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Oct 20, 2011 - Composition of essential oil and biological activity of extracts of Viola odorata L. from central Iran. Maryam Akhbari a. , Hossein Batooli b.
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Composition of essential oil and biological activity of extracts of Viola odorata L. from central Iran a

b

Maryam Akhbari , Hossein Batooli & Fereshteh Jookar Kashi

a

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The Essential Oil Research Center, University of Kashan , Kashan , I.R. Iran b

Isfahan Research Center for Natural Resources and Agriculture , Kashan Station , I.R. Iran Published online: 20 Oct 2011.

To cite this article: Maryam Akhbari , Hossein Batooli & Fereshteh Jookar Kashi (2012) Composition of essential oil and biological activity of extracts of Viola odorata L. from central Iran, Natural Product Research: Formerly Natural Product Letters, 26:9, 802-809, DOI: 10.1080/14786419.2011.558013 To link to this article: http://dx.doi.org/10.1080/14786419.2011.558013

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Natural Product Research Vol. 26, No. 9, May 2012, 802–809

Composition of essential oil and biological activity of extracts of Viola odorata L. from central Iran Maryam Akhbaria*, Hossein Batoolib and Fereshteh Jookar Kashia a

The Essential Oil Research Center, University of Kashan, Kashan, I.R. Iran; Isfahan Research Center for Natural Resources and Agriculture, Kashan Station, I.R. Iran

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(Received 28 June 2010; final version received 24 January 2011) Essential oil composition of the leaves of Viola odorata L. growing wild in Kashan, central Iran, was extracted by hydro distillation–solvent extraction method and analysed using GC–MS technique. The analysis revealed the presence of 25 identified compounds, representing 92.77% of the oil with butyl-2-ethylhexylphthalate (30.10%) and 5,6,7,7a-tetrahydro-4,4,7a-trimethyl-2(4H)-benzofuranone (12.03%) being the two main components. Several components were identified for the first time in this chemotype of V. odorata. Antioxidant and antibacterial activities of the oil, methanol and chloroform extracts were also evaluated for the first time in this research work. Keywords: essential oil; Viola odorata; antibacterial; antioxidant

1. Introduction The genus Viola belongs to the family Violaceae and contains about 500 species. There are 19 endemic species of this plant in Iran; however, about 30 known species grow wild in northern and northwestern regions. The most important medicinal plants of this genus are Viola tricolor, Viola arvensis and Viola odorata (Karimi, 2002; Mozafarian, 1996; Tutin, et al., 1968). Viola odorata L. is also known as sweet violet and is a herb with stout rootstocks. Stems are short with slender stolons. Leaves are crowded at the end of the stems, orbicular to subreniform, 5–8 cm long, heart-shaped base, round tipped, with toothed margins. Flowers (1.5–1.8 cm) are fragrant. Sepals are green, about 1 cm long; petals are violet with the throat marked with white spots or lines (Mozafarian, 1996). It is reported to be used more for therapeutical purpose and perfumery. The leaves and flowers of V. odorata L. are employed in high grade in ‘French-type’ perfumery. Solvent extraction is the best way for essential oil isolation. The leaf oil is dark-green in colour with earthy and leafy scent; flower oil is yellow in colour with rich, sweet and floral scent. Its perfume aroma is middle to top note. Essential oils of V. odorata (sweet violet) which contain citronella, geraniol, salicylaldehyde and linalool are known as anti-inflammatory, healing and soothing agents (Drozdova & Bubenchikov, 2004). In traditional

*Corresponding author. Email: [email protected]

ISSN 1478–6419 print/ISSN 1478–6427 online ß 2012 Taylor & Francis http://dx.doi.org/10.1080/14786419.2011.558013 http://www.tandfonline.com

