rutin complex

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disodium hydrogen phosphate (Na2HPO4), ammo- nium molybdate (NH4)2MoO4, and sodium phos- phate (Na3PO4) were purchased from Merck (Ger- many).
Chemical Papers 68 (5) 614–623 (2014) DOI: 10.2478/s11696-013-0494-6

ORIGINAL PAPER

Synthesis, characterisation, and antioxidant study of Cr(III)–rutin complex a,b

a Dr.

Qadeer K. Panhwar, b Shahabuddin Memon

M. A. Kazi Institute of Chemistry, b National Centre of Excellence in Analytical Chemistry, University of Sindh, Jamshoro 76080, Pakistan Received 9 April 2013; Revised 7 August 2013; Accepted 7 August 2013

The article describes the synthesis and characterisation of the Cr(III)–rutin complex along with an estimate of its antioxidant activity. The complex was characterised using elemental analysis, UV-VIS, IR, conductance data, thermal, and gravimetric analyses. In the UV-VIS study, a bathochromic shift of approximately 98 nm indicates the formation of a rutin complex by more than one chelating site. The FT-IR spectra clearly show the formation of the Cr—O bond between rutin and Cr(III) at 494 cm−1 , while the thermal study shows the presence of eight coordinated water molecules in the complex. The gravimetric analysis quantitatively proves the presence of four chloride ions. From these data, the formula of the Cr(III)–rutin complex was deduced as [Cr2 (C27 H28 O16 )(H2 O)8 ]Cl4 . Moreover, the antioxidant study of the complex was evaluated by using 2,2 –diphenyl–1–picrylhydrazyl (DPPH) free-radical, ferric-reducing, and phosphomolybdenum assays, which show that the complex has a higher antioxidant activity than rutin. c 2013 Institute of Chemistry, Slovak Academy of Sciences  Keywords: chromium(III), rutin, reducing power, chelation, antioxidant

Introduction Flavonoids are known to be the largest group of polyphenolic compounds in the plant kingdom, with 9000 known structural variants. The common structure of flavonoids is based on a phenylbenzopyrone arrangement of C6-C3-C6. They contain A and C phenyl rings bonded by three carbon atoms that make a closed structure of pyran ring B. On the basis of their saturation level and central pyran ring opening, flavonoids are categorised into six groups (Balasuriya & Rupasinghe, 2011; Buer et al., 2010), i.e. flavones, isoflavones, flavanols, and flavanones. They occur as glycosides, aglycones, and methylated derivatives. This highly diverse nature of flavonoid structures is useful in playing several functions in many biological systems. All dietary plants (Ren et al., 2003; Ray et al., 2001) such as vegetables, fruits, herbs, seeds, nuts, spices, flower stems, red wine, tea, apples, as well as onions, are rich in flavonoids. They are

also the main components of citrus fruits e.g. grapes, oranges, and other food sources. Because of their dietary importance, they are regularly consumed in considerable amounts in the human diet (Middleton et al., 2000). Flavonoids perform multiple functions in plants such as producing beautiful colours in flowers, protecting them from UV light, defence, and inhibiting auxin transport. In addition, they protect plants from fungal, bacterial, and viral infections. They also attract or repel insects through the colour of the flowers, leaves, and fruits, etc. (Balasuriya & Rupasinghe, 2011; Buer et al., 2010; Ren et al., 2003). Above all, flavonoids are known as the pigments giving rise to the autumnal hues. Being good chromophores, they impart many shades such as orange, red, and yellow to food and flowers. They exhibit numerous biological activities such as antioxidant, anti-inflammatory, antimutagenic, anti-HIV, anti-allergic, and anti-platelet activities. These properties have stimulated considerable interest in flavonoids. Their strong free radical-

*Corresponding author, e-mail: [email protected]

