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Chemical Examination and Evaluation of Antioxidant and Antimicrobial Activities of Essential Oil from Gymnema sylvestre R. Br. Leaves

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The present study reports chemical examination and in vitro antioxidant and antimicrobial activities of essential oil isolated from leaves of a well known antidiabetic plant, Gymnema sylvestre R. Br. Essential oil was isolated from leaves by
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  12  /Journal of Essential Oil Research Vol. 23, May/June 2011 Chemical Examination and Evaluation of Antioxidant and Antimicrobial Activities of Essential Oil from Gymnema sylvestre R. Br. Leaves. Dattatraya G. Naik,* Chitra N. Dandge and Shobha V. Rupanar Chemistry Group, Agharkar Research Institute, Pune, India  Abstract The present study reports chemical examination and in vitro antioxidant and antimicrobial activities of essential oil isolated from leaves of a well known antidiabetic plant, Gymnema sylvestre R. Br. Essential oil was isolated from leaves by hydrodistillation using a Clevenger apparatus. The essential oil was found to contain 40 volatile compounds by GC/MS analysis. Hitherto unknown major components of essential oil identified by GC/MS analysis were found to be palmitic acid (17.8%), hydroquinone (13.9%), phytol (6.9%), pentadecanoic acid (6.8%), 4-vinyl guaiacol (4.5%), and eugenol (4.0%). Essential oil exhibited strong 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity (IC 50  28 µg/mL). Dose dependent antioxidant activity was shown by essential oil in b -carotene bleaching and 2,2-azinobis-(3-ethyl benzothiazoline-6-sulphonic acid) diammonium salt (ABTS) radical scavenging assays. The total phenolic content of essential oil was found to be 34 mg GAE/g fresh leaves. The essential oil inhibited the growth of Pseudomonas aeruginosa, Pseudomonas asplenii, Bacillus subtilis, Proteus mirabilis, Escherichia coli and   fungus  Candida albicans  with minimal inhibitory concentration (MIC) varying between 22 mg/mL and 28 mg/mL. Key Word Index Antimicrobial activity, antioxidant assays, essential oil composition, 4-ethyl guaiacol, eugenol, Gymnema sylvestre  R. Br., hydroquinone, palmitic acid, total phenolic content, 4-vinyl guaiacol. 1041-2905/11/0001-03$14.00/0 —© 2011 Allured Business Media *Address for correspondence: dgnpune@yahoo.co.in Introduction A woody climber,  Gymnema sylvestre  R. Br. (Asclepia-daceace), is located in central and western India, tropical Africa and Australia. The leaves have a unique property of inhibiting ability to taste sweet substances (1). Besides this, antidiabetic property of leaves is also reported (2). It is well known that the antidiabetic plants possess antioxidant activity. Antioxidant properties of leaf extract and root extract of G.  sylvestre  have been recently reported (3, 4). The ethanolic extract and saponin fractions of G. sylvestre leaves were found to possess antimicrobial activity (5-7). In vivo antiinflammatory activity of G. sylvestre leaf extract in rats has been recently demonstrated (8).Large number of reports on the pharmacological activi-ties of essential oil from plants have been recently published. The essential oil from endemic plant Zataria multiflora (9) and Thymus pectinatus  (10) indicated good antioxidant properties. The essential oil from Pistacia atlantica  showed antioxidant effects (11). The essential oil from the leaves of  Lippia sidoides  and one of its main constituents, thymol, exhibited antioxidant activity besides having a good potential as a gastroprotective and topical antiinflammatory agent (12). The essential oil from Croton sonderianus  (13),  Ammomum  subulatum (14),  Nigella sativa (15) and Croton cajucara  (16) exhibited protective activities on gastric mucosa. Anethole, a constituent present in many essential oils, showed gastro-protective activity on ethanol-induced gastric lesions (17). Essential oils showing antimicrobial activities are proposed as a natural preservative agent for cosmetic preparations (18). The essential oils containing terpenes are also reported to possess antimicrobial activity (19). Although the plants from the Asclepiadaceace family are known to possess medicinal properties, very few attempts have been made to study their essential oil and biological properties (20). This prompted the authors to investigate the chemical constituents of the es-sential oil of   G. sylvestre  and its antioxidant and antimicrobial activity against different microbial strains.Recent reviews describe the antimicrobial, antihypercho-lesterolemic, hepatoprotective and anti-inflammatory activities of leaves of G. sylvestre  plant and state that they are used for making antidiabetic formulations in folk, ayurvedic and homeopathic medicines (21, 22). Molecular fingerprinting Naik et al.   