substances. The kinetic parameters of the process are obtained using the linear form of the following equation: ln

g (α ) T

2

 AR  2 RT  E AR E 1 –  – = ln  – ≅ ln , E  RT qE RT  qE 

(1)

where g(α) is an integral form of the conversion function, the expression of which depends on the kinetic model of the occurring reaction. If the correct g(α) function is used, a plot of ln[g(α)/T2] against 1/T should give a straight line from which the values of the activation energy E and the pre-exponential factor A in the Arrhenius equation can be calculated. Using the Arrhenius equation: k = A exp(–E/RT)

(2)

the values of the rate constant k can be calculated for any temperature in the temperature interval where the process combustion of TFE–HFP copolymer occurs17,20. From the theory of the activated complex (transition state) of Eyring27, the following general equation may be written: k = (χkBT/h) exp(–∆G*/RT),

(3)

where χ is transmission coeficient, which is unity for monomolecular reactions; kB – the Boltzmann constant (1.381 ×10–23 J K–1); h – the Plank constant (6.626 ×10–34 J s), and ∆G* – the change of Gibbs free energy for the formation of the activated complex from the reagents. Using the thermodynamic equation: ∆G* = ∆H* – T∆S*,

(4)

equation (3) may be rewritten as follows: k=

χk BT h

ΔS ∗   ΔH ∗    exp  exp –  R   RT   

   , 

(5)

where ∆S* and ∆H* are the changes of entropy and enthalpy for the formation of the activated complex from the reagent, respectively. Taking logarithm from the both sides of equation (5) and rearranging we obtain: ln

kh χkBT

=

∆S* R

–

∆H* RT

.

(6)

Plotting ln ((kh)/(χkBT)) versus 1/T and using a linear regressive of least square method, the values of the changes of entropy ∆S* and enthalpy ∆H* can be calculated from the intercept and the slope of the straight line.

408

reSULtS anD DiSCUSSion TFE–HFP is perluorinated copolymer of TFE with HFP and its macromolecules have spiral conformation and properties characteristic for PTFE. In copolymer macromolecule, however, the presence of side (–CF3) group changes the crystal lattice parameters and disturbs the location of every ifth carbon atom of the main chain, so its diameter is by 11% higher than that of the PTFE macromolecule28. The macromolecule of the copolymer is less twisted so its hardness is lower. The main advantage of TFE–HFP is the possibility for processing from melt and its modiication with suitable illers like WRHA could impart speciic rheological properties and widen its ields of application. The melt low index of the composites studied decreased with the increase of WRHA content from 3.9 for initial copolymer to 2.2 g 10 min–1 at 10 mass.% WRHA. At higher iller concentrations, the melt low index is supposed to decrease further to values at which the copolymer processing would become hard. The change of the physicomechanical parameters – tensile strength (σ), elongation (ε) and the Young modulus (Y) of the composites depending on the content of the iller WRHA is shown in Fig. 1.

400

(MPa), ε (%), Y (MPa)

300

200

100 Y ε

0 0

2.5 C (mass.%)

5

10

fig. 1. Dependence of some physicomechanical parameters of studied composites on the content (С) of WRHA

It can be seen from the igure that the tensile strength and especially the elongation sharply decreased with the increase of WRHA content compared to these values for the initial TFE–HFP copolymer. The Young modulus (Y) of the composite materials on iller content did not change signiicantly and remained in the range 300−360 МРа. The studies on the kinetics of degradation of the initial TFE–HFP copolymer and its composites with BRHA showed21 that the degradation occurs in two stages. The irst one comprises the pyrolysis of the copolymer which begins at 400°С, shows exothermic effect at 510°С and ends at 530°С with weight loss of the sample ca. 40 mass.%. 409

The second stage was also accompanied by an exothermal effect at 560°С and ended at about 590°С. It was due to the burning of luorocarbon residues formed as a result of the pyrolysis. The same degradation stages were observed also for the composites illed with BRHA but at temperatures lower by about 20°С. The thermooxidative degradation of the TFE–HFP composites with 5 and 10 mass.% WRHA was carried out under non-isothermal heating at four temperatures. The corresponding TG curves observed for the initial copolymer and its composites taken at heating rate 6°С min–1 are presented in Fig. 2.

fig. 2. TG curves of thermooxidative degradation (heating rate 6°С min–1): 1 – TFE–HFP, 2 – TFE–HFP + 5 mass.% WRHA, 3 – TFE–HFP + 10 mass.% WRHA

As can be seen from Fig. 2, the thermal degradation of the composites started at temperature lower than that of the pure polymer. During the irst stage of degradation (380–490°С), the composites studied behaved similarly up to about 470°С. With the increase of iller content, the thermal stability of the pure polymer decreased, thus to composite illed with 10 mass.% WRHA continued decomposing at the lowest temperatures. The second stage occurred in the temperature interval 490 – 590°С where the shape of the TG curves was the same for all the samples. This is explained with the full destruction of the iller and combustion of the luorocarbon residue from the pyrolysis. Each stage is characterised by its own mechanism and kinetic equation and, respectively, different values of the activation energy Е and pre-exponential factor А in the Arrhenius equation24. The most probable mechanism of thermal degradation of the samples and the kinetic parameters characterising the process were determined using the method of Coats–Redfern23 and the algebraic expressions of the most commonly used differential f (α) and integral g(α)-functions presented in Ref. 17. For the thermal degradation of the samples studied, single heating rate plots ln[g(α)/T 2] against 1/T were calculated according to equation (1). The kinetic curves of degradation obtained for the composite illed with 10 mass.% WRHA are presented in Fig. 3. 410

n = 0.33

–10

n = 0.5 n = 0.67 n = 0.75 n = 1.0 n = 1.25

2

ln[g(α)/T ]

–12

n = 1.5

–14

n = 1.75 n = 2.0

–16

–18 1.15

1.20

1.25

1.30

1.35

1.40

1.45

103 / (K–1)

fig. 3. Kinetic curves of degradation of TFE–HFP + 10 mass.% WRHA according to equation (1) at different values of n and heating rate 6°С min–1

It should be noted for all the samples studied that the highest values of linear regression coeficient R2 were obtained when kinetic equations for the Fn mechanism with different values of n were used. To ind the values of n with which the highest value of R2 was obtained, the dependence R2 = f(n) was drawn. The maximum value of R2 for both composites was determined: irst stage at n = 1.5; second stage at n = 0.33. The values of the kinetic parameters calculated are shown in Table 1. table 1. Kinetic parameters of thermooxidative degradation of the initial TFE–HFP copolymer and its composites

Samples n TFE–HFP TFE–HFP+5 mass.% WRHA TFE–HFP+10 mass.% WRHA

0.67 1.50 1.50

I stage A E (kJ mol–1) (min–1) 164.5 1.42×1010 158.1 3.25×107 165.6

6.57×107

n 0.33 0.33 0.33

II stage E A (kJ mol–1) (min–1) 79.73 1.23×104 80.61 3.58×101 82.05

4.88×101

As can be seen from Table 1, the reaction order found for the composites was higher than that of the pure polymer. The values of the activation energy for both stages of degradation are very close which means that the amount of iller used did not have substantial effect on the mechanism of matrix degradation. However, the same can not be said about the values of the pre-exponential factor. Values in the range 1010–1012 min–1 observed for the irst stage of degradation of the pure polymer mean that the active complex formed was strongly limited in rotation compared to the initial reagent29. At values lower than 106, it is assumed that the reagent is in equilibrium with the surface adsorption layer and after the degradation of the active complex it transforms into product. This was the situation during the second stage of the degradation of the copolymer and its composites. The values of the kinetic parameters found are presented in Table 2. 411

table 2. Most probable kinetic parameters of thermal degradation of TFE-HFP copolymer and its composites

Samples TFE–HFP TFE–HFP + 5 mass.% WRHA TFE–HFP + 10 mass.% WRHA

I stage ∆S* ∆G* –1 –1 (J mol K ) (kJ mol–1) –60.3 217.1

II stage ∆H* ∆S* ∆G* –1 –1 –1 ( kJ mol ) (J mol K ) (kJ mol–1) 171.0 –242.5 286.4

∆H* ( kJ mol–1) 86.8

–151.4

269.8

151.7

–266.1

296.0

73.7

–145.5

272.5

159.1

–263.5

220.1

75.1

The values of ∆H* express the difference between the energies of the reagent and the active complex and if it is small then the formation of the active complex is favoured. Low values of ∆S* mean that the material passes through physical or chemical process and is in a state close to thermodynamic equilibrium. Negative values mean that the formation of the active complex is accompanied by decrease of entropy, i.e. the complex is ‘more organised’ structure than the initial reagent. The kinetic parameter ∆G* characterises the total increase of the energy of the system by the transition of the reagents into active complex. Only the composite illed with 10 mass.% WRHA showed decrease in this parameter for the second stage of degradation. To compare the degradation behaviour of the TFE−HFP copolymer and its composites illed to the same extent with BRHA and WRHA, the corresponding TG curves taken at heating rate of 6°С min–1 are presented in Fig. 4.

fig. 4. Experimental TG curves for thermooxidative degradation of: 1 – TFE–HFP copolymer, 2 – TFE– HFP + 10 mass.% WRHA, 3 – TFE–HFP+10 mass.% BRHA

412

It can be seen from the igure that the initial copolymer (curve 1) began degrading at temperature higher than that of its composites which have the same degradation behavior (curves 2 and 3) until 40% weight loss which is the irst stage of degradation. After the pyrolysis of the copolymer, the degradation of the composite containing 10 mass.% BRHA continued at temperatures lower than these for the one containing 10 mass.% WRHA. It indicates that the illers used decreased the thermal stability of the initial copolymer to different extents. The second stage of combustion of the luorocarbon residues of the BRHA containing composite occurred at temperatures about 20°С lower than these for the pure copolymer and 10°С than the WRHA containing composite. Due to the pronounced thermal stability of TFE–HFP copolymer, the iller contents studied did not have signiicant effect on the thermal stability of the initial copolymer, in contrast to composites based on TFE–E copolymer illed with the same amount of illers17. ConCLUSionS Some physicomechanical properties of composites based on TFE–HFP illed with 5 and 10 mass.% WRHA were studied. Melt low index, tensile strength, elongation and the Young modulus of the studied composites decreased with the increase of WRHA content. The decrease of these values was similar to that observed for the composites illed with the same amount of BRHA. The thermogravimetric analyses of the TFE– HFP-based composites with white rice husk ash carried out at four heating rates – 6, 10, 12 and 15°С min–1, as well as the calculations on the kinetics of their thermal degradation revealed two degradation stages – pyrolysis and combustion, which are characterised by different values of the activation energy and pre-exponential factor in the Arrhenius equation. The pure copolymer TFE–HFP degrades at temperature higher than that of the composites studied. The kinetics of thermal degradation of the samples can be best described by kinetic equations of n-th order (Fn-mechanism). For both composites, illed with 5 or 10 mass.% WRHA, the most probable mechanisms for the irst and second stages of pure copolymer thermal degradation were found to be F3/2 and F1/3, respectively. reFerenCeS 1. yu. A. PANSHIN, S. G. MALKEVICH, Z. S. DUnaevSKaya: Fluoropolymers. Khimiya, Leningrad, 1978 (in Russian). 2. L. A. WALL: Fluoropolymers. John Wiley & Sons, New York-London-Sydney-Toronto, 1972. 3. r. K. eBy, F. C. WILSON: Relaxation in Copolymers of Tetraluoroethylene and Hexaluoropropylene. J Appl Phys, 33 (10), 2951 (1962). 4. i. J. van der WALT, H. W. J. P. NEOMAGUS, J. T. NEL, O. S. L. BRUINSMA, Ph. L. CronSe: A Kinetic Expression for the Pyrolytic Degradation of Polytetraluoroethylene. J Fluorine Chem, 129 (4), 314 (2008). 5. H. ISMAIL, Z. MOHAMAD, A. A. BAKAR: A Comparative Study on Processing, Mechanical Properties, Thermo-oxidative Aging, Water Absorption, and Morphology of Rice Husk Powder and

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7. 8. 9. 10. 11. 12. 13.

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18. 19. 20.

21.

22. 23. 24.

25.

Silica Fillers in Polystyrene/Styrene Butadiene Rubber Blends. Polym Plast Technol Eng, 42 (1), 81 (2003). H. G. B. PREMALAL, H. ISMAIL, A. BAHARIN: Comparison of the Mechanical Properties of Rice Husk Powder Filled Polypropylene Composites with Talc Filled Polypropylene Composites. Polym Test, 21 (7), 833 (2002). H. D. BANERJEE, S. SEN, H. N. ACHARYA: Investigations on the Production of Silicon from Rice Husks by the Magnesium Method. Mater Sci Eng, 52 (2), 173 (1982). A. CHAKRAVETY, P. MISHRA, K. BANERJEE: Investigation of Thermal Degradation of Rice Husk. Thermochim Acta, 94 (2), 267 (1985). M. A. HAMAD: Thermal Characteristics of Rice Hulls. J Chem Tech Biot, 31 (10), 624 (1981). D. S. CHAUDHARY, M. C. JOLLANDS, F. CSER: Understanding Rice Hull Ash as Fillers in Polymers: A Review. Silicon Chem, 1 (4), 281 (2002). T. H. LioU: Evolution of Chemistry and Morphology during the Carbonization and Combustion of Rice Husk. Carbon, 42 (4), 785 (2004). G. Crini: Non-conventional Low-cost Adsorbents for Dye Removal: A Review. Bioresour Technol, 97 (9), 1061 (2006). S. CHANDRASEKHAR, K. G. SATYANARAYANA, P. N. PRAMADA, P. RAGHAVAN, T. N. GUPTA: Review Processing, Properties and Applications of Reactive Silica from Rice Husk. An Overview. J Mater Sci, 38 (15), 3159 (2003). M. Y. AHMAD FUAD, Z. ISMAIL, M. S. MANSOR, Z. A. MOHD ISHAK, A. K. MOHD OMAR: Mechanical Properties of Rice Husk Ash/Polypropylene Composites. Polym J, 27 (10), 1001 (1995). R. PUCCIARIELLO, V. VILLANI: Room-temperature Transitions and Melting of Tetraluoroethylene-hexaluoropropylene Copolymers with Low Comonomer Contents. Thermochim Acta, 227, 145 (1993). V. VILLANI, R. PUCCIARIELLO: Calorimetric Study of the Room-temperature Transitions of Tetraluoroethylene-hexaluoropropylene Copolymer: Thermal History and Crystalline state. Thermochim Acta, 199, 247 (1992). A. ATANASSOV, S. GENIEVA, L. VLAEV: Study on the Thermooxidative Degradation Kinetics of Tetraluoroethylene-ethylene Copolymer Filled with Rice Husks Ash. Polym Plast Technol Eng, 49, 541 (2010). J. M. CroSBy, C. a. Carreno, K. L. taLLey: Melt Processible Fluoropolymer Composites. Polym Comp, 3 (2), 97 (1982). J. t. CHERIAN, D. G. CaStner: ESCA Characterization of Fluoropolymer Film Residue on Carbon-iber-reinforced Plastic Components. Surf Interf Anal, 29 (11), 729 (2000). S. D. GENIEVA, L. T. VLAEV, A. N. ATANASSOV: Study of the Thermooxidative Degradation Kinetics of Poly(Tetraluoroethene) Using Iso-conversional Calculation Procedure. J Therm Anal Calorim, 99, 551 (2010). a. atanaSSov, S. Genieva, L. vLaev, D. KiryaKova: Study on the Properties and the Thermooxidative Degradation Kinetics of Composites Based on Tetraluoroethylene-hexaluoropropylene Copolymer Composites with Black Rice Husk Ash. Oxid Commun, 35 (4), 869 (2012). L. VLAEV, P. PETKOV, A. DIMITROV, S. GENIEVA: Cleanup of Water Polluted with Crude Oil or Diesel Fuel Using Rice Husks Ash. J Taiwan Inst Chem Eng, 42, 957 (2011). A. W. COATS, J. P. REDFERN: Kinetic Parameters from Thermogravimetric Data. Nature (London), 201, 68 (1964). G. E. ZAIKOV, S. M. LOMAKIN, E. V. KUVARDINA, L. A. NOVOKSHONOVA, N. G. SHILKINA, R. KOZLOWSKI: Thermal and Combustion Behaviour of PP/MWCNT Composites. Oxid Commun, 35 (2), 438 (2012). t. OZAWA: A New Method of Analyzing Thermogravimetric Data. Bul Chem Soc Jpn, 38, 881 (1965).

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26. J. SeStaK, G. BerGGren: Study of the Kinetics of the Mechanism of Solid-state Reactions at Increasing Temperatures. Thermochim Acta, 3, 1 (1971). 27. L. vLaev, v. GeorGieva, S. Genieva: Product and Kinetics of Non-isothermal Decomposition of Vanadium(IV) Oxide Compounds. J Therm Anal Calorim, 88 (3), 805 (2007). 28. T. G. CHUAH, A. JUMASIAH, I. AZNU, S. KATAYON, S. Y. CHOONG: Rice Husks as a Potentially Low-cost Biosorbent for Heavy Metal and Dye Removal: An Overview. Desalination, 175, 305 (2005). 29. S. Ch. tUrManova, S. D. Genieva, a. S. DiMitrova, L. t. vLaev: Non-isothermal Degradation Kinetics of Filled with Rice Husk Ash Polypropene Composites. Express Polym Lett, 2 (2), 133 (2008). Received 20 January 2014 Revised 2 March 2014

415

Oxidation Communications 37, No 2, 416–423 (2014) Ionic oxidation in liquid phase

kinetiCS and meChaniSm of oxidation of 5-SulPhoSaliCyliC aCid By dihydroxydiPeriodatoniCkelate(iv) in aqueouS medium JINHUAN SHAN*, ZIWEI ZHANG,YI LI, JIN HAN College of Chemistry and Environmental Science, Hebei University, Baoding, 071 002 Hebei, China E-mail: [email protected]

aBStraCt The kinetics of oxidation of 5-sulphosalicylic acid by dihydroxydiperiodatonickelate(iv) (DPN) had been studied spectrophotometrically in alkaline medium in the temperature range of 293.2 to 313.2 K. The reaction rate showed irst order dependence in DPN and fractional order in 5-sulphosalicylic acid. the rate increased with the increase of [OH–] and decreased with the increase of [IO4–]. In addition, the reaction has negative salt effect and no free radical was detected. A mechanism based on experimental results was proposed, and the rate constants of the rate-determining step in the mechanism were evaluated. The rate equations derived from mechanism can explain all experimental observations. Keywords: dihydroxydiperiodatonickelate(iv), 5-sulphosalicylic acid, oxidation, kinetics and mechanism. aiMS anD BaCKGroUnD 5-Sulphosalicylic acid is a kind of poisonous substituted aromatic compounds, which is dificult to oxidation and hardly biodegradable. It often is used for synthetic drug intermediates and synthetic dyes. It also is used as catalysts. In the synthetic process, these substances produce some wastewater which contains 5-sulphosalicylic acid. It causes pharmaceutical pollution, and the appearance of turbidity wastewater containing 5-sulphosalicylic acid which is dificult to purify by conventional methods. Transition metals in a higher oxidation state generally can be stabilised by chelation with suitable polydentate ligands. Diperiodatonickelate(IV) (Refs 1 and 2) is a good oxidant in a medium with an appropriate pH value. Ni(IV) complexes have *

For correspondence.

416

been employed as oxidising agents for the investigation of some organic compounds. Currently using diperiodatonickelate(IV) for oxidation of amino acid3,4, drugs5 and catalytic oxidation has become a research hotspot. In addition, a new chemiluminescence (CL) (Ref. 6) reaction that occurs between luminol and diperiodatonickelate in alkaline medium had been reported. In the present paper, the mechanism of oxidation of 5-sulphosalicylic acid by Ni(IV) is reported. EXPERIMENTAL Reagents and instrumentation. All the chemicals used were of AR grade. All solutions were prepared with doubly distilled water. The solution for oxidation was prepared and standardised by the method of Murthy7. The concentration of DPN was derived from its absorption at 410 nm. The solution for oxidation and 5-sulphosalicylic acid was always freshly prepared before use. Its UV spectrum was found to be consistent with that reported in literature. The ionic strength was maintained by adding KNO3 solution and the pH value of the reaction mixture was regulated with KOH solution. Measurements of the kinetics were performed on a UV-vis. spectrophotometer (TU1901, Beijing Puxi Inc., China), which had a cell holder kept at constant temperature (± 0.1oC) by circulating water from a thermostat (DC-2010, Baoding, China). All other species did not show absorption signiicantly at this wavelength. Kinetics measurements. All kinetics measurements were carried out under pseudo-irst order conditions. 2 ml of the oxidation solution (2 ml) containing required concentration of Ni(IV), OH–, io4– and ionic strength and reductant solution (2 ml) of requisite concentration were transferred separately to the upper and the lower branch type 2-cell reactor. After thermal equilibration at the desired temperature in a thermostat, the two solutions were mixed well and immediately transferred into a 1-cm thick rectangular quartz cell in a constant temperature cell-holder (±0.1oC). The reaction process was monitored automatically by recording with a TU-1900 spectrophotometer. The complete fading of the DPN colour (wine red) marked the completion of the reaction. reSULtS anD DiSCUSSion Evaluation of pseudo-irst order rate constants. Under the conditions of [5-sulphosalicylic acid]0 >> [Ni(IV)]0, the plots of ln(At – A∞) versus time were straight lines, indicating that the reaction was irst order to Ni(IV),where At and A∞ were the absorbency at time t and at ininite time, respectively. The pseudo-irst order rate constants kobs were calculated by the method of least squares (r > 0.998). Generally, kobs values were the averaged values of at least 3 independent experiments, and reproducibility was within ±5%. Dependence of rate on the concentration of 5-sulphosalicylic acid. At ixed concentration of Ni(IV), OH–, io4– and μ constant, the values of kobs were determined at different temperatures and [5-sulphosalicylic acid] concentrations. The plots of 417

kobs versus [5-sulphosalicylic acid] were straight lines through the origin at different temperatures according to the following equation: kobs = m [R′].

(1)

From equation (1) we can indicate that the reaction order dependence on 5-suphoO

salicylic acid was irst order and R′ represents 5-sulphalicylic acid ( (Fig. 1, r ≥ 0.997).

O–

OH

)

2H2O HO3S

313.2 K 2.5

kobs × 102 (s–1)

2.0

308.2 K

1.5 303.2 K

1.0

298.2 K 0.5 293.2 K 0.0 0

1

2

3

4

5

[5-suphosalicylic acid] × 102 (mol l–1)

fig. 1. Plots of kobs versus [5-sulphosalicylic acid] (r ≥ 0.997) [DPN] = 4.75×10–6 mol l–1; [IO4–] = 1.00×10–3 mol l–1; [OH–] = 1.00×10–2 mol l–1; µ = 1.71×10–1 mol l–1

Dependence of rate on IO4– concentration. At ixed concentration of Ni(IV), 5-sulphosalicylic acid, OH–, μ and temperature, the experimental results indicate that the values of kobs decrease with the increase of the concentration of IO4−. The order with respect to [IO4−] was negative fractional and the plot of 1/kobs versus [IO4−] was linear which corresponds to the equation at different temperatures: 1/kobs = a + b [IO4–]

(2)

Equation (2) shows that there was a pre-equilibrium involving the process of dissociation of H2io63– from the Ni(IV) complex (Fig. 2, r ≥ 0.997).

418

293.2 K 360 298.2 K

1/kobs (s)

300 240 303.2 K 180 308.2 K 120 313.2 K 60 0.5

1.0

1.5

2.0

2.5

[IO4–] × 103 (mol l–1)

fig. 2. Plots of 1/kobs versus [IO4–] (r ≥ 0.997) [DPN] = 4.75×10–6 mol l–1; [5-sulphosalicylic acid] = 3.00×10–2 mol l–1; [OH–] = 1.00×10–2 mol l–1; µ = 1.71×10–1 mol l–1

Dependence of rate on OH− concentration. At constant [Ni(IV)], [5-sulphosalicylic acid], [IO4−], μ and temperature, the value of kobs increased rapidly with the increase in [OH–].The order with respect to [OH–] was fractional and the plot of 1/kobs versus f([OH–])/[OH–] was linear with a positive intercept (Fig. 3). φ([OH–])/[OH–] (mol l–1) 180

0.5

0.6

0.7

0.8

0.9

170 160

1/kobs (s)

150

搬

140 帆

130 120 110 100 90 0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

f([OH–])/[OH–] (mol l–1)

fig. 3. Plots of 1/kobs versus f([OH–])/[OH–] (I) compared with 1/kobs versus φ([OH–])/[OH–] (ii) at 303.2 K (r >0.997) [DPN] = 4.75×10–6 mol l–1; [5-sulphosalicylic acid] = 3.00×10–2 mol l–1; [IO4–] = 1.00×10–3 mol l–1; µ = 1.71×10–1 mol l–1

419

Dependence of rate on ionic strength. With other conditions ixed, kobs increased with increase in ionic strength when 5-sulphosalicylic acid was oxidised by Ni(IV), indicating that there was a negative salt effect to 5-sulphosalicylic acid which is consistent with the common kinetics regularities8 (Table 1). table 1. Rate dependence on ionic strength µ at 303.2 K [DPN]= 4.96×10–6 mol l–1; [5-sulphosalicylic acid]= 1.00×10–2 mol l–1; [OH–]=1.00×10–2 mol l–1; [IO4–] =1.00×10–3 mol l–1

10 μ (mol l–1) 5-sulphosalicylic acid (kobs ×103(s–1)

1.61 11.18

2.61 10.92

3.61 10.42

4.61 10.06

5.61 9.85

DISCUSSION ON THE REACTION MECHANISM

In alkaline solution, equilibria (3)–(5) were observed and the corresponding equilibrium constants at 298.2 K were determined by Aveston9 as follows: H2i2o104– lgβ1 = 15.05

(3)

io4 + OH +H2o

H3io6 lgβ2 = 6.21

(4)

io4– + 2OH–

H2io63– lgβ3 = 8.67

(5)

2io4– + 2OH– –

–

2–

The distribution of all periodate species in alkaline solution can be calculated from equilibria (3)–(5). The amount of dimer H2i2o104– and io4– species can be neglected, the main species of periodate are H3io62– and H2io63–. This is consistent with the result calculated from the Crouthamel data8,10 by Murthy. Based on such a distribution, the formula of Ni(IV) periodate complex is represented by the less protonated ionic species [Ni(OH)2(H2io6)2]4–. We preferred to use [Ni(OH)2(H2io6)2]4– to present DPN because it is close to the formula suggested by Mukherjee10 and is supported by the kinetic studies. According to the above experimental results and discussion, a plausible reaction mechanism was proposed: K

[Ni(OH)2(H2io6)2]4– + OH– –––––→ [Ni(OH)2(HIO6)]2– + H2io63– + H2o DPN MPN

(6)

k

[Ni(OH)2(HIO6)]2– + R′ –––––→ adduct MPN

(7)

fast

adduct –––––→ Ni(IV) + product

(8)

Reaction (6) is dissociation and deprotonation, whose reaction rate is generally fast. Reaction (7) belongs to an electron-transfer reaction11, so reaction (7) is the rate-determining step.

420

As the rate of the disappearance of Ni(IV) was monitored, the rate of the reaction can be derived as follows: –

d[Ni(IV)t

kK[OH–][R′][Ni(IV)]t

=

dt

= kobs[Ni(IV)]t

[H2io63–] + K[OH–] kK[OH–][R′]

kobs =

[H2io63–] + K[OH–]

,

(9)

(10)

where [Ni(IV)]t = [DPN]e + [MPN]e = [MPN]e([H2io63–] + k[OH–])/(k[OH–]); subscripts t and e stand for total concentration and concentration at equilibrium, respectively. Neglecting the concentration of ligand dissociated from Ni(IV) and the species of periodate other than H2io63– and H3io62–, equations (11) and (12) can be obtained from equations (4) and (5): [H2io63–] =

[H3io62–] =

β3[OH–][IO–]ex β2 + β3[OH–] β2[IO–]ex β2 + β3[OH–]

= f([OH–])[IO4–]ex

(11)

= φ([OH–])[IO4–]ex,

(12)

where [IO4–]ex represents the original overall entering periodate and equals approximately to the sum of [H2io63–] and [H3io62–]. Substituting equation (11) into equation (10), we can get the expression of pseudoirst order rate constants as follows: 1 kobs 1 kobs

=

1 k[R′] 1

=

k[R′]

[IO4–]ex f([OH–])

+

+

kK[R′]

(13)

[OH–]

f([OH–]) kK[R′][OH–]

[IO4–]ex.

(14)

Equation (10) suggests that the plot of kobs versus [R′] should be linear, and equation (13) shows that the plot of 1/kobs versus f([OH–])/[OH–] should also be linear, which are consistent with the experimental results (Fig. 2). If the formula of DPN was [Ni(OH)2(H3io6)2]2– , equation (15) would be obtained instead of equation (14): 1 kobs

=

1 k[R′]

+

[IO4–]ex

φ([OH–])

kK[R′]

[OH–]

.

(15)

The plot of 1/kobs versus φ([OH–])/[OH–] should also be linear, but the linearity was not straight (Fig. 2), which substantially denies equation (13). Therefore, it seems advisable to represent DPN by [Ni(OH)2(H2io6)2]4–, which is consistent with the experimental result. 421

Meanwhile, the plots of 1/kobs versus [IO4–] were linear at different temperatures. From their slopes, the rate-determining step constants k were evaluated. The activation parameters data of 5-sulphosalicylic acid were obtained12,13 (Table 2). table 2. rate constants (k) and activation parameters of the rate-determining step at 298.2 K

T (K) 293.2 298.3 303.2 308.2 313.2 9.07 14.51 24.65 42.74 76.33 k×102 (mol–1 l s–1) –1 * –1 Thermodynamic activation parameters Ea= 81.46 kJ mol , ΔH = 79.98 kJ mol , ΔS*= –224.729 J K–1 mol–1

The plots of ln k versus 1/T have the following intercept (A), slope (B) and relative coeficient (r): A = –9797.93; B = 30.96; r = 0.998. ConCLUSionS In this paper, using dihydroxydiperiodatonickelate(iv) oxide 5-sulphosalicylic acid and the study of its mechanism, monoperiodatonickelate is considered as the active species. Based on the above discussion and results, we know that the rate constant k of the rate-determining step and the activation parameters for 5-sulphosalicylic acid are contiguous. The mechanism described here can well explain the experimental rules. In addition14, the experimental study of medicine provides a new direction for the wastewater treatment of 5-sulphosalicylic acid; for the development of the whole ield of chemical dynamics provides the basis data, as well as it provides an important theoretical basis for the application of environmental governance. reFerenCeS 1. S. S. RAMESH, T. N. SHARANAPPA: Kinetic, Mechanistic and Spectral Investigations of Ruthenium(III)-catalysed Oxidation of 4-Hydroxycoumarin by Alkaline Diperodatonickelate(IV) (Stopped Flow Technique). J Mol Catal A: Chem, 234, 137 (2005). 2. V. H. CHANABASAYYA, C. H. DEEPAK, T. N. SHARANAPPA: Ruthenium(III) Catalysed Oxidation of Gabapentin (Neurontin) by Diperiodatonickelate(IV) in Aqueous Alkaline Medium. A Kinetic and Mechanistic Study. J Mol Catal A: Chem, 269, 246 (2007). 3. J. H. SHAN, H. Y. WEI, L. WANG, B. S. LIU, S. G. SHEN, H. W. SUN: Kinetics and Mechanism of Oxidation of Some Amino Acids by Diperodatonickelate(IV) Complex in Aqueous Alkaline Medium. Indian J Chem A, 40, 865 (2001). 4. P. J. TIMY, M. T. SURESH, W. J. NIU, Y. ZHU, K. C. HU, C. L. TONG, H. S. YANG: Kinetics of Oxidation of SCN− by Diperiodato Cuprate(III) (DPC) in Alkaline Medium. Int J Chem Kinet, 28, 12 (1996). 5. C. Y. YANG, Z. J. ZHANG, J. L. WANG: A New Luminol Chemiluminescence Reaction Using a Tetravalent Nickel–Periodate Complex as the Oxidant. Microchim Acta, 167, 91 (2009). 6. U. CHANDRAIAH, C. P. MURTHY, D. K. SUSHAMA: Kinetics of Oxidation of Lactic, Mandelic and Glycollic Acids by Diperiodatonickelate(IV) in Alkaline Medium. Indian J Chem A, 28, 162 (1989).

422

7. L. C. W. BAKER, S. B. MUKHERJEE: Synthesis and Characterization of Lithium Hexaorthoperiodate Nickelate(IV). Indian J Chem A, 21, 618 (1982). 8. R. T. MAHESH, P. D. POL, S. T. NANDIBEWOOR: Kinetics and Mechanism of Oxidation of L-leucine by Alkaline Diperiodatonickelate(IV): A Free Radical Intervention, Deamination, and Decarboxylation. Monatsh Chem, 134, 1341 (2003). 9. J. AVESTON: Hydrolysis of Potassium Periodate, Ultracentrifugation Potentiometric Titration and Raman Spectra. J Chem Soc A, 273 (1969). 10. H. G. MUKHERJEE, B. MANDAL, S. DE: Preparation and Studies of the Complex Periodatoferrate(III) Hexahydrare and Periodatonickelate(IV) Monohydrate. Indian J Chem A, 23, 426 (1984). 11. Z. T. LI, F. L. WANG, A. Z. WANG: Kinetics and Mechanism of Oxidation of Tetrahydrofuryl Alcohol by Dihydroxydiperiodatonickelate(IV) Complex in Aqueous Alkaline Medium. Int J Chem Kinet, 24, 933 (1992). 12. J. H. SHAN, S. Y. HUO, S. G. SHEN, H. W. SUM: Kinetics and Mechanism of Oxidation of 1,3butylene Glycol by Dihydroxyditellutoargentate(III) in Alkaline Medium. Turkish J Chem, 30, 65 (2006). 13. K. M. NAIK, S. T. NANDIBEWOOR: Kinetic and Mechanistic Study of Oxidation of Succinamide by Diperiodatocuprate(III). Oxid Commun, 35, 545 (2012). 14. J. H. SHAN, Y. LI, H. JIN, Z. W. ZHANG: Oxidation of Valine and 2-Aminoisobutyric Acid by Diperiodatocuprate(III) in Alkaline Medium. A Kinetic and Mechanistic Study. Oxid Commun, 36, 720 (2013). 15. C. P. MURTHY, B. SETHURAM, T. RAO: Oxidation by Tetravalent Nickel. Part 1: Kineties of Electron Transfer from Some Aliphatic Alcoholos to Ni (IV) in Aqueous Alkaline Media. Z Phys Chem. (Leizig), 287, 1212 (1986). 16. W. J. NIU, Y. ZHU, K. C. HU, C. L. TONG, H. S. YANG: Kinetics of Oxidation of SCN– by Diperiodatocuprate(III) (DPC) in Alkaline Medium. Int J Chem Kinet, 28, 899 (1996). 17. J. QIAN, M. Z. GAO, J. H. SHAN, S. G. SHEN, H. W. SUN: Kinetics of Oxidation of Ethanolamine by Dihydroxydiperiodatonickelate(IV) in Alkaline Media. J Guangxi Normal Univ, 21 (1), 248 (2003). Received 15 October 2013 Revised 20 December 2013

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Oxidation Communications 37, No 2, 424–439 (2014) Oxidation in the presence of Cr- and Mn-containing oxidants

kinetiCS and Correlation analySiS of reaCtivity in the oxidation of SuBStituted BenzaldehydeS By Benzimidazolium diChromate R. KUMAR, D. PANDEY, S. KOTHARI* Department of Chemistry, J N V University, 4F/13 New Power House Road, 342 001 Jodhpur, India E-mail: [email protected]; [email protected]

aBStraCt Kinetic and mechanistic studies of the oxidation of a number of para-, meta- and ortho-substituted benzaldehydes by benzimidazolium dichromate (BIDC), in dimethyl sulphoxide, were discussed with an emphasis on correlation of structure and reactivity. The product of the oxidation is the corresponding benzoic acid. The reaction is irst order with respect to BIDC, however, the dependence is of second order with respect to hydrogen ion. The Michaelis–Menten type kinetics was observed with respect to aldehyde. The deuterium isotope effect for the oxidation of [2H] benzaldehyde (kH/kD = 6.17 at 293 K) indicated an α-C–H bond cleavage in the rate-determining step. Based on the kinetic data, analyses of the solvent effect and results of structure–reactivity correlation along with some non-kinetic parameters suggested a mechanism involving rate-determining oxidative decomposition of an aldehyde–BIDC complex via a cyclic transition state to give a carbocationic species through hydride–ion transfer from the aldehyde to the oxidant. Keywords: kinetics, oxidation, substituted benzaldehydes, benzimidazolium dichromate, correlation analysis. aiMS anD BaCKGroUnD The aim of the research was to study the kinetics and mechanism of the oxidation of a number of para-, meta- and ortho-substituted benzaldehydes by benzimidazolium dichromate (BIDC), in dimethyl sulphoxide, with an emphasis of correlation of structure and reactivity. Selective oxidation of organic compounds under non-aqueous conditions is an important transformation in synthetic organic chemistry. For this, a number of different *

For correspondence – 4F/13, New Power House Road, 342 001 Jodhpur, India.

424

chromium(VI) derivatives have been reported1–5. In 1998, Meng et al.6 reported a new Cr(VI) derivative–benzimidazolium dichromate (BIDC). It is neither hygroscopic nor light sensitive, therefore, it is more stable and easily stored as compared to other Cr(VI) reagents. BIDC is reported3 to convert benzylic and allylic alcohols to corresponding carbonyl compounds in yield ranging from 75 to 98%. We have been interested in the kinetics and mechanism of the oxidation by newer Cr(VI) derivatives. There seems to be only 3 reports available on the kinetic and mechanistic aspects of the oxidation by BIDC (Refs 7–9), published from our laboratory. In the continuation of our studies, we report in this paper the kinetics and correlation analyses of organic reactivity in the oxidation of a number of monosubstituted benzaldehydes by BIDC in dimethyl sulphoxide (DMSO) as the solvent. The mechanistic aspects are discussed. EXPERIMENTAL Materials. BIDC was prepared by the reported method6 and its purity was checked by an iodometric method. The preparation and puriication of the aldehydes have been reported earlier10. [2H]Benzaldehyde (PhCDO) was prepared by the reported method11. Toluene p-sulphonic acid (TsOH) was used as a source of hydrogen ions. The solvents were puriied by the reported methods12. Amongst the solvents, CS2 is a lammable toxic liquid. Product analysis. The product analysis was carried out under kinetic conditions. In a typical experiment, freshly distilled benzaldehyde (0.05 mol) and BIDC ( 0.01 mol) were made up to 100 ml in DMSO. The reaction mixture was allowed to stand for ca.20 h to ensure completion of the reaction. It was rendered alkaline with NaOH, iltered, and the iltrate evaporated to dryness under reduced pressure. The residue was dissolved in minimum quantity of dil. HCl and cooled in crushed ice to yield crude acid. This was further recrystallised from hot water to produce pure benzoic acid (3.33 g, 91%, m.p. 120°C). Stoichiometry. To determine the stoichiometry, BIDC (0.005 mol) and benzaldehyde (0.001–0.003 mol) were made up to 100 ml in DMSO in the presence of 1.0 mol dm−3 TsOH. The reaction was allowed to stand for ca. 20 h to ensure the completion of the reaction. The residual BIDC was determined spectrophotometrically at 364 nm. Several determinations, with differently substituted benzaldehydes with their different concentrations showed that the stoichiometry is 3:1, i.e. 3 mol of aldehyde are consumed by 1 mol of oxidant. The results with benzaldehyde are presented in Table 1. BIDC thus acts as a 6-electron oxidant and is reduced to Cr(III).