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medicine, the aerial parts are used for their anti-inflammatory, expectorant and diuretic properties and to treat various skin conditions, bronchitis, cystitis and rheumatism (Anca, Philippe, Ilioara, & Mirceal, 2009). Drozdova and Bubenchikov (2004) published a report about the determination of the antioxidant activity of ethanol extract from V. odorata from Russia by checking for spots on thin-layer chromatograms, which were capable of inhibiting peroxide formation. To the best of our knowledge, there is no similar report in the literature. There are some information about the antimicrobial activity of V. odorata in the literature. This plant has been introduced as the most effective antibacterial plant in a research article between 10 Indian medicinal plants (Arora & Kaur, 2007). Recently, cyclotide cycloviolacin O2 from V. odorata has been reported, which has potent bactericidal activity against gram-negative bacteria (Pra¨nting, Lo¨o¨v, Burman, Go¨ransson, & Andersson, 2010). There is limited information on the essential oil composition of Viola species in the literature and only one is related to V. odorata L. in which 23 volatile components of V. odorata leaves are reported, mostly containing aliphatics (1dodecanol, pentadeca-5,10-dien-1-ol and 1-octadecene) or shikimic acid derivatives (2-pentyl-furan and ionone) (Cu, Perineau, & Gaset, 1992). In spite of the common application of this plant in local traditional medicine for treating headache, fever and chills, inflammatory and throat sore, etc., there is no information about volatile chemistry or biological activity of the Iranian V. odorata L. The purpose of this study is to analyse the chemical composition of the essential oils and measuring the biological activity of Iranian V. odorata L.

2. Results and discussion There is a limited information about volatile chemistry of this genus because of the low extraction yield of essential oils from Viola leaves. Extraction of essential oils using new SDE technique enabled us to obtain a high amount of essential oil (1.2% yield). GC–MS analysis revealed 25 identified components constituting 92.77% of the total oil. Some compounds are found in significant quantity in the oil of the Iranian V. odorata L. which were not generally reported in the literature for other Viola species (Table 1). Considering Table 1, it is found, at a glance, that compounds with high molecular weight exist more than the other volatile compounds such as monoterpenes with small and simple molecular structure in the essential oil of this chemotype of V. odorata. Some compounds such as 3-hexenol, -pinene, -pinene, 2-hexenal, salicylaldehyde, linalool, citronellal, methyl salisylate, undecanal, dodecanol, spathulenol, geraniol, tridecane, hexadecane, heneicosane and 1,8-ocimene were reported in different species of Viola genus (Anca, et al., 2009; Cu, et al., 1992; Flamini, Cioni, & Morelli, 2002). 1-Hexadecene, 1-octadecene, 1-ecosene and hexadecanoic acid seem to be specific to V. odorata (Cu et al., 1992). 3-hexenol and 2-hexenal are important aroma components for V. odorata green note. Six components were identified in this chemotype of V. odorata for the first time, of which butyl-2-ethylhexylphtalate (30.10%) and 5,6,7,7 a-tetrahydro-4,4,7 a-trimethyl-2(4 H)-benzofuranone (12.03%) are the major components.

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Table 1. Chemical composition of essential oil of V. odorata L. leaves from Iran. RIa

Comp.b

No.

cis-3-Hexenol -Pinene -Pinene Salicylaldehyde cis-2-Hexenal 1,8-Cineole Linalool

871 939 984 1031 1091 1041 1105

1.42 1.31 0.62 0.46 1.12 1.92 3.06

14 15 16 17 18 19 20

Camphor Citronellal Methyl salicylate 9-19-Cycloanost6-ene-3,7-diol Geraniol Tridecane

1146 1160 1195 1215

0.92 2.96 2.32 2.88

21 22 23 24

1259 1300

3.21 2.23

No. Compound name

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1 2 3 4 6 5 7 8 9 10 11 12 13

RIa

Compound name Undecanal 1-Dodecanol Spathulenol 1-Hexadecene Hexadecane Veridiflorol 5,6,7,7a-Tetrahydro-4, 4,7 a-trimethyl-2 (4 H)-Benzofuranone 1-Octadecene 1-Eicosene Heneicosane Hexadecanoic acid