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scavenging and antioxidant properties (Balasuriya & Rupasinghe, 2011; Buer et al., 2010; Middleton et al., 2000; Es-Safi et al., 2007) are useful for their pharmacological activities including anti-cancer and antiageing. They have also gained recognition as potential risk-reducing elements for cardiovascular as well as neurodegenerative diseases. (Balasuriya & Rupasinghe, 2011; Sharma, 2006). Rutin is known as a prominent flavonoid and also as a model glycoside, consisting of a saccharide unit and an aglycone part (Špačková & Pazourek, 2013). There are many sources of rutin such as Ruta graveolens and Sophora japonica etc. It is a widely recognised antioxidant. Antioxidant activity is mainly related to two groups of compounds, i.e. flavonols and phenolic compounds (Filipiak-Szok et al., 2012); rutin is a natural compound belonging to the flavonol group and shows significant antioxidant property. It also has medicinal importance. It reducess capillary fragility, bruising, and swelling. It can be used for curing venous insufficiency (varicose veins, diabetic retinopathy, diabetic vascular disease, and haemorrhoids) and improving micro-vascular blood flow (pain, night cramps, tired, and restless legs). It is used as a food additive and flavouring agent for different drinks and food preparations. It is also used in cosmetics as well as for colouring purpose (Fathiazad et al., 2006). Chromium is a unique toxic element present in two stable oxidation states, i.e. Cr(III) and Cr(VI), the latter being more toxic. Cr(III) is the most stable under acidic conditions but it is readily oxidised to Cr(VI) in alkaline solution. Cr(III) usually forms a very stable type of octahedral complexes with six coordination numbers. This stability stems from its d 3 electronic configuration, which affords a high crystal field stabilisation energy (CFSE) value. Cr(VI) compounds demonstrate carcinogenicity and corrosion to tissues as well as toxicity to plants, animals, and bacteria. In humans, they affect the liver, kidneys, and cause gastric damage and lung cancer. Chromium is used extensively in alloying and metal plating to improve corrosion resistance. It is used as component for inorganic pigments, nuclear and high-temperature research, and chromium-based stainless steel protective coating for automotive and equipment accessories (Lee, 1996; Aroua et al., 2007). In industry, it acts as a corrosion inhibitor for water pipes used in drinking water and acts as the major source of Cr(III) and Cr(VI) in the drinking water distribution system (Tang et al., 2004). Humans are directly exposed to these sources, with the resultant health problems. Chromium supplementation is highly useful for weight gain in protein energy malnutrition (PEM) states (Khade et al., 2011). Trivalent chromium is considered as essential trace element. Its higher concentrations in body may be regarded as toxic. Cr(III) has an important role in maintaining the normal glucose tolerance by regulating the

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action of insulin (Krejpcio, 2001). Due to its specific transport mechanism, limited amounts may penetrate the cells. But higher concentrations in the cells may lead to DNA damage. Acute toxicity may appear in the range of 1.5–3.3 mg kg−1 (Eastmond et al., 2008; Katz & Salem, 1993). In such a case, it becomes necessary to reduce the metal content in the body using strong complexing agents (Tian et al., 2006) such as rutin in the transport process of chromium. Thus, the interaction of rutin and Cr(III) may prove to be of biochemical and biological importance. In order to reduce the metal-induced toxicity, chelation therapy is the ideal method, where chelating agents (like rutin) may bind the toxic or excess metal ions strongly by forming complex structures that may be easily excreted from the body (Dehghan & Khoshkam, 2012). Similarly, Cr may be excreted from the body mainly in urine, as well as in sweat, hair, and bile in minute amounts. But, after absorption, faecal excretion is considered as the main route. Hence, urinary excretion acts as the main elimination route (Krejpcio, 2001). Thus, rutin (as a metal chelating agent) can play a major role in the bioavailability of essential metals as well as in metal detoxification of toxic metal ions (Dehghan & Khoshkam, 2012). In the event of chromium deficiency, chromium picolinate may be used as a nutritional supplement present in various forms, e.g. chewing gum, pills, nutrition bars, and drinks (Dubey et al., 2008). It may activate the insulin receptor’s kinase activity in the presence of insulin. However, chromium picolinate has been proved to be toxic, hence flavonoid chromium complexes may be alternatives and can act as safer supplements for human consumption in future. They can act as poor DNA cleaving agents (Yazdanbakhsh et al., 2009) with milder toxic effects that need to be explored in subsequent studies. This is because insufficient dietary chromium has become an extensive health problem that may cause glucose intolerance as a source of diabetes, while chromium supplements may improve the sugar metabolism in hyperglycemic, hypoglycemic, and diabetic patients (Krejpcio, 2001). The complexing reactions can also be used in the determination of either rutin or chromium from a variety of samples (Kunti´c et al., 2000). Hence, formation of the Cr–rutin complex may be useful in the colorimetric determination of metal ions, e.g. Cr(III) from foods and water. Besides, it may be useful in the identification, determination, and quantification of rutin from the nectar of honey, waste tobacco leaves (Fathiazad et al., 2006), rutinion forte tablets, beverages, food, drugs, etc. (Kunti´c et al., 1998). The Cr(III)–rutin complex can have many biological as well as industrial applications. It acts as highly antioxidant compound relative to rutin. It produces a deeply coloured complex, which can be useful for the textile and dye industries. Such compounds can also be used for cosmetics purposes, due to their having no