425

table 1. Stoichiometry of the oxidation of benzaldehyde by BIDC

[BIDC] ×103 (mol dm–3) 5.0 5.0 5.0 Mean = 3.02

[Benzaldehyde] × 103 [Residual BIDC] ×103 (mol dm–3) (mol dm–3) 1.0 4.67 2.0 4.32 3.0 4.03

[Benzaldehyde]/ [consumed BIDC] 3.03 2.94 3.09

Kinetic measurements. Pseudo-irst order conditions were attained by keeping a large excess (× 20 or greater) of the aldehyde over the oxidant. The reactions were carried out at constant temperature (±0.1 K). The solvent was DMSO, unless stated otherwise. The reactions were followed by monitoring the decrease in the concentration of BIDC at 364 nm for up to 80% of the reaction. The Beer law is valid for BIDC within the concentration range used in our experiments. The pseudo-irst order rate constant, kobs, was evaluated from the linear (r2 > 0.995) plots of lg [BIDC] versus time. Duplicate kinetic runs showed that the rate constants were reproducible to within ±3%. In correlation analyses, we have used coeficient of determination (R2 or r2), standard deviation (SD) and the Exner parameter13, ψ, as measures of the goodness of it. reSULtS The rate and other experimental data were obtained for all the aldehydes. Since the results are similar, only representative data are reproduced here. The oxidation of benzaldehyde resulted in the formation of the corresponding benzoic acid. The product analysis and stoichiometric determinations indicated the following overall reaction: 3 PhCHO + Cr2o72– + 8 H+ → 3 PhCOOH + 4 H2o + 2 Cr3+.

(1)

Test for free radical. The oxidation of benzaldehyde by BIDC, in an atmosphere of nitrogen, failed to induce polymerisation of acrylonitrile. In blank experiments, with the aldehyde absent, no noticeable consumption of BIDC was observed. The addition of acrylonitrile had no effect on the rate of oxidation (Table 2). To further conirm the absence of free radicals in the reaction pathway, the reaction was carried out in the presence of 0.05 mol dm–3 of 2,6-di-t-butyl-4-methylphenol (butylated hydroxytoluene or BHT). It was observed that BHT was recovered unchanged, almost quantitatively.

426

table 2. Rate constants for the oxidation of benzaldehyde by BIDC at 313 K

[PhCHO] (mol dm–3) 0.10 0.20 0.30 0.50 0.80 1.50 0.50 0.50 0.50 0.50 0.50 1.50 1.50 *

and

**

[BIDC] ×103 (mol dm–3) 1.0 1.0 1.0 1.0 1.0 1.0 0.5 0.8 2.0 3.0 5.0 1.0 1.0

[H+] (mol dm–3) 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0

kobs ×105 (s−1) 8.96 33.3 61.9 130 187 240 123 132 141 127 136 233* 245**

contained 0.001 and 0.005 mol dm−3 acrylonitrile.

Rate laws. The reactions were found to be irst order with respect to BIDC. In individual kinetic runs, plots of lg [BIDC] versus time were linear (r2 > 0.995). Further, it was found that the observed rate constant, kobs, does not depend on the initial concentration of BIDC. The order with respect to aldehyde was more than one but less than two (Table 2). A plot of 1/kobs versus 1/[aldehyde]2 was linear with an intercept on the rate ordinate. Thus, the Michaelis–Menten type kinetics was observed with respect to the aldehyde. This leads to the postulation of the following overall mechanism and the rate law: K

2 aldehyde + BIDC

[complex]

k2

(2)

[complex] → products

(3)

rate = k2 K [aldehyde]2 [BIDC]t/(1 + K [aldehyde]2)

(4)

(rate/[BIDC]t )–1 = 1/kobs = 1/k2K[aldehyde]2 + 1/k2

(5)

or where [BIDC]t = [BIDC] + [complex]. The dependence of kobs on the concentration of aldehyde was studied at different temperatures and the values of K and k2 were evaluated from the double reciprocal plots using equation (5). The thermodynamic parameters for the complex formation and the activation parameters for decomposition of the complexes were calculated from the values of K and k2, respectively, at different temperatures (Tables 3 and 4).

427

table 3. Formation constants and thermodynamic parameters for benzaldehyde–BIDC complexes

Subst. H p-Me p-OMe p-F p-Cl p-Br p-CF3 p-CO2Me p-NO2 p-NHAc p-CN p-SMe p-NMe2 m-Me m-OMe m-Cl m-Br m-F m-NO2 m-CO2Me m-CF3 m-CN m-SMe m-NHAc o-Me o-OMe o-NO2 o-CO2Me o-NHAc o-Cl o-Br o-I o-CN o-SMe o-F o-CF3 PhCDO

428

K (dm3 mol−1) 293 K 303 K 313 K 323 K 4.61 3.95 3.34 2.83 4.30 3.78 3.13 2.72 4.90 4.20 3.61 3.14 4.19 3.60 3.20 2.81 3.91 3.47 3.09 2.78 4.95 4.28 3.70 3.25 4.32 3.60 3.11 2.65 4.06 3.64 3.23 2.93 4.61 4.05 3.61 3.27 4.50 3.94 3.40 3.03 4.30 3.65 3.19 2.77 4.25 3.72 3.30 3.00 4.35 3.73 3.22 2.83 4.65 4.05 3.51 3.08 4.25 3.77 3.33 2.95 4.87 4.14 3.55 3.06 4.15 3.68 3.28 2.96 4.00 3.50 3.08 2.77 4.61 3.99 3.43 2.97 4.85 4.15 3.55 3.05 4.51 3.85 3.37 2.96 4.65 4.05 3.62 3.20 4.31 3.70 3.18 2.79 4.51 3.93 3.44 3.06 4.63 3.90 3.40 2.96 4.71 4.13 3.59 3.12 4.00 3.55 3.14 2.83 4.38 3.75 3.30 2.93 4.80 4.15 3.61 3.15 4.45 3.93 3.44 3.02 4.27 3.76 3.31 2.96 4.91 4.25 3.70 3.23 4.58 4.08 3.62 3.19 4.67 4.00 3.48 3.01 4.81 4.10 3.53 3.11 4.08 3.65 3.20 2.88 4.65 3.90 3.31 2.85

∆H (kJ mol–1) −15.3±0.2 −14.6±0.1 −14.1±0.1 −12.9±0.2 −11.5±0.1 −13.6±0.1 −15.2±0.3 −13.5±0.4 −11.5±0.3 −13.0±0.2 −13.9±0.2 −11.7±0.2 −13.8±0.1 −13.3±0.2 −12.1±0.2 −14.7±0.1 −11.4±0.1 −12.2±0.1 −14.1±0.2 −14.7±0.1 −13.5±0.2 −12.2±0.2 −14.0±0.1 −12.7±0.1 −14.1±0.2 −13.3±0.2 −11.6±0.2 −13.0±0.2 −13.5±0.1 −12.7±0.2 −12.1±0.1 −13.5±0.3 −12.0±0.4 −14.0±0.4 −14.0±0.2 −14.0±0.2 −15.3±0.1

∆S (J mol–1 K–1) −31±1 −30±1 −27±1 −24±2 −20±1 −25±1 −32±2 −26±1 −19±1 −24±1 −27±2 −20±1 −27±1 −25±1 −21±2 −29±1 −19±1 −22±2 −27±1 −29±1 −26±2 −21±1 −27±3 −23±2 −28±3 −24±2 −20±1 −24±1 −25±1 −23±1 −21±1 −25±2 −20±1 −27±1 −27±1 −20±1 −32±1

∆G (kJ mol–1) −6.09±0.2 −5.91±0.1 −6.24±0.1 −5.86±0.2 −5.73±0.1 −6.28±0.1 −6.28±0.1 −5.90±0.3 −6.12±0.3 −6.05±0.3 −5.91±0.3 −5.91±0.2 −5.95±0.2 −6.13±0.2 −5.93±0.2 −6.22±0.1 −5.87±0.1 −5.76±0.2 −6.10±0.2 −6.21±0.2 −6.03±0.3 −6.14±0.2 −5.92±0.3 −6.06±0.2 −6.08±0.4 −6.77±0.3 −5.78±0.2 −5.96±0.1 −6.20±0.1 −6.04±0.2 −5.93±0.1 −6.26±0.2 −6.12±0.4 −6.12±0.3 −6.19±0.2 −5.84±0.3 −6.08±0.1

table 4. Rate constants for the decomposition of benzaldehyde–BIDC complexes and their activation parameters

Subst. H p-Me p-OMe p-F p-Cl p-Br p-CF3 p-CO2Me p-NO2 p-NHAc p-CN p-SMe p-NMe2 m-Me m-OMe m-Cl m-Br m-F m-NO2 m-CO2Me m-CF3 m-CN m-SMe m-NHAc o-Me o-OMe o-NO2 o-CO2Me o-NHAc o-Cl o-Br o-I o-CN o-SMe o-F o-CF3 PhCDO kH/kD

k2 × 105 ( s–1) ∆S* ∆H* –1 293 K 303 K 313 K 323 K (kJ mol ) (J mol–1 K–1) 41.7 107 278 677 70.7±0.7 −69±2 136 325 780 1760 64.8±0.5 −79±2 855 1660 3510 7250 53.8±1.3 −102±4 98.8 215 523 1210 63.5±1.5 −87±5 57.9 138 341 785 66.1±0.8 −82±3 57.5 137 339 775 66.0±0.8 −82±3 6.35 17.6 52.5 143 79.5±1.2 −55±4 10.3 29.5 83.5 183 73.7±1.5 −70±5 3.00 9.23 26.8 69.1 79.9±0.5 −59±2 330 699 1560 3280 58.0±0.9 −95±3 4.95 14.0 42.3 107 78.7±0.9 −59±3 540 1100 2390 4830 55.3±0.9 −100±3 11900 19700 35500 63100 41.4±1.3 −122±4 69.8 173 420 1010 67.5±0.8 −76±3 53.7 132 355 881 71.2±1.3 −65±4 11.3 33.6 96.5 255 79.3±0.2 −50±1 11.4 33.5 96.0 255 79.1±0.3 −51±1 11.6 34.9 99.1 263 79.4±0.2 −50±1 1.89 6.80 21.8 64.1 89.8±0.5 −29±2 9.22 24.8 75.3 199 78.6±1.4 −54±5 5.64 18.5 55.5 154 84.2±0.3 −39±1 3.29 10.2 30.7 90.8 84.4±0.8 −43±3 46.0 120 311 766 71.3±0.6 −66±2 39.6 101 269 673 72.0±1.0 −65±3 22.0 62.5 173 411 74.6±0.7 −61±2 97.6 247 639 1460 68.8±0.6 −68±2 0.95 3.61 11.2 32.3 89.7±1.1 −35±4 2.28 7.61 22.9 61.7 84.0±0.6 −47±2 16.4 53.5 151 355 78.3±1.6 −50±5 6.15 18.7 56.1 142 80.2±0.7 −52±2 4.33 15.6 47.2 122 85.1±1.6 −38±5 3.75 12.4 35.6 97.0 82.6±0.5 −48±2 1.24 5.08 15.4 43.1 90.1±2.0 −31±6 30.2 85.5 225 537 73.1±0.4 −63±2 19.5 58.0 163 489 81.6±1.3 −38±4 0.43 1.79 5.83 16.8 93.4±1.8 −29±6 6.76 18.6 50.8 129 75.0±0.5 −69±2 6.17 5.75 5.47 5.25

∆G* (kJ mol–1) 91.1±0.5 88.3±0.4 84.0±1.1 89.2±1.2 90.4±0.6 90.4±0.6 95.7±0.9 94.4±1.2 97.4±0.4 86.2±0.7 96.2±0.7 85.0±0.7 77.6±1.0 89.9±0.6 90.5±1.0 94.2±0.2 94.1±0.3 94.1±0.1 98.4±0.4 94.8±1.1 95.8±0.2 97.2±0.7 90.8±0.4 91.2±0.8 92.6±0.5 89.0±0.5 100±0.9 98.0±0.5 93.1±1.3 95.6±0.6 96.3±1.2 96.8±0.4 99.3±1.6 91.8±0.3 92.8±1.1 102±1.4 95.5±0.4

429

Effect of hydrogen ions. The reaction rate increases with an increase in the concentration of hydrogen ions. The dependence on the hydrogen ion concentration is of second order (Table 5). The dependence of the reaction rate on the concentration of benzaldehyde was studied at [H+] = 0.2, 0.4 and 1.0 mol dm−3. It was observed that the formation constant, K, does not vary appreciably with the hydrogenion–ion concentration. This leads to the conclusion that the change in the hydrogen-ion concentration affects only the rate of decomposition of the complex, k2. Similar observations have been made in the oxidation of aldehydes14,15 and oxyacids of phosphorus16 by butyltriphenylphosphonium dichromate (BTPPD). table 5. Dependence of the reaction rate on hydrogen–ion concentration [Benzaldehyde] = 1.5 mol dm−3; [BIDC] = 0.001 mol dm−3; temperature 313 K

[H+] (mol dm−3) kobs × 105 (s−1)

0.1 2.31

0.2 9.71

0.3 21.0

0.4 39.6

0.6 82.7

1.0 240

Kinetic isotope effect. To ascertain the importance of the cleavage of the aldehydic C−H bond in the rate-determining step, the oxidation of [2H]benzaldehyde (PhCDO) by BIDC was studied. The results (Tables 3 and 4) showed that the formation constants of the complexes of ordinary and deuteriated benzaldehydes are almost similar but the rates of their decomposition exhibited a substantial kinetic isotope effect (kH/kD = 6.17 at 293 K). Solvent effect. The oxidation of benzaldehyde by BIDC was studied in 19 organic solvents. The solubility of the reactants and the reaction of BIDC with primary and secondary alcohols limited the choice of solvents. There was no reaction with the chosen solvents. The kinetics was similar in all the solvents. The values of K and k2 are recorded in Table 6. table 6. Effect of solvent on the oxidation of benzaldehyde by BIDC at 313 K

Solvent 1 Chloroform 1,2-Dichloroethane Dichloromethane DMSo acetone Dimethylformamide Butanone Nitrobenzene Benzene Cyclohexane Toluene

430

K (dm3mol−1) 2 3.79 3.19 3.59 3.34 3.50 3.75 3.11 3.23 3.47 3.53 3.80

k2 × 105 (s–1) 3 68.5 94.5 85.9 278 84.4 150 63.1 112 32.2 3.45 25.7

to be continued

Continuation of Table 6

1 Acetophenone Tetrahydrofurane tert-Butyl alcohol Dioxane 1,2-Dimethoxyethane acetic acid Ethyl acetate Carbon disulphide

2 3.29 3.49 3.20 3.50 3.75 3.43 3.51 3.03

3 127 43.6 27.0 44.0 24.1 8.12 35.0 13.7

DiSCUSSion The entropies and enthalpies of the activation of the oxidation of 36 benzaldehydes exhibited satisfactory correlation (r2 = 0.9728). The value of isokinetic temperature is 504±14 K. The correlation was tested and found genuine by applying the Exner criterion17. The Exner plot between the values of lg k2 at 293 and 323 K, for the 36 aldehydes, is linear (slope = 0.8066±0.008; r2 = 0.9970). The value of isokinetic temperature, determined by the Exner method, is 577±17 K. A linear isokinetic relationship is a necessary condition for the validity of linear free energy relationships17. It also implies that all the reactions, so correlated, follow a similar mechanism. BIDC seems to be an ionic compound as a result of proton transfer. To ind out the state of BIDC in our reaction conditions, conductivity measurements have been carried out. It was observed that DMSO has very low conductivity and the addition of BIDC in DMSO shows negligible change in the conductivity value. Therefore, BIDC can be considered to be remained as non-ionised under our reaction conditions and does not dissociate as dichromate and benzimidazolium ions. No effect of added benzimidazolium ion on the rate of oxidation also supports the postulation that BIDC remain as nonionised5. The crystal structure study of BIDC, reported by Meng et al.18 supports the non-ionic nature of the oxidant in the reaction system. The dichromate ion connects 2 benzimidazolium rings via hydrogen bonds. With the effective hydrogen donor (N–H) and hydrogen acceptor (O) in the molecule, BIDC forms a number of hydrogen bonds. Furthermore, an intermolecular hydrogen bridge is remarkably formed between 2 neighboured dichromate ions. The molecules are then linked into ininite chains by these hydrogen bridges which controlled the releasing process of dichromate ions to reaction system and thus the compound behaves as non-ionic in our reaction system18. Solvent effect. The data recorded in Table 6 indicate that the equilibrium constant, K, is fairly insensitive to the change in solvent, however, k2 varies appreciably. Therefore, the values of the rate constant of the decomposition of complexes, k2, in 18 solvents (CS2 was not considered as the complete range of the solvent parameters are not 431

available), were correlated in terms of linear solvation energy relationship (LSER) of Kamlet et al.19 But the correlations were insigniicant. The data on solvent effect were then analysed in terms of the Swain equation20 (6), where A represents the anion-solvating power of the solvent and B – the cationsolvating power; C – the intercept term, and (A + B) – the solvent polarity. lg k = aa + bB + C

(6)

The results of the correlation analyses in terms of equation (6), individually with A and B, and with (A + B) are given below. lg k2 = 0.28±0.02 A + 1.79±0.01 B – 4.58

(7)

R2 = 0.9993, SD = 0.01, n = 19, ψ = 0.03 lg k2 = 0.02±0.59 A – 3.35

(8)

r2 = 0.0001, SD = 0.48, n = 19,ψ = 1.03 lg k2= 1.77±0.05 B – 4.49

(9)

r = 0.9863, SD = 0.06, n = 19,ψ = 0.12 2

lg k2 = 1.29±0.19 (A + B) – 4.54

(10)

r = 0.7235, SD = 0.25, n = 19,ψ = 0.54 2

The data on solvent effect showed an excellent correlation in terms of the Swain equation20 with both anion- and cation-solvating powers contributing to the observed solvent effect. However, the role of cation-solvation is major, it alone accounts for ca. 99% of the data. There is no signiicant collinearity between A and B for the 19 solvents (r2 = 0.0108; SD = 0.27). The solvent polarity, represented by (A + B) accounted for ca. 72% of the data. In view of the fact that ca. 72% of the data are accounted for by (A + B), an attempt was made to correlate the data with the relative permittivity of the solvents. A plot of lg k against the inverse of relative permittivity, however, is not linear (r2 = 0.4597). Correlation analysis of reactivity. A perusal of values in Tables 3 and 4 shows that the formation constants, K, of the aldehyde−BIDC complexes are not much sensitive to the substitution in the aromatic ring. Similar observations have been made earlier in the oxidation of benzaldehydes by butyltriphenylphosphonium dichromate (BTPPD) (Ref. 15) and pyridinium lurochromate (PFC) (Ref. 21). But the rates of decomposition of the complexes, k2, show considerable variation with the substitution. Since the discovery of signiicant effects of substituents on reactivity, many workers have attempted correlations with the Hammett equation22 or with dual substituent-parameter equations23,24. In the late 1980’s, Charton25 introduced a triparametric LDR equation for the quantitative description of structural effects on chemical reactivities. This triparametric equation results from the fact that substituent types differ in their mode 432

of electron delocalisation. This difference is relected in a different sensitivity to the electronic demand for the phenomenon being studied. It has the advantage of not requiring a choice of parameters as the same 3 substituent constants are reported to cover the entire range of electrical effects of substituents. We have, therefore, begun a study of structural effects on reactivity by means of the LDR equation. In this work we have applied the LDR equation to the rate constants, k2: lg k = L σl + D σd + R σe + h

(11)

where h is the intercept term; σl – a localised (ield and/or inductive) effect parameter; σd – the intrinsic delocalised (resonance) electrical effect parameter when active site electronic demand is minimum, and σe – the sensitivity of the substituent to changes in electronic demand by the active site. The latter two substituent parameters are related by the following equation: σD = ησe + σd

(12)

where η represents the electronic demand of the reaction site and is given by η = R/D, and σD – the delocalised electrical parameter of the diparametric LD equation. For ortho-substituted compounds, it is necessary to account for the possibility of steric effects and Charton25 therefore, modiied the LDR equation to generate the LDRS equation (13), to account for the steric effects: lg k = L σl + D σd + R σe + S V + h

(13)

where V is the well-known Charton steric parameter based on the van der Waals radii26. The rates of oxidation of ortho-, meta- and para-substituted benzaldehydes show excellent correlations in terms of the LDR/LDRS equations (Table 7). All the three series of substituted benzaldehydes meet the requirement of a minimum number of substituents for analysis by LDR and LDRS equations25. The comparison of the L and D values for the substituted benzaldehydes showed that the oxidation of ortho- and para-substituted benzaldehydes is more susceptible to the delocalisation effect than to the localised effect. However, the oxidation of meta-substituted compounds exhibited a greater dependence on the ield effect. In all cases, the magnitude of the reaction constants decreases with an increase in the temperature, pointing to a decrease in selectivity with an increase in temperature. All the three regression coeficients, L, D and R, are negative indicating an electron-deicient centre in the transition state of the reaction. The positive value of η adds a negative increment to σd, increasing the donor effect of the substituent where σd is negative and decreasing the acceptor effect where σd is positive. The substituent is, therefore, better able to stabilise a cationic reaction site. This also supports the presence of an electron-deicient centre in the transition state of the rate-determining step. The large magnitude of η, which represents the electronic demand of the reaction, indicates a pronounced charge separation in the transition state and supports a mechanism involving formation of a carbocationic ion in the rate-determining step. 433

table 7. Temperature dependence of the reaction constants for the oxidation of substituted benzaldehydes by BIDC

T (K)

L

D

R

293

−1.42 (±0.01) −1.36 (±0.02) −1.29 (±0.02) −1.24 (±0.02)

−2.71 (±0.01) −2.48 (±0.01) −2.30 (±0.01) −2.19 (±0.02)

−3.81 (±0.03) −3.61 (±0.05) −3.36 (±0.03) −3.03 (±0.06)

−1.88 (±0.02) −1.69 (±0.02) −1.56 (±0.02) −1.44 (±0.01)

−1.07 (±0.01) −0.98 (±0.02) −0.92 (±0.01) −0.86 (±0.01)

−1.49 (±0.07) −1.25 (±0.11) −1.20 (±0.08) −1.15 (±0.06)

−1.58 (±0.02) −1.40 (±0.02) −1.32 (±0.02) −1.24 (±0.02)

−2.05 (±0.02) −1.88 (±0.02) −1.81 (±0.02) −1.71 (±0.01)

−2.86 (±0.11) −2.67 (±0.10) −2.43 (±0.11) −2.29 (±0.09)

303 313 323 293 303 313 323 293 303 313 323

η S R2 para-Substituted 1.41 0.9999 −

SD

ψ

PD

Ps

0.01

0.011

65.6

−

−

1.46

0.9998

0.01

0.016

64.6

−

−

1.46

0.9998

0.01

0.016

64.1

−

−

1.38

0.9997

0.02

0.02

63.8

−

meta-Substituted 1.39 0.9996 −

0.01

0.023

36.3

−

−

1.28

0.9990

0.02

0.037

36.7

−

−

1.30

0.9994

0.01

0.028

37.1

−

−

1.34

0.9996

0.01

0.023

37.4

−

0.02

0.021

56.5

26.2

0.01

0.021

57.3

26.0

0.02

0.027

57.8

25.8

0.01

0.021

58.0

26.3

ortho-Substituted −1.29 1.40 0.9997 (±0.02) −1.15 1.42 0.9997 (±0.02) −1.09 1.34 0.9995 (±0.02) 1.05 1.34 0.9997 (±0.02)

The negative value of S indicates that the reaction is subjected to steric hindrance by the ortho-substituent. This may be due to steric hindrance of the ortho-substituent to the approach of the oxidising species. We have evaluated the signiicance level for all the three/four independent variables by determining the Student t function for each coeficient27. The signiicance level was found to be >99.9%. Thus, all the parameters are required to explain the effect of structure on the reactivity in the oxidation of benzaldehydes by BIDC. There is no signiicant colinearity between the various substituent constants in all the three series. The percent contribution25 of the delocalised effect, PD, is given by the following equation: PD =

434

(|D| × 100) (|L| + |D|)

.

(14)

Similarly, the percent contribution of the steric parameter25 to the total effect of the substituent, PS, was determined by using the following equation: PS =

(|S| × 100) (|L| + |D| + |S|)

.

(15)

The values of PD and PS are also recorded in Table 7. The value of PD for the oxidation of para-substituted benzaldehydes is ca. 64% whereas the corresponding values for the meta- and ortho-substituted benzaldehydes are ca. 37 and 57%, respectively. This shows that the balance of localisation and delocalisation effects is different for differently substituted benzaldehydes. The less pronounced resonance effect from the ortho-position than from the para-position may be due to the twisting away of the aldehydic group from the plane of the benzene ring. The magnitude of the PS value shows that the steric effect is signiicant in this reaction. Mechanism. An one-electron oxidation, giving rise to free radicals, is unlikely in view of the failure to induce polymerisation of acrylonitrile. BHT is an excellent trap for free radicals28. The fact that BHT was recovered unchanged also goes against the occurrence of an one-electron oxidation. The formation constants of the complexes of ordinary and deuteriated benzaldehydes are almost similar, however, the rate of its decomposition exhibited a substantial kinetic isotope effect. This indicates that the aldehydic C−H bond is cleaved in the rate-determining step. The negative values of L, D, and R point to an electron-deicient reaction centre in the transition state of the rate-determining step. It is further conirmed by the positive value of η, which indicates that the substituent is better able to stabilise a cationic or electron-deicient reactive site. Thus, the transition state of the aldehyde−BIDC complex approaches a carbocation in character. This postulation is supported by the analyses of the solvent effect indicating much more contribution of the cation solvation on the rate of decomposition of the complex. The order of reactivity also supports it. Therefore, the removal of hydrogen as hydride-ion resulting in an electron-deicient species in the rate-determining step, is indicated. However, the observed Michaelis−Menten type kinetics with respect to the aldehyde led us to suggest the formation of 2:1 complex of benzaldehyde and BIDC in a rapid pre-equilibrium, involving a nucleophilic attack of aldehydic oxygen on chromium. The nature of the complex suggested is similar to that reported in the oxidation of aromatic aldehydes by BTPPD (Ref. 15) and PFC (Ref. 21). The observed order of reactivity indicated that the electron-releasing groups accelerated the oxidation process. This is accounted in terms of an increase in the electron-availability at the oxygen of the aldehydic group resulting in the facilitation of the complex formation. The observed independence of K on the change in the concentration of hydrogen ion, indicated a rapid reversible protonation of the intermediate complex prior to its disproportionation. A mechanism depicted in the Scheme accounts for the experimental results. 2 mol of aldehyde react with 1 mol of BIDC in a pre-equilibrium to give an intermediate complex, which undergoes protonation 435

to produce a doubly protonated species. This protonated complex then decomposes in a rate-determinig step via hydride-ion transfer from aldehyde to oxidant to form a carbocation, which in a fast step results in a inal product of the reaction (Scheme). Scheme

BI = benzimidazole. 436

The rate law based on the mechanism (Scheme) can be written as follows: rate =

kH K1 K [BIDC]t [aldehyde]2 [H+]2 1 + K [adehyde]2

.

(16)

Comparing equations (4) and (16), we get k2 = kH K1[H+]2. The analysis of the temperature dependence of the kinetic isotope effect by the method of Kwart and Nickle29 showed that the loss of hydrogen proceeds through a concerted cyclic process. The data for protio- and deuterio-benzaldehyde were itted to the following familiar expression: kH/kD = AH/AD exp (–∆Ea/RT)

or lg (kH/kD) = lg (AH/AD) – ∆Ea/RT.

(17)

The results showed that the activation energy difference for kH/kD is 4.3 kJ which agrees well with the zero-point energy difference for the respective C–H and C–D bonds (ca. 4.5 kJ mol–1) and the entropy of activation of the respective reactions are equal. This directly corresponds to the properties of a symmetrical transition state30,31. Similar phenomenon have been observed earlier in the oxidation of alcohols by BIDC (Ref. 7). Bordwell32 has given cogent evidence against the occurrence of concerted one-step bimolecular process of hydrogen transfer and it is clear that in the present reaction also, the hydrogen transfer does not occur by an acyclic bimolecular process. The only truly symmetrical processes involving linear transfer of hydrogen are intrinsically concerted sigmatropic reactions characterised by transfer with a cyclic transition state9. The second step of the reaction was the transfer of 2 electrons in a cyclic system. This electrocyclic mechanism for the oxidation of aldehyde by BIDC involved 6 electrons, being a Hückel type system, is an allowed process33. Therefore, one can safely conclude that in the oxidation of benzaldehyde by BIDC, the hydrideion transfer occurs via a cyclic transition state. The manner of electron transfer has to be established. The irst step involved the nucleophilic attack of aldehydic-oxygen electrons on electron-deicient chromium atom to form an intermediate complex. This complex then undergoes unimolecular decomposition in the slow step. The transition state involves the bonding of hydrogen atom both to the aldehydic-carbon and the OH group attached to chromium. The electron low in a cyclic transition state has been considered assuming that the hydrogen atom is removed as hydride ion. Thus, the process of electron transfer takes place through the carbon–hydrogen–oxygen–chromium bond. This would facilitate the formation of a carbocationic species by reverting back the nucleophilic attack of aldehydic oxygen. A similar type of mechanism is reported for the oxidation of aromatic aldehydes by BTPPD (Ref. 15). It may be mentioned that though the formation of complex and its diprotonation are shown as single steps, these must be taking place in two steps each.

437

The proposed mechanism is, however, supported by the observed negative entropy of activation. As the charge separation takes place in the transition state, the two ends become highly solvated. This results in an immobilisation of a large number of solvent molecules, relected in the loss of entropy. The negative activation entropy additionally accounts for the inluence of solvent. Initially Cr(VI) is reduced to Cr(IV). It is likely to react with another Cr(VI) to generate Cr(V) which is then reduced in a fast step to the ultimate product Cr(III). Such a sequence of reactions in Cr(VI) oxidations is well known34. ACKNOWLEDGEMENTS We thank the University Grants Commission (India) and Council of Scientiic and Industrial Research (India) for the inancial support. reFerenCeS 1. E. J. COREY, W. J. SUGGS: Pyridinium Chlorochromate. An Eficient Reagent for Oxidation of Primary and Secondary Alcohols to Carbonyl Compounds. Tetrahedron Letts, 2647 (1975). 2. M. M. BHATTACHARYA, M. K. CHAUDHURI, H. S. DASGUPTA, N. ROY, D. T. KATHING: Pyridinium Fluorochromate. A New and Eficient Oxidant for Organic Substrates. Synthesis, 588 (1982). 3. M. LI, M. E. JOHNSON: Oxidation of Certain 4-substituted Phenethyl Alcohols with Collin’s Reagent: On the Mechanism of a Carbon–Carbon Bond Cleavage. Synth Commun, 25, 533 (1995). 4. H. FIROUZABADI, A. SHARIFI: Chromium (VI)-based Oxidants. IV. Zinc Chloro-chromate Nonahydrate as an Eficient and Mild Oxidizing Agent. Synthesis, 999 (1992). 5. I. M. BALTROKE, M. M. SADEGHI, N. MAHMOODI, B. KHARMESH: n-Butyltriphenylphosphonium Dichromate: An Eficient and Selective Oxidizing Agent. Indian J Chem B, 36, 438 (1997). 6. Q. H. MENG, J. C. FENG, N. S. BIAN, B. LEU, C. C. LI: Benzimidazolium Dichromate – A New Reagent for Selective Oxidation under Microwave Irradiation. Synth Commun, 28, 1097 (1998). 7. D. PANDAY, S. KOTHARI: Kinetics and Mechanism of the Oxidation of Aliphatic Alcohols by Benzimidazolium Dichromate. Oxid Commun, 32, 371 (2009). 8. D. PANDAY, S. KOTHARI: Kinetics and Mechanism of the Oxidation of DL-methionine by Benzimidazolium Dichromate. Prog React Kinet Mech, 34, 199 (2009). 9. R. KUMAR, D. PANDAY, S. KOTHARI: Kinetics and Mechanism of the Oxidation of Secondary Alcohols by Benzimidazolium Dichromate. Prog React Kinet Mech, 36, 01 (2011). 10. K. B. WIBERG, R. STEWART: The Mechanisms of Permanganate Oxidation: I. The Oxidation of Some Aromatic Aldehydes. J Am Chem Soc, 77, 1786 (1955). 11. K. B. WIBERG: The Deuterium Isotope Effect of Some Ionic Reactions of Benzaldehydes. J Am Chem Soc, 76, 5371 (1954). 12. D. D. PERRIN, W. L. ARMAREGO, D. R. PERRIN: Puriication of Laboratory Chemicals. Pergamon, Oxford, 1980. 13. O. EXNER: Additive Physical Properties. I. General Reactions and Problems of Statitical Nature. Collect Czech Chem Commun, 31, 3222 (1966). 14. K. M. DILSHA, S. KOTHARI: Kinetics and Mechanism of the Oxidation of Some Aliphatic Aldehydes by n-butyltriphenylphosphonium Dichromate. Oxid Commun, 32, 874 (2009). 15. D. PANDEY, S. KOTHARI: Kinetics and Mechanism of the Oxidation of Aromatic Aldehydes by n-butyltriphenylphosphonium Dichromate. Prog React Kinet Mech, 33, 293 (2008).

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16. A. KOTHARI, S. KOTHARI, K. K. BANERJI: Kinetics and Mechanism of the Oxidation of Oxyacids of Phosphorus by n-butyltriphenylphosphonium Dichromate. Oxid Commun, 23, 93 (2000). 17. J. SHORTER: Correlation Analysis of Organic Reactivity with Particular Reference to Multiple Regression. Research Studies Press, New York, 1982, p. 215. 18. Q. MENG, W. YAN, S. XU, D. HUANG: Crystal Structure of Benzimidazolium Dichromate. J Chem Crystallog, 34, 333 (2004). 19. M. J. KAMLET, J. L. M. ABBOUD, M. H. ABRAHAM, R. W. TAFT: Linear Solvation Energy Relationships. A Comprehensive Collection of the Solvatochromic Parameters, .pi.*, .alpha., and .beta., and Some Methods for Simplifying the Generalized Solvatochromic Equation. J Org Chem, 48, 2877 (1983) and references therein. 20. C. G. SWAIN, M. S. SWAIN, A. L. POWELL, S. ALUMNI: Solvent Effects on Chemical Reactivity. Evaluation of Anion- and Cation-solvation Components. J Am Chem Soc, 105, 502 (1983). 21. S. AGARWAL, K. CHOWDHURI, K. K. BANERJI: Kinetics and Mechanism of the Oxidation of Aromatic Aldehydes by Pyridinium Fluorochromate. J Org Chem, 56, 5111 (1991). 22. K. B. WIBERG: Physical Organic Chemistry. John Wiley & Sons, New York, 1964, p. 416. 23. S. DAYAL, S. EHRENSON, R. W. TAFT: Substituent Effects, Electronic Transmission and Structural Dependence of π-delocalization as Studied with the π-luorophenyl Tag. J Am Chem Soc, 94, 9113 (1972). 24. C. G. SWAIN, S. H. UNGER, N. R. ROSENQUIST, M. S. SWAIN: Substituent Effects on Chemical Reactivity, Improved Evaluation of Field and Resonance Components. J Am Chem Soc, 105, 492 (1983). 25. M. CHARTON, B. CHARTON: Structural Effects on Radical Reactions. 1. Homolytic Aromatic Substitution. Bull Soc Chim Fr, 199 (1988) and references therein. 26. M. CHARTON: Nature of the Ortho-effect. XI. Reaction Rates of Carboxylic Acids with Diazodiphenylmethane. J Org Chem, 40, 407 (1975). 27. R. L. WINE: Statistics for Scientists and Engineers. Prentice Hall, New Delhi, 1966. 28. D. MOHAJER, S. TANGESTANINEJAD: Eficient Olein Epoxidation with Tetrabutyl-ammonium Periodate Catalyzed by Manganese Porphyrin in the Presence of Imidazole. Tetrahedron Lett, 35, 945 (1994). 29. H. KWART, J. H. NICKLE: Transition States in Chromium (VI) Oxidation of Alcohols. J Am Chem Soc, 95, 3394 (1973). 30. H. KWART, H. C. LATIMER: The Kinetic Deuterium Isotope Criterion for Concertedness. J Am Chem Soc, 93, 3770 (1971). 31. H. KWART, J. SLUTSKY: Transition-state Structure in Thermal β-cis-elimination of Esters. J Chem Soc Chem Commun, 1182 (1972). 32. F. G. BORDWELL: How Common are Base-initiated Concerted 1,2-eliminations. Acc Chem Res, 5, 374 (1974). 33. J. S. LITTLER: Oxidation of Oleins, Alcohols, Glycols and Other Organic Compounds by Inorganic Oxidants such as Chromium(VI), Manganese(VII), Iodine(VII), Lead(IV), Vanadium(V) and Halogens, Considered in the Light of the Selective Rules Electrocyclic Reactions. Tetrahedron, 27, 81 (1971). 34. K. B. WIBERG, W. H. RICHARDSON: Oxidation in Organic Chemistry. Vol. A. Academic Press, New York, 1965, p. 69. Received 5 July 2011 Revised 21 August 2011

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Oxidation Communications 37, No 2, 440–444 (2014) Oxidation in the presence of Cr- and Mn-containing oxidants

OxidatiOn Of 5-(4′-fluOrOphenyl)-5-OxOpentanOic aCid By aCid Permanganate S. SHREE DEVIa, P. KRISHNAMOORTHYb, B. MUTHUKUMARANc* a

Research and Development Centre, Bharathiar University, 641 046 Coimbatore, India E-mail: [email protected] b Department of Chemistry, Dr. Ambedkar Government Arts College, 600 039 Chennai, India E-mail: [email protected] c Department of Chemistry, Presidency College, 600 005 Chennai, India E-mail: [email protected]

aBStraCt The kinetics and mechanism of oxidation of δ-oxoacid by acid permanganate in aqueous acetic acid medium have been studied under varied experimental conditions. The reaction is irst order with respect to [oxoacid], [MnO4–] and [H+]. At low [H+] the reaction is zero order with respect to [MnO4–]. The reaction rate increases with decrease in dielectric constant of the medium. A mechanism consistent with the observed results has been suggested. Keywords: δ-oxoacid, oxidation, acid permanganate. aiMS anD BaCKGroUnD The kinetics of oxidation of a variety of organic compounds by permanganate has been studied extensively1–3. The oxidation of cyclohexanone and some open-chain ketones by acid permanganate was also studied4,5 and in this paper the kinetics and mechanism of oxidation of a δ-oxoacid by acid permanganate are reported. EXPERIMENTAL The δ-oxoacid, namely 5-(4′-luorophenyl)-5-oxopentanoic acid, was prepared by the Friedel–Crafts acylation of luorobenzene with glutaric anhydride. All the chemicals used were Analar grade. Potassium permanganate was used as such and acetic acid was distilled over chromic oxide before use. Perchloric acid was used as a source of H+ ions and sodium perchlorate was added to maintain the ionic strength. Sodium *

For correspondence.