Comp.b

1309 1.11 1475 1. 29 1581 2.54 1588 4.53 1592 4.25 1606 6.31 1616 12.03 1777 1994 2088 2131

0.92 1.81 1.41 1.01

25 Butyl-2-ethylhexylphthalate 2299 30.10 Total 92.77%

Notes: aRelative retention indices to C8–C24 n-alkanes on HP-5MS column. Composition of compounds (%).

b

Table 2. Antioxidant activity of V. odorata L. leaves from Iran. Sample Methanol extract Chloroform extract Essential oil BHT

DPPH assay (IC50, mg mL1)

-Carotene assay (I%)

31.53  0.43 54.72  1.11 NTa 20.24  0.25

68.35  0.94 35.87  0.96 29.86  1.12 85.12  0.49

Note: aNT: Not tested.

Results from the evaluation of antioxidant activity confirmed that although the antioxidant activity of the chloroform extract was weaker than that of the methanol extract, both have high ability as a natural antioxidant in the inhibition of -carotene/linoleic acid and 2,2-diphenyl-1-pycril hydrazyl (DPPH) bleaching assays (Table 2) compared with buthylated hydroxytoluene (BHT) standard antioxidant. However, the essential oil did not show antioxidant activity as we expected according to its inactive components; due to lack of flavonoids, phenolic and other antioxidative active components, DPPH assay was deemed to be unnecessary. Antimicrobial activities of methanol and chloroform extracts of V. odorata were evaluated against a set of 11 microorganisms and their potencies were assessed qualitatively and quantitatively by the presence or absence of inhibition zones, zone diameters and MIC values. The results are given in Table 3 and indicate that, at tested concentrations, methanol extract of the plant has considerable antimicrobial activity against some of the gram-positive and gram-negative microorganisms.

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Table 3. Antimicrobial activity of V. odorata L. leaves from Iran. Extracts

Antibiotics

Samples

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Tested microorganisms P. aeruginosa B. subtilis E. coli S. aureus K. pneumoniae S. epidermidis S. dysenteriae P. vulgaris S. paratyphi-A serotype C. albicans A. niger

CH3OH

CHCl3

Essential oil Rifampin Gentamicin Nystatin

DDa MICb DD MIC DD 10 – 11 – – 19 – 12 – – –

500 – 500 – – 250 – 250 – – –

– – – – – – – – – – 16 250 – – 11 500 – – – – – –

– 8 – – 12 9 – – – – –

MIC DD MIC DD MIC DD MIC – 500 – – 125 500 – – – – –

– 13 11 10 7 40 8 10 – NA NA

– 23 15 21 500 20 250 21 250 22 250 35 250 18 125 23 – 21 NA NA NA NA

500 500 500 500 250 500 500 500 500 NA NA

NA NA NA NA NA NA NA NA NA 33 27

NA NA NA NA NA NA NA NA NA 125 31

Notes: A dash (–) indicates no antimicrobial activity. NA: Not applicable. a Inhibition zone in diameter (mm) around the impregnated discs. b Minimal inhibition concentrations (as mg mL1).

However, less polar chloroform extract also showed antibacterial activity against Staphylococcus epidermidis and Proteus vulgaris.

3. Experimental 3.1. Plant materials The plants were collected from Karkas Mountains of Kashan in central Iran, at an altitude of 1985 m in June 2009. The voucher specimen of the plant were confirmed in the Herbarium of Kashan Botanical Garden (HKBG 431).

3.2. Essential oil separation The aerial parts of the examined plants were dried in shadow at room temperature. Simultaneous water steam distillation–organic solvent extraction (SDE) method was carried out as suggested by Filek, Bergamini, and Lindner (1995), in which the EOs of the species were distilled from 100 g of powdered leaves by steam distillation for 4 h, while simultaneously, extraction was carried out by n-pentane. The procedure was repeated thrice to obtain enough EOs for the corresponding biological evaluating tests. The n-pentane solution was then dehydrated by anhydrous sodium sulphate, the solvent was evaporated at room temperature under vacuum, nitrogen was blown into the sample vessel and sealed right away, and the oil was maintained in 5 C.