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adverse effects. Hence, the present study investigates the interaction between Cr(III) and rutin, due to its having the possible applications discussed above.

Experimental General All the reagents and solvents were of analytical grade or chemically pure. Rutin trihydrate and 2,2 – diphenyl–1–picrylhydrazyl radical (DPPH) were purchased from Sigma–Aldrich (USA). Chromium(III) chloride, sodium dihydrogen phosphate (NaH2 PO4 ), disodium hydrogen phosphate (Na2 HPO4 ), ammonium molybdate (NH4 )2 MoO4 , and sodium phosphate (Na3 PO4 ) were purchased from Merck (Germany). HPLC-grade quality methanol was obtained from Fisher Scientific (UK), KBr from Aldrich Chemical (Germany), potassium ferricyanide (K3 [Fe(CN)6 ) and trichloroacetic acid were purchased from Fluka (Switzerland), whereas iron(III) chloride was obtained from Acros Organics (Belgium). All the reagents were weighed within an accuracy of ±0.1 mg. UV-VIS spectra were obtained using a Perkin– Elmer (USA) Lambda 35 UV-VIS double beam spectrophotometer using standard 1.00 cm quartz cells. FT-IR spectra were recorded in the spectral range of 4000–400 cm−1 on a Thermo Scientific (USA) Nicolet iS10 FT-IR instrument using KBr pellets. 1 H NMR spectra were recorded on a Bruker (Germany) 500 MHz spectrometer in DMSO using TMS as internal standard. Chemical shifts are given in δ relative to TMS. Thermo-gravimetric differential thermal analysis (TG-DTA) curves were obtained on a PyrisTM Diamond TG-DTA (Perkin–Elmer) under a nitrogen atmosphere at a heating rate of 10 ◦C min−1 from ambient to 600 ◦C. Synthesis of the complex 0.332 g of rutin was dissolved in methanol, yielding a clear lemon-yellow solution after 5 min of stirring; subsequently approximately 0.266 g of CrCl3 · 6H2 O was added. The experimental assembly consisted of a 50 mL round bottom flask, an electromagnetic stirrer, a small magnetic bar, and a thermometer. The content was heated at 85 ◦C and refluxed for at least 6 h; the colour of the solution changed to dark magenta. The solution was transferred to a Petri dish and the solvent was removed by slow evaporation at ambient temperature. Finally, the dark magenta-coloured product was collected and washed with tert-butanol followed by acetone. The product was dried completely in a vacuum evaporator and used for further analyses as needed. The yield was calculated as 67 %. Elemental analysis found C: 31.95 %; H: 4.22 %. Anal. calc. for (Cr2 (C27 H28 O16 )(H2 O)8 )Cl4 : C: 32.48 %; H: 4.44 %, respectively.

DPPH free-radical scavenging assay The antioxidant activity of rutin and the Cr(III)complex was determined by the DPPH free-radical scavenging method. DPPH has a violet colour in methanolic solution and exhibits maximum absorbance at 515 nm (Limaye et al., 2010). The addition of a sample solution to the free-radical solution may convert the DPPH into a non-radical form, wherein it decolourises the DPPH and causes its absorbance to decrease. Hence, the antioxidant potential of a sample can be measured using the discolouration assay. In the experimental procedure, solutions of rutin and Cr(III)–rutin complex were prepared by dissolving 2 mg of each sample in 10 mL of methanol (Shyam et al., 2012). Almost 50 L of each sample was added to 2 mL of the DPPH solution (0.1 mM in methanol) and the solutions were properly mixed by shaking the reaction mixture. The solutions were analysed by using the spectrophotometer. The decrease in DPPH absorbance was noted and followed up to 40 min (Dehghan & Khoshkam, 2012) with the difference noted at 5 min intervals, i.e. 0 min, 5 min, 10 min, 15 min, 20 min, 25 min, 30 min, 35 min, and 40 min (Tan et al., 2011). Finally, the % scavenging activities for of rutin and Cr(III)-complex were calculated by applying the formula: scavenging activity = (Ac − At )/Ac × 100 %