440

luoride was added as a complexing agent to suppress the autocatalysis in permanganate oxidations6. The kinetics of the reaction was followed titrimetrically by standard iodometric procedure. The reaction was followed under pseudo-irst order conditions, maintaining always the concentration of the substrate in excess. Under kinetic conditions δ-oxoacid, H+ and permanganate were mixed together. After 48 h the excess permanganate was removed by the addition of sodium metabisulphite. The residual mixture was extracted with ether. The ether layer was separated and evaporated. The product was identiied as p-luorobenzoic acid. The product was further conirmed as 1 mol of δ-oxoacid required 3 mol of permanganate. 5F−C6H4−CO−(CH2)3−CO2H+ + 15Mno4– + 20H+ → 5F−C6H4−CO2H +20CO2 + 25H2o + 15Mn2+

reSULtS anD DiSCUSSion The rate of oxidation was dependent on the irst order on the concentration of δ-oxoacid, H+ and permanganate (Table 1). table 1. Oxidation of δ-oxoacid by acid permanganate at 35oC [NaF]= 5.0 ×10−2 mol dm−3; [NaClO4] =5.0 ×10−2 mol dm−3; solvent – AcOH (30% v/v)

[Oxoacid] × 102 (mol dm−3) 2.0 3.0 4.0 5.0 2.0 2.0 2.0 2.0 2.0 2.0* 2.0** 2.0***

[KMnO4] × 103 (mol dm−3) 2.0 2.0 2.0 2.0 3.0 4.0 5.0 2.0 2.0 2.0 2.0 2.0

[H+] (mol dm−3)

k′ × 105 (s−1)

1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.5 2.0 1.0 1.0 1.0

7.011 10.344 13.685 17.044 6.963 6.920 6.894 18.332 24.021 7.634 10.165 12.584

* 40%; ** 50%; *** 60% of acetic acid.

The reaction rate increases with increase in percentage of acetic acid. The reaction rate was not affected by the addition of acrylonitrile, hence the free radical pathway was ruled out. The oxidation rate increases slightly with the increase in ionic strength of the medium. The protonation of permanganate ion leads to the formation of permanganic acid (PA). The rate-determining step involves the attack of permanganic acid on the enol form of the δ-ketoacid. Thus the mechanism involves 2 protons, one for the formation of HMnO4 and another – for the enolisation of oxoacid (EA). The experimental 441

results indicate that the reaction is irst order with respect to [MnO4–] at high mineral acid concentration and zero order dependence on [MnO4–] at low [H+] (Table 2). table 2. Oxidation of δ-oxoacid by acid at low [H+] [NaF] = 5.0 × 10−2 mol dm−3; [NaClO4] = 5.0 ×10−2 mol dm−3; [oxoacid] = 5.0 × 10−2 mol dm−3; [H+] = 5.0 × 10−2 mol dm−3; solvent – AcOH (30% v/v); temperature 35oC

[KMnO4] × 103 (mol dm−3) 1.0 2.5 3.5 4.5

[Zero order rate constant] × 103 (mol dm–3 s−1) 3.916 3.930 3.907 3.921

Thus under conditions of high [H+] the enol removal is less rapid than its formation, but at lower [H+] the enol removal is faster than enol formation. This type of difference in order with respect to [H+] has been already reported7,8. Mn7+ is reduced to Mn5+ as hypomanganate ester (HPO) (step 4), which on hydrolysis and oxidation gives the acid. The formation of hypomanganate is believed to be a reaction intermediate in the acid medium permanganate oxidation of many organic and inorganic compounds9,10. (1)

(2)

(3)

(4)

where Ar = p-F–C6H4−. 442

RATE LAW

rate = –

d[MnO4–] dt

=

K2K3k4 [H+][S][HMnO4] K–2K–3 [H+] + k4(K–2 + K3) [HMnO4]

at high acid concentration, K–2 K–3 [H+] > k4(K–2 + K3) [HMnO4] rate =

K2K3k4 [S][HMnO4] K–2K–3

=

K2K3k4 [S][HMnO4] K–2K–3 [H+]

where ka = [MnO4–][H+]/[HMnO4], S = δ-oxoacid. Observed rate at high [H+] rate =

K2K3k4 K–2K–3ka

at lower H+ concentration k4 (K–2 + K3) [HMnO4] > K–2 K–3 [H+] rate =

K2K3k4[S][HMnO4][H+] k4 (K–2 + K3) [HMnO4]

=

K2K3 [S][H+] (K–2 + K3)

.

ConCLUSionS Kinetics and mechanism of oxidation of δ-oxo acid by acid permanganate in aqueous acetic acid medium have been studied. The experimental results indicate that the reaction is irst order with respect to [MnO4–] at high mineral acid concentration and zero order dependence on [MnO4–] at low [H+]. The protonation of permanganate ion leads to the formation of permanganic acid. Variation in ionic strength has little inluence on the reaction rate. The reaction rates are enhanced on lowering the dielectric constant of the reaction medium. A mechanism consistent with the observed kinetic data has been proposed and discussed. A suitable rate law has been derived based on the mechanism. reFerenCeS 1. J. W. LADBURY, C. P. CULLIS: Kinetics and Mechanism of Oxidation by Permanganate. Chem Rev, 58 (2), 403 (1958). 2. W. A. WATERS: Mechanisms of Oxidation by Compounds of Chromium and Manganese. Q Rev Chem Soc, 12, 277 (1958). 3. M. P. NATH, K. K. BANERJI: Kinetics and Mechanisms of the Oxidation of Methyl Aryl Ketones by Acid Permanganate. Austr J Chem, 29 (9), 1939 (1976). 4. A. MOONDRA, A. MATHUR, K. K. BANERJI: Kinetics and Mechanism of the Oxidation of Phosphinic, Phenylphosphinic, and Phosphorous Acids by Pyridinium Fluorotrioxochromate(VI). J Chem Soc Dalton, 2697 (1990).

443

5. P. N. MARIGANGAIAH, NATH, K. K. BANERJI: Kinetics and Mechanism of Oxidation of Acetone, Butanone, Pentan-2-one, 3-methylbutan-2-one, Hexan-2-one and 4-methylpanean-2-one by Acid Permanganate. Indian J Chem, 14a, 660 (1976). 6. R. M. BARTER, J. S. LITTLER: Hydride Ion Transfer in Oxidations of Alcohols and Ethers. J Chem Soc B, 205 (1967). 7. R. CECIL, J. S. LITTLER, G. EASTON: Electron Transfer Oxidation of Organic Compounds. Part III. Oxidation of Cyclohexanone by the Hexachloroiridate(IV) Anion and by Related Species. J Chem Soc B, 626 (1970). 8. A. J. AUDSLEY, G. R. QUICK, J. S. LITTLER: Electron Transfer Oxidation of Organic Compounds. Part 5. Oxidation of Cyclohexanone by the tris-2,2′-bipyridylruthenium(III) Cation. J Chem Soc Perk T 2, 557 (1980). 9. F. A. COTTON, G. WILKINSON: Advanced Inorganic Chemistry. 5th ed. Wiley Interscience, New York, 1988, p. 172. 10. N. N. GREENWOOD, A. EARNSHAW: Chemistry of the Elements. Pergamon Press, Oxford, 1984. Received 17 September 2011 Revised 25 October 2011

444

Oxidation Communications 37, No 2, 445–460 (2014) Oxidation in the presence of heterogeneous catalysts

oPtimiSation of the ProCeSS ParameterS of ePoxidation of Crotyl alCohol with hydrogen Peroxide on ti-mww CatalySt A. FAJDEK, A. WROBLEWSKA*, E. MILCHERT Institute of Organic Chemical Technology, West Pomeranian University of Technology of Szczecin, 10 Pulaskiego Street, 70 322 Szczecin, Poland E-mail: [email protected]

aBStraCt The epoxidation of crotyl alcohol (CA) with 30 wt.% hydrogen peroxide over the titanium-silicalite Ti-MWW catalyst under atmospheric pressure with methanol as a solvent was investigated. The inluence of: temperature in the range of 10–60oC, the molar ratio of CA/H2o2 0.2:1–3.0:1, methanol concentration 5–90 wt.%, catalyst concentration 1.0–9.0 wt.%, and the reaction time 30–300 min, has been studied. The process was described by the following functions: the selectivity of transformation to 2,3-epoxybutane-1-ol (2,3EB1O) in relation to CA consumed, the selectivity of transformation to organic compounds in relation to H2o2 consumed and the conversions of CA. The major product of epoxidation is 2,3-epoxybutane-1-ol (2,3EB1O), a compound with many applications. Keywords: 2,3-epoxybutane-1-ol, Ti-MWW catalyst, optimisation. aiMS anD BaCKGroUnD The development of zeolite catalysts in many cases enabled the replacement of homogeneous catalysts with acidic properties (HF, HCl, H2So4). As a result, the amount of waste generated and the environmental impact was reduced. Moreover, these catalysts can be regenerated and the period of their exploitation can be prolonged without a decrease of the selectivity of transformation to the desired product. The selectivities and yields obtained in their presence are many times higher than those obtained in the presence of classical acidic catalysts1,2. One of the most important catalysts of this type is titanium-silicalite Ti-MWW, analogous in terms of the topology with zeolite MCM-22 (Refs 3–5). Ti-MWW possesses a pore system consisting of two independent systems of 10-membered sinusoidal channels. One of these pore systems is formed *

For correspondence.

445

by 2-dimensional sinusoidal channels surrounded by the double 6-member rings which link supercages. The other pore systems consist of large supercages having inside dimensions of 7.1 × 7.1 × 18.2 Å, whose inner free diameter 7.1 Å is deined by 12-member ring with height of 18.2 Å. In the supercage entrance windows are limited by 10-ring apertures and the surface pockets joined together through double 6-member rings. The diffusion to the internal pores proceeds through 10-memberring windows. The volume and the depth of pockets, limited by 12-member ring, is suficient to hold large, organic molecules. Owing to such a catalyst is promoting the reactions which could not occur, due to the restrictions appearing in the case of 10-member ring apertures5–9. The existence of tetrahedrally coordinated Ti in the crystal structure of titanium silicalites enables the activation of hydrogen peroxide. Due to this feature they are becoming promising catalysts of selective oxidation of different organic compounds. Ti-MWW catalyst have also been found to exhibit the catalytic performance superior to conventional titanium silicalites such as TS-1 and Ti-Beta, for example, the epoxidation of linear and cyclic alkenes10. The product of epoxidation of crotyl alcohol – 2,3-epoxybutane-1-ol is of signiicant technical importance. It was applied for the modiication of properties of epoxy resins, preparation of intermediates to the synthesis macrolide-class antibiotics: erythromycin A (Ref. 11), and tylosin12. (2R,3S)-Epoxybutane-1-ol was applied in the synthesis D,L-valine, useful in the synthesis of penicillin and cephalosporin antibiotics and sex pheromone of cigarette beetle – serricornine13. The objective of this work was to investigate the course of epoxidation of crotyl alcohol to 2,3-epoxybutane-1-ol with 30 wt.% hydrogen peroxide, in the presence of Ti-MWW catalyst, and the determination of optimum process parameters. The inluence of the following parameters: temperature, the molar ratio of crotyl alcohol to hydrogen peroxide, methanol concentration, Ti-MWW catalyst concentration, and reaction time was investigated. The course of the process was evaluated using the following functions: the selectivity of transformation to 2,3-epoxybutane-1-ol in relation to crotyl alcohol consumed, conversion of crotyl alcohol, selectivity of transformation to organic compounds in relation hydrogen peroxide consumed. Each function was presented in the form of the mathematical equation and the optimum process parameters for each function were established. The optimum parameters of the course of epoxidation process were also determined. EXPERIMENTAL In the epoxidation of crotyl alcohol (CA) the following raw materials were used: crotyl alcohol (95 wt.%, Fluka), hydrogen peroxide (30 wt.% water solution, P. O. Ch. Gliwice, Poland) and methanol (analytical grade, P. O. Ch. Gliwice, Poland). The titanium-silicalite Ti-MWW catalyst was obtained at the Institute of Organic Chemical Technology. The methods of catalyst preparation and its characterisation by 446

X-ray diffraction spectroscopy (XRD), scanning electron micrographs (SEM), infrared spectroscopy (IR), UV-vis. method, X-ray microanalysis were presented earlier14. The epoxidation was performed under atmospheric pressure (in the presence of air), in a glass reactor with mechanic stirrer and relux condenser. The determined amounts of reactants were introduced into the reactor in the following sequence: the titanium-silicalite catalyst, crotyl alcohol, methanol (solvent) and a 30 wt.% hydrogen peroxide. The process was carried out for a speciied period of time. After the process was completed, a post-reaction mixture was weighed, and analysed. In order to perform the mass balances of the syntheses performed, the following analyses were made: unreacted hydrogen peroxide was iodometrically determined, the organic products and unreacted crotyl alcohol were determined by gas chromatography. The quantitative chromatographic analyses were performed on a FOCUS apparatus equipped with a lame-ionization detector (FID), using a capillary column Quadrex 30 m × 250 μm × 0.25 μm packed with methylsiloxane modiied with phenyl groups. The parameters of chromatographic separation were as follows: helium pressure 50 kPa, sensitivity 10, temperature of the sample chamber 150˚C, detector temperature 250˚C. The thermostat temperature was programmed in the following way: isothermally 55˚C for 3 min, followed by an increase at the rate 10˚C/min to 250˚C, isothermally 250˚C for 5 min, cooling to 60˚C. reSULtS The optimisation of the process parameters of crotyl alcohol epoxidation by 30 wt.% hydrogen peroxide over Ti-MWW catalyst was performed according to the mathematical method of experimental design using a rotatable-uniform design. In the preliminary studies crotyl alcohol demonstrated a high reactivity in the reaction system: Ti-MWW catalyst − 30 wt.% hydrogen peroxide−methanol. However, the reactions resulted in the formation of by-products were also proceeded. They are presented in Fig. 1. Figure 1 shows that the epoxidation of crotyl alcohol by hydrogen peroxide and in the presence of Ti-MWW catalyst causes the formation of 2,3-epoxybutane-1ol. In the reaction medium the epoxide compound very easily undergoes hydration to 1,2,3-butanetriol. The etheriication of the last compound with crotyl alcohol molecule causes formation of 4-(2,3-butenoxy)-butane-2,3-diol. The second direction of 1,2,3butanetriol transformation is solvolysis (with methanol molecules) and creation of 2,3-dimetoxybutane-1-ol. In the reaction medium the molecule of crotyl alcohol can undergoes also oxidation of –OH group, as a result of this reaction crotyl aldehyde and crotonic acid are formed. In suitable conditions also etheriication of crotyl alcohol molecules can take place and formation of bis(2-butene)ether is observed.

447

H3C CH CH CH2

O CH CH CH3

OH OH 4-(2,3-butenoxy)-butane-2,3-diol CH3 CH CH CH2 H3C CH CH CH2

OH

H2O

OH

–H2O

H3C CH CH CH2

OH

OH OH

O

1,2,3-butanetriol

2,3-epoxybutane-1-ol

–2H2O

2CH3OH

H3C CH

CH CH2

OH

OCH3 OCH3

H2O2

Ti-MWW –H2O

2,3-dimetoxybutane-1-ol

CH3 CH CH CH2

OH

1/2O2 CH3 CH CH –H2O

crotyl alcohol, CRA

CHO

1/2O2

CH3 CH CH COOH

crotyl aldehyde

crotonic acid

–H2O

H3C CH CH CH2

O CH2

CH CH CH3

bis(2-butene)ether

fig. 1. Reactions during the epoxidation process of crotyl alcohol over titanium silicalite Ti-MWW catalyst

The mathematical functions describing the process were: the selectivity of transformation to 2,3-epoxybutane-1-ol (2,3EB1O) in relation to crotyl alcohol (CA) consumed (S2,3EB1O/CA): S2,3EB1O/CA =

number of moles of 2,3EB1O number of moles of crotyl alcohol consumed

× 100%

conversion of crotyl alcohol (CCa): CCa =

number of moles of crotyl alcohol consumed number of moles of crotyl alcohol introduced into reactor

× 100%

selectivity of transformation to organic compounds in relation to hydrogen peroxide consumed (Sorg. /H2o2): Sorg./H2o2 =

number of moles of organic compounds obtained number of moles of H2o2 consumed

× 100%.

In all experiments the conversion of hydrogen peroxide amounted to at least 97%. In order to develop an appropriate mathematical description of the course of process, ive independent factors (parameters) were taken into consideration. The studies were performed over the following ranges of changes of technological parameters: temperature – x1 = 10−60°C, the CA/H2o2 molar ratio – x2 = 0.2−3.0:1, methanol

448

concentration – x3 = 5−90 wt.%, Ti-MWW catalyst concentration – x4 = 1−9.0 wt.%, reaction time – x5 = 30−300 min. In order to simplify the calculations the real values of the input variables x1–x5 were recalculated into the normalised values (dimensionless) according to the following equation: Xi = [2α(xk – xk min.)/(xk max. – xk min.)] – α,

where Xi ∈ (–α; α); Xi – normalised input variable, i = 1…n; α – star arm, α = 2; xk – real input variable; xk max. – maximum value of the real input variable; xk min. – minimum value of the real input variable; k = 1...n, n – the number of input variables, n = 5. Therefore, an universal standardised design of changes of parameters of experiments in the dimensionless range [–2; 2] was obtained. The real and normalised (coded) input variables at levels resulting from experimental design are presented in Table 1. Experimental design and the calculations results were performed by computer, applying the program Cadex: Esdet 2.2 (Ref. 15). table 1. Normalised (coded) and real values of the parameters of experimental design

Level

Basic Higher Lower Star higher Star lower

Coded Real parameters (factors) parameter temperature CA/H2o2 methanol Ti-MWW reaction (°C) molar ratio concentra- concentra- time (min) mol/mol tion (wt.%) tion (wt.%) x1 x2 x3 x4 x5 Xi 0 1 –1 2 –2

35 48 23 60 10

1.6 2.3 0.9 3.0 0.2

47.5 68.7 26.2 90 5

5 7 3 9 1

165 232.5 97.5 300 30

For successive collections of parameters of the design presented in Table 2 the experiments were carried out and the value of functions describing the process were calculated (response function z1−z3). The inluence of coded independent parameters (X1−X5) of the epoxidation process on the values of the response functions was presented in the form of second order polynomial (regression function) containing the linear and square components and the double products (interactions): Z = Z(Xk)=b0 + b1 X1+ ... + bi Xi + b11 X12 + ... + bii Xi2 + b12 X1X2 + ... + bi–1,i Xi–1 Xi

for Xk ∈ [–2,2]; bi – normalised coeficients of the approximation function; Nb = 1/2 (i + 1)(i + 2),

where Nb is number of polynomial coeficients; i – number of the input variables Xk; k = 1…5.

449

table 2. Design matrix and experimental values of response functions

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32

X1

X2

X3

X4

X5

–1 1 1 1 1 –1 –1 –1 1 –1 –1 –1 –1 1 1 1 –2 2 0 0 0 0 0 0 0 0 0 0 0 0 0 0

–1 –1 –1 –1 1 1 1 1 –1 –1 –1 –1 1 1 1 1 0 0 –2 2 0 0 0 0 0 0 0 0 0 0 0 0

–1 1 –1 –1 –1 1 –1 –1 1 –1 1 1 1 –1 1 1 0 0 0 0 –2 2 0 0 0 0 0 0 0 0 0 0

–1 –1 –1 1 –1 –1 –1 1 1 1 1 –1 1 1 1 –1 0 0 0 0 0 0 –2 2 0 0 0 0 0 0 0 0

–1 –1 1 –1 –1 –1 1 –1 1 1 –1 1 1 1 –1 1 0 0 0 0 0 0 0 0 –2 2 0 0 0 0 0 0

S23EB1O/CA Sorg./H2o2 z1 (%) z2 (%) 3.8 96.3 17.9 88.9 5,1 95.0 22.9 97.1 9.6 95.9 1.3 96.6 5.8 96.5 98.5 97.2 8.3 98.3 19.9 98.0 87.6 98.5 79.6 98.7 29.4 99.9 84.4 99.9 1.6 99.7 78.9 99.4 38.7 97.4 34.5 99.4 34.1 57.2 43.2 98.5 89.4 97.7 86.9 71.2 13.5 75.0 86.3 96.5 37.7 77.3 43.7 80.0 97.3 81.3 23.9 79.4 19.5 74.1 48.3 80.7 52.4 79. 6 38.2 82.4

CCa z3 (%) 95.5 86.8 94.0 96.0 94.3 92.5 94.8 95.4 95.6 96.8 95.7 95.9 98.5 98.0 95.8 95.2 95.3 97.3 56.6 95.6 96.6 64.7 73.8 94.4 75.6 78.3 79.5 77.7 72.5 78.9 77.9 80.6

To obtain the response functions containing the real coeficient of function and real independent parameters xk (process parameters), the coded values of the independent parameters Xk, were recalculated into the real values using the following relationships: X1 = 0.08 (x1 – 10) – 2,

where x1 ∈ [10; 60]; X2=1.43(x2 – 0.2) – 2, where x2 ∈ [0.2; 3]; X3=0.047(x3 – 5) – 2, where x3 ∈ [5; 90]; X4 = 0.5 (x4 – 1) – 2, where x4 ∈ [1;9]; X5 = 0.015(x5 – 30) – 2, where x5 ∈ [30; 300].

450

The coeficients of the regression function for the normalised input variables (process parameters) were determined by the least square method. After the determination of the functions approximating the experimental results, a veriication of the adequacy of obtained function was performed based on the Fisher–Snedecor test by a comparison with the critical values of F(α) taken from Tables16. Moreover, the measurements and calculations performed enabled to determine of the relative errors of the approximation, the measure of which is the coeficient of multidimensional correlation R. The value for each function was also calculated. The variance of repeatability was calculated from the relationship: n0

S

2 repeat .

=

∑ (z i =1

0 i

− z i )2

f repeat.

where zi0 – the experimental value of the response function in the i-th experiment in the design centre (i = 1…n0); zi – the average experimental value of the response function in the design centre; frepeat. – the number of degrees of freedom of repeatability variance (frepeat.= n0 – 1); n0 – the number of experiments in the design centre. By rejecting unimportant coeficients, the algebraic polynomials (regression equations) were determined again and the variance of adequacy was calculated – S2adeq.. In this way, the agreement of experimental results and appropriate results calculated from the regression functions was tested. 2 S adeq . =

N − n0  1  0 0 2 − + n ( z ž ) (z i − ž i ) 2  ∑  0 i i f adeq.  i =1 

where fadeq. is freedom degrees number of variance of adequacy (fadeq.= N – Nb – 1); N – the total number of experiments in experimental design; Nb −฀the number of coeficients in the regression equation; ži0 − the average value of the response function for experiments in the design centre; zi0 – the value of the response function in the design centre – obtained experimentally (i = 1…n0); zi – the average value of the response function for experiments in the design centre; ži − the value of the response function in the i-th experiment calculated by means of the regression equation. The maximum absolute error of approximation was calculated from the following relationship: ∆zmax = max [∆zu]

where Δzu = |zu – Zu|, Δzu – absolute error of the design of experiments arrangement; zu – measured value; Zu – real value. Table 3 presents the coeficients of the regression equations for the three investigated functions (Z1 – Z3) and values of the other very important statistical parameters for these functions, for example: R and Δzmax.. The highest value of Δzmax. was obtained for function Z1. It is probably connected with very high reactivity of the epoxide 451

compound. The values of the coeficient of multidimensional correlation R were very close for all investigated functions and amounted to 0.79–0.90. table 3. Coeficients of regression equation in the normalised form and statistical parameters of equations Z1 − Z3

b00 b01 b02 b03 b04 b05 b11 b12 b13 b14 b15 b22 b23 b24 b25 b33 b34 b35 b44 b45 b55 S2repeat. frepeat. S2adeq. fadeq. F R Δzmax

S2,3EB1O/CA Z1 49.72 –4.40 3.44 2.07 12.34 3.34 –5.62 11.01 –5.32 –8.7 11.32 –5.11 –1.43 5.37 6.67 7.26 –15.76 6.71 –2.3 –12.84 –4.6 785.5 5 198 10 0.25 0.90 47.58

Sorg./H2o2 Z2 77.6 –0.14 4.04* –2.04* 2.68* 0.87 6.68 1.06 –0.46 0.64 0.41 1.54* 0.51 –0.29 –0.18 3.19* 0.27 0.61 3.52* –0.52 1.74* 8.446 5 133.5 10 15.80 0.80 18.5

CCa Z3 75.85 –0.22 3.59* –3.03* 2.67* 0.92 6.61* 0.85 –0.56 0.46 0.19 1.56* 0.49 –0.06 0.01 2.70* 0.47 0.75 3.56* –0.30 1.77* 8.00 5 135.7 10 16.95 0.79 18.31

* essential coeficient; R – coeficient of multiple correlation; Δzmax. – the maximum error of approximation; F – the Fisher–Snedecor test value.

The maximum values of obtained algebraic polynomials (regression equations) were determined by computer, applying the program Cadex:Esdet 2.2. The optimisation of obtained algebraic polynomials was performed with the application of the methods: Box-Wilson and Hooke-Jeeves. The maximum values of the response functions (regression equations) and corresponding to them values of the process parameters were summarised in Table 4. 452

table 4. Process parameters determining the maximum values of the response functions Z1−Z3

Parameter or function

Unit

Maximum value of function Temperature CA/H2o2 molar ratio Methanol concentration Ti-MWW catalyst concentration reaction time

% °C mol/mol wt.% wt.% min.

S2,3EB1O/CA Z1 100 43 2.9:1 5 9 167

Sorg./H2o2 Z2 100 59 2.8:1 19 8 285

CCa Z3 100 59 2.8:1 10.8 8.4 175

It results from this compilation, that each function describing the epoxidation of crotyl alcohol achieves a maximum value 100% for a given set of the reaction parameters differing among themselves. These investigations provide the additional information about the process, because they enable to establish the course of changes of considered functions during changes of two parameters. Moreover, they allow to establish the range of variation of the process parameters, in which high values of function can be obtained. Inluence of process parameters on the selectivity of transformation to 2,3-epoxybutane-1-ol in relation to crotyl alcohol consumed (S2,3EB1O/CA) − Z1 function. The inluence of temperature and the CA/H2o2 molar ratio on the course of changes of the selectivity of transformation to 2,3EB1O in relation to crotyl alcohol consumed – S2,3EB1O/CA is presented in Fig. 2. 3

molar ratio CA/H2O2 (mol/mol)

2.5

2

1.5

1

0.5

10

15

20

25

30 35 40 temperature (°C)

45

50

55

60

fig. 2. Effect of temperature and the CA/H2o2 molar ratio on the course of variation of the selectivity of transformation to 2,3EB1O in relation to CA consumed constant process parameters: methanol concentration 5 wt.%, Ti-MWW catalyst concentration 9 wt.%, reaction time 167 min

453

The course of the isolines indicates that there is a wide range of changes of temperature and the CA/H2o2 molar ratio in which the function achieve a maximum value – 100%. It concerns the range of temperatures 10−34°C and the entire range of changes of the molar ratio of reactants. The increase of temperature in the range from 34°C to 60°C and the CA/H2o2 molar ratio from 0.2:1 to 1.4:1, signiicantly decreases the selectivity of the transformation to 2,3EB1O in relation to CA consumed, in limiting cases from 95 to 15 mol.%. The molar ratio of CA/H2o2 = 1:1 was established as the most advantageous. Running the epoxidation at the CA/H2o2 molar ratios higher than 1:1 is not beneicial due to the necessity of recovery and recycling of unreacted CA to the process. From Fig. 2 it also results that the most advantageous temperature of running the epoxidation of crotyl alcohol is located in the range of 10−34°C. Taking into consideration the costs of energy and the consumption of raw materials, the temperature 20°C was recognised to be the most advantageous. 90

80

methanol concentration (wt.%)

70

60

50

40

30

20

10 10

15

20

25

30

35

40

45

50

55

60

temperature (°C)

fig. 3. Effect of temperature and methanol concentration on the selectivity of transformation to 2,3EB1O in relation to CA consumed constant process parameters: the molar ratio of CA/H2o2 = 2.9:1, Ti-MWW catalyst concentration 9 wt.%, reaction time 167 min

The inluence of changes of temperature and the concentration of methanol as a solvent on the selectivity of the transformation to 2,3EB1O in relation to CA consumed was presented in Fig. 3. In the temperature range from 10 to 20°C, the appropriate concentration of methanol is 10−14 wt.%. When the reaction temperature exceeded 20°C and for the temperature range from 20 to 60°C the application of methanol concentration of 25 wt.% is more favourable. For these concentrations of methanol in the range of temperatures mentioned, the selectivity of transformation to 2,3EB1O does not depend on the temperature or it undergoes minor changes. There is the threshold concentration of methanol 52−58 wt.%, above which the epoxide compound is not

454

formed or it is formed in very small amounts, regardless of temperature. It results from Fig. 3 that the appropriate concentration of the methanol is in the range from 5 to 17 wt.% and its concentration also depends on the temperature. In the case of temperature 20°C the reaction proceeds with the high selectivity already at methanol concentration of 5 wt.%. Due to this, a relatively small amount of methanol is recycled. At constant temperature, chosen from the range 10−60°C, and during the increase of methanol concentration, the selectivity of transformation to 2,3EB1O is lowering in a similar way. Such a course of changes is a result of consecutively proceeding reactions of 2,3EB1O with methanol. The presence of 2,3-dimetoxybutane-1-ol was found in the reaction product (Fig. 1) with increasing methanol concentration. 9

8

catalyst concentration (wt.%)

7

6

5

4

3

2

1 10

15

20

25

30

35

40

45

50

55

60

temperature (°C)

fig. 4. Effect of temperature and Ti-MWW catalyst concentration on the selectivity of transformation to 2,3EB1O in relation to CA consumed constant process parameters: the molar ratio of CA/H2o2 = 2.9:1, methanol concentration 5 wt.%, reaction time 167 min

Figure 4 shows the inluence of changes of temperature and the Ti-MWW catalyst concentration on the course of variation of the selectivity of transformation to 2,3EB1O in relation to CA consumed (S2,3EB1O/CA). The interaction effect of temperature and the Ti-MWW catalyst concentration on the selectivity of transformation to 2,3EB1O in relation to CA consumed demonstrates that the selectivity – S2,3EB1O/CA decreases as the temperature and catalyst concentration is reduced. At temperature 10°C the function S2,3EB1O/CA achieves the highest value at the catalyst concentration of 8.1 wt.%. At temperature 60°C, this value is obtained at signiicantly lower concentration of 4.8 wt.%. It was found that 2,3EB1O is not formed, if the catalyst concentration is below the threshold concentration, which is different depending on the temperature. At temperature 10°C this concentration of catalyst is equal to 5.3 wt.%, at 20°C – 4.3 wt.%, at 30°C – 3.3 wt.%, and at 40°C – 2.1 wt.%. It results from Fig. 4 that the most 455

advantageous concentration of Ti-MWW catalyst at temperature 20°C is 7.8 wt.%, but it can be higher (to 9.0 wt.%). The process proceeds with high selectivities of transformations to 2,3EB1O in the temperature range 10–60°C. However, in this case it is necessary to increase the catalyst concentration in the range 4.7–9.0 wt.% and to decrease the temperature as it was shown in Fig. 4. 300

250

reaction time (min)

200

150

100

50

10

15

20

25

30

35

40

45

50

55

60

temperature (°C)

fig. 5. Effect of temperature and reaction time on the selectivity of transformation to 2,3EB1O in relation to CA consumed constant process parameters: the molar ratio of CA/H2o2 = 2.9:1, methanol concentration 5 wt.%, Ti-MWW catalyst concentration 9 wt.%

Analysis of the inluence of temperature and the reaction time on the selectivity of transformation to 2,3EB1O in relation to CA consumed (Fig. 5) demonstrates that after the reaction time of 190 min at temperature up to 10°C, the selectivity is about 95 mol.% and decreases to 5 mol.% after 300 min. This selectivity of transformation amounts to 100 mol.% at temperature 20°C, when the reaction time is in the range from 30 to 230 min. At temperature 10°C, the reaction time should not exceed 190 min, at 20°C – 220 min, at 30°C – 270 min. Running the CA epoxidation over the time period from 30 to 190 min is the most advantageous. Then, the selectivities of transformations to 2,3EB1O are also high, over a wide temperature range. The studies performed also demonstrate that the reaction time 30 min is suficient to achieve the maximum selectivity. In this case highest selectivity of transformation to 2,3EB1O – S2,3EB1O/CA is obtained after the application of the following parameters: temperature 20°C, molar ratio of CA/H2o2 = 1:1, methanol concentration 5 wt.%, Ti-MWW catalyst concentration 7.8 wt.%, reaction time 30 min.

456

Inluence of process parameters on the conversion of crotyl alcohol (CCa) − Z2 function. The interaction of temperature and the CA/H2o2 molar ratio, temperature and methanol concentration, temperature and catalyst concentration on the conversion of CA indicates that over the range of variation of mentioned parameters, this function also assumes the maximum values – 100 mol.%. The inluence of temperature and the reaction time on the conversion of CA in the epoxidation process is presented in Fig. 6. Only in the temperatures range 20−50°C and after exceeding the reaction time 22 min, the crotyl alcohol conversion is slightly lower (below 95%). 300

250

reaction time (min)

200

150

100

50 10

15

20

25

30 35 40 temperature (°C)

45

50

55

60

fig. 6. Effect of temperature and reaction time on the conversion of crotyl alcohol constant process parameters: the molar ratio of CA/H2o2 = 2.8:1, methanol concentration 10.8 wt.%, Ti-MWW catalyst concentration 8.4 wt.%

From the point of view of the highest conversion of CA, it is not important whether the process will be conducted at temperature 20°C, or at other temperature chosen from the range 10–60°C. Due to the possibility of the limitation of energy consumption it is advantageous to keep the temperature close to the ambient temperature. For similar reasons the application of equimolar ratio of reagents is purposeful. The methanol concentration should be 5 wt.% but it can be within the range 5−90 wt.%, at the catalyst concentration 1−9 wt.% (most favourably 1 wt.%), reaction time 30−300 min (most favourably 30 min). Inluence of process parameters on the selectivity of transformation to organic compounds in relation to hydrogen peroxide consumed (Sorg./H2o2) − Z3 function. The interaction of temperature and the CA/H2o2 molar ratio, temperatures and methanol concentration, temperature and catalyst concentration on the selectivity of the transformation to organic compounds in relation to hydrogen peroxide consumed – Sorg./H2o2 indicates that over the range studied of variation of examined parameters the function assumes the maximum values – 100 mol.%. Only the interaction of temperature and 457

the reaction time on the selectivity of transformation to organic compounds in relation to hydrogen peroxide consumed demonstrates the range in which a decrease in the value of this function (Fig. 7) at up to 85 mol.% is observed. 300

250

reaction time (min)

200

150

100

50

10

15

20

25

30

35

40

45

50

55

60

temperature (°C)

fig. 7. Effect of temperature and reaction time on the selectivity of transformation to organic compounds in relation to organic compounds in relation hydrogen peroxide consumed constant process parameters: the molar ratio of CA/H2o2 = 2.8:1, methanol concentration 19 wt.%, Ti-MWW catalyst concentration 8 wt.%

From the studies of the inluence of process parameters on the course of function − Sorg./H2o2 it results that the maximum selectivity of transformation to organic compounds in relation to hydrogen peroxide consumed can be achieved when the process will be run at temperature chosen from the range 10−60°C, favourable at a temperature close to the ambient temperature (20−25°C). The CA/H2o2 molar ratio can be varied in the range 1:1−3:1. However, the application of the equimolar ratio of reagents is advantageous. The methanol concentration can be in the range 5−90 wt.% (most favourable 5 wt.%), catalyst concentration in a range 1−9 wt.% (most favourable 1 wt.%), reaction time 30−300 min (most favourable 30 min). ConCLUSionS The optimisation of process parameters of epoxidation of crotyl alcohol with 30 wt.% hydrogen peroxide in the presence of the Ti-MWW catalyst under atmospheric pressure allows to establish the parameters for which the functions describing the process achieve the maximum value and to determine the interactions among the parameters. Taking into consideration the courses of isolines of function describing the selectivity of transformation to 2,3EB1O in relation to CA consumed, the selectivity 458

of transformation to organic compounds in relation to hydrogen peroxide consumed and the CA conversion, the following values of parameters were recognised as optimum: temperature 20°C, molar ratio of CA/H2o2 = 1:1, methanol concentration 5 wt.%, the Ti-MWW catalyst concentration 7.8 wt.% and reaction time 140 min. A certain discrepancy between the parameters was only observed in the selection of catalyst concentration. The highest conversion of crotyl alcohol and the selectivity of transformation to organic compound in relation to hydrogen peroxide was achieved at the Ti-MWW catalyst concentration 1 wt.%. The highest value of function of the selectivity of transformation to 2,3EB1O in relation to CA consumed was found at the catalyst concentration 7.8 wt.%. However, the optimum parameters can be extended over the ranges in which the mentioned functions achieve the high values. These ranges are as follows: temperature 10−34°C, the CA/ H2o2 molar ratio from 1:1 to 3:1, methanol concentration 5−12 wt.%, the Ti-MWW concentration 1−8 wt.% and the reaction time 30−140 min. Hence, the interaction of parameters indicates that there is a possibility of their changes over wider intervals without lowering the values of the functions mentioned or to establish the parameter ranges, in which lowering of the function values is negligible. reFerenCeS 1. B. K. MarCUS, W. E. CorMier: Going Green with Zeolites. Chem Eng Prog, 95 (6), 47 (1999). 2. n. FenG, G. PENG: Applications of Natural Zeolite to Construction and Building Materials in China. Constr Build Mater, 19, 579 (2005). 3. F. SonG, y. LiU, H. WU, P. WU, t. tatSUMi: A Novel Titanosilicate with MWW Structure: Highly Effective Liquid-phase Ammoximation of Cyclohexanone. J Catal, 237, 359 (2006). 4. S. L. LAWTON, M. e. LEONOWICZ, r. D. PARTRIDGE, P. CHU, M. K. rUBin: Twelve-ring Pockets on the External Surface of MCM-22 Crystals. Micropor Mesopor Mat, 23, 109 (1998). 5. t. F. naGy, S. D. MAHANTI, J. L. Dye: Computer Modeling of Pore Space in Zeolites. Zeolites, 19, 57 (1997). 6. a. CorMa, C. MARTINEZ, a. MARTINEZ: Isobutene/2-butene Alkylation on MCM-22 catalyst. Inluence of Zeolite Structure and Acidity on Activity and Selectivity. Catal Lett, 28, 187 (1994). 7. r. ROQUE-MALHERBE, r. WENDELBO, a. MiFSUD, a. CorMa: Diffusion of Aromatic Hydrocarbons in H-ZSM-5, H-Beta, and H-MCM-22 Zeolites. J Phys Chem, 99, 14064 (1995). 8. M. e. LEONOWICZ, S. L. LAWTON, J. a. LAWTON, M. K. rUBin: MCM-22: A Molecular Sieve with Two Independent Multidimensional Channel Systems. Science, 264, 1910 (1994). 9. a. CorMa, C. CoreLLi, J. PEREZ-PARIENTE, r. GUIL-LOPEZ, S. NICOLOPOULOS, J. GONZALEZ: Adsorption and Catalytic Properties of MCM-22: The Inluence of Zeolite Structure. Zeolites, 16, 7 (1996). 10. X. CHEN, Z. Fan, X. QUan, K. WIE: Epoxidation of Allyl Alcohol to Glycidol on Ti-MWW Molecular Sieves. Chin J Catal, 27, 285 (2006). 11. D. ABERHART, L. J. Lin: Studies on the Biosynthesis of Beta-lactam Antibiotics. Part I. Stereospeciic Syntheses of (2RS,3S)-[4,4,4-2-H3]-, (2RS,3S)-[4-3-H]-,(RS,3R)- [4-3-H]-, and (2RS,3S)[4-13C]-valine. Incorporation of (2RS,3S)-[4-13C]-valine into Penicillin V. J Chem Soc Perk T, 1, 320 (1974).