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3.3. GC-MS analysis The isolated EOs were analysed using GC/MS on a Hewlett-Packard 6890 gas chromatograph coupled with a mass detector (Hewlett-Packard model 6973 HP). A fused silica HP-5 column, 60 m length, 250 mm I.D. and 0.25 mm film thickness was used as the oil analyser. The mass spectra were obtained by electron ionisation at 70 eV. The oven was operated isothermally with its temperature maintained first at 60 C for 30 min. It was then raised to 250 C at a rate of 5 C min1. The injection temperature was 250 C. The carrier gas (helium) flow rate was 1 mL min1. The sample (1 mL) was injected with a split ratio of 1/90. Retention indices were calculated for all components using a homologous series of n-alkanes injected in conditions equal to those of the samples. Identification of components of EO was based on retention indices (RI) relative to n-alkanes and computer matching with the Wiley275.L library. Also, comparisons of the fragmentation pattern of the mass spectra were made with data published in the literature (Adams, 2001; Baser, Ozek, Nuriddinov, & Demirci, 2002).

3.4. Preparation of extracts Thirty grams of powdered aerial parts of the plant were Soxhlet-extracted with 300 mL solvent. Solvent removal using rotary evaporation and drying the residue using a vacuum oven at 50 C yielded 7.68 g (25.57% w/w) of dried methanol extract and 5 g (16.65% w/w) chloroform extract. The extracts were kept in the dark at 4 C until tested.

3.5. Antioxidant activity For evaluating antioxidant activities of samples, DPPH bleaching and -carotene/ linoleic acid assays were used.

3.5.1. -Carotene/linoleic acid assay In this assay the antioxidant capacity was determined by measuring inhibition of -Carotene oxidation using the organic compounds of the extracts in the presence of linoleic acid hydro peroxide. A reagent solution were prepared by adding -carotene (0.7 mg) and linoleic acid (37.5 mL) and 300 mg Tween 40 (as co-solvent) to 150 mL of oxygen-saturated distilled water, then the resulting mixture was stirred well until a homogenous clear solution was obtained (solution R). Three types of samples were prepared in this investigation; the first (type a) contained 2.5 mL solution R to which 1 mL of the samples dissolved in ethanol with a concentration of 2 mg mL1 was added; the second sample (type b) was used as a blank and contained only 2.5 mL of solution R and the third sample (type c) used for positive control contained 1 mL of BHT solution in ethanol with the concentration of 2 mg mL1. All these samples (types a–c) were totalled in a 5 mL volumetric balloon using ethanol. They were then incubated in hot water (50 C) for 2 h except type b samples, for which the absorbance reading was taken immediately after it was totalled to 5 mL. The absorbance was measured at 470 nm for all the above samples.

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Antioxidant capacities (Inhibition percentage, I%) of the tested solutions were calculated using the following equation: I % ¼ ðAbsorbance after 2 h=initial absorbanceÞ  1000

ð1Þ

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where the numerator indicates the absorbance made from the types a or c samples and the denominator indicates the absorbance made from type b samples (t ¼ 0 min). Tests were carried out in triplicate for accuracy. Inhibition percentage of the type a samples was compared to that of the positive control from type c samples. It should also be mentioned that in the experiments with BHT, the yellow colour was maintained during the incubation period.