(1)

where Ac and At represent the absorbance of the control and test samples, respectively (Choudhary et al., 2011). Ferric-reducing power The ferric-reducing power assay for rutin and the Cr(III)–rutin complex was carried out using the Yen and Chen method. Approximately a 0.5 mL aliquot of various concentrations, i.e. 0.5 mg mL−1 , 1.0 mg mL−1 , 1.5 mg mL−1 , and 2.0 mg mL−1 of rutin and complex was each mixed with 2.5 mL of the phosphate buffer (0.2 M, pH 6.6) and potassium ferricyanide (1 %), respectively. The solutions were incubated at 50 ◦C for 20 min (Ebrahimzadeh et al., 2010a); subsequently a portion of trichloroacetic acid (2.5 mL, 10 %) was added to each solution to quench the reaction. Then, the resultant solutions were centrifuged at 3000 min−1 for approximately 10 min (Ebrahimzadeh et al., 2010b). Finally, 2.5 mL from the upper layer of the solutions was mixed with 2.5 mL of distilled water and a freshly prepared solution of FeCl3 (0.5 mL, 0.1 %), respectively. The solutions were analysed by using the spectrophotometer and the absorbance measured at 700 nm. A higher absorbance corresponds to the higher reducing power (Lugasi et al., 2003).

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Fig. 1. UV-VIS spectra of rutin (a) and its Cr(III)-complex (b). The complex spectrum shows bathochromic shift in bands (I–IV) of rutin after coordination with Cr(III).

Determination of total antioxidant capacity The total antioxidant capacity of rutin and its corresponding Cr(III) complex was assessed using the method devised by Prieto (Zheng et al., 2012). Both the samples were prepared in concentrations of 0.5 mg mL−1 . They were mixed with 0.5 mL of reagent solution (0.6 M H2 SO4 , 28 mM sodium phosphate, and 4 mM ammonium molybdate), then the samples were incubated at 95 ◦C for 150 min. The mixture solutions were cooled to ambient temperature and their absorbance was measured at 595 nm. The blank solution consisted of all the other reagents except for the sample. The reading was recorded at 30 min intervals to investigate the interaction of the samples with the reagent solution at 20 min, 40 min, 60 min, 80 min, 100 min, and 120 min (Tan et al., 2011; Prieto et al., 1999).

Results and discussion UV-VIS spectroscopy Flavonoids exhibit two characteristic absorption peaks at approximately 200–400 nm for the cinnamoyl and benzoyl parts. The rutin flavonoid similarly exhibits two absorption peaks at 354 nm and 257 nm for band I and band II, respectively (Fig. 1). The addition of Cr(III) to the rutin solution produces the bathochromic shift in both bands (i.e. I & II, Fig. 1) of rutin due to the formation of new chelate rings with increased conjugation (Malesev & Kunti´c, 2007). In effect, rutin possesses two potential chelating sites, i.e. 4-C—O/5-OH and catechol. Hence, some clues as to the character of the chelate, can be deduced from the degree of bathochromic shift. A very small shift corresponds to complexation at a single site, while a very large shift corresponds to complexation at more than one site, forming multiple rings. Thus, Cr(III) forms a 5-member ring in 4-C—O/5-OH position in rutin. In addition, the possibility exists that 3 ,4 -dihydroxy groups may also bind a second metal ion (de Souza et al., 2003). In the resultant complex formation, the