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12. y. KOBAYASHI, y. Kitano: Diastereo- and Enantioselective Preparation of β-alkylhomoallylic Alcohols: Synthesis of Serricornin and Corynomycolic Acid. Tetrahedron, 42, 2937 (1986). 13. H. oKaMUra, S. KUroDa: Synthesis of Aplysiatoxin: Stereoselective Synthesis of Key Fragments. Tetrahedron Lett, 32, 5137 (1991). 14. a. WROBLEWSKA, a. FaJDeK, e. MILCHERT, B. GRZMIL: The Ti-MWW Catalyst – Its Characteristic and Catalytic Properties in the Epoxidation of Allyl Alcohol by Hydrogen Peroxide. Pol J ChemTechnol, 12, 29 (2010). 15. Z. POLANSKI, r. GORECKA-POLANSKA: R. Cadex: Esdet 2.2. Krakow, 1992 (in Polish). 16. r. ZIELINSKI: Statistical Tables. PWN, Warszawa, 1992 (in Polish). Received 15 October 2013 Revised 3 December 2013

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Oxidation Communications 37, No 2, 461–473 (2014) Oxidation in the presence of heterogeneous catalysts

PreParation of v/k-γ-al2o3 CatalySt and itS CatalytiC PerformanCeS in the oxidative dehydrogenation of CyClohexane HAINA WANGa, ZHIWU GUOa, HAIBO JINa*, SUOHE YANGa, GUANGXIANG HEa, XIAOQIAN ZHUb a

Department of Chemical Engineering, Beijing Institute of Petro-chemical Technology, 102 617 Beijing, China E-mail: [email protected] b State Grid Xinyuan Company Ltd., 100 761 Beijing, China aBStraCt The catalyst materials of the V/K-γ-Al2o3 are prepared and used in the oxidation dehydrogenation of cyclohexane to cyclohexene. The inluence of the different calcination temperature on the properties of catalyst is discussed using X-ray diffraction, XPS and SEM. The purity and grain size of the V/K-γ-Al2o3 catalyst and the dispersion of active components are presented in this study. According to the experimental results, the optimum reaction conditions were obtained. The cyclohexane conversion is 10.7% and cyclohexene selectivity is 47.8% under the reaction conditions for 450oC, hydrocarbon/oxygen mol ratio is 6/1 and contact time is 0.95 s. Moreover, the cyclohexene selectivity is higher with a decrease of the contact time. Keywords: catalyst materials, cyclohexane, oxidative dehydrogenation, cyclohexene. aiMS anD BaCKGroUnD Cyclohexene is an important organic chemical raw material that is widely used in medicine, food, agricultural chemicals, polyester, and other ine chemical products1. Cyclohexene is not only used to produce cyclohexanol by direct hydration, but is also an important intermediate in organic synthesis and being widely used to synthesise various ine chemicals, such as adipic acid, nylon-6, nylon-66, polyamide, and polyester. Cyclohexene and its downstream products thus have important industrial uses and broad market prospects2. To produce nylon-6 and nylon-66 as an example, there are 2 kinds of different methods. The irst involves oxidation of cyclohexane to cyclohexanone. This route *

For correspondence.

461

is associated with certain disadvantages: cyclohexane conversion and selectivity in favour of the inal product are low, considerable waste is generated, and the process consumes much energy3. The second route involves partial hydrogenation of benzene to cyclohexene and hydration of the latter to cyclohexanol. This process generates no waste or environmental pollution, is more atom-economic and environmental-friendly, and represents an advanced level of technology and green process development. With cyclohexene being the intermediate product of this process, the need for more active and selective catalysts is one of the limiting factors in the development of this technology. Therefore, an effective means of performing the oxidative dehydrogenation of cyclohexane to cyclohexene process should greatly expedite the eficiency of the process, and thus has broad application prospects4. For the oxidation of cyclohexane to cyclohexene, the selection of a suitable oxidant and catalyst is critical. The oxidants used have included S, O2, air, hydrogen peroxide, and tert-butyl hydrogen peroxide. The catalyst is a bimetallic complex catalyst5,6 (alloy or metal salt) or a metal oxide composite catalyst. If too strong an oxidising agent is used, besides the desired cyclohexene, some benzene, cyclohexanol, cyclohexanone, and COx may also be produced7. Anaerobic catalysts have also many disadvantages; investigators have used air as the oxidant for the oxidative dehydrogenation reaction8. O’Connor9 used air as the oxidant in conjunction with a noble metal active catalyst attached to a wire mesh, which gave a cyclohexane conversion rate of 25% and a selectivity in favour of cyclohexene of up to 45.3%. Jibril10 used MgxV2o5 as the catalyst for the alkane oxidative dehydrogenation reaction, while the selectivity and conversion were not high. Therefore, the main task is to ind the most eficient and economic catalyst to balance the relationship between the conversion rate of cyclohexane and the selectivity in favour of cyclohexene. Literature reports have documented the use of ZnO, TiO2 (Ref. 11), lanthanum oxide12, and alumina13 as supports for catalysts. Vanadium oxide shows good oxidation performance, and is used for the oxidative dehydrogenation reaction. Meanwhile, the strong alkalinity of potassium hydroxide can neutralise acidic sites on the catalyst, which is conducive to product formation. High surface area alumina is the possibility of a better dispersion of the active sites on the surface of the support. On the other hand, the acidic character of alumina can not negatively inluence the catalytic behaviour of the catalysts14. In this article, we represent the impregnation of γ-Al2o3 with V and K to produce a 3%V/6%K-γ-Al2o3 catalyst, and the results obtained when it was deployed in the oxidative dehydrogenation of cyclohexane. EXPERIMENTAL Catalyst preparation. Al2o3 powder was calcined in a mufle furnace at 550°C for 4 h to obtain γ-Al2o3. A weighed amount of ammonium met vanadate was dissolved in the appropriate volume of deionised water. A certain amount of potassium nitrate 462

was added to the solution, followed by γ-Al2o3 powder. After stirring at room temperature for 5 h, the mixture was impregnated overnight. Water was then evaporated in a rotary evaporator and the resulting solid powder was dried in an oven at 100°C for 10 h, calcined in a mufle furnace, then allowed to cool and ground to 80−100 mesh. In this way, 3%V/6%K-γ-Al2o3 catalyst was obtained. Experimental method and apparatus for the oxidation. Oxidative dehydrogenation of cyclohexane was performed in a ixed-bed tubular reactor. In the central portion of a 1/3 inch tubular reactor were placed a certain amount of the catalyst (0.5 g) and quartz sand (2 g). The feed gas low rate was controlled by means of a mass low-meter. All tested catalysts were pretreated at a certain temperature in air for 1 h. Cyclohexane was introduced into the gasiication chamber at a certain low rate, mixed with air, and transferred to the reactor. After a few hours under the above conditions, the product was analysed by gas chromatography to calculate the selectivity and conversion of cyclohexane to cyclohexene. The gas chromatograph used was a Shimadzu GC-14C. The chromatographic conditions were as follows: nitrogen as carrier gas, a gasiication chamber temperature of 200°C, an initial column temperature of 50°C, a heating rate of 15°C/min to a inal temperature of 110°C, and an injection volume of 2 μl. Characterisation of the catalyst. Powder X-ray diffraction (XRD) patterns were obtained on a Shimadzu XRD-7000 diffractometer using Cu-Kα radiation (λ = 40 kV, 30 mA); the scan rate was 4° min−1, the scan step was 0.02°, and the scan range was 15−80°. The morphology and size of the catalyst particles were assessed on a Hitachi S4700 series scanning electron microscope. X-ray photo electron spectroscopy (XPS) measurements were performed with a PHI5300/ESCA instrument equipped with a charge neutraliser and a Al K Alpha source. Spectra were recorded from 0 to 1350 eV, with an analyser pass energy of 100 eV and the scan step is 1 eV. Narrow spectrum scan step is 0.5 eV, and pass energy is 30 eV. Binding energies were referenced to C1s of adventitious carbon at 285 eV. reSULtS anD DiSCUSSion CHARACTERISATION OF THE CATALYST

XRD characterisation. Figure 1 shows the XRD spectra of 3% V/6%K-γ-Al2o3 catalyst samples calcined at different temperatures. It can be seen from Fig.1 that the main diffraction peaks are those attributable to γ-Al2o3 (2θ =32.65°, 37.91°,45.88°, and 67.27°). For the sample calcined at 600°C, the XRD spectrum is relatively broad, and the diffraction peak intensity is weak. For samples calcined at above 650°C, the XRD spectra are sharper, showing that an appropriate calcination temperature can contribute to the formation of a good crystal structure.

463

intensity (a.u.)

e d c b a 20

30

40

50

60

70

80

2θ (degree)

fig. 1. XRD patterns of catalysts calcined at different temperatures: a – 600°C; b – 650°C; c – 700°C; d – 750°C; e – 800°C

Figure 2 shows the XRD patterns of samples obtained after different calcination times. It can be seen from Fig. 2 that the calcination time does not have a signiicant impact on the crystalline form of the catalyst. Under typical calcination times, the crystalline phase and structure do not show signiicant changes. Although the crystalline phase diffraction peaks become slightly sharper and the diffraction intensity is gradually increased after an extended calcination time. The crystalline phase diffraction peaks were sharpest after a calcination time of 7 h.

intensity (a.u.)

e d c b a

20

30

40 50 60 2θ (degree)

70

80

fig. 2. XRD patterns of catalysts calcined at different time: a – 4 h; b – 5 h; c – 6 h; d – 7 h; e – 9 h

XPS characterisation. Figure 3 shows XPS full-scan spectrum in 3% V/6%K-γ-Al2o3 catalyst (calcined at 650°C, 5 h) surface. By comparing the standard binding energy table, located in 292.9, 295.7, 517, and 530 eV near-by spectrum peak corresponding to K (1s), v (2p) and o (1s) binding energy, it illustrated that K2o and v2o5 exist in the sample. O (1 s)spectrum peak is stronger than V (2p) spectrum peak. Due to the active species highly dispersed or the component capacity below the instrument valve value, XRD characterisation results did not present the diffraction peak of V2o5 and K2Os. Figure 4 shows the 3% V/6%K-γ-Al2o3 catalyst surface XPS spectra. Figure 4a is K (1s) spectrum, there are 2 strong peaks and the binding energy is 293 and 296 eV. There are two strong peaks of 516 and 524 eV corresponding to the V (2p3/2) and v (2p1/2) in the V (2p) spectrum in Fig. 4b. Figure 4c shows the O (1s) spectrum; 464

binding energy is 530–533 eV, and the spectrum peak is asymmetrical. In addition, there is obvious a tailing phenomenon in high binding energy direction.

intensity (CPS)

O (1s)

K (1s) V (2p)

0

200

400

600

800

1000

binding energy (eV)

fig. 3. XPS scanning spectrum of 3%V/6%K-γ-Al2o3 catalyst a

intensity (CPS)

intensity (CPS)

b

290

295 300 305 310 binding energy (eV)

315

505

510

515 520 525 binding energy (eV)

530

intensity (CPS)

c

525

530

535 540 545 binding energy (eV)

550

fig. 4. Fine XPS spectra of elements of the surface of catalyst: a – K(1s); b – V(2p); c – O(1s)

In oxidative dehydrogenation reaction, gas phase oxygen will irst be absorbed on the catalyst surface, through the V4+ and v5+ REDOX into lattice oxygen then to reaction. According to reports in literature15, oxygen species electron binding energy varies in general between 529.0 and 533.0 eV. The values of the binding energy amount to 530.4, 531.8 and 532.9 eV, which correspond to O2–, o– and o2– oxygen species. So, we separate the O (1s) spectrum peak as shown in Fig. 5a. From Fig. 5a one can see that O (1s) to high binding energy direction has trailing, this means that the original O– and o2– oxygen species can easily become lattice oxygen species O2–. Vanadium species exist as V4+ and v5+, v4+ exists as V2o4, and the values of the elec465

tron binding energy (BE) are 515.5, 515.7 and 516.3–516.6 eV, V5+ exists as V2o5, and the electron binding energy is 517.1 and 518.0 eV (Refs 16–18). We separate the V spectrum peak in Fig. 5b. The results are shown in Table 1. table 1. Results of peak itting for 3%V/6%K-γ-Al2o3 catalyst XPS spectra

Species

Be (ev) 530.4 531.8 532.9 515.7 524.3 517.1 524.9

o2– o– o 2– V′(2p3/2) V′(2p1/2) v(2p3/2) v(2p1/2)

o

v4+ v5+

Mol. fraction (%) 30.87 52.41 16.72 28.70 71.30

b

intensity (CPS)

intensity (CPS)

a

525

530

535 540 binding energy (eV)

545

550

508 510 512 514 516 518 520 522 524 526 528 530 binding energy (eV)

fig. 5. Fitting curves of XPS spectra for 3% V/6% K-γ-Al2o3 catalyst: a – O (1s); b – V (2p)

SEM characterisation. Figure 6 shows SEM images of catalyst samples calcined at different temperatures. It can be seen that different degrees of dispersion of the catalyst samples were obtained at the different temperatures. For the catalysts calcined at 600 and 650°C, relatively uniform particles of size about 0.5−1.5 µm were obtained. When the calcination temperature was 700°C, the surface of the active species was granular, with a particle size of about 1.5−2.5 µm. For the sample calcined at 750°C, the granules were more obvious and showed some agglomeration. When the calcination temperature was increased to 800°C, the active component on the sample surface showed a signiicant degree of agglomeration.

466

b

a

d

c

e

fig. 6. SEM micrographs of catalysts calcined at different temperatures: a – 600°C; b – 650°C; c – 700°C; d – 750°C; e – 800°C

EFFECT OF PREPARATION CONDITIONS ON THE PROPERTIES OF THE CATALYST

Inluence of calcination temperature. A series of experiments was performed using catalysts calcined for 5 h at different temperatures. The reaction temperature was 450°C, the cyclohexane feed rate was 0.84 ml/min, and the air low rate was 150 ml/min.The experimental results are shown in Fig. 4. The calcination temperature is selected according to the temperature at which the catalyst is to be deployed, the temperature at which the active substance forms a speciic crystal type, the temperature of matrix decomposition of the active substance, and the thermal stability of the carrier. The calcination temperature directly affects the lattice, the crystal type at the active centers, and the pore structure of the catalyst. From Fig. 7, it can be concluded that when the catalyst was calcined at 650°C, its activity was good, the conversion rate was 11.6%, and the selectivity was 47.7%. When the calcination temperature was higher, a part of the catalyst particles agglomerated and the catalyst surface area was reduced leading to catalyst sintering and deactivation. When the calcination temperature was lower, because the speciic crystal type of the active substance had not been formed, the conversion and selectivity were lower. Therefore, the optimum calcination temperature was 650°C.

467

12

conversion (%)

10 8 6 4 2 600

650

700 temperature (°C)

750

selectivity (%)

50 48 46 44 42 40 38 36 34 32 30 28 26 24 22 20 18 16

14

800

fig. 7. Effect of different calcined temperature on cyclohexane conversion and cyclohexene selectivity ■ – cyclohexane conversion;▲ – cycloalkene selectivity,▼ – benzene selectivity, ● – COx selectivity

Inluence of calcination time. A series of experiments were performed with catalyst samples calcined for different times at 650°C. The reaction temperature was 450°C, the feed rate of cyclohexane was 0.84 ml/min, and the air low rate was 150 ml/min. The results of these experiments are shown in Fig. 8. It can be seen that with increasing calcination time the activity of the catalyst remained largely unchanged. When the calcination time exceeded 7 h, the selectivity for cyclohexene decreased. In general, all of the catalysts gave good results. 14

12

8

6

4

2 3

4

5

6

7

8

9

selectivity (%)

conversion (%)

10

50 48 46 44 42 40 38 36 34 32 30 28 26 24 22 20 18 16 14 10

time (h)

fig. 8. Effect of different calcined times on cyclohexane conversion and cyclohexene selectivity ■ – cyclohexane conversion,▲ – cycloalkene selectivity,▼ – benzene selectivity, ● – COx selectivity

468

OPERATING CONDITIONS FOR OXIDATIVE DEHYDROGENATION

Inluence of reaction temperature. Figure 9 shows the effect of different reaction temperatures on the dehydrogenation of cyclohexane to cyclohexene. Within a certain temperature range, the selectivity in favour of cyclohexene is improved by increasing the reaction temperature. From Fig. 9 it is evident that on raising the reaction temperature, the conversion rates of cyclohexane and the selectivity in favour of the by-products were signiicantly increased. With increasing the temperature from 425 to 525°C, the conversion rate of cyclohexane rose from 6 to 38.3%. The selectivity for benzene changed little, while that for COx increased from 34.4 to 51.3%. When the temperature exceeded 500°C, the selectivity for cyclohexene decreased from 46.5 to 28.1%. 40 45

30

40

25

35 30

20 25 15 20 10

selectivity (%)

conversion (%)

50 35

15

5 420

440

460

480

500

520

10 540

temperature (°C)

fig. 9. Effect of reaction temperature on cyclohexane oxidative dehydrogenation depending on residence time ■ – cyclohexane conversion, ▲ – cycloalkene selectivity, ▼ – benzene selectivity, ● – COx selectivity

Cyclohexane reacts with the metal oxide catalyst as follows: C6H12+[Mn+on2–]

[C6H11–Mn+on–1(OH)]

[C6H11–Mn+on–1(OH)] → [C6H11–M(n–1)+on–12–] → C6H11– + [M(n–1)+on–12–(OH)]–

The generated cyclohexane free radicals can continue to react with the catalyst, forming cyclohexene. Cyclohexene will be further dehydrogenated to generate 1,3cyclohexadiene, benzene, and COx. The main reaction is oxidative dehydrogenation to generate cyclohexene.The main and side reactions are irreversible, and reach a thermodynamic equilibrium balance. From the curves of the selectivities for cyclohexene, benzene, and COx, it can be seen that with increasing reaction temperature, the conversion rate of cyclohexane increased accordingly. Because cyclohexene on the catalyst surface will undergo secondary reaction, the rates of generation of benzene

469

and Cox will also be enhanced. Based on these results, 450°C was selected as the optimum reaction temperature. Inluence of residence time. Figure10 shows the effect of different residence times when the reaction temperature was 450°C. It can be seen from Fig. 10 that when the residence time was increased from 0.76 to 1.05 s, the conversion rate of cyclohexane increased from 6 to 15.3%, the selectivity for COx increased from 30.7 to 37.1%, and the selectivity for cyclohexene decreased from 58 to 46.1%. At shorter residence times, cyclohexene is the principal product. With the increase of residence time, more cyclohexane will reside on the catalyst surface, and cyclohexene is more likely to undergo side reactions. When cyclohexane remains on the catalyst surface for a long time, the selectivity for COx increases, and the conversion rate of cyclohexane is improved. Therefore, cyclohexane conversion rate and selectivity for COx are higher and the selectivity for cyclohexene is lower at longer residence times. 60 16 50

12

40

10

30

8

selectivity (%)

conversion (%)

14

20 6 10 0.75

0.80

0.85

0.90 time (s)

0.95

1.00

1.05

fig. 10. Effect of residence time of cyclohexane oxidative dehydrogenation ■ – cyclohexane conversion,▲– cycloalkene selectivity,▼– benzene selectivity, ● – COx selectivity

The oxidative dehydrogenation of cyclohexane involves a series of chain reactions. Cyclohexene is an unstable intermediate product, hence it can be further oxidised to benzene and COx at long contact times. Therefore, the shorter the residence time, the higher the selectivity for cyclohexene. Inluence of the hydrocarbon/oxygen ratio. Figure 11 shows the inluence of hydrocarbon/oxygen molar ratio on the oxidative dehydrogenation of cyclohexane. From Fig. 11, it can be seen that at 450°C, as the hydrocarbon/oxygen ratio was increased from 2:1 to 6:1, the cyclohexane conversion rate was reduced from 18 to 11.6%, the selectivity for COx decreased from 75.7 to 35.4%, and the selectivity for cyclohexene increased from 15.4 to 47.7%. The Mars–van Krevelen reaction mechanism suggests that oxygen is irst adsorbed on the catalyst surface, and then through redox reactions of V4+ and v5+ it is 470

20 19 18 17 16 15 14 13 12 11 10 9 8 7 6 5

70 60 50 40 30

selectivity (%)

conversion (%)

transformed from adsorbed oxygen into lattice oxygen, which then participates in the oxidative dehydrogenation of cyclohexane. When excess oxygen is supplied with the reactant, it can be adsorbed on the catalyst surface, generating the electrophilic oxygen species o2−, o−, and o2−. These species are conducive to the combustion reaction, so that the selectivity for COx is high. Conversely, when fewer electrophilic oxygen species are adsorbed on the catalyst surface, lattice oxygen can participate in the oxidative dehydrogenation reaction more readily, and so the selectivity for cyclohexene is higher19. The reaction order of the oxidative dehydrogenation of hydrocarbons to generate COx is higher than that of the main reaction20,21, and so increasing the partial pressure of oxygen does not usually increase the reaction rate. However, it decreases the selectivity for the target product.

20 10 2:1

3:1

4:1 5:1 n(cyclohexane)/n(O2)

6:01

fig. 11. Effect of hydrocarbon oxygen ratio of cyclohexane oxidative dehydrogenation ■ – cyclohexane conversion, ▲ – cycloalkene selectivity, ▼ – benzene selectivity, ● – COx selectivity

Since the reaction is performed at 450°C, in addition to the catalytic reaction on the surface, the presence of oxygen will lead to extensive oxidation of gaseous cyclohexane, thus leading to a decrease in the selectivity for the target product and the generation of more by-products. ConCLUSionS 1. The optimum preparation conditions for 3% V/6%K-γ-Al2o3 are obtained as calcination at 650°C for 5 h. 2. Different operating conditions for the oxidative dehydrogenation of cyclohexane inluence the product distribution. The optimum reaction temperature has been identiied as 450°C. The contact time and hydrocarbon/oxygen ratio also affect the conversion rate of cyclohexane and the selectivities for cyclohexene and COx. Under reaction conditions of 450°C, a hydrocarbon/oxygen molar ratio of 6:1, and a contact time of 0.95 s, the cyclohexane conversion was 10.7% and the selectivity for

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cyclohexene was 47.8%. The shorter the residence time is, the higher the selectivity for cyclohexene. To improve the selectivity for cyclohexene, an appropriate residence time and hydrocarbon/oxygen ratio should be selected. ACKNOWLEDGEMENTS This research was supported by the National Natural Science Foundation of China under agreement number 21073020, the Beijing Municipal Natural Science Foundation under agreement number 2093034, and The Importation and Development of High-Caliber Talents Project of Beijing Municipal Institutions Agreement Number CIT&TCD20130325. reFerenCeS 1. M. JIN, G. X. YU, Z. M. LI: Research Progress of Catalysts for the Gas-phase Oxidative Dehydrogenation of Cyclohexane to Cyclohexene. Chem Bioeng, 28 (3), 13 (2011). 2. L. F. ZHAO, J. B. GUO: Development in Synthesis of Cyclohexene. Adv Fine Petrochem, 5 (7), 19 (2004). 3. H. B. JIN, W. W. YUAN, Z. W. GUO: Catalytic Oxidation Reaction of Cyclohexane on Co3o4 Supported Carbon Nanotube Catalysts. Petroleum Processing Section (Act A Petrolei Sinica), 27 (1), 47 (2011) (in Chinese). 4. Z. W. GUO, H. B. JIN, Z. M. TONG: Advances in Techniques for Production of Cyclohexanone and Cyclohexanol. Chem Industry Eng, 25 (8), 852 (2006). 5. N. A. GAIDAI, A. L. LAPIDUS. Inhibition of Catalytic Reactions Related to Process Mechanism. Oxid Commun, 32 (2), 285 (2009). 6. E. HERACLEOUS, A. A. LEMONIDOU: Ni–Me–O Mixed Metal Oxides for the Effective Oxidative Dehydrogenation of Ethane to Ethylene. Effect of Promoting Metal. Met J Catal, 270, 67 (2010). 7. H. JIN, D. LI, ZH. WU: Catalytic Oxidation Conditions of 2,6-diisopropylnaphthalene in a Liquid Phase Reactor with Co-Mn-Br Catalyst. Oxid Commun, 33 (2), 283 (2010). 8. B. Y. JIBRIL, N. O. ELBASHIR, S. M. Al-ZAHRANI, A. E. ABASAEED: Oxidative Dehydrogenation of Isobutane on Chromium Oxide-based Catalyst. Chem Eng Process, 44 (8), 835 (2005). 9. R. P. O’CONNOR, E. J. KLEIN, D. HENNING, L. D. SCHMIDT: Tuning Millisecond Chemical Reactors for the Catalytic Partial Oxidation of Cyclohexane. Appl Catal A General, 238 (1), 29 (2003). 10. B. Y. JIBRIL, A. Y. ATTA, K. MELGHIT, Z. M. El-HADIANDA. H. Al-MUHTASEB: Performance of Supported Mg0.15v2o5.152.4H2O Nanowires in Dehydrogenation of Propane. Chem Eng J, 193&194, 391 (2012). 11. E. HERACLEOUS, M. MACHLI, A. A. LEMONIDOU, I. A.VASALOS: Oxidative Dehydrogenation of Ethane and Propane over Vanadia and Molybdena Supported Catalysts. J Mol Catal A – Chem, 232, 29 (2005). 12. A. A. SHUKLA, P. V. GOSAVI, J. V. PANDE, V. P. KUMAR, K. V. R. CHARY, R. B. BINIWALE: Eficient Hydrogen Supply through Catalytic Dehydrogenation of Methylcyclohexane over Pt/Metal Oxide Catalysts. Int J Hydrogen Energy, 35, 4020 (2010). 13. E. GBENEDIO, Z. WU, I. HATIM, B. F. K. KINGSBURY, K. LI: A Multifunction Pd/Alumina Hollow Fiber Membrane Reactor for Propane Dehydrogenation. Catal Today, 156, 93 (2010).

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14. G. MITRAN, I. C. MARCU, A. U. I. SANDULESCU: Oxidative Dehydrogenation of Isobutane over Supported V–Mo Mixed Oxides. J Serb Chem Soc, 75 (8), 1115 (2010). 15. A. J. XU, G. T. ZHAO, M. L. JIA, Q. LIN: Study on Performance of Ni3v2o8 Catalyst and Analysis of X-ray Photoelectron Spectroscopy. Spectrosc Spect Anal, 27, 2134 (2007). 16. S. H. SAM, D. V. SOENEN, J. C. VOLTA: Oxidative Dehydrogenation of Propane over V–Mg–O Catalysts. J Catal, 123, 417 (1990). 17. X. T. GAO, Z. P. RUI, Q. XIN, X. X. GUO, B. DELMON: Effect of Coexistence of Magnesium Vanadate Phases in the Selective Oxidation of Propane to Propene. J Catal, 148, 56 (1994). 18. S. SINGH, S. B. JONNALAGADDA: V2o5 Supported on Cobalt Hydroxyapatite for the Partial Oxidation of n-pentane. Oxid Commun, 34 (4), 768 (2011). 19. M. JIN: Oxidative Dehydrogenation of Cyclohexane over Mg–V–O. East China University of Science and Technology, Shanghai, China, 2010. 20. S. H. GE: Oxidative Dehydrogenation of Butane over V–Mg–O Catalyst and Selective Oxidation of Lower Hydrocarbons Using Inert Inorganic Membrane Reactor. Dalian University of Technology, Dalian, China, 2000. 21. JI-DONG LOU, FANG LIN, QIANG WANG: Selective Oxidation of Benzoins with Chromic Acid Supported on Silica Gel under Viscous Conditions. Oxid Commun, 34 (3), 616 (2011). Received 12 March 2013 Revised 2 May 2013

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Oxidation Communications 37, No 2, 474–482 (2014) Antioxidants in biological systems

ProteCtive effeCt of Allium cepa l. anthoCyanin extraCt on the oxidative StaBility of Sunflower oil S. oanCeaa*, C. GroSUb a

Department of Agricultural Sciences and Food Engineering, ‘Lucian Blaga’ University of Sibiu, 7–9 Ion Ratiu Street, 550 012 Sibiu, Romania E-mail: [email protected] b Department of Environmental Sciences, ‘Lucian Blaga’ University of Sibiu, 5–7 Ion Ratiu Street, 550 012 Sibiu, Romania aBStraCt The paper describes the great potential of anthocyanin extracts from dry skins of red onions (Allium cepa L.) to stabilise sunlower oil in comparison to the effect generated by tocopherols mixture. Total phenolics content in the investigated red onion samples is 1346 mg GAE 100 g–1 fresh mass (FM), while total anthocyanins content is 99.66 mg 100 g–1 FM. The anthocyanin extract shows activity against Streptococcus pyogenes. The effectiveness of the natural antioxidant on the oxidative stability of sunlower oil was assessed by evaluation of primary and secondary oxidation products, using peroxide value and thiobarbituric acid reactive substances tests. Our results indicate that sunlower oil containing small amounts of red onion anthocyanin extract exhibits lower levels of lipid oxidation at 40oC during 10-day storage compared to control and samples containing tocopherols. Also, a signiicant decrease of thiobarbituric acid reactive substances (TBARS) values of sunlower oil treated with red onion anthocyanin extract was observed compared to the control sample. These results may contribute to the future consideration of anthocyanins from red onion by-products as economically advantageous sources of natural antioxidants and antimicrobials to be used in edible oils. Keywords: red onion, anthocyanins, sunlower oil, peroxide value, TBARS, antimicrobial activity. aiMS anD BaCKGroUnD Sunlower oil is an edible oil rich in unsaturated fatty acids, in particular oleic and linoleic acids, and is traditionally consumed in Eastern Europe. Ukraine and Russian *

For correspondence.

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Federation are the top producers of sunlower seeds, while Romania ranks on fourth place (http://faostat.fao.org/site/339/default.aspx). Several health effects generated by consumption of sunlower oil, most of them being associated with a reduced risk of cardiovascular diseases, are sustained by clinical studies1. Sunlower oil has also found application as cosmetical product based on its anti-wrinkling and anti-ageing properties2. In natural form, sunlower oil is highly susceptible to autoxidation and thermal oxidation under air, light and temperature. This oxidative degradation inluences not only the quality of sunlower oil such as lavour, colour and texture, but also the nutritional quality and the potential generation of toxic compounds3,4. Consequently, antioxidant protection is required. Effective synthetic antioxidants such as butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), propyl gallate (PG), and tertiary butylhydroquinone (TBHQ) are frequently used to delay lipid oxidation in oils, fats and foods. Nowadays, there is an increased concern regarding the safety of synthetic antioxidants, so that alternative strategies based on identiication of natural sources of antioxidants have been the hot topic of research in the ield. Natural antioxidants are found in a wide range of plants. In literature are described several free radical scavengers isolated from natural sources, such as tocopherols, carotenoids (including β-carotene, lycopene and lutein), polyphenols (including catechins and lavonoids), amino acids, peptides, proteins, urate and ascorbate, which act as singlet oxygen quenchers5–7. Among plant bioactives, anthocyanins which are water-soluble pigments, display beneicial health effects based on their free-radical scavenging and antioxidant capacities8. Most of the reported studies regarding the application of anthocyanins in food industry have been focused on their stability and colour change in aqueous media9,10 and limited data regarding their behaviour in a polar media is available. Therefore, it is highly required to stabilise these pigments in food systems in order to manufacture food products enriched with anthocyanin extracts. Among sources rich in anthocyanins, dry skin of red onion (Allium cepa L.) may ind useful economic and eco-friendly application for obtaining natural food ingredients and for improving the use of such wastes resulted from the red onion manufacturing. Different extraction parameters may be optimised in order to obtain an anthocyanin enriched crude extract with good stability11. Besides culinary and medicinal applications, onions are also used for cosmetic purposes or as herbal dyestuffs in textile dyeing12. These practical considerations were of great interest to us to investigate some properties of crude extracts obtained from this cheap source in terms of potential food ingredients or antimicrobials, which are reported here for the irst time as part of our studies and research on anthocyanins. Thus, the present paper describes a study on the improvement of sunlower oil oxidative stability by addition of a red onion anthocyanin extract. The aims of this investigation were: (i) to characterise red onion dry skins through total anthocyanins and total phenolics content; (ii) to determine the antioxidant effect of a crude anthocyanin extract from Allium cepa L. added to 475

sunlower oil in an emulsion system through 2 different analytical methods; (iii) to compare the results to those exhibited by control and by sunlower oil treated with tocopherols (TOCOMIX) additive, and (iv) to test the antimicrobial activity of the crude anthocyanin extract on standard bacterial and fungal strains. EXPERIMENTAL Reagents and solutions. Chemical reagents of analytical grade without further puriication were used for preparing the solutions. Ethanol (> 96% v/v), chloroform (min. 99%), hydrochloric acid (37%) and glacial acetic acid were obtained from Adrachim (Romania), potassium iodide, potassium chloride, sodium acetate (trihydrate) and 0.1 N sodium thiosulphate from Chimopar (Romania), gallic acid from Fluka (Germany), propyl gallate (98%) and malonaldehyde bis(diethylacetal) (97%) from Acros Organics (Belgium), thiobarbituric acid (98%) from Alfa Aesar (Germany), while starch of synthesis grade, sodium monophosphate and sodium diphosphate were purchased from Scharlau (Spain). Buffer solutions were prepared in distilled water. Extraction and assay of total anthocyanins. Anthocyanins were extracted from dry skins of red onion (Allium cepa L.) grown in Romanian region, in a solvent mixture of ethanol/acetic acid/water/ (25/4/21), overnight, at 4oC. The extract was iltered and centrifuged at 4oC at 8000 rpm for 10 min. The refrigerated centrifuge (Nuve NF 800R, Turkey) was used. The total content of anthocyanins in red onion extract was spectrophotometrically determined by the pH differential method13. Measurements were done in 2 replicates. The Specord 200Plus UV-vis. spectrophotometer (Analytik Jena, Germany) was used. Total anthocyanins were expressed as cyanidin-3-O-glucoside (Cyn-3-O-G) in milligrams 100 g–1 fresh mass (mg 100 g–1 FM). Extraction and assay of total phenolics. Total phenolics were extracted from dry skins of red onion (Allium cepa L.) in 90% (v/v) methanol solution. The total phenolics content in the methanol extract was determined spectrophotometrically according to the Folin–Ciocalteu method14. Gallic acid was used as standard for the calibration curve. The total phenolics content was expressed in milligrams of gallic acid equivalents 100 g–1 fresh mass (mg GAE 100 g–1 FM). Reverse micelles preparation. The commercially purchased cold-pressed sunlower oil was used in the present investigation. Reverse micelles (RM) were prepared by dissolving liquid soy lecithin in sunlower oil to obtain 0.5% concentration. To the mixture, 100–200 µl of red onion anthocyanin extract were added, under vigourous stirring conditions. Aliquots from the obtained emulsions were used for TBARS analysis. All emulsions were stored in duplicate in Petri dishes in dark at 40oC over a 10-day period. Two aliquots of each were removed periodically for peroxide value (PV) analysis. Similar preparation was done when using tocopherols as fat-soluble