3.5.2. DPPH bleaching assay In this assay the antioxidant activity was determined by measuring DPPH free radical scavenging ability. Extract samples with various concentrations were prepared in methanol. 1 mL from each such sample solution was mixed with 1 mL of 85 mg mL1 DPPH solution and totalled in a 5 mL volumetric balloon by methanol. The resulting solution was kept in dark at room temperature for 35 min. Absorbance was measured at 517 nm for each solution sample, and inhibition of DPPH free radical in percent (I%) was calculated as follows: I % ¼ ðAblank  Asample =Ablank Þ  100

ðAÞ

where Ablank is the absorbance of the control reaction, containing all reagents except the test compound. IC50, concentration for 50% inhibition, was read from the graph plotting inhibition percentage against log of extract concentration. The number of samples for each extract was chosen so that a sigmoid curve was resulted. A similar procedure was used to calculate IC50 for standard antioxidant Buthylated Hydroxy Toluene (BHT), as a positive control. All tests were carried out in triplicate to improve the accuracy.

3.6. Antimicrobial activity 3.6.1. Microbial strains Methanol and chloroform extracts of V. odorata were tested against a set of 11 microorganisms. The following microbial strains were provided by Iranian Research Organization for Science and Technology (IROST) and used in this research: Pseudomonas aeruginosa (ATCC 27853), Escherichia coli (ATCC 10536), Bacillus subtilis (ATCC 6633), Staphylococcus aureus (ATCC 29737), Klebsiella pneumoniae (ATCC 10031), S. epidermidis (ATCC 12228), Shigella dysenteriae (PTCC 1188), P. vulgaris (PTCC 1182), Salmonella paratyphi-A serotype (ATCC 5702), Candida albicans (ATCC 10231) and Aspergillus niger (ATCC 16404). Bacterial strains were cultured overnight at 37 C in Nutrient Agar (NA) and yeast and mould were cultured overnight at 30 C in Sabouraud Dextrose Agar (SDA).

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3.6.2. Disk diffusion assay Determination of the antimicrobial activity of samples was accomplished by agar disk diffusion method (Gulluce et al., 2007; National Committee for Clinical Laboratory Standard, 1997). Extracts were dissolved in DMSO to a final concentration of 30 mg mL1 and filtered by 0.45 mm Millipore filter for sterilisation (Murray, Baron, Pfaller, Tenover, & Yolke, 1995). 100 mL of suspension containing 108 CFU mL1 of bacteria, 106 CFU mL1 of yeast and 104 spore mL1 of mould were spread on the Nutrient Agar (NA), Sabouraud Dextrose Agar (SDA) and Potato Dextrose Agar (PDA) media, respectively. The disks (6 mm in diameter) impregnated with 10 mL of the extract solution (300 mg per disk) and DMSO (as negative control) were placed on the inoculated agar. The inoculated plates were incubated for 24 h at 37 C for bacterial strains and 48 and 72 h at 30 C for yeast and mould isolates, respectively. Gentamicin (10 mg per disk) and rifampin (5 mg per disk) were used as positive controls for bacteria and nystatin (100 U per disk) for yeast and mould. The diameters of inhibition zones were used as a measure of antimicrobial activity and each assay was repeated twice. 3.6.3. Micro-well dilution assay Bacterial strains and yeast, which were sensitive to the extracts of the plant in disk diffusion assay, were studied for their minimal inhibition concentration (MIC) values using micro-well dilution assay method; mould was not sensitive, and therefore it was not studied for MIC. The inocula of the microbial strains were prepared from 12 h broth cultures, and suspensions were adjusted to 0.5 McFarland standard turbidity. Extracts of V. odorata were dissolved in 10% DMSO in highest concentration, 500 mg mL1, serial twofold dilutions were made in a concentration range from 7.8 to 500 mg mL1 in 10 mL sterile test tubes containing Brain Heart Infusion (BHI) for bacterial strains and Sabouraud Dextrose Broth (SDB) for yeast. The 96-well plates were prepared by dispensing 95 mL of the cultures media and 5 mL of the inoculum into each well. A 100 mL aliquot from the stock solution of the plant extract initially prepared at the concentration of 500 mg mL1 was added into the first well. Then, 100 mL from their serial dilutions was transferred into six consecutive wells. The last well containing 195 mL of the cultures media without the test materials and 5 mL of the inoculum on each strip was used as negative control. The final volume in each well was 200 mL. Gentamicin and rifampin for bacteria and nystatin for yeast were used as standard drugs for positive control in conditions identical to test materials. The plates were covered with sterile plate sealers. The contents of each well were mixed on a plate shaker at 300 rpm for 20 s and then incubated at appropriate temperatures for 24 h. Microbial growth was determined by the presence of a white pellet on the well bottom and confirmed by plating 5 mL samples from clear wells on NA medium. The MIC value was defined as the lowest concentration of the plant extracts required for inhibiting the growth of microorganisms. All tests were repeated twice.