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peaks appeared at 271 nm and 452 nm and were denoted as bands IV and III, respectively, where the shift is observed in both parts. The red shift of approximately 98 nm suggests the simultaneous chelate ring formation at both sites, i.e. 4-C—O/5-OH and catechol moiety. In the earlier reports, it was observed that the chelation of metal ions taking place at only the catechol moiety caused the bathochromic shift of 30 nm and 52 nm for Al(III) and Zn(II), respectively (de Souza & de Giovani, 2005), while Ti(C2 O4 )2− 2 and VO2+ may form complexes at only the 4-C—O/5-OH position and exhibit red shifts of 72 nm and 32 nm, respectively (Uivarosi et al., 2010; Kunti´c et al., 2000). If these individual values are combined, they will then be equal to the red shift caused by Cr(III). Thus, the value of 98 nm is considerably higher than that obtained with those metal ions involved in chelation at individual sites. Hence, these observations strongly suggest that Cr(III) is simultaneously chelated by both the 5-OH/4-C—O and o-dihydroxyl groupings (Sekhon et al., 1983). Consequently, the rutin shows 2 : 1 stoichiometric composition in the Cr(III) complex. In effect, CrCl3 on dissolution may form a highly stable complex with the solvent molecules, acting as an inert complex. As a result, it is difficult for the Cr(III)–rutin complex to form at ambient temperature. Thus, unlike other flavonoid metal complexes formed at ambient temperature (de Souza et al., 2003), this complex is formed at 85 ◦C, albeit the interaction was very slow and completed within 6 h to 7 h under constant and vigorous stirring. In the case of the rutin ligand, the peaks arise from intra-ligand transitions of π–π* type, whereas in the case of the complex, the newly formed peaks may arise either from d–d transition within the metal orbitals (Medvidovi´c-Kosanovi´c et al., 2011) or due to the ligand-to-metal charge transfer (LMCT) (Uivarosi et al., 2010). Physical properties of the complex The Cr(III)–rutin complex was soluble in water, methanol, ethanol, dimethylsulphoxide, and N,Ndimethylformamide, and insoluble in hexane, acetone, tert-butanol, tetrachloromethane, and diethyl ether. The conductance study also described the electrolytic nature of the complex. IR spectroscopy IR spectroscopy is a useful analytical tool for characterising various functional groups as well as the changes in them after chemical reactions. It is also useful in the characterisation of metal complexes/chelates. A comparison of the IR spectra of rutin and its Cr(III)-complex afforded evidence of coordination between Cr(III) and rutin (Fig. 2) (Medvi-

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it clearly indicates the coordination of rutin with the Cr(III) metal ion. This band does not appear in the spectrum of rutin. The additional bands in the spectrum of rutin appear at 1600 cm−1 , 1504 cm−1 for C—C, 1362 cm−1 , 1294 cm−1 for C—O, and 1204 cm−1 , 1064 cm−1 , 1010 cm−1 for C—O—C, respectively. However, in the complex C—C (benzene ring skeleton, stretching vibration) bands appear at 1593 cm−1 , 1518 cm−1 / 1486 cm−1 , C—O stretching, and OH in plane deformation coupling at 1347 cm−1 and 1272 cm−1 , while vinyl ether C—O—C exhibits stretching vibrations at 1206 cm−1 and 1095 cm−1 (de Mello et al., 2004; Yang et al., 2005; Niu et al., 2008). Thermal analysis

Fig. 2. IR spectra of rutin (a) and Cr(III)–rutin complex (b). The complex spectrum illustrates spectral changes upon coordination of rutin to Cr(III).

dovi´c-Kosanovi´c et al., 2011). The IR spectrum of rutin shows a band at 1654 cm−1 for C—O, which shifted to 1627 cm−1 upon coordination to Cr(III). This is regarded as a significant spectral shift, which shows the involvement of C—O in chelation with metal ion. Along with C—O, 5-OH is also involved in chelation because the 3-O site is blocked by sugar moiety (rutinose), hence it does not take part in the complexation. In addition, the complex spectrum exhibits an important band at 3330 cm−1 , similar to rutin which exhibits the same band at 3420 cm−1 ; these bands correspond to the stretching of OH/H2 O groups. Another useful band in the complex spectrum is observed at 494 cm−1 ; this is assigned to the Cr—O stretching vibration. Thus,