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antioxidants (Tocomix, liquid mixed tocopherols) added to sunlower oil samples, in concentration of 0.1%. Lipid hydroperoxides determination (PV). Peroxide values were determined by iodometric standard procedure and expressed as meq kg–1 (Ref. 15). Data from the PV measurements were plotted against time. Thiobarbituric acid reactive substances (TBARS) determination. The thiobarbituric acid reactive substances (TBARS) method16 with some modiications was used to measure the antioxidant activity of red onion anthocyanin extract related to the lipid oxidation in a sunlower oil system. Briely, 1 g of prepared sunlower oil samples was treated with 4 ml of the 50 mM phosphate buffer–trichloracetic acid (TCA) solution and 4 ml of 20 mM thiobarbituric acid (TBA) solution. The mixture was heated in a boiling water bath for 20 min. After cooling the mixture at room temperature, 2 ml of chloroform were added. After centrifugation, the chloroform layer was separated and the absorbance of the supernatant was measured at 530 nm. Malonaldehyde standard curve was prepared using 1,1,3,3-tetramethoxypropane and TBARS were expressed as mg of malonaldehyde kg–1 oil sample (meq MDA kg–1). Antimicrobial activity assay. Antimicrobial activity of the anthocyanin extract from red onion dry skins was tested against gram-positive/gram-negative bacteria and fungi by the standardised disk diffusion method17 using the Müller–Hinton culture medium and 5-mm diameter disks. In the case of streptococci, a sheep-blood agar medium was used. An inoculum size of 105–107 CFU (colony forming units) was swabbed on the test medium. Extract-impregnated disks were placed on the inoculated plates and incubated at 37oC. The results were read after 24 h by measuring the inhibition zones (in mm). The following microbial strains were investigated: Staphylococcus aureus ATCC 25923, Streptococcus pyogenes ATCC 19615, Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, Enterobacter aerogenes, Bacillus cereus, Bacillus subtilis, Cronobacter muytiensis, Lysteria monocitogenes and Candida albicans ATCC 10231, where ATCC stands for American Type Culture Collection. reSULtS anD DiSCUSSion Characterisation of red onion dry skin samples was done by evaluation of relevant antioxidant chemical compounds (total anthocyanins and total phenolics). Total phenolics content was found of 1346 mg GAE 100 g–1 FM, while total anthocyanins content was found of 99.66 mg 100 g–1 FM. Extraction of anthocyanins from red onion was conducted in a solvent mixture of ethanol/acetic acid/water/ (25/4/21), so that better anthocyanins recovery and slower anthocyanins degradation is obtained. Commonly, extraction of anthocyanins is carried out in acetone or acidiied methanol solutions in order to obtain the red stable lavylium cation13. Considering the toxic effects of methanol and the inal potential food application of the obtained crude extract, we have substituted methanol with ethanol. 477

An increased number of research studies are described in literature for assessing the antioxidant activity of anthocyanins in vivo and in vitro. Such investigative methodologies refer to oxygen radical absorbance capacity assay (ORAC), total reactive antioxidant potential (TRAP), ferric-reducing antioxidant power assay (FRAP), Trolox equivalent antioxidant capacity (TEAC), the Folin–Ciocalteu (FC) or total phenolics assay, thiobarbituric acid reactive substances assay (TBARS), DPPH radical scavenging activity, β-carotene-linoleic acid assay, generation of the radical anion superoxide with xanthine oxidase-hypoxanthine system, and generation of the hydroxyl radical by means of the system hydrogen peroxide–peroxide. These assays are based either on the hydrogen atom transfer or a single-electron transfer reactions18. Our investigation aimed to assess the antioxidant effect by simple assays based on the evaluation of primary and secondary oxidation products in a sunlower oil system that has relevant practical applications. Through this methodology it is possible both to determine the antioxidant potential of anthocyanins and to exploit their application in food/dietary supplements industry to stabilise edible oils. In Table 1, nutritional information of investigated sunlower oil declared as biological, is presented as described by the manufacturer. Tocopherols were not removed from the samples. table 1. Composition of the investigated sunlower oil

Composition Saturated fatty acids (g) Monounsaturated fatty acids (g) Polyunsaturated fatty acids (g)

100 g fats 10 25 65

As sunlower oil is subject to oxidative deterioration, we investigated its stabilisation by addition of the red onion anthocyanin extract in a reverse micelle (RM) system. We selected red onion anthocyanin extracts as natural antioxidants because they are rich in phytochemicals, which may preserve unsaturated fatty acids during storage. Moreover, from the economic point of view, the wastes in terms of dry skins of red onion which were used in the present study for bioactive compounds extraction, may ind useful application as food ingredients. We investigated a RM system which we prepare from sunlower oil and acidiied hydroethanolic anthocyanin extract, in the presence of an emulsiier (soy lecithin). The inhibition of lipid oxidation was measured by peroxide value (PV) and thiobarbituric acid reactive substances (TBARS). PV measurement is a well-established method (AOCS Oficial Method) for the determination of the primary oxidation products in fats and related compounds, while TBARS is a widely used test for measuring the secondary oxidation products (e.g. aldehydes). In our study, we have monitored the oxidative stability of sunlower oil over a 10-day period at 40°C as follows: (1) control sample; (2) samples treated with red

478

onion anthocyanin extract (water soluble antioxidant); and (3) samples treated with Tocomix (fat soluble antioxidant). The obtained PV results indicate that samples of sunlower oil containing red onion anthocyanin extract had lower levels of lipid peroxidation compared to samples treated with Tocomix during 10-day of storage at 40oC, as shown in Fig. 1. The inhibition of hydroperoxides formation in sunlower oil was 82% in case of addition of red onion anthocyanin extract compared to control sample, while oxidation reactions were increased by about 10% in case of samples treated with Tocomix, in the irst 5 days. After this period, PV values were found similar both for control and samples treated with Tocomix, while a steady state regarding the improvement of oxidative stability was registered for the samples treated with red onion anthocyanin extract. The mechanism of action regarding the stable PV values of anthocyanins-treated oil after 5-day storage at 40oC needs to be further investigated; slow decomposition of hydroperoxides to endoperoxides and other products in the presence of anthocyanins crude extract might be involved. On the other hand, the time-related decrease of inhibition of oxidative reactions by added tocopherols in polyunsaturated oils and their pro-oxidant action at higher concentration was also shown by other workers19. We do not exclude a sinergistic effect between anthocyanins and tocopherols present naturally in sunlower oil as minor components, which might occur in the investigated system, as naturally tocopherols were not removed from the original oil samples.

fig. 1. Effect of red onion anthocyanin extract and synthetic tocopherols on the oxidative stability of sunlower oil at a storage temperature of 40oC

The results of TBARS screening test of the three type of samples indicate a signiicant increase of these values for the sunlower oil sample treated with Tocomix and a signiicant decrease for the sunlower oil sample treated with red onion anthocyanin extract, compared to the control sample as shown in Fig. 2. Therefore, one would recommend adding small amounts, such as 25 µg of total anthocyanins

479

from red onion extract to 100 g oil in order to increase its oxidative stability, without much affecting colour characteristics.

fig. 2. Changes in the TBARS values of sunlower oil treated with red onion anthocyanin extract and Tocomix

These results highlight the great potential of red onion dry skins to be used as an economic source of antioxidant bioactive compounds for development of functional foods. To our knowledge, literature is scarce in such studies on anthocyanins, but successful attempts were done by our group by using bilberry anthocyanin extract which exhibited remarkable improvement of oxidative stability of cod liver oil20. Other authors reported good results with different natural antioxidants such as tea catechins21 or rosemary extract added to ish oils22 and mulberry leaves extract added to rice bran oil23. It is well known that extracts of Labiatae (Lamiaceae) plant family such as oregano, savory, sage, rosemary, thyme, and basil have high total phenolics content24, which determined most experiments to investigate these herbs in stabilising sunlower oil25. The antioxidant components of rosemary, sage, basil, black pepper, and garlic appear to be relatively stable26. Moreover, by testing the antimicrobial activity of the red onion anthocyanin extract on 10 microbial strains (Staphylococcus aureus, Streptococcus pyogenes, Escherichia coli, Pseudomonas aeruginosa, Enterobacter aerogenes, Bacillus cereus, Bacillus subtilis, Cronobacter muytiensis, Lysteria monocitogenes and Candida albicans) by disk diffusion test, we have found an antistreptococcal activity of 8-mm inhibition diameter against the standard strain Streptococcus pyogenes ATCC 19615. The exhibited bioactivity may further contribute to the preservation of the sunlower oil enriched with the anthocyanin extract from red onion dry skins. Also, this inding may be of future interest for the design of new potential antibiotics of plant origin.

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ConCLUSionS Due to the current trends facing the replacement of synthetic antioxidants with natural ones in edible oils, and based on the health beneits of anthocyanins, this paper describes the effect of anthocyanins extracted from dry skins of red onion (Allium cepa L.) on the oxidative stabilisation of sunlower oil at 40 oC. Total phenolics content in dry skins of red onion investigated samples was found of 1346 mg GAE 100g–1 FM, while total anthocyanins content was found of 99.66 mg 100 g–1 FM. Peroxide PV and TBARS values were investigated in control sample, samples with anthocyanin extract and samples with synthetic tocopherols. The obtained PV results indicate an improvement on the oxidative stability of sunlower oil by addition of the anthocyanin crude extract from red onion skins compared to the added synthetic tocopherols and to the control sample. The results of TBARS screening test of the three type of samples indicate a signiicant decrease of TBARS values for the sunlower oil sample treated with red onion anthocyanin extract, compared to the control. Therefore, one would recommend adding small amounts such as 25 µg of total anthocyanins from red onion extract to 100 g oil in order to delay its oxidative degradation, without much affecting colour characteristics. Antistreptococcal activity of 8-mm inhibition diameter against the standard strain Streptococcus pyogenes ATCC 19615 was found for the anthocyanin acidifed hydroethanolic extract. The antibacterial activity of natural extracts to be incorporated in foods is important as it adds a multifunctional value and because food spoilage is becoming of great concern. The results may contribute to future applications of anthocyanins obtained from cheap by-products of red onions manipulation, for the stabilisation of edible oils and may allow the development of natural food ingredients and potential antibiotics. ACKNOWLEDGEMENTS This work was supported by a grant of the Romanian National Authority for Scientiic Research CNCS–UEFISCDI, project number PN-II-ID-PCE-2011-3-0474. reFerenCeS 1. A. ESMAILLZADEH, L. AZADBAKHT: Different Kinds of Vegetable Oils in Relation to Individual Cardiovascular Risk Factors among Iranian Women. Br J Nutr, 105, 919 (2011). 2. A. K. MISHRA, A. MISHRA, P. CHATTOPADHYAY: Herbal Cosmeceuticals for Photo-protection from Ultraviolet B Radiation: A Review. Trop J Pharm Res, 10, 351 (2011). 3. e. n. FranKeL (ed.): Lipid Oxidation. The Oily Press, Dundee, Scotland, 1998. 4. S. KUBOW: Routes of Formation and Toxic Consequences of Lipid Oxidation Products in Foods. Free Radic Biol Med, 12, 63 (1992). 5. M. DeKKer (ed.): Food Lipids: Chemistry, Nutrition, and Biotechnology. New York, 2002. 6. N. GOUGOULIAS: Comparative Study on the Antioxidant Activity and Polyphenol Content of Some Salvia Species (Salvia L.). Oxid Commun, 35, 404 (2012).

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7. V. YANEVA, D. BABRIKOV, N. MASHEV: Study on Polyphenols Content and Antioxidant Activity of Selected Seedless Grape Varieties (V. vinifera). Oxid Commun, 33, 661 (2010). 8. J.-M. KONG, L.-S. CHIA, N.-K. GOH, T.-F. CHIA, R. BROUILLARD: Analysis and Biological Activities of Anthocyanins. Phytochem, 64, 923 (2003). 9. J. BAKKER, P. BRIDLE, A. KOOPMAN: Strawberry Juice Color: The Effect of Some Processing Variables on the Stability of Anthocyanins. J Sci Food Agric, 60, 471 (1992). 10. L. CaBrita, t. FoSSen, o. M. anDerSen: Colour and Stability of the Six Common Anthocyanin 3-glucosides in Aqueous Solutions. Food Chem, 68, 101 (2000). 11. S. OANCEA, O. DRAGHICI: pH and Thermal Stability of Anthocyanin-based Optimised Extracts of Romanian Red Onion Cultivars. Czech J Food Sci, 31 (3), 283 (2013). 12. P. S. VANKAR, R. SHANKER: Dyeing of Cotton, Wool and Silk with Extract of Allium cepa. Pigm Resin Technol, 38, 242 (2009). 13. R. E. WROLSTAD (Ed.): Food Analytical Chemistry. John Wiley & Sons, New York, 2001, p. 1. 14. V. L. SINGLETON, J. A. ROSSI, Jr.: Colorimetry of Total Phenolics with PhosphomolybdicPhosphotungstic Acid Reagents. Am J Enol Vitic, 16, 144 (1965). 15. Farmacopeea romana. Edition X, Edit. Medicala, Bucharest, 1993. 16. D. FIRESTONE (Ed.): Oficial Methods and Recommended Practices of the American Oil Chemists’ Society. 5th ed. AOCS, Champaign, Ill, 1998. 17. A.W. BAUER, W. M. KIRBY, J. C. SHERRIS, M. TURCK: Antibiotic Susceptibility Testing by a Standardized Single Disk Method. Am J Clin Pathol, 45, 493 (1966). 18. D. HUANG, B. OU, R. L. PRIOR: The Chemistry behind Antioxidant Capacity Assays. J Agric Food Chem, 53, 1841 (2005). 19. A. J. ST. ANGELO: Lipid Oxidation in Foods. Crit Rev Food Sci Nut, 36, 175 (1996). 20. S. oanCea, C. GroSU: effect of Vaccinium myrtillus Anthocyanin Extract on Lipid Oxidation in Cod Liver Oil. Rom Biotech Lett, 18 (1), 7897 (2013). 21. A. M. O’SULLIVAN, N. B. SHAW, S. C. MURPHY, J. P. KERRY: Use of Natural Antioxidants to Stabilize Fish Oil Systems. J Aquat Food Prod Technol, 14, 75 (2005). 22. H. K. SHIN, D. S. HAN, O. S. YI: US pat. 5 084 289, 1992. 23. L. G. ROY, S. ARABSHAHI-DELOUEE, A. UROOJ: Antioxidant Eficacy of Mulberry (Morus indica L.) Leaves Extract and Powder in Edible Oil. Int J Food Prop, 13, 1 (2009). 24. H. Y. CHEN, Y. C. LIN, C. L. HSIEH: Evaluation of Antioxidant Activity of Aqueous Extract of Some Selected Nutraceutical Herbs. Food Chem, 104, 4 (2007). 25. M. STOIA, S. OANCEA: Health Reasons for Improving the Oxidative Stability of Sunlower Oil. Review. Oxid Commun, 36 (3), 636 (2013). 26. M. S. BREWER: Natural Antioxidants: Sources, Compounds, Mechanisms of Action, and Potential Applications. Compr Rev Food Sci Food Saf, 10, 221 (2011). Received 6 October 2013 Revised 15 November 2013

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Oxidation Communications 37, No 2, 483–491 (2014) Antioxidants in biological systems

antioxidant, antimiCroBial and mineralS analySiS StudieS of Corchorus depressus Stem T. H. BOKHARIa*, a. aSLaMa, N. B. RIZVIb, n. raSooLa, M. J. SaiFc, I. H. BUKHARIa, M. ZUBAIRa, a. JaBara, M. RIAZd, S. HINAe, M. iQBaLf, A. I. HUSSAINc a

Department of Chemistry, Government College University, 38 000 Faisalabad, Pakistan E-mail: [email protected] b Institute of Chemistry, New Campus, University of the Punjab, Lahore, Pakistan c Department of Applied Chemistry, Government College University, 38 000 Faisalabad, Pakistan d Department of Chemistry, University of Sargodha, Women Campus, 38 000 Faisalabad Pakistan e Department of Bioinformatics and Biotechnology, Government College University, Faisalabad, Pakistan f Department of Chemistry and Biochemistry, University of Agriculture, Faisalabad, Pakistan

aBStraCt The present study was carried out to examine the minerals, antioxidant and antimicrobial activities of extracts Corchorus depressus stem. The ground stem was extracted with solvent–solvent extraction method with increasing polarity-based solvents, i.e. n-hexane, ether, acetone, ethanol and methanol. The Corchorus depressus stem extracts contained signiicant level of minerals, total phenolic contents (8.29–32.18 catechin equivalent (CE, mg/100 g), ascorbic acid contents (0.036–0.063 mg/g) and total lavonoid contents 5.95–37.06 gallic acid equivalent (GAE, mg/100g). Corchorus depressus stem extracts showed very good DPPH radical scavenging activity, showing IC50 ranged within 16.39–65.70 µg/ml and % inhibition linoleic acid peroxidation ranged from 19.6 to 54.37. All the results of Corchorus depressus stem extracts demonstrated signiicant (p < 0.05) variations. Keywords: Corchorus depressus, DPPH, total phenolic, total lavonoids.

*

For correspondence.

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aiMS anD BaCKGroUnD An important phenomenon that attracted the attention of many scientists is oxidation. Free radicals are considered to initiate oxidation that leads to ageing and causes diseases in human beings. Oxidation widely occurs in food systems. It is mediated by oxygen free radicals or reactive oxygen species. Among organic compounds lipids are prone to autoxidation reaction by oxygen. Oxidation widely occurs in food systems and is mainly mediated by oxygen free radicals or reactive oxygen species. Lipid peroxidation is a major deterioration reaction in food and is responsible for signiicant changes in texture and nutritive value. Excessive amount of free radicals are produced during oxidation process which help the progression of many clinical diseases. Dietary antioxidant might play positive role in delaying or inhibiting the oxidation reactions1. Antioxidants play an important role in scavenging free radicals both in vivo and in vitro. The use of synthetic antioxidants such as butylated hydroxyl toluene, butylated hydroxyl anisol, propyl gallate and tertiary butyl hydroquinone has led to promote negative health effects2. The interest in replacing synthetic antioxidants with natural antioxidants is increasing in the world day by day. It has been observed that synthetic antioxidants are carcinogenic, pathogenic and toxic. Enzymes, lipids and reproductive processes are affected by them3. In the present plant-based natural antioxidants like phenol, lavonoids and tocopherols have been gaining high recognition due to their antioxidant activities. They show anticarcinogenic and potential and various health promoting effects. The food manufacturers prefer natural antioxidants over synthetic antioxidants for the perfection of health foods4. Corchorus depressus belongs to a family Mavaceae (formally under Tiliaceae) and genus is Corchorus. The common name of this plant is bauphali. It is a perennial herb. It is distributed throughout the Pakistan, India, Africa and Cape Verde islands. It grows from sea level to the altitude 1000 m in arid and semi-arid regions throughout the Pakistan5. It is 6–9 inches in length. Its stem is diffusely branched. Its leaves are ecliptic 4–18 mm long and 2–9 mm broad. Its lowers are yellowish and 1mm long. The growth of leaves and fruits is much stunted in saline and rocky soils6. This plant is regarded as good sand binder in the desert. Its seeds are minute and their colour is like chocolate. Its fruits are like capsule and their length is in the range 8–15 mm. Its branches are radiating from woody crown7. It is reported that Corchorus depressus contains triterpenoids, sterols, phenolics, fatty acids, cardiac glycosides and carbohydrates8. Approximately 100 species of corchorus are found. South Africa is richest in species of corchorus which are 16. Tanzania represents 13 species, Ethiopia contains 12 species, Kenya contains 11 species and Pakistan has 6 species. Wild species are mostly found in Africa, America, Brazil, Mexico, Bolivia, Venezuela, West Indies, Australia, China, Taiwan, India, Japan and Sri Lanka9.

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EXPERIMENTAL Collection of sample. Chochorus depressus (Barphali) stem was collected from district Nankana, Pakistan. The sample was identiied and authenticated by Dr. Muhammad Naeem, Assistant professor, Department of Botany, Government College, University, Faisalabad, Pakistan. The collected stem was dried under ambient temperature and crushed to powder form by pestle and mortar. Chemicals and reagents. Gallic acid, 2,2-diphenyl-1-picrylhydrazyl radical (90.0%), the Folin–Ciocalteu reagent, butylated hydroxytoluene (99.0 %), linoleic acid, ascorbic acid, aluminum chloride, ferric chloride, ferrous chloride, sodium nitrite, trichloroacetic acid, potassium ferricyanate were purchased from Sigma Chemicals Co (St. Louis, MO, USA). All other analytical grade chemicals such as ammonium thiocyanate, methanol and anhydrous sodium carbonate were obtained from Merck (Darmstadt, Germany). Preparation of Corchorus depressus extract. The extracts of Corchorus depressus stem were prepared by using the solvents: methanol, ethanol, acetone, ether and nhexane by increasing polarity. Determination of total phenolic contents (TPC). The Folin–Ciocalteu reagent was used to determine the amount of total phenolic contents and the method followed already reported method10. Determination of total lavonoid contents (TFC). The method used for the determination of amount of total lavonoid contents as described by the method11. Estimation of antioxidant activity by linoleic acid system. The antioxidant activity was determined in terms of percentage inhibition of peroxidation of linoleic acid system by using the method already reported12. The percentage inhibition of linoleic acid peroxidation was calculated to exhibit antioxidant activity from the following equation: % inhibition of peroxidation = 100-[(absorbance increase of sample at 175 h/abs. increase of control at 175 h) × 100]. DPPH free radical scavenging assay. The 2,2-diphenyl-1-picrylhydrazy radical (DPPH) assay was carried out spectrophotometrically as reported in Ref. 13. The IC50 values were calculated. Three replicates were recorded for each sample. The percentage scavenging by DPPH calculated from the following equation: inhibition (%) = 100 × (Ablank – Asample/Ablank)

Antimicrobial activity. In order to study the antimicrobial activity of selected bacterial, i.e. Pasturella multocida (locally isolated), Escherichia coli, Bacillus subtilis and Staphylococcus aureus and fungal strains, i.e. Aspergillus niger, Aspergillus lavus, Fusarium solani and Rhizopus solani by using plant extracts analysed by disc diffusion method14–16.

485

reSULtS anD DiSCUSSion Percentage yield of stem extracts. The present study shows that the % yield extracts with different solvents was found in the range of 1.62 –4.26% (Table 1). table 1. % yield of different extracts of Corchorus depressus stem

Extracts of stem Methanol Ethanol acetone Ether n-Hexane

% Yield (g/100g dry matter) 4.26 4.09 2.53 2.19 1.62

Minerals composition. To investigate the quantity of minerals in Corchorus depressus stem wet digestion method was used and atomic absorption spectrophotometer (AAS) apparatus was used to analyse minerals. The results of mineral composition are described in Fig. 1

fig. 1. Concentration of metals in Corchorus depressus

Total phenolic contents (TPC). The Folin–Ciocalteu method was used for the investigation of the quantity of TPC of Corchorus depressus extracts. This method was preferred because of its high sensitivity, lower interference and rapidity to quantify the phenolics rather than other competitive test17. Table 2 represents the amount of TPC (mg/100 mg of dry weight as GAE) and TFC (mg/100 mg of dry weight as CE) of extracts. The present indings regarding TPC of Corchorus depressus stem (38.60 mg GAE/100 g fresh weight) using pure methanol solvent system was found in close agreement with those reported for the antioxidant activity of phenolic fractions of Corchorus depressus stem in methanol using FTC model (HUDA) (Refs 18 and 19) reported the total phenolic contents as using ethanol water mixture.

486

table 2. Total phenolic contents (TPC) of Corchorus depressus stem

Extracts Methanol Ethanol acetone Ether n-Hexane

TPC (mg/100 g of dry matter measured as GAE) 37.06±0.05 31.04±0.02 18.06±0.02 9.62±0.04 5.95±0.06

Total lavonoid contents (TFC). Table 3 represents the amount of total lavonoid contents (mg/100 g of dry weight as CE) of the stem extracts of Corchorus depressus. The present indings regarding TFC of Corchorus depressus stem using are in the range 8.29–32.18 mg/100 g of CE. table 3. Total lavonoid contents (TFC) of Corchorus depressus stem

Extracts Methanol Ethanol acetone Ether n-Hexane

TFC (mg/100 g dry matter measured as CE) 32.18±0.09 29.44±0.04 22.07±0.05 17.82±0.04 8.29±0.05

DPPH radical scavenging activity. The free radical DPPH, most stable, has deep violet colour and range of absorption maxima in-between 515–528 nm. As the DPPH accept proton, from hydrogen donating species, especially phenols, its colour changes from deep violet to yellow. The DPPH scavenging activity increases as the concentration and degree of hydroxylation increases. In the transformation of DPPH into its reduced form DPPH-H, the extracts behave as donor of hydrogen atoms or electrons. In a manner of concentration dependent, free radical scavenging capacity of the extracts was increased. Table 4 represents the data regarding the IC50 extracts of Corchorus depressus stem, which showed excellent radical scavenging activity. The pure methanol exhibited highest scavenging activity but slightly lower antioxidant activity of the extract was observed when compared with synthetic antioxidant butylated hydroxytoluene (BHT). Our result is comparable with the already reported investigations of plant extract20. The scavenging activity of Corchorus depressus statistically was signiicantly (ptomato>garlic> lettuce after chemical extraction (Table 3). Thus, it can be speculated that active compounds present in onion, tomato, and garlic have a lipophilic character. Additionally, it can be supposed that digestive enzymes and pH conditions caused the release of phenolic compounds with strong reducing power from the lettuce.

500

table 3. Comparison of antioxidative abilities and bioeficciency factors (BEF) between gastrointestinal digested (D) and chemically extracted (CE) vegetable samples, N=9a

Sample Concentration (mg FW/ml) Lettuce 0.2 0.1 0.05 0.02 tomato 0.2 0.1 0.05 0.02 onion 0.2 0.1 0.05 0.02 Garlic 0.2 0.1 0.05 0.02

Reducing power (µg QE/mg FW) D Ce BeFc 55.30±0.18aa 11.41±0.06a 4.85 27.15±0.05Ba 4.81±0.02Ba 14.01±0.06Ca 2.91±0.04Ca 5.33±0.02Da 1.07±0.01Da 17.00±0.07ea 41.34±0.04ea 0.41 9.01±0.03Fa 21.57±0.07Fa 4.13±0.01Ga 10.32±0.05Ga 1.55±0.01Ha 4.55±0.01Ha 49.82±0.15ia 60.71±0.20ia 0.82 24.41±0.11Ja 29.45±0.12Ja 12.36±0.08Ka 15.15±0.09Ka 4.69±0.02La 5.96±0.03La 19.47±0.06Ma 29.65±0.08Ma 0.66 10.23±0.08na 15.72±0.09na 4.90±0.02oa 7.39±0.05oa 2.01±0.01Pa 3.21±0.02Pa

Antiradical abilitiesb (%) D Ce BeFc 43.83±0.07ab 10.77±0.02a 4.07 21.71±0.00Bb 5.28±0.00Bb 11.06±0.00Cb 2.55±0.00Cb 4.12±0.00Db 1.11±0.00Db 25.79±0.05eb 30.14±0.08eb 0.86 13.09±0.03Fb 15.18±0.09Fb 6.55±0.03Gb 7.65±0.04Gb 2.38±0.01Hb 3.02±0.01Hb 60.26±0.12ib 14.51±0.04ib 4.15 29.93±0.10Jb 7.14±0.04Jb 15.27±0.07Kb 3.53±0.02Kb 6.11±0.04Lb 1.47±0.01Lb 51.93±0.13Mb 15.69±0.04Mb 3.31 26.16±0.00nb 7.96±0.05nb 12.58±0.00ob 4.05±0.02ob 5.52±0.00Pb 1.51±0.01Pb

Chelating power (%) D Ce 40.59±0.11ac 89.68±0.13a 20.16±0.00Bc 44.54±0.00Bc 10.22±0.00Cc 22.39±0.00Cc 4.11±0.00Db 9.01±0.00Dc 88.33±0.17ec 2.04± 0.01ec 44.29±0.11Fc 1.32±0.01Fc 22.11±0.08Gc 0.61±0.01Gc 8.92±0.06Hc 0.18±0.00Hc 69.84±0.04ic 48.35±0.11ic 34.79±0.09Jc 23.88±0.11Jc 17.32±0.09Kc 12.11±0.07Kc 6.75±0.02Lc 4.55±0.03Lc 88.97±0.13Mc 72.19±0.21Mc 44.61±0.12nc 36.41±0.18nc 22.29±0.06oc 17.97±0.08oc 9.01±0.04Pc 6.99±0.03Pc

BeFc 0.45

43.25

1.44

1.23

a Results are expressed as mean ± SD; b antiradical activity was measured using ABTS assay; c BEF was calculated according to formula: BEF =AD/ACh, where AD – activity of extracts obtained after simulated gastrointestinal digestion, ACh – activity of extracts obtained after chemical extraction; A,B,C – the same capital letters in columns represent statistically not signiicant differences (plettuce> tomato as compared to tomato>onion ≈garlic> lettuce after chemical extraction. Antiradical compounds present in tomato showed the lowest bioeficiency (BEF=0.86) (Table 3). Furthermore, these results might suggest that the most bioaccessible antiradical phytochemicals were probably quercetin and its derivatives from onion as well as sulphur-containing compounds from garlic, whereas the most commonly used solvent for chemical extraction (50% MeOH) was the most eficient for extracting antiradical compounds from tomato. Transition metal ions possess the ability to move single electrons and by virtue of it allow the formation and propagation of several radical reactions, even starting with relatively non-reactive radicals. Food is often contaminated with transition metal ions that might be introduced by various processing methods. Bivalent transition metal ions play an important role as catalysts of oxidative processes, leading to the formation of hydroxyl radicals and hydroperoxide decomposition reactions via the Fenton reaction44. It can be observed from Table 3 that bioaccessible compounds with strong chelating abilities were released from tomato, garlic and onion (approximately 88, 89 and 70%, respectively) during simulated gastro-intestinal digestion. Probably, the digestive enzymes and/or pH conditions caused the release of chelating compounds from the food matrices. The lowest activity (approximately 40%) was observed for a lettuce-digested sample, and the highest chelating activity for chemical extracts (approximately 90%); however, the chemical extract of tomato exhibited the lowest activity (Table 3). In view of these results it appears that lettuce was the best source of bioaccessible compounds with high reducing power, onion contained antiradical – active phytochemicals, while tomato and garlic contained compounds with strong chelating activity (Table 3). It can be suggested that in the case of tomato the hydrophilic compounds were mainly responsible for chelating activity, whereas the lipophilic ones mostly exhibited antiradical activity. In addition to this, lipophilic compounds extracted from lettuce and onion possessed strong chelating activity. Although antioxidant activities determined by different methods differ signiicantly, this could be probably explained on the basis of the chemical nature and reactivity of the compounds and the nature of the solvents. Antioxidant activity of the combination of vegetables. An important aspect when studying the role of antioxidants in human health is the evaluation of their bioavailability from foods. However, the bioavailability of these substances is not always well known. Some classes of antioxidants, such as some vitamins, carotenoids and polyphenols, merit speciic attention, not only because they are well represented in our diet but also because they are differently absorbed and metabolised and can exert diverse func-

502

tions with a signiicant impact on human health45. Thus, the biological properties of antioxidants might depend on their release from the food matrix during the digestion process (bioaccessibility) and might differ quantitatively and qualitatively from those produced by the chemical extraction employed in most studies1. The antioxidant capacity of individual vegetable extracts indicated a dose-response relationship at various concentrations (Table 3), which allowed the theoretical calculations. The reducing-powers data (Table 4) indicated a statistically-signiicant (p≤0.05) synergy when the measured values were compared with the theoretical ones for onion+garlic and an ‘all vegetables’ combination (IF=1.16 and 1.17, respectively). In addition to this, the antagonistic effect of bioaccessible compounds was found for an onion+tomato combination (IF=0.83) (Fig. 1A, Table 4). In the chemical extracts, slight synergistic interactions were observed in the onion+garlic and ‘all vegetables’ combinations (IF = 1.37 and 1.20, respectively). Additive interactions were observed in other samples with statistically-insigniicant differences found between the measured and the theoretical reducing power (IF about 1). In addition, bioaccessible reducing compounds were found to be present in the lettuce/tomato mixture (BEF = 1.57) and the mixture of all vegetables (BEF = 0.96) (Fig. 1B, Table 4).

B

A 40

measured

summated

a

a

70

a reducing power (μg QE)

reducing power (μg QE)

45 35

b

30 25 20 15 10 5 0

onion + tomato

lettuce + tomato sample

mix

summated

60 b

50

d

40 30

c

20 10 0

onion + garlic

measured a

onion + garlic

onion + tomato

lettuce + tomato

mix

sample

fig. 1. Measured and theoretical reducing power values of extracts obtained after digestion (A) and chemical extraction (B) of mixed vegetables mix – all vegetables combination; a, b, c, d – the same letters represent non statistically signiicant differences (p>0.05)

503

504

table 4. Comparison of interaction (IF) and the bioeficiency (BEF) factors of vegetable mixtures, N=9a

Sample

Reducing power iFd

BeFd

Dc

Cec

Onion+garlic (1:1)

1.16±0.06aa

1.37±0.05aa

onion+tomato (1:1)

0.83±0.02Ba

Lettuce+tomato (1:1)

1.09±0.02aa

All vegetables (1:1:1:1) 1.17±0.07

aa

Chelating power iFd

BeFd

Dc

Cec

0.65

1.06±0.07aa

1.22±0.06ab

0.97±0.01Ba

0.56

0.98±0.05ab

0.95±0.01Ba

1.57

Ca

0.96

1.20±0.05

Antiradical activityb iFd

BeFd

Dc

Cec

1.14

1.24±0.18ab

2.43±0.09ac

1.90

0.59±0.01Bb

5.16

1.04±0.18Bb

1.79±0.08Bc

1.12

1.02±0.02ab

0.82±0.01Cb

1.74

1.17±0.18aBa

0.70±0.06Cc

2.84

Bb

Db

1.38

3.20±0.11

0.86

0.75±0.01

0.74±0.02

1.07±0.18

Ba

Dc

Results are expressed as mean ± SD; antiradical activity was measured using ABTS assay; D – digested, CE – chemically extracted vegetable samples; IF – calculated according to formula: IF= AM/At, where AM – measured activity of the mixture of samples, At – theoretical calculated mixture activity; BEF – calculated according to formula: BEF =AD/ACh, where AD – activity of extracts obtained after simulated gastrointestinal digestion, ACh – activity of extracts obtained after chemical extraction; A, B, C – the same capital letters in columns represent statistically not signiicant differences (p0.05)

Taking into account chemically-extracted active compounds, it was observed that onion+garlic combinations had the highest chelating power (over 70%) and showed signiicant synergistic interactions (IF = 1.22). However, statistically-signiicant antagonistic interactions were observed in other combinations (Table 4). The lowest chelating power (about 15%) was determined for the onion+tomato mixture. The strongest antagonism (IF = 0.74) was observed in chemical extract obtained from the ‘all-vegetables’ combination (Fig. 3B, Table 4). The vegetables used as the material in this study were mixed in the usuallyconsumed combinations. It is important to note that in the case of bioaccessible compounds the most often occurring interactions were synergistic and additive effects (Figs 1, 2 and 3A, Table 4). Antagonism was observed in a tomato+onion combination 506

in the case of reducing power assays and in an ‘all-vegetables’ combination sample for the chelating activity assay. These results are in opposition to those obtained by Hidalgo et al.47, who demonstrated that lavonoid interactions partially reduced the total antioxidant activity (DPPH assay, in a model system) found in food matrices, and in general in plant foods. In the chemical approach, the extractable antioxidant compounds were found to interact in a different manner (Figs 1, 2 and 3B, Table 4). In antiradical assays strong synergisms were observed in all samples, except the lettuce+tomato combination. Extractable phytochemicals exhibit a synergistic or additive effect in the reducing-power assay, with mainly antagonistic interactions in the chelating power study. The results obtained by Pinelo et al.25 indicated that the addition of a new polyphenol to a complex phenolic system does not always promote a positive effect on its overall antioxidant capacity. In fact, in contrast, it could be considerably reduced. However, in contrast to this, as can be seen in this study, in most cases the mixing of vegetables caused an increase or a summation of their antioxidant activities. In investigations involving the antioxidant capacities of individual and combined phenolics in a model system, Heo et al.48 stated that the addition of chlorogenic acid to cyanidin, peonidin-3-glucoside, quercetin, and quercetin 3-galactoside caused a decrease in antioxidant capacity as compared to the theoretically-calculated capacity. On the other hand, Hildago et al.47 suggested that the lavonol quercetin and quercetin3-glucoside triggered a noticeable increase in antioxidant activity when mixed in solution with another lavonoid (in a FRAP assay). Some authors49,50 have demonstrated a linear correlation between the total content of phenolic compounds and their antioxidant capacity, while others51 have indicated a poor linear correlation. In the present study it was found that only the antiradical activity of extracts obtained after simulated gastrointestinal digestion had a positive statistically-signiicant correlation with the total phenolics content (r = 0.86, p≤0.05), whereas in the case of chemical extract a statistically-signiicant positive correlation was found between the reducing-power and the total phenolics content (r = 0.52, p≤0.05). On the other hand, the total lavonoids content, after digestion in vitro, was positively and statistically correlated with the reducing power and antiradical activity (r = 0.81 and 0.55, respectively, p≤0.05), while a negative statistically-signiicant correlation was found with chelating power (r= –0.50, p≤0.05) post-digestion in vitro. Moreover, chelating power was negatively correlated with the phenolic acid content (r = –0.90, p≤0.05) post-digestion in vitro, whereas a positive statistically-signiicant correlation was found between the phenolic acids content and reducing power (r = 0.71, p≤0.05) post-digestion in vitro. It is important to note that in the case of chemical extracts only reducing power was positively correlated with the total phenolics, lavonoids, and phenolic acids content (Table 5).