References Adams, R.P. (2001). Identification of essential oil components by gas chromatography-mass spectroscopy. Carol Stream, IL: Allured Publishing Co.

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Anca, T., Philippe, V., Ilioara, O., & Mirceal, T. (2009). Composition of essential oils of Viola tricolor and Viola arvensis from Romania. Chemistry of Natural Compounds, 45, 91–92. Arora, D.S., & Kaur, G.J. (2007). Antibacterial activity of some Indian medicinal plants. Journal of Natural Medicines, 61, 313–317. Baser, KH.C., Ozek, T., Nuriddinov, H.R., & Demirci, A.B. (2002). Essential oils of two Hypericum species from Uzbekistan. Chemistry of Natural Compounds, 38, 54–572. Cu, J.Q., Perineau, F., & Gaset, A. (1992). Volatile components of Violet leaves. Phytochemistry, 31, 571–573. Drozdova, I., & Bubenchikov, R. (2004). Antioxidant activity of Viola odorata L. and Fragaria vesca L. polyphenolic complexes. Rastitel’nye Resursy, 40, 92–96. Filek, G., Bergamini, M., & Lindner, W. (1995). Steam distillation-solvent extraction, a selective sample enrichment technique for the gas chromatographic electron capture detection of organochlorine compounds in milk powder and other milk products. Journal of Chromatography, 712, 355–364. Flamini, G., Cioni, P.L., & Morelli, I. (2002). Analysis of the essential oil of the aerial parts of Viola etrusca from Monte Labbro (South Tuscany, Italy) and in vivo analysis of flower volatiles using SPME. Flavour and Fragrance Journal, 17, 147–149. Gulluce, M., Sahin, F., Sokmen, M., Ozer, H., Daferera, D., Sokmen, A., . . . , Ozkan, H. (2007). Antimicrobial and antioxidant properties of the essential oils and methanol extract from Mentha longifolia L. ssp. Longifolia. Food Chemistry, 103, 1449–1456. Karimi, H.A. (2002). Dictionary of Iran’s vegetation plants (p. 203). Tehran, Iran: Parcham. Mozafarian, V. (1996). A dictionary of Iranian plant names. (Farhange Moaser: Iran) (p. 112). Tehran, Iran: Tehran Nashr-e Markaz. Murray, P.R., Baron, E.J., Pfaller, M.A., Tenover, F.C., & Yolke, R.H. (1995). Manual of clinical microbiology (7th ed., p. 1773). Washington, DC: ASTM. National Committee for Clinical Laboratory Standard (1997). Performance standards for antimicrobial disk susceptibility test. Sixth approved standard. M2-A6, Wayne, PA. Pra¨nting, M., Lo¨o¨v, C., Burman, R., Go¨ransson, U., & Andersson, D.I. (2010). The cyclotide cycloviolacin O2 from Viola odorata has potent bactericidal activity against Gramnegative bacteria. Journal of Antimicrobial Chemotherapy, 65, 1964–1971, Jun 17. Tutin, T.G., Heywood, V.H., Burges, N.A., Moore, D.M., Valentine, D.H., Walters, S.M., & Webb, D.A. (1968). Flora Europaea (Vol. 2, pp. 270–282). Cambridge: University Press.