The thermal study was considered as worth performing to determine and verify the nature and number of water molecules and stoichiometry, thereby determining the complex composition. In the case of a complex formed from the result of the reaction between rutin and Cr(III) (Fig. 3), thermal patterns show both the dehydration and decomposition process. The complex decomposes in two mass-loss stages for coordinated water and rutin molecules, respectively, where the former shows endothermic and the latter shows exothermic mass loss. In the first step, elimination of water molecules takes place by the release of eight molecules in the temperature range of 145–250 ◦C. This higher value indicates their coordinated nature, as shown by both the TG and DTA curves. This endothermic step is followed by the exothermic degradation of rutin at 330 ◦C and above. This decomposition occurs as the last step with the formation of Cr metal as the final residue because formation of the metal oxide is not possible under the nitrogen atmosphere. The mass loss for coordinated water molecules is 14.7 %, while for rutin it is 58.2 %. On the basis of the above data, a complex structure which contains only coordinated water molecules was proposed (Uivarosi et al.,

Fig. 3. Chemical reaction between rutin and CrCl3 · 6H2 O metal salt.

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Fig. 5. Structure of rutin showing important features for defining its classical antioxidant property based on catechol moiety/B-ring (shaded in maroon). The catechol group and other functions such as 4-oxo may represent its ability to chelate metal ions such as chromium (shaded yellow/light blue). Fig. 4. Provisional structure proposed for [Cr2 (C27 H28 O16 ) (H2 O)8 ]Cl4 complex.

2010). Gravimetric analysis of chloride An appropriate quantity of [Cr2 (C27 H28 O16 ) (H2 O)8 ]Cl4 complex was dissolved in water and approximately 0.5 mL of HNO3 (68 %) was added. The solution was heated to approximately 70 ◦C then 5 mL of AgNO3 was added under constant stirring. The precipitates of AgCl appeared. The solution was heated further and subsequently cooled in the dark. Complete precipitation of the chloride ions was checked repeatedly by adding AgNO3 to the supernatant liquid from the sides of the beaker. The process continued until no further precipitates appeared. The solution containing precipitates was held overnight in the dark. Next, the precipitates were filtered and washed with diluted HNO3 (0.01 N) and water then dried at above 100 ◦C. They were cooled in a desiccator and then combusted in ashless filter paper. Finally, the precipitates were weighed (Panhwar et al., 2010). The percentage of chloride ions was found to be 14.6 %, which is in good agreement with the theoretically calculated value of 14.2 % from the formula proposed for the complex, i.e. [Cr2 (C27 H28 O16 )(H2 O)8 ]Cl4 , as shown in Fig. 4. Antioxidant activity by DPPH The evaluation of antioxidant activity by the DPPH free-radical method is based on the hydrogen atom transfer (HAT) reaction. Rutin possesses a catechol (o-dihydroxyl groups) moeity on the aromatic ring, an important structural element that forms intra-molecular hydrogen bonds with free radicals and contributes to the high antioxidant activity (Fig. 5).

It is presumed that DPPH may be scavenged by an antioxidant compound through donation of the hydrogen atom (H) to form a stable DPPH-H molecule, which does not absorb at 517 nm. In the reaction of rutin and the complex with DPPH, they donate H via homolysis of one of the -OH bonds in hydroxyl groups to produce another radical in which a conjugated system may lead to its stability. In effect, there is an intramolecular hydrogen bond between the o-dihydroxyl groups in rutin which undoubtedly hinders the abstraction of H by DPPH, hence the homolysis of -OH becomes more difficult (Fig. 6). Thus, rutin exhibited a lower antioxidant ability (Li & Chen, 2012) than its corresponding complex because this intra-molecular hydrogen bond is not present in the complex, hence the proton on 4 position may be readily abstracted by DPPH; thus this proton acts as the most labile proton and increases the potential of rutin in the Cr(III) complex as shown in Fig. 7. Ferric-reducing power of Rut and Cr(III)– rutin complex DPPH and ferric reducing antioxidant power (FRAP) are two well-known methods, to some degree complementary, which indicate the host’s total capacity to withstand free-radical stress. Both are seen to be very easy, reproducible, speedy, and inexpensive methods for measuring the antioxidant potential of compounds. The reducing power of a bioactive compound reflects its electron-donating capacity, thereby serving as a significant indicator of its potential antioxidant activity (Li & Chen, 2012). In this assay, the test solution changes from yellow to green or blue depending on the reducing power of the antioxidant samples (Koksal et al., 2011):

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Fig. 6. Proposed mechanism for rutin (a) and its Cr(III)–rutin complex (b) for scavenging DPPH free radical.