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table 5. The Person correlation coeficients between total phenolics, total lavonoids, total phenolic acids and antioxidant activities expressed as a correlation coeficientsa

Sample Reducing power Chelating power Antiradical activity c

Total phenolics Db Ceb 0.52 0.13 0.18 0.30 0.86 0.22

Total lavonoids Db Ceb 0.81 0.80 –0.50 0.03 0.55 0.11

Total phenolic acids Db Ceb 0.71 0.55 –0.90 0.30 –0.18 0.36

Statistically signiicant results with p origano > mint > basil sweet > dill > bay tree >celery > parsley. The comparison of the three assay methods reveals that the test using the radical cation aBtS•+ leads to higher values of ТЕАС and ААЕ than the other two methods. Keywords: herbs, spices, polyphenols, DPPH, ABTS, FRAP activity. aiMS anD BaCKGroUnD Many scientiic and epidemiological studies demonstrate that foods rich in natural antioxidants suppress strongly the appearance of the so-called oxidative stress in the human organism. At oxidative stress proceed biochemical processes leading to the generation of reactive oxygen- and nitrogen-containing compounds (ROS, RNS), which damage major biological molecules – lipids, proteins and nucleic acids, and ipso facto, thus seriously increase the risk of many diseases of the human organism1,2. Fruits, vegetables, nuts, legumes, seeds of many plants, as well as many medicinal 512

and culinary species being rich in natural antioxidants exert a number of favourable health effects on the human organism3,4. The signiicant antioxidant impact of the plant foods and spices is associated with the high content of vitamins C and E, carotenoids, as well as polyphenols and a lot of other biologically active compounds5–7. It was established that extracts obtained from many plant spices, herbs and medicinal plants manifest not only antioxidant, but also antimicrobial, anti-inlammatory, antivirus and anticancer effects due to the presence of various kinds of biologically active polyphenol compounds7–9. Particularly rich in polyphenols antioxidants are various plant spices used from ancient times till today for medicinal and culinary purposes of the botanical families – Lamiaceae and Ubelliferae. They have been intensively researched in view of obtaining new antioxidants replacing ВHA, ВHТ and other synthetic antioxidants in the food technology to prevent the fats from oxidation (rancidity) and to increase the durability and quality of food products8,10,11. Particular interest in this respect are basil, oregano, parsley, celery, which are used worldwide in the kitchen as spices and are rich in polyphenols with high antioxidant eficiency12–15. It was of interest (1) to study and compare the major polyphenol fractions – total polyphenols (TP), nonlavonoid phenols (NFP), lavonoid phenols (FP) and total lavanols (F-3-ols ), and (2) to determine and compare the antiradical activity using two free stable radicals – DPPH• and aBtS•+, and ferric reducing power with FRAP reagent of methanol extracts of the most commonly used spices and herbs of the Lamiasea, Ubelliferae and Lauraceae families. EXPERIMENTAL Plant material. The plant material consisted of the leaf mass of 4 species of the Lamiaceae family – Origanum vulgare L. (Greek oregano), Ocimum basilicum L. (basil sweet), Ocimum basilicum purpurescens L. (basil purple) and Menta spicata L. (mint), 3 species of the Ubelliferae family – Apium graveolens L. (celery), Petroselium crispum (parsley), Anethum graveolens (dill), and 1 species of the Lauraceae family – Laurus nobilis (bay tree). The plants were grown in the experimental ield of TEI–Larissa, Greece. The leaves were collected in the summer of 2010, dark-dried, at room temperature, inely ground and kept at 4oC in dark until tested. Preparation of the methanol extracts. 500 mg of the inely ground sample were 2-fold treated by 20 ml 80% aqueous methanol. At the irst treatment the samples was incubated for 24 h in the extragent at stirring and the second one – continued for 2 h at stirring at ambient temperature. The extract was collected after centrifugation or iltration and the volume was made up to 50 ml with aqueous methanol. DETERMINATION OF POLYPHENOLS

Total polyphenols (TP). Total polyphenols (TP) contents were determined with the Folin–Ciocalteu (F.–C.) reagent according to the method of Singleton and Rossi16

513

using the microvariant proposed by Badenschneider et al.17, and were expressed as gallic acid equivalent (GAE) in mg/g dry weight (dw). Nonlavonoid phenols (NFP). They were determined with the F.–C. reagent after removal of the lavonoid phenols (FP) with formaldehyde according to the method of Kramling18. NFP content was expressed as gallic acid equivalent (GAE) in mg/g dw. Flavonoid phenols (FP). Flavanoid phenols were determined as a difference between the content of total phenols (TP) and nonlavonoid phenols (NFP). Their amount was evaluated as gallic acid equivalent in mg/g dw. Total lavanols (F-3-ols). The determination of the content of total lavanols (catechins and procyanidins) was performed using р-dimethylaminocinnamaldehyde (p-DMACA) reagent after the method of Li et al.19 and was presented as catechin equivalent (CE), in μg/g dw. DeterMination oF antioXiDant aCtivity

DPPH• assay. The antiradical activity of the methanol extracts was determined on the basis of the method of Brand–Williams20, using the stable free radical 2,2′-diphenyl-1pycrylhydrazyl (DPPH•), as a reagent. The activity was expressed in μmols DPPH• /g dw, as well as in mg/g dw and μmol Trolox (synthetic vitamin Е)/g dw. ABTS assay. The activity was determined by bleaching the colour of the stable free cation aBtS•+ (2,2-аzinobis-(3-ethylbenzothiazolin-6-sulphonic acid) using the method of Re et al.21, and expressed in μmol Trolox (TAEC)/g dw. Ferric reducing antioxidant power assay (with FRAP reagent). The ferric reducing antioxidant power (FRAP) was evaluated according to the method of Benzie et al.22 and was expressed as µmol FRAP reagent/g dw. The activity was also presented as a Trolox equivalent (TEAC) and ascorbic acid equivalent (AAE) in μmol/ g dw. The inhibition coeficient (IC50), represents 50% reduction in the colour intensity of the radicals DPPH and ABTS by the total phenols (µg/ml) in the studied extracts after plotting the dependence of the TP content on the bleaching of DPPH• and aBtS•+ solutions. The inhibition coeficient (IC50) was calculated by the following equation: % inhibition = [(E0 – Ex)/E0 ] × 100 ,

where Е0 is the extinction of the radical solution before the reaction; Ех – after polyphenols addition23. Data were reported as mean arithmetic for at least three replications. The statistical analysis of the results was performed by well-accepted methods with the help of a program estimating the mean side deviation (±SD). The correlation coeficients (R2) were determined using dispersion analysis.

514

reSULtS anD DiSCUSSion The content of total phenols – nonlavonoid and lavonoid phenols using the F.–C. reagent is expressed in one and the same units (gallic acid equivalent – GAE) for comparison purposes, whereas total lavanols assayed with the help of p-DMACA reagent are presented as catechin equivalent (CE). The content of total phenols (ТР) in the methanol extracts of the leaf mass of the selected plant species varies from 10.30 tо 18 mg/g dw (Table 1), being the highest in the leaf mass of Ocimum basilicum purpurescens and Origanum vulgare, 18.00 and 17.35 mg/g dw, and the lowest – in the leaves of Petroselium crispum – 10.30 mg/g dw. The selected species of the Lamiaceae family are characterised by higher TP content as compared with those of the Ubelliferae family. The irst ones contain on average 16.42, and the second ones – 12.30 mg/g dw. Of the two species the basil coloured in purple is richer in ТР than the sweet one by 23% (р< 0.05). Of the Ubelliferae family, Anethum graveolens has higher content of ТР and is superior to Apium graveolens by 17%, and Petroselium crispum – by 30% (р < 0.05). Among the species of the Lamiaceae family, Laurus nobilis is distinguished by lower average content of TP (12.35), which is equal to that of the Ubelliferae family species. Regarding the TP content, the species of the Lamiaceae family can be arranged in the following sequence: O. basilicum purpurescens L.> Origanum vulgare > Menta spicata > Ocinum basilicum, and those of the Ubelliferae family: Anethum graveolens > Apium graveolens >Petroselium crispum. table 1. Content of total phenols (ТР) in methanol extracts

no

Species

1 2 3 4 5 6 7 8

Origanum vulgare L. (Greek oregano) Ocimum basilicum L. O. basilicum purpurescens L. Menta spicata L. Apium graveolens L. Petroselium crispum Anethum graveolens Laurus nobilis

Gallic acid equivalent (GAE) (mg/g dw) (µmol/ml extract) 17.35 ± 0.22 0.9228 14.68 ± 0.18 0.7695 18.00 ± 0.26 0.9575 15.65 ± 0.19 0.8325 11.96 ± 0.14 0.6365 10.83 ± 0.12 0.5760 14.08 ± 0.15 0.7500 12.35 ± 0.17 0.6570

The content of nonlavonoid phenols in the studied plant species ranges from 3.12 to 6.07 mg/g dw (Table 2). The leaves of the O. basilicum purpurescens of the Lamiaceae family has the highest TP content, while those of the Petroselium crispum – the lowest one. The difference in their content is close to 45%. The content of NFP in the species of the Lamiaceae family is in the range of 30%, while that of the Ubelliferae family – 20%. The amount of NFP in the leaves of Laurus nobilis is close to the average content of the plant species of the Ubelliferae family (р > 0.05). Regarding the NTP content, the species of the Lamiaceae family can be arranged in the following order: O. basilicum purpurescens L.> Origanum vulgare (Greek oregano) > Menta spicata 515

> Ocimum basilicum, and those of the Ubelliferae family – Anethum graveolens > Apium graveolens >Petroselium crispum. Таble 2. Content of nonlavonoid phenols (NFP) in methanol extracts

no 1 2 3 4 5 6 7 8

Species Origanum vulgare L. (Greek oregano) Ocimum basilicum L. O. basilicum purpurescens L. Menta spicata L. Apium graveolens L. Petroselium crispum Anethum graveolens Laurus nobilis

Gallic acid equivalent (GAE) (mg/g dw) (μmol/ml extract) 5.53 ± 0.06 0.2544 4.66 ± 0.08 0.2480 6.07 ± 0.12 0.3230 5.31 ± 0.13 0.2825 3.36 ± 0.05 0.17.85 3.12 ± 0.04 0.1660 3.74 ± 0.05 0.1990 3.77 ± 0.04 0.2005

The lavonoid phenols (FP) are the dominant fraction of phenol compounds in the studied plant species and their amount is nearly 70% of the TP (Table 3). The leaves of Ocimum basilicum purpurescens and Origanum vulgare are distinguished by the highest FP content of 11.93 and 11.83 mg/g dw, respectively, while that of Petroselium crispum – by the lowest (7.71 mg/g dw). The average content of FP of the selected species of the Lamiaceae family amounts to 11.03 mg/g, and that of the Ubelliferae family – 8.58 (р < 0.05). The content of FP in the leaves of Laurus nobilis is close to the average content in the species of the Ubelliferae family. table 3. Content of lavonoid phenols (FP) in methanol extracts

no

Species

1 2 3 4 5 6 7 8

Origanum vulgare L. (Greek oregano) Ocimum basilicum L. O. basilicum purpurescens L. Menta spicata L. Apium graveolens L. Petroselium crispum Anethum graveolens Laurus nobilis

Gallic acid equivalent (GAE) (mg/g dw) (µmol/ml extract) 11.82 ± 0.16 0.6285 10.02 ± 0.13 0.5330 11.93 ± 0.13 0.6435 10.34 ± 0.12 0.5300 8.84 ± 0.10 0.4700 7.71 ± 0.11 0.4100 10.34 ± 0.12 1.5335 8.58 ± 0.09 0.4565

With respect to the FP content, the species of the Lamiaceae family follow the sequence: O. basilicum purpurescens > Origanum vulgare (Greek oregano) > Menta spicata > Ocimum basilicum, and those of the Ubelliferae family: Anethum graveolens > Apium graveolens >Petroselium crispum. The fraction of total lavanols (F-3-оls) in the selected plant species constitute a relatively small part of the total phenols and their ratio is 1/100 (Table 4). Although in small amounts the phenols such as catechin, epicatechin, proanthocyanidins and 516

the speciic for these plants species lavanols have strong antioxidant properties24,25. Their content is highest in the leaves of O. basilicum purpurescens and Origanum vulgare, 206 and 197 μg/g dw, respectively, and the lowest and very close (р > 0.05), in the leaves of Petroselium crispum and Apium graveolens, 86 and 88.4 µg CE/g dw, respectively. It should be noted that the selected species of the Lamiaceae family are enriched in F-3-ols (on average 165.4 µg/g), in comparison with those of the Ubelliferae family (on average 96 µg/g). With respect to the content of F-3-ols, the leaves of Menta spicata are superior to all selected species of the Ubelliferae family and occupy the third place after O. basilicum purpurescens and Origanum vulgare (140.3 µg/g). table 4. Content of total lavanols (F-3-ols) in methanol extracts

no

Species

1 2 3 4 5 6 7 8

Origanum vulgare L. (Greek oregano) Ocimum basilicum L. O. basilicum purpurescens L. Menta spicata L. Apium graveolens L. Petroselium crispum Anethum graveolens Laurus nobilis

Catechin equivalent (CE ) (µg/g dw) (µmols/l extract) 196.5 ± 3.16 6.77 118.6 ± 2.54 4.09 206.2 ± 2.95 7.11 140.3 ± 2.09 4.84 88.4 ± 1.79 3.03 86.0 ± 1.57 2.95 113.8 ± 2.35 3.93 111.7 ± 2.23 3.85

Recently the phenol compounds in a lot of plant species of the Lamiaceae and Ubelliferae families are object of many investigations due to their high content and antioxidant properties, which can be used for medicinal and food purposes. Many authors indicate the diversity of phenol compounds and their antioxidant properties, particularly in the leaves of various species of basil and oregano11,24–26. Zeng and Wang27, established that among the different herbs, oregano exerts from 3 to 20 times higher antioxidant activity and has the highest content of total phenols. Our results also conirm that oregano and basil have higher amount of TP in comparison with the other selected species14. Besides this, O. basilicum purpurescens has higher amount of phenol compounds in comparison with Ocimum basilicum, which supports the data of other authors studying similar basil species12,24,26. We have found out that lavonoid phenols amount to 70% of TP. Flavonoids are a class of phenol compounds with characteristic С6–С3–С6 coniguration and functional groups. For this reason their antioxidant action proceeds via different mechanisms – radical scavenging, hydrogen donating and metal chelating and inhibition of lipid oxidation28. Тhis determines the high antioxidant effects of the studied selected food species. The results on the antiradical activity of the selected species in µmols DPPH, µmols Trolox and IC50 are demonstrated in Table 5. The extract of O. basilicum

517

purpurescens displays the highest antiradical activity – 11.61 µmols TEAC/g dw, whereas that of Petroselium crispum – the lowest one (8.24 µmols TEAC/g dw). This dependence is also conirmed by the determination of the inhibition coeficient (iC50) of the two spices, which amounts to 184 and 460 µg/ml extract, respectively. The spices of the Lamiaceae family show higher antiradical eficiency than those of the Ubelliferae family, which amounts on average to 10.63 and 8.70 µmols Trolox/g dw, respectively (p < 0.05). This is also conirmed by the values of IC50 of the spices of the two families. table 5. Antiradical activity (DPPH) of methanol extracts

no 1 2 3 4 5 6 7 8

Species Origanum vulgare L. (Greek oregano) Ocimum basilicum L. O. basilicum purpurescens L. Menta spicata L. Apium graveolens L. Petroselium crispum Anethum graveolens Laurus nobilis

DPPH (µmol/g dw) 29.89 ± 0.39 27.34 ± 0.33 31.71 ± 0.44 28.70 ± 0.37 25.02 ± 0.40 23.72 ± 0.33 25.58 ± 0.41 23.98 ± 0.36

teaC iC50 (µmol/g dw) (µg/ml extract) 10.86 ± 0.13 251 9.76 ± 0.12 320 11.61 ± 0.11 184 10.29 ± 0.17 302 8.78 ± 0.10 380 8.24 ± 0.09 460 9.02 ± 0.12 442 9.16 ± 0.11 458

The antiradical activity of the selected extracts, determined by bleaching the colour of the stable free cation ABTS•+, varies from 12.68 tо 17.95 µmol TEAC/g dw (Table 6 ). The leaves of basil purple have the highest antiradical activity and those of parsley – the lowest one. Once again should be noted the higher activity of the extracts from the plant species of the Lamiaceae family in comparison with those of the Umbelliferae family. The mean inhibition capacity of the extracts of the selected species of the Lamiaceae family is 129, and that of the Umbelliferae family –184 µg/ml extract, i.e. nearly 43% higher antioxidant activity. table 6. Antiradical activity (ABTS) of methanol extracts

no 1 2 3 4 5 6 7 8

Species Origanum vulgare L. (Greek oregano) Ocimum basilicum L. O. basilicum purpurescens L. Menta spicata L. Apium graveolens L. Petroselium crispum Anethum graveolens Laurus nobilis

518

teaC iC50 1/IC50 (µmol/g dw) (µg/ml extract) (µl extract/ml) 16.23±0.23 135 ± 3 7.41×10–3 16.30±0.18 151 ± 4 6.62×10–3 17.95±0.20 95 ± 5 10.53×10–3 16.34±0.18 134 ± 6 7.46×10–3 14.03±0.17 173 ± 5 5.78×10–3 12.68±0.17 197 ± 4 5.08×10–3 13.97±0.15 181 ± 3 5.52×10–3 14.38±0.19 171 ± 5 5.85×10–3

The ferric reducing power of the methanol extracts of the selected species ranges from 3.18 tо 5.97 µmol TEAC/g dw and from 4.05 до 7.69 µmol ААЕ/g dw (Table 7). table 7. Ferric reducing power (FRAP) of methanol extracts

no 1 2 3 4 5 6 7 8

Species Origanum vulgare L. (Greek oregano) Ocimum basilicum L. O. basilicum purpurescens L. Menta spicata L. Apium graveolens L. Petroselium crispum Anethum graveolens Laurus nobilis

FRAP (µmol/g dw) 14.45±0.33 11.24±0.21 15.76±0.32 12.48±0.23 9.50±0.24 7.95±0.21 10.36±0.20 9.12±0.17

teaC (µmol/g dw) 5.28±0.07 4.08±0.05 5.97±0.60 4.64±0.50 3.83±0.04 3.18±0.03 3.96±0.05 3.56± 0.04

aae (µmol/g dw) 7.08 5.35 7.69 6.50 4.12 4.05 5.18 4.61

The antioxidant activity of the selected plant species determined with the FRAP assay shows that the species of the Lamiaceae family exert higher activity than those of the Umbelliferae family. It has been also established that basil purple possesses both higher antioxidant and ferric reducing power in comparison with the common basil, which is in agreement with the indings by other authors24,29. Very good correlations (R2) have been established between TP content of the selected species and DPPH, ABTS and FRAP activity (Table 8). table 8. Correlation coeficient (R2) between TР and antioxidant activity determined by DPPH•, aBtS•+ and FRAP assay

TP/µmol DPPH TP/µmol TEAC TP/µmol/IC50 TP/µmol TEAC TP/IC50 TP/µmol FRAP TP/µmol TEAC TP/µmol AAE

DPPH• – R2

aBtS•+ – R2 FRAP – R2

0.9523 0.9492 0.8369 0.9266 0.8629 0.9779 0.9376 0.9691

The use of three assay methods for the determination of antioxidant activity (with DPPH, ABTS and FRAP) of the selected food spices makes possible more reliable assessment of their real antioxidant potential as well as its differentiation in the separate species. Although the antioxidant activity determined by the three methods is different, the relative activity of the separate species assessed by the three methods 519

is preserved. The values expressed as TEAC using the ABTS assay are lower than those that by the ABTS assay, which is in agreement with the observations of other authors9,29. The explanation of this fact could be found in the studies of Wang et al.30, Arts et al.31, who established that some compounds, which display scavenging of ABTS, do not show activity towards DPPH, since the mechanisms of the reactions with the participation of the two radicals are different. ConCLUSionS The results on studying the most widely used food spices, mainly of two families – Lamiaceae and Ubelliferae, and one selected species of the Lauraceae family show that the species of the Lamiaceae family are enriched in ТР, FP and NFP, and display higher DPPP, ABTS and FRAP antioxidant activity than those of the Ubelliferae family. Bay tree is characterised by higher amount of polyphenols and antioxidant activity than the leaves of celery and parsley. Considering TP and antioxidant activity, the selected plant spices follow the order: basil purple > oregano > mint > basil sweet > dill > bay tree >celery > parsley. Strong correlations were established between ТР and DPPH, ABTS and FRAP activity of the studied spices. reFerenCeS 1. B. HALLIWELL, J. M. C. GUTTERIDGE: Free Radicals in Biology and Medicine. 2nd ed. Clarendon Press, Oxford, 1989, 1–21. 2. H. SIES: Oxidative Stress. Introduction. In: Oxidative Stress (Ed. H. Siess). Academic Press, 1999, 3. L. BRAVO: Polyphenols: Chemistry, Dietary Sources, Metabolism and Nutritional Signiicance. Nutr Rev, 56 (11), 317 (1998). 4. C. A. RICE-EVANCE, N. J. MILLER: Antioxidants: The Case for Fruit and Vegetables in the Diet. Br Food J, 97 (9), 35 (1995). 5. J. J. MACHIEX, A. FLURIENT, J. BILLOT: Fruit Phenolics. CRC Press, Boca Raton, FL, 1990. 6. D. E. PRATT: Natural Antioxidants not Exploited Commercially. In: Food Antioxidants (Ed. B. H. F. Hodson). Elsevier Applied Science, 1990. 7. X. HAN, T. SHEN, H. LOU: Dietary Polyphenols and Their Signiicance. Int J Mol Sci, 8, 950988 (2007). 8. N. V. YANISHLIEVA-MASLAROVA, I. M. HEINONEN: Sources of Natural Antioxidants: Vegetables, Fruits, Herbs, Spices and Teas. In: Antioxidants in Food (Eds J. Pokorny, N. Yanishlieva, M. Gordon). CRC Press, England, 2001. 9. A. WOJDYLO, J. OSZMIANSKI, R. CZEMERYS: Antioxidant Activity and Phenolic Compounds in 32 Selected Herbs. Food Chem, 105, 940 (2007). 10. J. POKORNY, N. YANASHLIEVA, M. GORDON: Antioxidants in Food. CSC Press, England, 2001. 11. F. SHAHIDI: Natural Antioxidants: Chemistry, Health Effects and Applications (Ed. F. Shahidi). AOCS Press, Champaign Illinois, 1997. 12. J. JAVANMARDI, C. STRUSHUOFF, E. LOCKE, J. M. VIVANCO: Antioxidant Activity and Total Phenols Content of Iranian Ocimum Accessions. Food Chem, 83, 547 (2003).

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13. М. CIOROI, D. DIMITRIU: Studies of Total Polyphenols Content and Antioxidant Activity of Aqueous Extracts from Selected Lamiaceae Species. Ann Univ Dunarea de Jos Galati, Romania, Fascicle – Food Technology, 34 (1), 42 (2009). 14. M. MUCHUWETI, E. KATIVU, C. H. MAPURE, C. CHIDIWE, A. R. NDHLALA, M. A. N. BEKHURA: Phenolic Composition and Antioxidant Properties of Some Spices. Am J Food Technol, 2 (5), 414 (2007). 15. B. J. F. HUDSON: Food Antioxidants (Ed. B. Hudson). Elsevier Applied Science, London, 1990. 16. v. L. SinGLeton, S. a. roSSi: Colorimetry of Total Phenolics with Phosphomolibdic-phosphotungstic Acid Reagents. J Enol Viticult, 16, 144 (1965). 17. B. BADENSCHNEIDER, D. LUTHRIA, A. L. WATERHOUSE, P. WINTERHALTER: Antioxidants in White Wine (Cv. Riesling): 1. Comparison of Different Testing Methods for Antioxidant Activity. vitis, 38 (3), 127 (1999). 18. t. e. KraMLinG, v. L. SinGLeton: An Estimate of the Nonlavonoids Phenolics in Wines. Am J Enol Viticult, 20, 86 (1969). 19. Y.-G. LI, G. TANNER, P. LAKIN: The DMACA–HCL Protocol and the Threshold Proantocyanidin Content for Bloat Safety in Forage Legumes. J Sci Food Agr, 70, 89 (1996) 20. W. BRAND-WILLIAMS, M. E. CUVELLIER, C. BERSET: Use of Free Radical Method to Evaluate Antioxidant Activity. Lebensm Wiss Technol, 28, 25 (1995). 21. R. RE, N. PELLEGRINI, A. PROTEGGENTE, A. PANNALA, C. MIN YANG, C. RICE-EVANS: Antioxidant Activity Applying an Improved ABTS Radical Cation Decolorization Assay. Free Radical Biol Med, 26 (9/10), 1231 (1999). 22. F. F. BENZIE, J. J. STRAIN: Ferric Reducing (Antioxidant Power Assay). Meth Enzymol, 299, 15 (1999); F. F. BENZIE, J. J. STRAIN: Red Wines. J Agr Food Chem, 48, 220 (2000). 23. G. C. YEN, P. D. DUH: Scavenging Effect of Methanolic Extracts of Peanut Hulls on Free-radical and Active-oxygen Species. J Agr Food Chem, 42, 629 (1994). 24. H. R. JULIANI, J. E. SIMON: Antioxidant Activity of Basil. In: Trends in New Crops and New Uses (Eds J. Janick A. Whpke). ASNS Press, Alexandria,VA, 2002, 575–579. 25. E. CAPECKA, A. MARECZEK, M. LEJA: Antioxidant Activity of Fresh and Dry Herbs of Some Lamiaceae species. Food Chem, 93, 223 (2005). 26. Z. KRUMA, M. ANDJELKOVIC, R. VERHE, V. KREICBERGS: Phenolic Compounds in Basil, Oregano and Thyme. Foodblat, Latvia, April 17–18, 2008. 27. W. ZHENG, Z. Y. WANG: Antioxidant Activity and Phenolic Compounds in Selected Herbs. J Agr Food Chem, 49, 5165 (2001). 28. F. SHAHIDI, R. D. PEGG, Z. O. SALEEMI: Stabilization of Meat Lipids with Ground Spices. J Food Lipids, 2,145 (1995). 29. W. WANGCHAROEN, W. MORAZUK: Antioxidant Activity and Phenolic Content of Holy Basil. Songklanakarin J Sci Technol, 29 (5), 1407 (2007). 30. M. F. WANG, Y. SHAO, J. G. LI, N. Q. ZHU, M. RANGARAJAN, E. J. LAVOIE, C. T. HO: Antioxidative Phenolic Compounds from Sage (Salvia oficinalis). J Agr Food Chem, 46, 4869 (1998). 31. J. T. M. J. ARTS, G. R. M. M. HAENEA, H. P. VOSS, A. BAST: Antioxidant Capacity of Reaction Products Limits. The Applicability of the Trolox Equivalent Antioxidant Capacity (TEAC) Assay. Food Chem. Toxicol., 42, 45 (2004). Received 16 November 2011 Revised 19 February 2012

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Oxidation Communications 37, No 2, 522–532 (2014) Antioxidants in biological systems

antioxidant aCtivity and ContentS of PhenoliCS and flavonoidS in the whole Plant and Plant PartS of Teucrium botrys l. M. S. StanKoviCa*, D. JaKovLJeviCa, M. TOPUZOVICa, B. ZLATKOVICb a

Department of Biology and Ecology, Faculty of Science, University of Kragujevac, 12 Radoja Domanovica Street, 34 0 00 Kragujevac, Republic of Serbia E-mail: [email protected] b Department of Biology and Ecology, Faculty of Sciences and Mathematics, University of Nis, 33 Visegradska Street, 18 000 Nis, Republic of Serbia

aBStraCt This paper presents the results of a screening of twenty different extracts from the whole plant and plant parts (leaves, lowers and stems) of Teucrium botrys for total phenolic content, concentration of lavonoids and in vitro antioxidant activity. Мain reason for this study is the determination for irst time of these parameters for T. botrys and their variability among plant parts and plant extracts obtained by different solvents, respectively. Obtained amounts for total phenolic content ranged from 17.7 to 88.14 mg GA/g. The concentration of lavonoids varied from 7.10 to 187.45 mg RU/g. iC50 values of antioxidant activity ranged from 99.25 to 4501.82 µg/ml. Parallel to the analysis of T. botrys plant parts, Ginkgo biloba and chlorogenic acid are analysed for comparison. Results obtained from the whole plant compared with different plant parts were of uneven value. Great variability of the studied parameters was observed comparing the effectiveness of the used solvents. The acetone and methanol extracts from leaves and lowers contain the greatest concentrations of phenolics compounds, especially lavonoids and showed high antioxidant activity. According to our research, plant parts from T. botrys can be regarded as promising candidates for natural plant sources with high value of biological compounds. Keywords: Teucrium botrys, phenolic compounds, lavonoids, natural antioxidans. aiMS anD BaCKGroUnD Teucrium botrys L. (family Lamiaceae, section Scordium (M i l l e r) B e n t h a m) is an annual herbaceous plant with upright or partially prostrate, little branched stems, *

For correspondence.

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10 to 40 cm of height. Leaves are with petioles, cut into an oblong-shaped lobes. The leaves and shoots are covered with glandular and non glandular trichomes. The lowers are light purple, zygomorphic, cymosely clustered. T. botrys is the Central European species, widespread in southern, central and eastern parts of Europe, as well as at several localities in the extreme north-west Africa. The plant are adapted to the limestone surface, inhabits open, sunny places, dry rocky habitats, riverbank sediments, etc.1 Species belonging to the genus Teucrium have been used for over 2000 years in traditional and herbal medicine2. These plants are applied in treatment of respiratory and digestive disorders, abscesses, gout and conjunctivitis, in the stimulation of fat and cellulite decomposition, and possess anti-inlammatory, antioxidative, antimicrobial, antidiabetic and antihelminthic effects. Elimination of some problems in digestive tract was the most signiicant therapeutic effect3–5. The species of the genus Teucrium are very rich in phenolic compounds with very strong biological activity6,7, but in literature there are little data of the quantitative and qualitative characteristics of secondary metabolites from the species of T. botrys. in previous studies on the best popular species of genus Teucrium in the lora of Europe8–11 are suggested very high levels of phenolic compounds, especially lavonoids as well as strong biological activity. The basic aim of the presented research was to determine the contents of phenolics and concentrations of lavonoids in various extracts of the species of T. botrys, as well as to examine the antioxidant activity of plant extracts in vitro using standard model system. In addition, we compared the results obtained from the whole plant extracts with the results of analysis of extracts from different plant parts such as leaves, lowers, and stems in order to determine whether there are differences between the whole plant and individual plant parts. The obtained results of antioxidant activity were also compared with the values of chlorogenic acid as a standard synthetic antioxidant, and those related to extract of Ginkgo (Ginkgo biloba) as one of the most popular plant rich in natural antioxidants. EXPERIMENTAL Chemicals. Acetone, methanol, petroleum ether, ethyl acetate and sodium hydrogen carbonate (NaHCO3) were purchased from ‘Zorka pharma’ Sabac, Serbia. Standards of phenolic acids (gallic acid) and lavonoids (rutin hydrate), chlorogenic acid and 2,2-diphenyl-1-picrylhydrazyl (DPPH) were obtained from Sigma Chemicals Co., St. Louis, MO, USA. The Folin–Ciocalteu phenol reagent and aluminium chloride (AlCl3) were from Fluka Chemie AG, Buchs, Switzerland. All other solvents and chemicals were of analytical grade. A standardised extract of Ginkgo biloba was obtained from Pharmaceutical Company ‘Ivancic i Sinovi’, Belgrade, Serbia. Plant material. In August 2009 aerial lowering parts of T. botrys were collected from the region of Mokra Gora, Serbia. The voucher specimen of T. botrys was conirmed 523

and deposited in Herbarium at the Department of Biology and Ecology, Faculty of Science, University of Kragujevac. The collected plant material was air-dried in darkness at room temperature (20°C). Preparation of plant extracts. The prepared plant material (10 g) was coarsely crushed in small pieces of 2–6 mm by using the cylindrical crusher and extracted with water, methanol, acetone, ethyl acetate and petroleum ether. The extract was iltered through a paper ilter (Whatman, No 1) and evaporated under reduced pressure by the rotary evaporator. The obtained extracts were stored in dark glass bottles for further processing. Determination of total phenolics in the plant extracts. Total soluble phenolic compound in the different extracts of T. botrys were determined with the Folin–Ciocalteu reagent12 using gallic acid as a standard. Methanol extract was diluted to the concentration of 1 mg/ml and 0.5 ml of the soluted extract were mixed with 2.5 ml of the Folin–Ciocalteu reagent (previously diluted 10-fold with distilled water) and 2 ml of NaHCO3 (7.5%). The samples were incubated at 45°C for 15 min. The absorbance was determined at λmax = 765 nm. The samples were prepared in triplicate and the mean value of absorbance was obtained. Blank was concomitantly prepared, with methanol instead of extract solution. The same procedure was repeated for the gallic acid and the calibration line was construed. The total phenolic content was expressed in terms of gallic acid equivalent (mg GA/g extract). Determination of lavonoid concentration in the plant extracts. The total lavonoid contents were determined spectrophotometrically13. Briely, 1 ml of 2% solution of AlCl3 in methanol was mixed with the same volume of extract (1 mg/ml). The samples were incubated for an hour at room temperature. The absorbance was determined at λmax = 415 nm. The samples were prepared in triplicate and the mean value of absorbance was obtained. The same procedure was repeated for the rutin and the calibration line was construed. The total lavonoid content was expressed in terms of gallic acid equivalent (mg Ru/g extract). Evaluation of antioxidant activity. The ability of the plant extract and reference substance to scavenge DPPH free radicals was assessed using the method described by Tekao et al.14, adopted with suitable modiications by Kumarasamy et al.15 The stock solution of the plant extract was prepared in methanol to achieve the concentration of 1 mg/ml. Dilutions were made to obtain concentrations of 500, 250, 125, 62.5, 31.25, 15.62, 7.81, 3.90, 1.99 and 0.97 µg/ml. Diluted solutions (1 ml each) were mixed with 1 ml of DPPH methanol solution (80 µg/ml). After 30 min in darkness at room temperature (23 °C), the absorbance was recorded at 517 nm. The control samples contained all the reagents except the extract. The percentage inhibition was calculated using equation: % inhibition = 100 × ((A of control – A of sample)/A of control), whilst IC50 values were estimated from the % inhibition versus concentration

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sigmoidal curve, using a non-linear regression analysis (Origin 8 Pro). The data were presented as mean values ± standard deviation (n = 3). Statistical analysis. Statistical analysis was done using a SPSS (Chicago, IL) statistical software package (SPSS for Windows, ver. 17, 2008) and Origin 8 Pro (OriginLab Corp.). reSULtS anD DiSCUSSion Twenty different extracts from the whole plant and plant parts (leaves, lowers and stems) are prepared using water, methanol, acetone, ethyl acetate and petroleum ether in order to examine the total soluble phenolic compounds, lavonoid concentration and in vitro antioxidant activity. Various solvents are used to achieve the extraction of active substances with diversity in their polarity. Technique of solvent extraction, as a selective process of separation, offers plenty opportunities in many ields of universal importance – health and nutrition, foods and some other substances of vital use16. The solvents chosen for this experiment proved to be very effective in earlier studies8. The total phenolic content in the examined plant extracts using the Folin–Ciocalteu reagent was expressed in terms of gallic acid equivalent, GAE (the standard curve equation: y = 7.026x – 0.0191, r2 = 0.999) as mg of GA/g of extract (Table 1). table 1. Total phenolic contents in the plant extracts of T. botrys expressed in terms of gallic acid equivalent (mg of GA/g of extract)

Type of extract Whole plant Leaves Flowers Stems

Water

Methanol

acetone

Ethyl acetate

19.07±0.27 31.15±1.12 24.90±0.74 17.70±0.99

55.68±0.82 85.87±1.85 75.05±0.82 29.55±0.80

62.34±1.13 88.14±0.46 77.80±0.83 75.99±0.67

30.82±0.50 36.90±1.28 60.68±0.62 56.88±1.17

Petroleum ether 27.28±0.98 27.30±0.60 35.28±0.92 41.27±1.16

Note: Each value is the average of 3 analyses ± standard deviation.

The total phenolic content in the examined extracts ranged from 17.7 to 88.14 mg GA/g. The high total phenolic content was measured in acetone and methanol extracts from leaves (88.14 mg GA/g in acetone and 85.87 mg GA/g in methanol), followed by acetone and methanol extracts of lowers (77.8 and 75.05 mg GA/g). All water, ethyl acetate and petroleum ether extracts had considerably smaller concentration of phenols. Results of the statistical test (see further in Table 4) showed signiicant differences between the obtained values for total phenolic content in the comparison of different plant parts, different solvents effectiveness as well as in the comparison of plant parts and solvents effectiveness simultaneously. According to these results, leaves and lowers are parts of the T. botrys that are richest with phenolic compounds, also the greatest concentration of these metabolites are obtained by extraction with acetone and methanol. Unlike previous studied Teucrium species8–10, water extracts

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of the T. botrys are very ineficient solvent in the case of whole plant, leaves, lowers and stems because give the least value of phenolic compounds. The concentration of lavonoids in various extracts of T. botrys is determined using spectrophotometric method with AlCl3. The content of lavonoids is expressed in terms of rutin equivalent, RuE (the standard curve equation: y = 17.231x – 0.0591, r2 = 0.999), mg of Ru/g of extract. The summary of quantities of lavonoids identiied in the tested extracts is shown in Table 2. table 2. Flavonoid concentrations in the plant extracts of T. botrys expressed in terms of rutin equivalent (mg of Ru/g of extract)

Type of extract Whole plant Leaves Flowers Stems

Water

Methanol

10.96±0.93 24.09±0.57 15.58±0.86 7.10±0.92

83.76±0.46 122.99±0.98 123.21±1.09 30.12±1.45

acetone

Ethyl acetate

151.68±1.46 74.05±0.91 187.45±1.23 132.96±1.08 153.65±1.18 107.24±1.36 109.03±0.70 97.27±1.05

Petroleum ether 23.83±0.66 35.94±0.69 24.00±0.86 24.83±0.66

Note: Each value is the average of 3 analyses ± standard deviation.

The concentrations of lavonoids in plant extracts range from 7.1 to 187.45 mg Ru/g. High concentrations of lavonoids were measured in acetone, and some ethyl acetate and methanol extracts. Far the highest lavonoid content was found in acetone extract from the leaves of T. botrys (187.45 mg Ru/g) and lowers with 153.65 mg Ru/g, followed by the acetone extract from whole plant (151.68 mg Ru/g ) and leaves ethyl acetate extract (132.96 mg Ru/g). The lowest lavonoid concentration was measured in water and petroleum ether in all types of extracts. Results of the statistical test (see further in Table 4) showed signiicant differences between the obtained values for lavonoid concentrations in the comparison of different plant parts, different solvents effectiveness as well as in the comparison of plant parts and solvents effectiveness, simultaneously. Acetone extracts obtained from T. botrys are clearly distinguished by its high content of lavonoids in comparison to other extracts. This suggests that the concentration of lavonoids in extracts depends on the solvent polarity, whereas for this plant species extracts of medium polarity were highly effective. High concentrations of lavonoids in acetone, ethyl acetate and methanol extracts is the result of lavonoids high solubility in the solvent, which is consistent with their chemical characteristics17,18. In previous studies of quantitative characteristics of secondary metabolites in the function of solvent polarity, acetone extract had the greatest quantity of total phenolic compounds, including lavonoids19,20. Based on the obtained values from our experiment, it has been found that primarily leaves and then lowers are parts of T. botrys that are richest with phenolic compounds and lavonoids. Extraction is most effective using particularly acetone and methanol, also ethyl acetate in order to obtain lavonoids from leaves. Whole plant extracts also have high lavonoid concentration, but extraction of lavonoids from whole plant is

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effective using only acetone as a solvent. This variability has been demonstrated in previously conducted comparative analysis. The results of the comparative analysis of the total phenolic content and lavonoid concentration of plant species Aegli marmelos L. C o r r. S e r r. show that the leaves contain a larger quantity of these secondary metabolites than lowers21. The antioxidant activity in whole plant and plant parts extracts of T. botrys was determined using methanol solution of DPPH reagent. DPPH method is based on the reduction of methanol solution of coloured free radical DPPH by free radical scavenger. The scavenging activity was measured as the decrease in absorbance of the samples versus DPPH standard solution22. The antioxidant activity is expressed in terms of iC50 (µg/ml) values (Table 3). Parallel to the examination of the antioxidant activity of the plant extracts, the values for well-known medicinal plant Ginkgo biloba and chlorogenic acid were obtained and compared with the values of the antioxidant activity (Table 5). table 3. Antioxidant activity of plant extracts from T. botrys presented as iC50 values (µg/ml)

Type of extract Whole plant Leaves Flowers Stems

Water

Methanol

1089.50±0.74 173.66±0.43 373.68±0.59 99.25±0.72 361.64±1.35 101.65±0.68 392.14±1.02 209.82±0.75

acetone

Ethyl acetate

206.85±1.52 288.03±0.60 106.95±1.00 118.61±0.62

710.46±0.66 377.48±0.45 240.20±0.55 335.76±0.70

Petroleum ether 4501.82±0.52 881.09±1.00 3492.16±1.59 634.74±0.64

Note: Each value is the average of three analyses ± standard deviation.

The obtained values of antioxidant activity examined by DPPH radical scavenging activity range from 99.25 to 4501.82 µg/ml. The largest capacity to neutralise DPPH radicals is measured in methanol extract from leaves of T. botrys, which neutralises 50% of free radicals at concentrations of 99.25 µg/ml, followed by methanol extract from lowers (101.65 µg/m) and acetone extract from lowers (106.95 µg/ml). Analysing the extracts from leaves, it may be noted that high antioxidant activity showed only extracts obtained using methanol as a solvent, while extracts from all other solvents did not show considerable ability to neutralise DPPH radicals. Extracts from lowers, similar to the leaves, demonstrated high ability to neutralise DPPH radicals only in acetone, respectively methanol extracts. Results of the statistical test (Table 4) showed signiicant differences between the obtained values for antioxidant activity in the comparison of different plant parts, different solvents effectiveness as well as in the comparison of plant parts and solvents effectiveness, simultaneously.

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table 4. Results of analysis of variance between the obtained values for different plant parts, different solvents effectiveness and plant parts and solvents effectiveness, simultaneously

Type of Total phenolic contents Flavonoid concentrations Antioxidant comparison activity Values F p F p F p pp 966.1 *** 6220.0 *** 4255257 *** s 6060.5 *** 38287.6 *** 13811350 *** pp × s 609.6 *** 1213.4 *** 2755329 *** pp – plant parts, s – solvent; * < 0.05, ** < 0.01, *** < 0.001.

The value of antioxidant activity of G. biloba standardised extract was 33.9 µg/ml (Table 5). Comparing the antioxidant activity of G. biloba and T. botrys, methanol extract from leaves (IC50 = 99.25 µg/ml) showed partially high free radical scavenging activity, close to the activity of G. biloba standardised extract. In comparison to antioxidant activity of chlorogenic acid as a pure standard antioxidant (IC50 = 11.65 µg/ml), methanol extract of T. botrys leaves proved to be several times less powerful to scavenge DPPH radicals. Regarding the fact that the extract is a mixture of a great number of components opposite to pure compounds used as a standards, antioxidant activity of the methanol extract of T. botrys could be considered as strong. Methanol and acetone extracts of T. botrys lowers also showed high antioxidant activity that is in accordance with their high total phenolic content and concentration of lavonoids. Due to the presence of various antioxidant compounds which have different chemical properties and the polarity, which may or may not be soluble in a certain solvent, the yield of the extract and the resulting antioxidant activity of plant material are strongly dependent on the nature of the extraction solvent23. Methanol has been generally found to be more eficient in extraction of lower molecular weight polyphenols10. table 5. Values for antioxidant activity of the G. biloba and chlorogenic acid obtained for comparison with the values of T. botrys

Type of substance Ginkgo biloba Chlorogenic acid

iC50 (µg/ml) 33.91 ± 1.16 11.65 ± 0.52

Note: Each value is the average of three analyses ± standard deviation.

Numerous investigations of the antioxidant activity of plant extracts have conirmed a high linear correlation between the values of phenolic content and antioxidant activity24. In the previous qualitative screening of T. botrys secondary metabolites such as new neo-clerodane diterpenoids – 19-deacetylteuscorodol and teubotrin; diterpenes – teucvidin, montanin D, teuchamaedrin C and 6β-hydroxyteuscordin25; phenolic acids – chlorogenic, rosmarinic and coumaric acids; lavonoids – rutin, quercetin and kaempferol as well as stilbene resveratrol26, were identiied as dominant active compounds. For some of these secondary metabolites, such as chlorogenic acid, rosmarinic acid27, rutin, quercetin28,29 and resveratrol30 in a number of studies signiicant 528

antioxidant activity has been demonstrated. These facts indicate that the secondary metabolites from the group of phenolic compounds, constitute the main components that contribute to the antioxidant activity of T. botrys extracts. Synthetic antioxidants such as butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT) and tertbutylhydroquinon (TBHQ) have been widely used as antioxidants, but concern about the safety during their use has led to the growth of interest in natural antioxidants31. Having in mind that synthetic antioxidants are substituted phenolic compounds, considerable researches on natural antioxidants are focused on phenolic compounds, in particular the lavonoids32. Phenolic compound possesses a wide range of physiological properties, associated with the health beneits33. The beneits of phenolic compound are attributed to their antioxidant activity34,35. One of the main problems in standardisation of medicinal plants is the huge variability of secondary metabolites. To obtain the improved phytochemical proiles, screening the wild plant population may be irst step36. Extraction eficiency is also practical aspect that needs to be considered since the use of naturally occurring antioxidants is particularly interesting. Synthesis and secondary metabolites accumulation in the plants are affected by numerous factors, among which the most important are: abiotic and biotic factors, current status of plants, the age of plants, and the particular stage of development, which all affect their qualitative and quantitative variability within space and time. Numerous factors that lead to differences in the quantitative and qualitative characteristics of phenolic compounds also cause a difference in the characteristics of secondary metabolites in various plant organs37. Variability of the quantity and activity of secondary metabolites between plant parts has been demonstrated in numerous comparative analyses10,38,39. Determining this variability is of great importance in the scientiic and practical aspects. Scientiic importance is relected in the contribution to the biology of the analysed plants, while practical importance related to the eficiency of exploitation, improving the extraction procedure. Results of similar comparative studies have applications in the use of medicinal plants as raw material because the selection of plant parts which are the richest of secondary metabolites, improves the process of its use. Therefore, information obtained from these work indicated that T. botrys is natural source of health promoting compounds for human use and our results may also help to select the most appropriate conditions to extract this substances with the minimum loss. ConCLUSionS In this study, the basic proile of phenolic compounds and biological activity by measuring antioxidant capacity of Teucrium botrys was determined for irst time. All parameters were determined for plant parts (leaves, lowers and stems) and whole plant and comparatively analysed. Investigated plant parts have different amounts and activity of secondary metabolites in relation to the investigated parameters for the whole plant. The acetone and methanol extracts from leaves and lowers con529

tain the greatest concentrations of phenolics compounds, especially lavonoids and showed high antioxidant activity. The comparison of the effectiveness of various solvents showed large variability. Polar solvents such as methanol and acetone have the highest extraction eficiency. The comparative analysis indicates that the amount of phenolic compounds and their activity depends on the plant parts and the solvent used for extraction. Large value of antioxidant activity of T. botrys was proven by comparing with the obtained results for Ginkgo biloba extract. The results of our study suggest that some T. botrys extracts have high concentrations of phenols and lavonoids, also noticeable effects on the scavenging of free radicals. The extracts of these plant species can be regarded as promising candidates for natural source of biologically active substances. ACKNOWLEDGEMENTS This work was supported by the Ministry of Science and Techological Development of the Republic of Serbia (III41010). reFerenCeS 1. T. G. TUTIN, D. WOOD: Teucrium. In: Flora Europaea III (Eds T. G. T. G. Tutin, V. H. Heywood, N. A. Burges, D. Moore, D. Valentine, S. Walters). Cambridge University Press, Cambridge, 1972. 2. A. FIORENTINO, B. D’ABROSCA, S. PACIFICO, M. SCOGNAMIGLIO, G. D’ANGELO, M. GALLICCHIO, A. CHAMBERY, P. MONACO: Structure Elucidation and Hepatotoxicity Evaluation against HepG2 Human Cells of Neo-clerodane Diterpenes from Teucrium polium L. Phytochem, 72, 2037 (2011). 3. R. JURISIC, K. S. VLADIMIR, Z. KALODJERA, J. GRGIC: Determination of Selenium in Teucrium Species by Hydride Generation Atomic Absorbtion Spectrometry. Z Naturforsch, 58, 143 (2003). 4. A. ARDESTANI, R. YAZDANPARAST, S. JAMSHID: Therapeutic Effects of Teucrium polium Extracts on Oxidative Stress in Pancreas of Streptozotocin-induced Diabetes Rats. J Med Food, 11, 525 (2008). 5. A. POURMOTABBED, A. FARSHCHI, G. GHIASI, M. P. KHATABI: Analgesic and Anti-inlammatory Activity of Teucrium chamaedrys Leaves Aqueos Extracts in Male Rats. Iran J Bas Med Sci, 13, 119 (2010). 6. G. YIN, H. ZENG, M. HE, M. WANG: Extraction of Teucrium manghuaense and Evaluation of the Bioactivity of Its Extract. Int J Mol Sci, 10, 4330 (2009). 7. S. HASANI-RANJBAR, N. NAYEBI, B. LARIJANI, M. ABDOLLAHI: A Systematic Review of the Eficacy and Safety of Teucrium Species: from Anti-oxidant to Anti-diabetic Effect. Int J Pharm, 6, 315 (2010). 8. M. STANKOVIC, M. TOPUZOVIC, S. SOLUJIC, V. MIHAILOVIC: Antioxidant Activity and Concentration of Phenols and Flavonoids in the Whole Plant and Plant Parts of Teucrium chamaedrys L. var. glanduliferum H a u s s k. J Med Plant Res, 4 (19), 2092 (2010). 9. M. STANKOVIC, N. NICIFOROVIC, M. TOPUZOVIC, S. SOLUJIC:Total Phenolic Content, Flavonoid Concentrations and Antioxidant Activity of the Whole Plant and Plant Parts Extracts from Teucrium montanum L. var. montanum, f. supinum (L.) R e i c h e n b. Biotechnol Biotechnol Eq, 25 (1), 2222 (2011).

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10. M. STANKOVIC, N. NICIFOROVIC, V. MIHAILOVIC, M. TOPUZOVIC, S. SOLUJIC: Antioxidant Activity, Total Phenolic Content and Flavonoid Concentrations of Different Plant Parts of Teucrium polium L. subsp. polium. Acta Soc Bot Pol, 81 (2), 117 (2012). 11. M. STANKOVIC, O. STEFANOVIC, Lj. COMIC, M. TOPUZOVIC , I. RADOJEVIC, S. SOLUJIC: Antimicrobial Activity, Total Phenolic Content and Flavonoid Concentrations of Teucrium Species. Cent Eur J Biol, 7 (4), 664 (2012). 12. V. L. SINGLETON, R. ORTHOFER, R. R. M. LAMUELA: Analysis of Total Phenols and Other Oxidation Substrates and Antioxidants by Means of Folin–Ciocalteu Reagent. Meth Enzymol, 299, 152 (1999). 13. D. C. QUITTER, B. GRESSIER, J. VASSEUR, T. DINE, C. BRUNET, M. LUYCKX, M. CAZIN, J. C. CAZIN, F. BAILLEUL, F. TROTIN: Phenolic Compounds and Antioxidant Activities of Buckwheat (F. esculentum M o e n c h) Hulls and Flour. J Ethnopharmacol, 72, 35 (2000). 14. T. TEKAO, N. WATANABE, I. YAGI, K. SAKATA: A Simple Screening Method for Antioxidant and Isolation of Several Antioxidants Produced by Marine Bacteria from Fish and Shellish. Biosci Biotechnol Biochem, 58, 1780 (1994). 15. Y. KUMARASAMY, M. BYRES, P. J. COX, M. JASAPARS, L. NAHAR, S. D. SARKER: Screening Seeds of Some Scottish Plants for Free-radical Scavenging Activity. Phytother Res, 21, 615 (2007). 16. M. ZUBAIR, F. ANWAR, S. A. ANWAR: Effect of Extraction Solvents on Phenolics and Antioxidant Activity of Selected Varieties of Pakistani Rice (Oryza sativa L.). Int J Agr Biol, 14, 935 (2012). 17. G. MIN, L. CHUN-ZAO: Comparison of Techniques for the Extraction of Flavonoids from Cultured Cells of Saussurea medusa M a x i m. World J Microb Biot, 21, 1461 (2005). 18. L. CHEBIL, C. HUMEAU, J. ANTHONI, F. DEHEZ, J. ENGASSER, M. GHOUL: Solubility of Flavonoids in Organic Solvents. J Chem Eng Data, 52 (5), 1552 (2007). 19. P. C. ANOKWURU, I. ESIABA, O. AJIBAYE, O. A. ADESUYI: Polyphenolic Content and Antioxidant Activity of Hibiscus sabdariffa C a l y x. Res J Med Plant, 5 (5), 557 (2011). 20. M. CURCIC, M. STANKOVIC, I. RADOJEVIC, O. STEFANOVIC, Lj. COMIC, M. TOPUZOVIC, D. S. ĐACIC, S. D. MARKOVIC: Biological Effects, Total Phenolic Content and Flavonoid Concentrations of Fragrant Yellow Onion (Allium lavum L.). Med Chem, 8, 46 (2012). 21. A. N. SIDDIQUE, M. MUJEEB, K. A. NAJMI, M. AKRAM: Evaluation of Antioxidant Activity, Quantitative Estimation of Phenols and Flavonoids in Different Parts of Aegle marmelos. Afr J Plant Sci, 4 (1), 1 (2010). 22. W. BRAND-WILLIAMS, E. M. CUVELIER, C. BREST: Use of a Free Radicals Method to Evaluate Antioxidant Activity. Food Sci Technol, 28, 25 (1995). 23. B. SULTANA, F. ANWAR, M. ASHRAF: Effect of Extraction Solvent/Technique on the Antioxidant Activity of Selected Medicinal Plant Extracts. Molecules, 14 (6), 2167 (2009). 24. G. PILUZZA, S. BULLITTA: Correlations between Phenolic Content and Antioxidant Properties in Twenty-four Plant Species of Traditional Ethnoveterinary Use in the Mediterranean Area. Pharm Biol, 49 (3), 240 (2011). 25. C. M. de la TORRE, F. FERNÁNDEZ-GADEA, A. MIVHAVILA, B. RODRIGUEZ, F. PIOZZIA, G. SAVONAA: Neo-clerodane Diterpenoids from Teucrium botrys. Phytochem, 25 (10), 2385 (1986). 26. M. STANKOVIC: Biological Effects of Secondary Metabolites of Species from the Genus Teucrium L. of Serbian Flora. Ph.D. Thesis, University of Kragujevac, Kragujevac, Republic of Serbia, 2012 (in Serbian). 27. A. Z. VELKOV, K. M. KOLEV, V. A. TAJDER: Modeling and Statistical Analysis of DPPH Scavening Activity of Phenolics. Collect Czech Chem Commun, 72 (11), 1461 (2007). 28. D. ZIELINSKA, D. SZAWARA-NOWAK, H. ZIELINSKI: Determination of the Antioxidant Activity of Rutin and Its Contribution to the Antioxidant Capacity of Diversifed Buckwheat Origin Material by Updated Analytical Strategies. Pol J Food Nutr Sci, 60 (4), 315 (2010).

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29. T. GUSNIDAR, R. HEROWATI, R. E. KARTASASMITA, I. K. ADNYANA: Anti-inlammatory and Antioxidant Activity of Quercetin-3,3′,4′-triacetate. J Pharm Toxicol, 6 (2), 182 (2011). 30. I. GULCIN: Antioxidant Properties of Resveratrol: A Structure–Activity Insight. Inov. Food Sci Emerg, 11 (1), 210 (2010). 31. U. N. WANASUNDARA, F. SHAHIDI: Antioxidant and Pro-oxidant Activity of Green Tea Extracts in Marine Oils. Food Chem, 63 (3), 335 (1998). 32. V. I. MARTINEZ, M. J. PERIAGO, G. PROVAN, A. CHESSON: Phenolic Compounds, Lycopene and Antioxidant Activity in Commercial Varieties of Tomato (Lycopersicum esculentum). J Sci Food Agr, 82, 323 (2002). 33. A. J. PARR, G. P. BOLWELL: Phenols in the Plant and in Man. The Potential for Possible Nutritional Encancement of the Diet by Modifying the Phenols Content or Proile. J Sci Food Agr, 80, 985 (2000). 34. K. E. HEIM, A. R. TAGLIAFFERO, D. J. BOBILYA: Flavonoid Antioxidants: Chemistry, Metabolism and Structure–Activity Relationships. J Nutr Biochem, 13, 572 (2002). 35. N. GOUGOULIAS: Comparative Study on the Polyphenol Content and Antioxidant Activity of Some Medicinal Plants. Oxid Commun, 35 (4), 1001 (2012). 36. C. CIRAK, J. RADUSIENE, V. JANULIS, L. IVANAUSKAS, N. CAMAS, K. A. AYAN: Phenolic Constituents of Hypericum triquetrifolium T u r r a (G u t t i f e r a e) Growing in Turkey: variation among Populations and Plant Parts. Turk J Biol, 35, 449 (2011). 37. A. RAMAKRKISHNA, G. A. RAVISHANKAR: Inluence of Biotic Stress Signaling on Secondary Metabolites in Plants. Plant Signal Behav, 6 (11), 1720 (2011). 38. V. SASIKALA, S. SARAVANA, T. PARIMELAZHAGAN: Evaluation of Antioxidant Potential of Different Parts of Wild Edible Plant Passilora foetida L. J Appl Pharm Sci, 1 (4), 89 (2011). 39. K. N. PURUSHOTHAM, H. V. ANNEGOWDA, N. K. SATHISH, B. RAMESH, S. M. MANSOR: Evaluation of Phenolic Content and Antioxidant Potency in Various Parts of Cassia auriculata L.: A Traditionally Valued Plant. Pak J Biol Sci, 2013 (in press). Received 5 August 2013 Revised 7 September 2013

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Oxidation Communications 37, No 2, 533–542 (2014) Antioxidants in biological systems

antioxidant and antimiCroBial aCtivitieS of Leontice eversmannii rootS Q. U. REHMANa, I. H. BUKHARIa*, n. raSooLa, T. H. BOKHARIa, n. aSLaMa, M. RIAZb, M. KHURRAM SHAHZAD QURESHIa, r. B. tareenc a

Department of Chemistry, Government College University, 38 000 Faisalabad, Pakistan E-mail: [email protected] b Department of Chemistry, University of Sargodha, Women Campus, 38 000 Faisalabad, Pakistan c Department of Botany University of Baluchistan, Quetta, Baluchistan, Pakistan aBStraCt Nature is a very important source of medicinal agents from the early times of human races. Plants do not only show medicinal properties but also contain secondary metabolites which are responsible for the medicinal characteristics of plants. Extracts of plants are used to cure infectious diseases from the ancient times. Extracts of plants do not show adverse effects as compared to the synthetic drugs. Keeping in view the medicinal importance of plants the present research project was designed to investigate biological activities of Leontice eversmannii roots by using different techniques. As a therapeutic agent there is limited information published on the biological activities of this plant. Antifungal and antibacterial activities were also studied and these studies revealed that Leontice eversmannii roots exhibited excellent antimicrobial potential. Antioxidant activities were also examined by using different latest reported method in literature. Total lavonoid contents, total phenolic contents and DPPH free radical scavenging were also determined. Overall research revealed that Leontice eversmannii can be used for medicinal purposes. Keywords: Leontice eversmannii, antioxidant and antimicrobial activities. aiMS anD BaCKGroUnD The use of plants as medications belonged to early man. Ancient Chinese, Indians, and North Africans civilisations provided written evidence of man knowledge in consuming plants for the treatment of various infectious and dangerous diseases. In *

For correspondence.

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ancient times of Greece, for example, some scholars classiied plants in a deinite order and gave detailed explanations of medicinal plants, thus made the identiication process of plants effective. Theophrastus has been considered as the father of botany by some scholars and botanists. He explained the characteristics of medicinal plants and forecasted the probability of discovering lavonoids in plants1. In some countries local herbs and shrubs are used in herbal medicines to get rid of diseases and also for the prevention from diseases2–4. Leontice eversmannii belongs to the Berberidaceae family. This family comprises of about 14 genera which contains herbs and shrubs. Majority of members of Berberidaceae family are of medicinal values having therapeutic applications and are used for the cure of infectious diseases. Plants of this family are used for the treatment of rheumatism, dropsy, colic, sore throat, hepatitis, cramps, epilepsy, hysterics and for the inlammation of uterus. Some plants of this family also have antispasmodic, diuretic and diaphoretic, parturifacient and expectorant effects5,6. It has been reported that genus Leontice has numerous important medicinal values. In the herbal or folk medicines of Turkey, tubers of these plants are used effectively for the treatment of epilepsy and brain diseases, rheumatism and joint pain7–10. Anti-inlammatory, analgesic and sedative effects were manifested when these extracts were applied to the biological systems. A number of compounds of medicinal importance along with the alkaloid and lavones have been isolated from these plants11,12. Tubers of genus Leontice are used for the treatment of tuberculosis, eczema and diseases of cardiovascular system effectively13,14. As per literature survey no reports have been available regarding antioxidant, antimicrobial activity of Leontice eversmannii extract and fractions. So antioxidant and antimicrobial activities of Leontice eversmannii roots were determined by using different parameters and techniques. EXPERIMENTAL The plant for the research project was selected on the foundations of intensive review and ethnopharmacological survey. The Leontice eversmannii roots were selected for research project. The plant was collected from the Quetta region and recognised by Dr. Rasool Bakhsh Tareen, from the Department of Botany, University of Balochistan, Quetta, Balochistan, Pakistan. Sample preparation. To remove dust specks and other superluous matter the roots of Leontice eversmannii were washed with cold water. The roots were dried under the shade and ground into a ine powder form. This powdered form sample was soaked in 100% methanol for 7 days at room temperature. The plant material was extracted 3 times. The extract obtained as a result of this procedure was concentrated with the help of rotary evaporator and was regarded as methanol extract. By using solvent extraction method, different fractions were made from methanol extract with the help of separating funnel with different solvents like n-hexane, chloroform, ethyl acetate and n-butanol on the polarity bases. Extracts were iltered, evaporated, concentrated 534

and freed of solvent under reduced pressure using a vacuum rotary evaporator at 50°C approximately. The whole process was repeated 3 times to obtain ample amounts of extracts. The semi-dried, crude, concentrated extracts were weighed to evaluate the yield and stored at –4°C till analysis14. Phytochemical screening. In order to determine phytochemical constituents of Leontice eversmannii roots different chemical tests were performed on powdered plant material and dried extracts by following already reported procedures15. Evaluation of antioxidant activity. The evaluation of antioxidant activity of Leontice eversmannii roots was done by using the following antioxidant assays: – determination of total phenolic contents (TPC). The total phenolic contents of different fractions of Leontice eversmannii roots extract were determined by applying reported method16; – determination of total lavonoid content (TFC). The total phenolic contents of different fractions of Leontice eversmannii roots extract were determined by applying reported method in Ref. 16; – determination of free DPPH radical scavenging assay. The free 1,1-diphenyl2-picrylhydrazyl radical (DPPH) scavenging of different fractions of Leontice eversmannii roots extract was determined by applying reported method17. Antimicrobial assay by zone of inhibition and minimum inhibitory concentration (MIC). The antimicrobial activities of the extract and all factions of Leontice eversmannii roots were tested against some microorganisms, comprising 4 strains of bacteria and three strains of fungi. Streptococcus, Bacillus subtilis, Escherichia coli and Staphylococcus were bacterial strains while strains of fungus were E. candida, Aspergillus lavus and Aspergillus niger. Determination of the antimicrobial activity with reference to the minimum inhibitory concentration (MIC) of the extract and fractions was also made by using the above-mentioned infective strains according to the method described in Ref. 18. reSULtS anD DiSCUSSion Phytochemical analysis. Phytochemical analysis of the Leontice eversmannii roots indicated the presence of alkaloids, lavonoids, glycosides, tannins, whereas phlobatannins and steroids were absent. Previous investigations of Leontice eversmannii showed the presence of alkaloids and saponins in it and they were isolated and characterised by using column chromatography and different spectroscopic techniques12. Lupanin and taspine along with many types of saponins have been isolated from the plants of the family Berberidaceae. In recent years different types of triterpene saponins isolated from the tubers of Leontice smirnowii – a member of Berberidaceae family19. after performing different types of qualitative tests for the determination of phytochemicals, it was revealed that alkaloids were almost present in fractions/extracts of Leontice eversmannii roots, but saponins were present only in the methanol, aqueous and ethyl 535

acetate extracts. Tannins were found in the extracts of methanol and residue only. It was observed that falvonoids, tannins, and saponins were present in highly polar solvents as compared to the other solvents20. Flavonoids were present in all extracts except in n-hexane. Results of different experiments revealed the presence of phytochemical constituents in the roots of Leontice eversmannii and results are shown in Table 1. table 1. Phytochemical analysis of various extracts of Leontice eversmannii roots

Sample name Methanol n-Hexane Chloroform Ethyl acetate n-Butanol Aqueous

Phloba- terpe- Saponins Glyco- Steroids Fla- tannins tannins noids sides vonoids – + + + – + + – – – – – – – – – – – – – – – – + – – + – – – – + – + + – + + + – + +

Alkaloids + – + + + +

Note: absence (–) and presence (+).

Percentage yield of extracts. The percentage yield of different extracts and fractions of Leontice eversmannii roots was found in the range of 1.12–21.45 g/100 g of ine dry powder. The maximum yield was found by absolute methanol extract which is 21.45 g; it was a good yield but minimum yield was given by chloroform extract –1.12 g. The percentage yield of different extract of fractions of Leontice eversmannii roots are shown in the decreasing order as follows: methanol > aqueous > n-hexane > n-butanol > ethyl acetate > chloroform. The results of average percentage of triplicate are shown in Table 2. table 2. Percentage yield, total phenolic, lavonoid contents, free radical scavenging activity and % inhibition in linoleic acid system of Leontice eversmannii roots

Sample Methanol n-Butanol Chloroform Ethyl acetate n-Hexane Aqueous BHT

% age Yield (g/100 g) 21.47±0.65 3.01±0.08 2.22±0.07 1.52±0.04 1.00±0.01 12.00±0.08 –

TFC (mg/100 g) TPC (mg/100 g) 107.20±1.01 33.21±0.06 31.53±0.02 8.26±0.09 2.64±0.01 43.67±0.65 –

115.93±0.45 16.70±0.09 13.72±0.26 32.03±0.04 11.35±0.01 45.82±0.08 –

iC50 (µg/ml) 9.84±0.02 18.25±0.03 10.93±0.01 13.98±0.01 14.43±0.03 12.10±0.02 8.91±0.01

Evaluation of antioxidant activity. It was reported earlier that extracts of genus Leontice has antioxidant potential along with important phenolic contents21,22. To evaluate antioxidant potential of Leontice eversmannii roots different kinds of experiments were performed and were elaborated as follows: 536

– total phenolic contents (TPC). Total phenolic contents shown by the extracts and fractions of Leontice eversmannii roots were in the range of 11.35–115.93 mg/100 g of all extracts. The TPC values shown by the plant for different extracts of fractions were as follows: methanol (115.93), n-hexane (11.35), chloroform (13.72), ethyl acetate (32.03), n-butanol (16.70) and aqueous (45.82). The order of TPC values of different fractions can be shown as given below: methanol > aqueous > ethyl acetate > n-butanol > chloroform > n-hexane

The maximum value of total phenolics contents was shown by methanol extract as it has maximum values out of all extracts of fractions whereas aqueous, n-butanol and ethyl acetate showed signiicant phenolic contents as compared to the methanol extracts. The chloroform and n-hexane extracts showed minimum phenolic contents whereas n-hexane fraction was found in the lower limit. The average results of TPC values as triplicate are shown in Table 2; – total lavonoid contents (TFC). The different extracts and fractions of Leontice eversmannii roots showed total lavonoid contents values were in the range of 2.64–107.2 mg/100 g of all extract. The TPC values shown by the plant for different extracts of fractions were as follows: methanol (107.2), n-hexane (2.64), chloroform (31.53), ethyl acetate (8.26), n-butanol (33.21) and aqueous (43.67). The order of TFC values of different fractions: methanol > aqueous > n-butanol > chloroform > ethyl acetate > n-hexane. The maximum value of total lavonoid contents was shown by methanol extract as it has maximum values out of all extracts of fractions whereas aqueous, n-butanol and chloroform showed signiicant lavonoid contents as compared to the methanolic extracts. The ethyl acetate and n-hexane extracts showed minimum lavonoid contents whereas n-hexane fraction was found in the lower limit. The average results of TFC values as triplicate are shown in Table 2. DPPH free radical scavenging. The IC50 assay was applied on the extracts and fractions of Leontice eversmannii roots, to check the DPPH free radical scavenging and its range was from 9.84 to 18.25%. A methanol extract and aqueous extract of root samples exhibited maximum free radical scavenging activity and so their values of IC50 were lower when compared to all other extracts of fractions of Leontice eversmannii roots. It was assessed that the range of IC50 value for roots were for n-hexane fraction showed the highest value (18.25), followed by chloroform (10.93%), aqueous (12.10%), ethyl acetate (13.98%), n-butanol (14.43%), and absolute methanol (9.84%) fractions. The order of DPPH free radical scavenging, according to the activities of extracts, was arranged as follows: n-hexane > n-butanol > ethyl acetate > aqueous > chloroform > methanol.

The IC50 values of extracts and fractions of Leontice eversmannii roots results are shown in Table 2.

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Antimicrobial activity of Leontice eversmannii roots. Some other studies have also shown the use of natural sources for antimicrobial studies23–25. Hence the extracts of fractions of Leontice eversmannii roots were used to determine the antibacterial and antifungal activities by using different strains of bacteria and fungi. The results, thus obtained were discussed below. Antibacterial activity of Leontice eversmannii roots. Antibacterial activity of different extracts of Leontice eversmannii roots was performed against 4 different bacterial strains with the help of samples of different extracts of fraction by using antibacterial assays. Hence the results obtained are shown in tabular form against each bacterial strain. The inhibition zone was noted in the millimetre scale. In all the extracts of fractions of Leontice eversmannii roots, excellent antibacterial activity against E. coli, B. cerus, Streptococcus and Staphylococcus was shown by the sample containing methanol extract. All the other remaining fractions also show better antibacterial activities but after methanol; aqueous extract of fraction shows good antibacterial activities against E. coli, B. cerus, Streptococcus and Staphylococcus. Samples comprising extracts/fraction of chloroform and ethyl acetate show overall minimum antibacterial activities against the above-mentioned 4 bacterial strains. Sample of n-hexane fraction also showed signiicant antibacterial activities against E. coli, B. cerus, Streptococcus and Staphylococcus. The order of antibacterial potential of extracts of different fractions against pathogenic bacterial strains can be arranged as follows: methanol > aqueous > n-butanol > n-hexane > ethyl acetate > chloroform.

The overall excellent antibacterial activities were shown against B. cerus by all the extracts/fractions of Leontice eversmannii roots and were for methanol (20.67±0.58), n-butanol (20.33±0.57), ethyl acetate (13.17±0.29), chloroform (12.33±0.29), n-hexane (15.17±0.76) and aqueous (17.33±0.57). The results as the mean of triplicate experiments are shown in Table 3. table 3. Antibacterial activity of Leontice eversmannii roots

Samples of extracts Methanol n-Butanol Ethyl acetate Chloroform n-Hexane Aqueous rifampicin

E. coli 15.17±0.29 10.67±0.57 11.10±0.17 11.17±0.29 13.33±0.57 14.50±0.50 25.67±0.57

Zones of inhibition of bacterial strains (mm) B. cerus Streptococcus Staphylococcus 20.67±0.58 18.17±0.28 20.50±0.50 20.33±0.57 17.33±0.57 11.83±0.76 13.17±0.29 15.33±0.57 11.33±0.57 12.33±0.29 12.17±0.28 12.50±0.50 15.17±0.76 11.50±0.50 10.33±0.57 17.33±0.57 12.16±0.76 15.33±0.57 25.67±0.57 25.67±0.57 25.67±0.57

Antifungal activity of Leontice eversmannii roots. Antifungal activity of different extracts of Leontice eversmannii roots was performed against 3 different fungal strains with the help of samples of different extracts of fraction by using antifungal assays. 538

Hence the results obtained are shown in tabular form against each bacterial strain. The inhibition zone was noted in the millimetre scale. In all the extracts of fractions of Leontice eversmannii roots, excellent antifungal activity against A. niger, A. lavus and E. candida was shown by the sample containing extract of n-butanol fractions. After the n-butanol, extracts of methanol and ethyl acetate showed good antifungal activities against the above-mentioned strains of pathogenic funguses. However, the sample comprising extracts of the n-hexane and aqueous fractions could not show any inhibition of the growth of E. candida fungus. Samples of extracts/fractions of chloroform also show signiicant inhibition of fungal growth in the culture media against the all 3 strains of fungi. The order of antibacterial potential of extracts of different fractions against pathogenic bacterial strains can be arranged as: n-butanol > methanol > ethyl acetate > n-hexane > aqueous > chloroform.

The overall excellent antifungal activities were shown against A. lavus by all the extracts/fractions of Leontice eversmannii roots – for methanol (18.5±0.50), nbutanol (20.83±0.76), ethyl acetate (17.5±0.50), chloroform (13.67±0.58), n-hexane (16.33±0.58) and aqueous (15.33±0.58). The results as the mean of triplicate experiments are shown in Table 4. table 4. Antifungal activity of Leontice eversmannii roots

Samples of extracts Methanol n-Butanol Ethyl acetate Chloroform n-Hexane Aqueous Fungone

Zones of inhibition of fungal strains (mm) A. niger A. lavus E. candida 15.17±0.58 18.50±0.50 16.70±0.57 21.16±0.29 20.83±0.76 15.17±0.73 11.67±0.57 17.50±0.50 13.83±0.29 16.17±0.76 13.67±0.58 10.33±0.57 13.33±0.29 16.33±0.58 – 15.33±0.57 15.33±0.58 – 26.04±0.58 26.04±0.58 26.04±0.58

Minimum inhibitory concentration (MIC) against fungi. MIC values were opposite to the antimicrobial values. During the antifungal assay n-butanol extract showed the maximum antifungal activity against A. niger as 21.16±0.29 and its minimum inhibitory concentration was observed as 1.73±0.01 which described that the assay of n-butanol extract of Leontice eversmannii roots could only inhibit the growth of fungus at this concentration. Same pattern was observed in methanol as it showed maximum inhibition of A. lavus and its inhibition zone against this strain was at 18.5±0.50 and its observed MIC value was 2.10±0.01 which was its minimum value of inhibition. In case of chloroform fraction, maximum antifungal activity was shown at 16.17±0.76 against A. niger but its MIC value was 1.80±0.02 which showed that it inhibited the growth of fungus at this concentration only. It was observed that in ethyl acetate fraction maximum antifungal activity was observed at 17.5±0.50 against A. lavus, its MIC was 1.45±0.01 which exhibited that it could inhibit the growth of 539

fungus at this concentration. The results as the mean of triplicate experiments are shown in Table 5. table 5. MIC of antifungal activity of Leontice eversmannii roots

Samples of extracts A. niger 1.50±0.01 1.73±0.01 1.76±0.01 1.80±0.02 1.90±0.01 1.75±0.02 0.81±0.01

Methanol n-Butanol Ethyl acetate Chloroform n-Hexane Aqueous Fungone

Fungal strains A. lavus 2.10±0.01 1.36±0.01 1.45± 0.01 1.60±0.02 1.92±0.01 1.61±0.01 0.78±0.01

E. candida 1.90±0.01 1.67±0.01 1.98±0.01 1.74±0.01 – – 0.89±0.01

Minimum inhibitory concentration (MIC) against bacteria. Bacterial strains were also subjected for the determination of minimum inhibitory concentration. MIC was determined against 4 pathogenic bacterial strains E. coli, B. cerus, Streptococcus and Staphylococcus. During the antibacterial assay extract of methanol showed the maximum antibacterial activity against B. cerus and was observed as 20.67±0.58 and its minimum inhibitory concentration was observed as 1.90±0.01 which described that the methanol extract of Leontice eversmannii roots could only inhibit the growth of bacteria at this concentration. Same pattern was observed in n-butanol as it showed maximum inhibition against B. cerus and its inhibition zone against this strain was at 20.33±0.57 and its observed MIC value was 1.76±0.01 which was its minimum value for inhibition. In case of aqueous extract/fraction maximum antibacterial activity was exhibited at 17.33±0.57 against B. cerus but its MIC value was 1.95±0.01 which showed that it inhibited the growth of bacteria at this concentration only. It was observed that in n-hexane fraction maximum antibacterial activity was observed at 15.17±0.76 against B. cerus and its MIC was 1.72±0.01 which exhibited that it could inhibit the growth of bacteria at this concentration. The results as the mean of triplicate experiments were shown in Table 6. table 6. MIC of antibacterial activity of Leontice eversmannii roots

Samples of extracts Methanol n-Butanol Ethyl acetate Chloroform n-Hexane Aqueous Ciproloxacin

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E. coli 1.72±0.01 1.98±0.01 1.87±0.01 1.89±0.01 2.00±0.02 1.78±0.01 1.02±0.01

Bacterial strains B. cerus Streptococcus 1.90±0.01 1.61±0.01 1.76±0.01 1.68±0.01 1.96±0.01 1.55±0.01 1.74±0.02 1.65±0.01 1.72±0.01 2.01±0.01 1.95±0.01 1.88±0.01 0.76±0.01 0.86±0.10

Staphylococcus 1.73±0.01 1.91±0.01 1.90±0.01 1.71±0..02 1.68±0.02 2.05±0.57 0.90±0.01

ConCLUSionS The research was focused on study of the antioxidant and antimicrobial activities of the Leontice eversmannii roots. The results of the total phenolic contents, total lavonoid contents, DPPH free radical scavenging assay and percentage inhibition in linoleic acid system showed that the plant has active antioxidant components in its chemical composition. Phytochemical analysis revealed that alkaloids, tannins, lavonoids, terpenoids and saponins are also present in it. It is concluded that Leontice eversmannii roots have very active component and showed an excellent antioxidant, antibacterial, antifungal activities. In short, the plant has many bioactive and secondary metabolites which can be used for the synthesis of drugs against the diseases provoked by pathogens and related to some oxidants. ACKNOWLEDGEMENTS We are highly thankful to Dr. Rasool Bakhsh Tareen, Department of Botany, University of Balochistan, Quetta, Balochistan, Pakistan, for suggesting and recognising the plant. reFerenCeS 1. J. D. PHILLIPSON: A Matter of Some Sensitivity. Phytochem, 38, 1319 (1995). 2. E. M. ABDALLAH, A. E. KHALID: A Preliminary Evaluation of the Antibacterial Effects of Commiphora molmol and Boswellia papyrifera Oleo-gum Resins Vapor. Int J Chem Biochem Sci, 1, 1 (2012). 3. T. H. BOKHARI, M. HUSSAIN, S. HINA, M. ZUBAIR, N. RASOOL, M. RIAZ, M. IQBAL, I. H. BUKHARI, A. I. HUSSAIN, M. SHAHID: Antioxidant, Antimicrobial, Cytotoxic Studies of Methanolic Extract, Fractions and Essential Oil of Curry Patta (Chalcas koeingii) from Pakistan. J Chem Soci Pak, 35, 468 (2013). 4. S. ADEEL, F. REHMAN, T. GULZAR, I. A. BHATTI, S. QAISER, A. ABID: Dyeing Behaviour of γ-irradiated Cotton Using Amaltas (Cassia istula) Bark Extracts. Asian J Chem, 25, 2739 (2012). 5. V. E. TYLER: Blue Cohosh in Tyler’s Honest Herbal. 1st ed. The Hworth Herbal Press, New York, 1999, 55–56. 6. M. S. ARAYNE, N. SULTANA, S. S. BAHADUR: The Berberis Story: Berberis vulgaris in Therapeutics. Pak J Pharma Sci, 20, 83 (2007). 7. T. BAYTOP: Therapy with Medicinal Plants in Turkey (Past and Present). 1st ed. Istanbul University Press, Istanbul, 1984, 294–295. 8. M. S. BUTTER: The Role of Natural Product Chemistry in Drug Discovery. J Nat Pro, 67, 2141 (2004). 9. A. GHORBANI: Studies on Pharmaceutical Ethnobotany in the Region of Turkmen Sahra, North of Iran. J Ethnopharmacol, 102, 58 (2005). 10. F. NAGHIBI, M. MOSADDEGH, M. S. MOTAMED, A. GHORBANI: Labiatae Family in Folk Medicine in Iran: From Ethnobotany to Pharmacology. Iran J Pharma Res, 2, 63 (2005). 11. Q. LIU, G. ZHU, P. HUANG: Anti-inlammatory, Analgesic and Sedative Effects of Leontice kiangnanensis. Zhong Zhong Zazh, 161, 50 (1991). 12. G. GRESSER, P. BACHMANN, L. WITTE, F. C. CZYGAN: Distribution and Taxonomic Signiicance of Quinolizidine Alkaloids in Leontice leontopetalum and L. eversmannii (Berberidacea). Biochem Syst Eco, 6, 679 (1993).