Fig. 7. Results of relative antioxidant activity of rutin ( ) and Cr(III)–rutin complex ( ) against DPPH. radical.

K3 [Fe(CN)6 ] + reductive antioxidant → → Fe(CN)4− 6 + FeCl3 → Fe4 [Fe(CN)6 ]3

(2)

It is measured by the direct reduction of Fe[(CN)6 ]3− to Fe[(CN)6 ]2− (Eq. (2)). The addition of free Fe3+ to the reduced product leads to the formation of an intense Perls’ Prussian blue Fe4 [Fe(CN)6 ]3 complex that exhibits strong absorbance at 700 nm. An increase in the absorbance of the reaction mixture would indicate an increase in the reducing capacity due to increased formation of the complex (G¨ ul¸cin et al., 2011). Thus, the ferric-reducing power of rutin

Fig. 8. Results for relative ferric-reducing power of rutin () and its corresponding Cr(III)-complex ().

and the Cr(III)-complex was investigated here, with the finding that rutin exhibits a slightly higher reducing power than the complex at a concentration of 1.5 mg mL−1 (Fig. 8). On the other hand, it is almost the same for the remaining three concentrations, i.e. 0.5 mg mL−1 , 1.0 mg mL−1 , 2.0 mg mL−1 . Total antioxidant capacity The total antioxidant capacity of the rutin and Cr(III)–rutin complex was assessed using the phosphomolybdenum method. The assay is based on the

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Fig. 9. Comparative total antioxidant capacity of rutin () and its corresponding Cr(III) complex ( ).

electron transfer (ET) reaction occurring at a particular redox potential value, which depends on the chemical structure of antioxidants. The assay is based on the principle where the sample analyte causes the reduction of Mo(VI) to Mo(V) (Eq. (3)) caused by the sample analyte (reductant/antioxidant) with the subsequent formation of a green phosphate/Mo(V) complex at acidic pH value. Essentially, the molybdenum is presumed to be more readily reduced in the complex (Huang et al., 2005; Marwah et al., 2007) so it is a useful method for examining the total antioxidant capacity of the range of compounds. It was measured spectrophotometrically, where the complex exhibited a maximum absorption at 695 nm (Abbasi et al., 2010): Mo(VI) + e− → Mo(V)

(3)

The total antioxidant activities of rutin and the Cr(III)–rutin complex were compared. Both the compounds exhibited high total antioxidant capacity (Raghu et al., 2011). They possess an almost equal capacity, but the total antioxidant capacity of the Cr(III) complex was observed to be a little higher than its corresponding rutin ligand, as shown in Fig. 9.

Conclusions The study established that a complex was formed between Cr(III) and rutin. The reaction was found to be relatively slow at ambient temperature because Cr(III) initially formed a highly inert complex with the solvent molecules. Hence, the reaction was performed at elevated temperature. The Cr(III) formed a stable binuclear complex with rutin; this was fully characterised by various techniques. The formula of the complex was provisionally determined as [Cr2 (C27 H28 O16 )(H2 O)8 ]Cl4 . The antioxidant potential of the rutin and Cr(III) complex was also explored by the DPPH free-radical, ferric-reducing power, and total antioxidant capacity methods. The Cr(III)–rutin complex possessed relatively more antioxidant power than rutin, which resulted from the combined hydro-

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gen atom as well as the electron transfer mechanism. This study presents many applications, such as the concentrations of toxic metal ions could be decreased when rutin binds to different metal ions in the human body, thereby inhibiting various diseases and ultimately prolonging life. In addition, the study shows an outstanding impact on designing anti-cancer drugs, combined with their cytotoxic potential and antioxidant activities. Thus, the anti-cancer drugs may selectively target cancer cells and increase the therapeutic index and afford additional advantages over other anti-cancer drugs. Acknowledgements. The authors wish to express their gratitude and appreciation to the National Centre of Excellence in Analytical Chemistry and the Institute of Advanced Studies and Research, University of Sindh, Jamshoro, Pakistan, for providing the necessary facilities and space to make this work possible.

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