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13. S. ABDALLA, M. ABU-ZARGA, S. SABRI: Cardiovascular Effects of Oblongine Chloride, an Alkaloid from Leontice leontopetalum, in the Anaesthetized Guinea-pig. Phyto Res, 9, 60 (2006). 14. I. GULCIN, V. MSHVILDADZE, A. GEPDIREMEN, R. ELIAS: Screening of Antiradical and Antioxidant Activity of Monodesmosides and Crude Extract from Leontice smirnowii T u b e r. Phytomed, 13, 343 (2006). 15. H. O. EDEOGA, D. E. OKWU, B. O. MBAEBIE: Phytochemical Constituents of Some Nigerian Medicinal Plants. Afr J Biotech, 4, 685 (2005). 16. M. RIAZ, N. RASOOL, I. H. BUKHARI, M. SHAHID, M. ZUBAIR, K. RIZWAN, U. RASHID: In vitro Antimicrobial, Antioxidant, Cytotoxicity and GC-MS Analysis of Mazus goodenifolius. Molecules, 17, 14275 (2012). 17. M. RIAZ, N. RASOOL, I. H. BUKHARI, M. SHAHID, F. ZAHOOR, M. GILANI, M. ZUBAIR: Antioxidant, Antimicrobial and Cytotoxicity Studies of Russelia equisetiformis. Afr J Microbial res, 6, 5700 (2012). 18. G. VALYA, A. RAGAN, V. S. RAJU: Screening for in vitro Antimicrobial Activity of Solanum americanum M i l l e r. J Rec Adv App Sci, 26, 43 (2011). 19. N. TABATADZE, B. TABIDZE, V. MSHVILDADZE, R. ELIAS, G. DEKANOSIDZE, G. BALANSARD, E. KEMERTELIDZE: Triterpene Glycosides from the Tubers of Leontice smirnowii. Chem Nat Comp, 45, 453 (2009). 20. M. RIAZ, N. RASOOL, S. RASOOL, U. RASHID, I. H. BUKHARI, M. ZUBAIR, M. NOREEN, M. ABBAS: The Chemical Analysis, Cytotoxicity and Antimicrobial Studies by Snapdragon – A Medicinal Plant. Asian J Chem, 25, 5479 (2013). 21. F. ASLAM, N. RASOOL, M. RIAZ, M. ZUBAIR, K. RIZWAN, M. ABBAS, T. H. BUKHARI, I. H. BUKHARI: Antioxidant, Haemolytic Activities and GC-MS Proiling of Carissa carandas. Int J Phytomed, 3, 567 (2011). 22. K. RIZWAN, M. ZUBAIR, N. RASOOL, M. RIAZ, M. ZIA-UL-HAQ, D. V. FEO: Phytochemical and Biological Studies of Agave attenuata. Int J Molec Sci, 13, 6440 (2012). 23. N. UPWAR, R. PATEL, N. WASEEM, N. K. MAHOBIA: Hypoglycemic Effect of Methanolic Extract of Berberis aristata Stem on Normal and Streptozotocin-induced Diabetic Rats. Int J Pharma Sci, 3, 222 (2011). 24. A. MUSHTAQ, N. RASOOL, M. RIAZ, R. B. TAREEN, M. ZUBAIR, U. RASHID, M. A. KHAN: Antioxidant, Antimicrobial Studies and Characterisation of Essential Oil, Fixed Oil of Clematis graveolens by GC-MS. Oxid Commun, 36, 1067 (2013). 25. M. RIAZ, N. RASOOL, I. H. BUKHARI, K. RIZWAN, F. JAVED, A. A. ALTAF, H. M. A. QAYYUM: Antioxidant, Antimicrobial Activity and GC-MS Analysis of Russelia equsetiformis Essential Oils. Oxid Commun, 36, 272 (2013). Received 7 November 2013 Revised 29 December 2013

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Oxidation Communications 37, No 2, 543–554 (2014) Antioxidants in biological systems

antioxidant and antimiCroBial Study of variouS PartS of Achillea wilhelmsii (a Medicinal plant) M. ASGHARa, n. raSooLa*, M. RIAZb, M. aBaSSc, a. U. MUStaFaa, H. M. ADEELa, M. ZUBAIRa, r. B. tareend, U. a. ranae, a. r. anSarif a

Department of Chemistry, Government College University, 38 000 Faisalabad, Pakistan E-mail: [email protected] b Department of Chemistry, University of Sargodha, Women Campus, 38 000 Faisalabad, Pakistan c Department of Chemistry and Biochemistry, University of Agriculture, 38 040 Faisalabad, Pakistan d Department of Botany University of Baluchistan, Quetta, Baluchistan, Pakistan e Deanship of Scientiic Research, College of Engineering, King Saud University, 11 421 Riyadh, Saudi Arabia f Independent University Hospital, 38 000 Faisalabad, Pakistan aBStraCt The methanol extract of various parts of Achillea wilhelmsii and their fractions were analysed for their antioxidant and antimicrobial activities. The methanol extract and the different fractions of various parts of the Achillea wilhelmsii are found to contain appreciable levels of total lavonoid and total phenolic contents. The DPPH radical scavenging activity and the inhibition linoleic acid peroxidation were also analysed. The plant extract and fractions were assessed against human blood erythrocytes for cytotoxic studies by haemolytic activity. The results of the present study have shown signiicant variations in the antioxidant activities of various parts of Achillea wilhelmsii and their fractions. Keywords: Achillea wilhelmsii, antioxidant activity, antimicrobial activity, cytotoxicity. aiMS anD BaCKGroUnD Achillea wilhelmsii belongs to Asteraceae, which possesses various chemical constituents1. The history of Achilles showed that the soldiers used yarrow to treat wounds, and therefore it was named as allheal and bloodwort2. Bioactive properties of genus Achil*

For correspondence.

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lea have been investigated through phytochemical studies. The irst anti-spasmodic lavonoids, cosmosiin and cynaroside were obtained from A. millefolium L.3 and the irst natural proazulene, achillicin, was conirmed from the genus Achillea4. It was reported that Achillea wilhelmsii also contains santolavone identiied in the methanol extract. Bumadaran is a popular name for several species of Achillea in Persian language which are used as anti-inlammatory, anti-spasmodic, diaphoretic, diuretic and emmenagogic agents and have been used for treatment of hemorrhage, pneumonia, rheumatic pain and wounds healing in Persian traditional literature5. Achillea is also known as plumajillo in New Mexico and southern Colorado. The name plumajillo refers to its leaf shape texture, while for practical purposes this was used for wound healing and anti-bleeding6. Herbal teas prepared from Achillea species are quite useful against abdominal pain7, whereas, the extract of Achillea species are very effective against hypertension8. Regardless of the numerous potential uses of Achillea, the wound healing property of Achillea has received a great deal of attention from the scientiic community9. Though, a few studies have been done to explore the chemical and biological characteristics of various plants extracts in Pakistan10–16, more serious efforts are still required to explore the wide variety of plant extracts with detailed investigations of their chemical and biological features. EXPERIMENTAL Collection of plant. The plant samples were collected from the Quetta region and were identiied at the Department of Botany, University of Balochistan, Quetta, Balochistan, Pakistan. The voucher for specimen (AW-RBT-06) was deposited in the Department of Botany, University of Balochistan, Quetta, Balochistan, Pakistan. Sample preparation. The stem, root and leaves of A. wilhelmsii were washed with cold water to remove any dust particles or extraneous matter. The shredded dried parts were further ground into powdered form and the ine samples were soaked into methanol for 4–5 days at room temperature. For systematic studies, different fractions of samples were made from methanol extract by using solvent extraction technique. A number of different solvents such as n-hexane, chloroform, ethyl acetate and n-butanol were also used on polarity basis. Once, the extraction is complete, the solvents were removed from the extracts using vacuum rotary evaporator (EYELA, SB-65, Rikakikai Co. Ltd., Tokyo, Japan) at 50°C. The process was repeated thrice to get adequate amount of extracts. The dried, crude and concentrated extracts were weighed to quantify the yield and later stored at –4°C until used for analysis17,18. Determination of total phenolic contents (TPC) and total lavonoid content (TFC). The Folin–Ciocalteu reagent was used for the determination of total phenolic contents according to the previously reported method19,20. The total lavonoid content (TFC) extract/fractions was calculated by the procedure used by Dewanto and co-workers21.

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DPPH radical scavenging assay. The 2,2-diphenyl-1-picrylhydrazyl radical (DPPH) assay was estimated by using spectrophotometric method22. Antioxidant activity in linoleic acid system. The antioxidant activity of various parts of A. wilhemsii was calculated in terms of measurement of percent inhibition of peroxidation in linoleic acid system by the method described in Refs 20 and 23. Haemolytic activity. Haemolytic activity of A. wilhelmsii was determined by using the already reported method17. Evaluation of antimicrobial activity. The antimicrobial activity of extracts and fractions of A. wilhelmsii was tested against the identiied bacteria (E. coli, B. cereus, S. aureus, S. typhi, R. pneumonea, B. subtilus, M. luteus and E. aerogens) and four fungal strains (Aspergillus niger, Aspergillus lavus, Fusarium oxysporium and Candila albicans). Antimicrobial assay (disc diffusion method) and minimum inhibitory concentration. The antimicrobial effect of different parts (root, stem and leaves) of A. wilhelmsii was determined by measuring the inhibition zone, using the wick paper disc (6 mm in diameter) by following disc diffusion method24. The minimum inhibitory concentration of different parts of A. wilhelmsii was determined by using the initially reported method described in Ref. 23. reSULtS anD DiSCUSSion Percentage yield of different fractions. The percentage yield of different extracts and fractions of A. wilhelmsii was calculated. The range of percentage yield of roots was determined between 2.20 and 24.41 g/100 g (Table 1). The maximum value of % yield was found in the methanol extract (24.41 g/100 g) of root. The least amount of yield was found in n-hexane (2.20 g/100 g) fraction of root as compared to all other fractions, which is very close to the chloroform (2.22), n-butanol (2.25) fraction. 80% methanol extract of root of A. wilhelmsii has also suitable quantity (8.42 g/100 g). The % yield calculated for ethyl acetate and aqueous fraction was 4.11 and 3.90, respectively. The range of percentage yield of stem was found between 0.66 and 12.36 g/100 g. The maximum value of yield in stem was found in methanol extract (12.36 g/100 g) and the least value was calculated for chloroform extract (0.66 g/100 g). The value of percentage yield for other fractions was also calculated, which was 3.28 for 80% methanol, 0.68 for ethyl acetate, 0.75 for n-butanol, 0.91 for n-hexane and 1.25 for aqueous fraction. The range of yield for leaves ranged within 0.50 and 16.62 g/100 g. The maximum yield was calculated in methanol extract (16.62 g/100 g) and least value of yield was found in n-butanol fraction (0.32 g/100 g). The value of percentage yield for other fractions was 6.02 for 80% methanol extract, 1.91 for n-hexane, 1.40 for chloroform, 0.50 for ethyl acetate and 1.22 for aqueous fraction. The overall results showed that the maximum value of percentage yield was found in 80% methanol and least value of yield was – found in other organic solvents.

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Total phenolic and lavonoid contents. Total phenolic contents were analysed in root, stem and leaves of A. wilhelmsii. The range of phenolic contents in roots was from 0.98 mg/100 g (n-hexane) to 654.41 mg/100 g (methanol) (Table 1). It was found that the methanol extracts of root have higher values of phenolic contents and least quantity of phenolic contents was determined in n-hexane fraction24. This least quantity of phenolic components in n-hexane might be due to the smaller polarity of n-hexane. Less phenolic contents were also calculated in chloroform (98), n-butanol (132) and ethyl acetate (106). A large quantity of phenolic contents was also calculated in aqueous fraction of the roots. So according to the results, methanol extract and aqueous fraction of roots of plant have higher values of phenolic contents as compared to all other fractions. It was assessed in the study of stem that methanol extract had high phenolic contents (197) and n-hexane fraction had least phenolic contents (0.75). In case of leaves, large phenolic contents were calculated in methanol extract (365) and aqueous fraction (246.15) and less quantities were observed in n-hexane (0.89), ethyl acetate (88), chloroform (82) and n-butanol (22). The overall investigation showed that leaves of plant contained high quantity of phenolics and n-hexane fraction contained less value of phenolics. The range of total lavoniod contents for leaves was 1.08 mg/100 g (n-hexane) to 784.08 mg/100 g (methanol), the range of root was found from aqueous fraction (31.95 mg/100 g) to methanol extract (710.7 mg/ 100 g) and the range of stem was found from ethyl acetate (66.14 mg/100 g) to 80% methanol extract (779.85 mg/100 g). From the results it was found that methanol extract has high value of lavonoids and lower values were found in aqueous fraction of root. 80% methanol extract has high conetnts of lavonoids and ethyl acetate has lower values of lavonoids in stem. In case of leaves n-hexane contained high quantity of lavonoids and 80% methanol extract contained least quantity. DPPH free radical scavenging activity and % inhibition in linoleic acid system. It was observed that the methanol extract and 80% methanol extract of root, stem, and leaves of A. wilhelmsii showed maximum free radical scavenging activity and hence their IC50 value is lower as compared to all other fractions (Table 2). It was noticed that the range of IC 50 values for roots was in-between 88.14 (methanol) and 812.43 (n-hexane). Similarly, the range of IC50 values for stem ranged from 102.59 (methanol) to 1004.23 (n-hexane), while the range of IC50 values for leaves was found between 96.51 (methanol) and 987.07 (ethyl acetate). The results also suggest that the methanol extract of roots, stem and leaves showed maximum free radical scavenging activity, while ethyl acetate, chloroform, n-hexane and n-butanol showed moderate free radical scavenging activity. The percentage inhibition in linoleic system was investigated in various parts of A. wilhelmsii. The range of % inhibition of leaves in linoleic acid system was from 0.87% (n-hexane) to 80.10% (80% methanol). The range of % inhibition of roots in linoleic acid system was from 0.75 % (n-hexane) to 75.30% (aqueous fraction). Similarly, the range of stem was 1.50% (n-hexane) to 82.50% (chloroform).

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table 1. Total phenolic and lavonoid contents of Achillea wilhelmsii

Sample name Methanol 80% Methanol n-Hexane Chloroform Ethyl acetate n-Butanol Aqueous fraction

root 108.09±0.40 74.41±0.33 0.98±0.74 92.68±0.37 132.55±0.44 106.89±0.40 51.06±0.28

TPC (mg/100 g) stem 109.30±0.40 97.56±0.38 0.75±0.50 89.37±0.36 86.48±0.36 160.96±0.49 95.04±0.57

leaves 365.38±0.74 80.85±0.35 0.89±0.69 82.94±0.35 288.71±1.00 22.82±0.18 246.15±0.60

root 710.7±1.01 134.42±0.44 0.74±0.93 270.28±0.95 562.21±0.90 222.08±0.56 31.95±0.21

TFC (mg/100 g) stem 526.1±0.87 779.85±1.06 1.21±0.26 270.07±0.62 66.14±0.31 582.11±0.91 184.39±0.51

leaves 106.03±0.39 97.97±0.57 1.08±1.06 332.34±0.58 626.71±0.95 220.96±0.56 727.97±1.02

root 42.50±2.16 21.25±1.25 0.75±2.5 67.50±2.16 29.16±0.72 48.75±1.25 75.3±2.16 88.87±0.02

% Inhibition stem 45.01±2.16 27.5±2.16 1.50±2.5 82.50±2.16 18.75±2.5 13.75±1.25 65.02±2.16 89.87±0.02

leaves 25.0±2.5 80.10±2.16 0.87±1.25 82.5±1.25 6.25±2.16 27.5±2.16 75.33±2.16 89.6±0.02

Values are expressed as mean of triplicate determination ±SD. table 2. Free radical scavenging activity and % inhibition in linoleic acid system

Sample Methanol 80% Methanol n-Hexane Chloroform Ethyl acetate n-Butanol Aqueous fraction BHT

root 88.14±0.02 106.06±0.01 812.43±0.03 570.8±0.015 421.97±0.01 389.11±0.03 228.81±0.02 8.91±0.01

iC50 stem 102.59±0.03 119.45±0.01 1004.2±0.02 313.5±0.015 653.17±0.01 811.05±0.02 621.1±0.03 8.78±0.02

Values are expressed as mean of triplicate determination ±SD.

leaves 96.51±0.02 114.23±0.01 987.07.10±0.01 384.67±0.01 679.29±0.01 213.7±0.01 160.2±0.01 8.87±0.01

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The higher values of percentage inhibition were determined in 80% methanol extract of leaves (80.25%) and in aqueous fraction of leaves (75.33%) and these values were found very close to the standard BHT (83.33%). Least values of percentage inhibition were found in n-hexane fraction of roots (0.75%). Aqueous fraction also showed good values of percentage inhibition for stem (65.02%), root (75.30%) and leaves (75.33%). All other fractions of A. wilhelmsii showed average percentage inhibition24. Haemolytic activity. The cytotoxic effects of various parts of A. wilhelmsii were observed on human blood. The maximum haemolytic activity was found in the methanol extract of leaves (9.49), stem (8.50) and roots (7.02). It was also observed that the chloroform fraction of stem (7.62), root (6.15) and leaves (5.18) showed good haemolytic activity. Least hemolytic activity was observed in leaves (1.62), root (2.0) of aqueous fraction and in stem (2.66) and leaves (2.79) of ethyl acetate fractions, while a substantial haemolytic activity was observed in all other fractions (Table 3). table 3. Hemolytic activity of A. wilhelmsii

Sample Methanol 80% Methanol n-Hexane Chloroform Ethyl acetate n-Butanol Aqueous fraction

root 7.02±0.038 4.10±0.026 3.38±0.030 6.15±0.047 3.82±0.020 4.69±0.028 3.82±0.025

Stem 8.53±0.025 7.16±0.033 3.04±0.055 7.62±0.042 2.66±0.031 2.37±0.025 2.01±0.025

Leaves 9.44±0.020 4.88±0.039 5.23±0.029 5.18±0.041 2.79±0.053 4.86±0.036 1.62±0.028

Values are expressed as mean of triplicate determination ±SD.

Evaluation of antimicrobial activity – Antifungal activity of A. wilhelmsii. The extracts and fractions of various parts of A. wilhelmsii were investigated against 4 fungal strains. It was observed that only methanol extract showed antifungal activity in all parts of A. wilhelmsii against all fungal strains. 80% methanol extract showed results against Aspergillus niger (9.2) and against Candila albicans (9.6) in root, and in stem 80% methanol extract was effective against A. niger (11.7), A. lavus (9.2), F. oxysporum (9.83) and C. albicans (9.13) while 80% extract of leaves were effective against all four fungus ranging from 9.13 in A. lavus to 13.63 in A. niger. Similarly, n-hexane fraction of root and stem were only effective against A. niger and A. lavus, but no result was found against F. oxysprium and C. albicans. n-Hexane fractions of leaves were effective against all observed fungus ranging from 9.33 (C. albicans) to 15.80 (A. lavus). Antifungal activity was not observed in chloroform fraction (root) and the chloroform fraction of stem was effective against only C. albicans (9.4) but chloroform fraction of leaves was effective against every fungus ranging from 9.6 (A. niger) to 11.66 (C. albicans). 548

Antifungal activities were not detected in root and stem of ethyl acetate fraction. The ethyl acetate fractions of leaves were not effective against F. oxysporum, but showed little activity against other fungal strain. No antifungal activity was observed in root and stem of n-butanol fraction, but n-butanol fraction of leaves was effective against all fungal strains ranging from 9.10 (A. niger) to10.31 (C. albicans). Aqueous fraction of root was effective against F. oxysporum (10.40) and C. albicans (9.9) in roots and aqueous fraction of stem showed antifungal activity against all fungal strain ranging from A. lavus (9.5) to C. albicans (12.4) and extracts of leaves were effective against three fungus as showed in Table 4. It was concluded that leaves of A. wilhelmsii were more effective against fungus as compared to stem and root. table 4. Antifungal activity of A. wilhelmsii

Sample Methanol (root) Methanol (stem) Methanol (leaf) 80% Methanol (root) 80% Methanol (stem) 80% Methanol (leaf) n-Hexane (root) n-Hexane (stem) n-Hexane (leaf) Chloroform (root) Chloroform (stem) Chloroform (leaf) Ethyl acetate (root) Ethyl acetate (stem) Ethyl acetate (leaf) n-Butanol (root) n-Butanol (stem) n-Butanol (leaf) Aqueous fraction (root) Aqueous fraction (stem) Aqueous fraction (leaf) Standard (fungone)

A. niger 11.30±0.10 13.33±0.15 15.03±0.15 10.60±0.15 11.70±0.20 13.13±0.15 9.43±0.20 9.90±0.10 9.93±0.20 – – 9.60±0.15 – – 9.73±0.15 – – 9.70±0.10 – 10.36±0.15 9.80±0.10 20.11

Strains A. lavus F. oxysporum 10.60±0.10 11.33±0.15 12.33±0.15 14.10±0.10 14.43±0.25 17.26±0.15 – – 9.20±0.10 9.83±0.15 9.93±0.15 12.36±0.20 – 10.60±0.10 10.33±0.15 9.90±0.30 15.80±0.10 13.50±0.10 – – – – 9.70±0.20 11.33±0.15 – – – – 9.23±0.15 – – – – – 9.40±0.20 9.53±0.15 – 10.40±0.10 9.50±0.10 9.70±0.15 – 12.53±0.15 20.87 21.03

C. albicans 11.20±0.10 14.63±0.20 17.06±0.15 9.60±0.10 9.13±0.15 11.66±0.20 – – 9.63±0.15 – 9.40±0.10 11.66±0.15 – – 10.70±0.15 – – 10.30±0.10 9.90±0.20 12.40±0.10 15.16±0.15 19.91

– Antibacterial activity of A. wilhelmsii. Effects of extracts and fractions of root, stem and leaves were also studied against 8 bacterial strains (Table 5). Methanol extract of root was effective against all observed bacterial strains ranging from M. luteus (10.23) to B. subtilus (16.26), but no activity was found in methanol extract of stem against S. typhi and Staphylococcus aureus, while it was effective against all other bacterial strains ranging from R. pneumonea (10.2) to E. coli (15.7) and methanol 549

extract of leaves showed antibacterial activity against all bacterial strains ranging from B. cereus (7.5) to M. luteus (17.6). In case of 80% methanol extract (root), only one result was found against S. aureus (11.3), but extracts and fractions of stem and leaves showed antibacterial activity against all bacterial strains ranging from Enterobacter aerogens (9.33) to M. luteus (17.13) for stem and from M. luteus (6.7) to S. aureus (15.33) for leaves. Antibacterial activity in all extracts and fractions was observed in n-hexane instead of against E. aerogens in stem and it was observed for S. aureus (10.23) to E. coli (19.25) in root and from B. subtilus (10.13) to B. cereus (17.96) in stem and from S. aureus (10.33) to B. subtilus (19.26). The range of chloroform fraction against bacteria was observed from 10.23 (leaves) against M. luteus to 19.20 (root) against B. subtilus. The range of ethyl acetate fraction was observed from E. coli (10.30) in stem to R. pneumonea (17.33) in leaves. In n-butanol fraction, the range was observed from B. cereus (6.1) in stem to S. aureus (19.16) in leaves. Aqueous fraction showed least effect as compared to all. Three fungul strains (A. niger, A. lavus, C. albicans) and three bacterial strains (E. coli, S. aureus, B. cereus) were used for this activity and results were compared with fungone (for fungus) and ciproloxacin (for bacteria). Minimum inhibitory concentration (MIC, mg/ml) against fungal strains by extract and fractions. In methanol extract the maximum antifungal activity was observed as ~ 17.26 mg/ml for leaves in C. albicans and its minimum inhibitory concentration was observed as 1.7 mg/ml that suggest that the methanol extract of leaves can inhibit the growth of fungus with this concentration. Similarly 80% methanol extract of leaves showed maximum antifungal activity, i.e. 13.13 against A. niger with MIC value 3.37. In the case of n-hexane fraction, the maximum antifungal activity was observed in leaves (15.8) against A. lavus with the MIC value ~ 4.34, which showed minimum inhibition. It was observed that for chloroform fraction the maximum antifungal activity was observed in leaves (11.66) with MIC value ~ 2.43 mg/ml. In the case of ethyl acetate, maximum antifungal activity was observed in leaves (9.23) against A. lavus and its MIC value was 2.73, which showed its minimum inhibition value (Table 6).

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table 5. Antibacterial activity in zone of inhibition (mm) by A. wilhelmsii

Sample

Methanol (root) Methanol (stem) Methanol (leaves) 80%methanol (root) 80%methanol (stem) 80%methanol (leaves) n-Hexane (root) n-Hexane (stem) n-Hexane (leaves) Chloroform (root) Chloroform (stem) Chloroform (leaves) Ethyl acetate (root) Ethyl acetate (stem) Ethyl acetate (leaves) n-Butanol (root) n-Butanol (stem) n-Butanol (leaves) Aqueous fraction (root) Aqueous fraction (stem) Aqueous raction (leaves)

E. coli

B. cereus

11.00±0.50 15.70±0.20 14.70±0.20 – 11.70±0.20 11.00±0.10 19.25±0.25 11.26±0.20 15.23±0.20 11.00±0.14 11.25±0.15 10.90±0.20 11.00±0.17 10.30±0.20 – 15.20±0.20 17.30±0.15 17.63±0.15 15.50±0.45 – –

12.00±0.15 14.00±0.20 7.50±0.15 – 14.33±0.15 14.40±0.10 18.36±0.15 17.96±0.25 16.63±0.26 14.43±0.10 12.33±0.15 16.26±0.26 – – 16.33±0.35 15.33±0.15 16.10±0.10 – – – –

Strains standard (Ciproloxacin) =19.90 M. luteus E. aerogens S. aureus S. typhi R. pneumo- B. subtilus nea 11.36±0.15 11.33±0.15 10.40±0.10 16.26±0.20 10.23±0.25 10.30±0.20 – – 10.20±0.20 12.40±0.10 13.10±0.10 11.16±0.15 15.33±0.45 15.60±0.10 15.00±0.26 12.23±0.25 17.60±0.10 12.60±0.10 11.30±0.30 – – – – – 17.13±0.32 11.33±0.35 11.66±0.15 13.23±0.25 15.16±0.15 9.33±0.15 15.13±0.15 13.13±0.15 11.53±0.15 15.23±0.20 10.20±0.26 11.16±0.15 10.26±0.25 17.16±0.15 17.25±0.25 13.23±0.25 15.16±.0.15 10.40±0.10 17.46±0.15 13.16±0.15 18.23±0.20 10.13±0.32 11.13±0.32 – 10.33±0.15 10.33±0.20 15.16±0.15 19.26±0.25 19.23±0.20 11.30±0.26 15.16±0.15 13.20±0.26 19.23±0.20 21.13±0.15 17.10±0.10 – – 13.13±0.27 15.23±0.20 19.20±0.20 10.25±0.25 – 11.25±0.25 13.26±0.25 11.16±0.10 23.10±0.36 10.23±0.25 – – 10.36±0.15 11.25±0.20 11.33±0.15 10.40±0.10 – – 11.25±0.20 15.36±0.40 10.60±0.10 – 13.20±0.26 17.40±0.10 13.13±0.20 17.33±0.35 19.23±0.20 13.23±0.20 13.23±0.25 19.10±0.26 11.86±0.98 15.36±0.40 18.33±0.20 18.23±0.25 11.16±0.15 19.16±0.13 11.16±0.15 17.00±0.30 12.13±0.15 11.26±0.25 – 17.06±0.20 19.23±0.20 – 12.46±0.20 15.13±0.15 – 13.36±0.15 – – – 15.40±0.40 – – – – – – – – – – – – –

551

552

table 6. Antimicrobial activity by minimum inhibitory concentration (MIC) of A. wilhelmsii against fungals and bacterial strains

Sample Methanol

Plant part

root stem leaves 80% Methanol root stem leaves root n-Hexane stem leaves Chloroform root stem leaves Ethyl acetate root stem leaves root n-Butanol stem leaves Aqueous fraction root stem leaves Standard fungone

A. niger 1.1±0.1 1.46±0.01 1.45±0.01 1.8±0.1 1.6±0.02 3.37±0.45 2.16±0.11 3.5±0.1 4.34±0.01 – – 1.34±0.04 – – 3.41±0.16 – – 3.66±0.15 – 2.66±0.1 – 0.93±0.01

Values are expressed as mean of triplicate ±SD.

Fungal strains A. lavus F. oxysporum 1.0±0.1 3.3±0.2 2.33±0.15 1.6±0.1 1.4±0.25 1.7±0.15 – – 1.34±0.01 1.36±0.05 2.54±0.01 1.34±0.3 – 2.3±0.3 4.23±0.01 3.53±0.3 4.4±0.1 2.5±0.1 – – – – 3.6±0.25 1.46±0.2 – – – – 2.73±0.02 – – – – – 1.6±0.1 2.4±0.1 3.34±0.2 3.3±0.2 – 2.76±0.15 – 7.7±0.2 0.89±0.01 0.85±0.01

Bacterial strains C. albicans E. coli B. cereus S. aureus 1.5±0.2 3.16±0.076 5.0±0.1 2.33±0.15 1.3±0.1 1.6±1.5 5.01±0.1 2.93±0.25 2.50±0.35 1.6±1.52 4.7±0.1 1.63±0.05 2.23±0.15 – – 1.3±0.1 1.43±0.15 4.6±1.52 3.33±0.15 – 3.8±0.1 5.23±0.05 2.7±0.1 1.33±0.15 – 1.4±0.1 2.63±0.15 3.14±0.01 – 3.3±0.2 1.43±0.15 4.13±0.01 2.43±0.23 4.50±0.2 2.23±0.15 2.2±0.01 – 1.53±0.2 2.9±0.07 2.63±0.02 3.5±0.2 5.43±0.15 2.33±0.15 – 2.46±0.2 1.33±0.15 6.33±0.01 3.20±0.2 – 0.046±0.001 – – – – – – – 2.43±0.01 7.05±0.01 7.26±0.01 2.23±0.01 – 2.66±0.2 3.52±0.01 4.46±0.2 – 2.63±0.15 2.2±0.01 4.46±0.2 1.33±0.15 – 2.50±0.2 2.56±0.15 2.73±0.15 – – 2.45±0.15 – – – – – – – 0.80±0.01 0.90±0.01 0.70±0.01 0.82±0.01

S. typhi 5.02±0.01 4.41±0.01 2.4±0.02 – 1.33±0.03 6.33±0.01 2.34±0.01 2.13±0.01 2.5±0.1 6.18±0.02 1.2±0.1 2.59±0.1 2.34±0.01 2.33±0.01 1.15±0.1 5.8±0.02 1.47±0.02 1.94±0.01 – – – 0.87±0.01

Minimum inhibitory concentration (MIC, mg/ml) against bacterial strains. Minimum inhibitory concentration was also determined against bacterial strains. MIC analysis was carried out against 4 bacterial strains (E. coli, B. cereus, S. aureus and S. typhi). In methanol extract, maximum antibacterial activity was observed in stem (15.7) and its MIC value was 1.63. In 80% methanol extract, maximum antibacterial was observed in stem (17.13) against S. aureus with MIC value 1.33, which suggest that the extract with this concentration can possibly inhibit the growth of S. aureus. in n-hexane fraction, maximum antibacterial activity was observed in root (19.25) against E. coli with the MIC value ~ 1.4. This amount (concentration) was suficient to inhibit the bacterial growth. Moreover, in chloroform fraction, maximum antibacterial activity was observed in leaves (16.58) against B. cereus and its MIC value was ~ 2.33, which showed that the bacterial growth was inhibited at this concentration. Similar trends were also seen on analysing the ethyl acetate, n-butanol and aqueous fractions (Table 6). ConCLUSionS Methanol extract and different fractions of various parts of Achillea wilhelmsii were found to contain appreciable levels of total lavonoids and total phenolics. The results from DPPH radical scavenging activity and inhibition in linoleic acid peroxidation suggest that the plant can potentially be used as an antioxidant. The plant extract and fractions were assessed against human blood erythrocytes for haemolytic activity. These cytotoxic studies suggest that the plant contain minor cytotoxicity. ACKNOWLEDGEMENTS The data present here is part of M. Ph. thesis of Muhammad Asghar. The authors gratefully acknowledge to the Deanship of Scientiic Research at King Saud University for funding through the Research Group Project No RGP-VPP-345. reFerenCeS 1. N. ALI, S. SHAH, I. SHAH, G. AHMED, M. GHIAS, I. KHAN: Cytotoxic and Anthelmintic Potential of Crude Saponins Isolated from Achillea wilhelmsii, C. koch and Teucrium stocksianum Boiss. BMC Compl Altern Med, 11, 106 (2011). 2. E. KUPELI, I. ORHAN, S. KUSMENOGLU, E. YESILADA: Evaluation of Anti-inlammatory and Antinociptive Activity of Five Anatolian Achillea species. Turk J Pharm Sci, 4, 89 (2007). 3. A. J. FALK, S. J. SMOLENSKI, L. BAUER, C. L. BELL: Isolation and Identiication of Three New Flavones from Achillea millefolium L. J Pharm Sci, 64, 1838 (1975). 4. C. BANH-NHU, E. GACS-BAITZ, L. RADICS, J. TAMAS, K. UJSZASZY, G. VERZAR-PETRI: Achillicin, the irst Proazulene from Achillea millefolium. Phytochem, 18, 331 (1979). 5. A. ZARGARI. Medicinal Plants. Tehran University Publications, Iran, 1996. 6. C. DODSON, W. W. DUNMIRE. Mountain Wildlowers of the Southern Rockies: Revealing Their Natural History. UNM Press, University of New Mexico, Japan, 2007.

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Oxidation Communications 37, No 2, 555–562 (2014) Antioxidants in biological systems

ProteCtive effeCt of alPha-liPoiC aCid againSt iSChemia-rePerfuSion injury in retina Z. YILDIRIMa*, H. EMMEZb, a. KaLeb a

Etimesgut Public Health Laboratory, 06 770 Ankara, Turkey E-mail: [email protected] b Department of Neurosurgery, Faculty of Medicine, Gazi University, 06 500 Ankara, Turkey

aBStraCt Radical oxygen species produced after injury counteracts antioxidant activity and frequently causes severe oxidative stress for the tissues. Alpha-lipoic acid (LA) is a powerful antioxidant. The aim of the study was to investigate the effects of LA supplementation on the levels of tumor necrosis factor-α (TNF-α), malondialdehyde (MDA), and total antioxidant status (TAS) in eye after spinal cord trauma. Twenty-four adults, male, New Zealand rabbits were divided into ischemia (n=8), control (n=8), and treatment groups (n=8). The abdominal aorta was clamped for 30 min by an aneurysm clip, approximately 1 cm below the renal artery and 1 cm above the iliac bifurcation in ischemia and treatment groups. In the control group, only laparotomy was performed. 25 cm3 of saline in control group and 100 mg/kg LA were administered intraperitoneally in the treatment group after closure of the incision. The animals were killed 48 h after the operation. Vitreous samples were collected for analysis. Vitreous levels of TNF-α, MDA, and TAS were analysed according to markers of oxidative stress and inlammation. The vitreous MDA and TNF-α levels were signiicantly higher in the ischemia group when compared to the control group (p