Vol. 23, May/June 2011 Journal of Essential Oil Research/  13 reveals that there is considerable variation in the Gymnema  germplasm collected from Kerala (23). Leaf extract of G. mon- tanum  has antiperoxidative and antioxidant effects on diabetic brain tissue in rats (24). Ethanol extract of   G. montanum is a potential   in vitro   antioxidant and has   protective effects against alloxan-induced oxidative stress in pancreatic b -cells (25). A literature search indicates that the isolation and evaluation of biological properties of the essential oil of G. sylvestre  is hitherto unknown. In the present study, antioxidant activity of essential oil from G. sylvestre  was determined by using DPPH, b -carotene bleaching   and ABTS radical scavenging assays. Antimicrobial activity of essential oil was evaluated against bacterial and fungal strains. To have an insight into the bioactive molecules, chemical examination of the essential oil was carried out by GC/MS analysis.   Experimental  Plant material:  Gymnema sylvestre  (200 g) was collected from ‘Hiranya Keshi,’ in the Amboli district of Maharashtra state in India in September 2009, August 2010 and September 2010. The samples were duly authenticated by the Botany Group of Agharkar Research Institute (ARI). The material has been deposited at AHMA Herbarium at ARI (Voucher No. 25516). Chemicals: Butylated hydroxy anisole (BHA), butylated hydroxy toluene (BHT) and tween-20 were purchased from Loba Chemicals, linoleic acid was purchased from SRL, b -carotene from HIMEDIA and Folin-Ciocalteu reagent  was purchased from Qualigens. These are Indian companies. 2,2-Diphenyl-1-picrylhydrazyl (DPPH), 2,2-Azinobis-(3-ethyl benzothiazoline-6-sulphonic acid) diammonium salt (ABTS), ammonium persulphate, eugenol, pentadecanol, limonene, heptadecane, hydroquinone, palmitic acid and undecanone  were purchased from Fluka, USA. Methyl palmitate was syn-thesized by esterification of palmitic acid with diazomethane using standard procedure. All the solvents used were of analytical grade. Cultures of bacteria and fungus :  Pure cultures of bac-teria  , Pseudomonas aeruginosa (Gram negative  , ARICHM-1),  Bacillus subtilis (Gram positive  ,  ARICHM-2)  , B. cereus  (Gram positive  ,  ARICHM-3),  Escherichia coli  (Gram nega-tive  ,  ARICHM-4), Staphylococcu s  aureus (Gram negative  ,  ARICHM-5)  , Proteus mirabilis (Gram negative  ,  ARICHM-6)  , Pseudomonas asplenii  (Gram   negative  ,  ARICHM-7) and   cul-ture of fungus  Candida albicans  (ARICHM-8) were obtained from the culture collection of ARI.  Isolation of essential oil:   The essential oil was obtained from the fresh leaves of G. sylvestre  (60 g) by hydrodistilla-tion for 3 h using a Clevenger apparatus. The isolation was carried out in three independent experiments and the average  yield was recorded. Gas chromatography/mass spectrometry analysis: GC/ MS analysis of the essential oil was carried out on a Shimadzu QP-5000 spectrometer equipped with a Supelcowax-10 col-umn, (30 m x 0.32 mm i.d., film thickness 0.25 µm composed of polyethylene glycol). Temperature program: 50 ° C (5 min), 50–200 ° C at 2ºC/min, 200 ° C (50 min). Injection temperature: 200ºC. Injection volume: 1.0 µl. Inlet pressure: 20 kPa. Carrier gas: He, linear velocity (  u ): 46.8 cm/s. Injection mode: split, ratio (1:29). MS interface temperature: 230 ° C; MS mode: EI; Figure 1. Gas chromatogram obtained in GC/MS analysis of the essential oil of G. sylvestre  leaf. G. sylvestre  14  /Journal of Essential Oil Research Vol. 23, May/June 2011 culation of their retention indices using  n -alkanes (C 10 -C 40 ) and comparison with the corresponding data published in literature (26-28). This was further confirmed by co-GC/MS analysis of the available authentic compounds. Quantitative analysis:  Calibration curve of each of the standard compounds, undecanone, pentadecanol, eugenol, Table I. Composition of G. sylvestre  leaf essential oil determined by GC/MS analysis.Sr. No. Compound RI a RI b  Concentration c, d  (%) Method of identification e 1 1,8-Cineole 1211 1214 t MS, RI, CO-I 2 Octanol 1551 1551 t MS, RI 3 b -Elemene 1568 1592 1.3 MS, RI 4 Acetophenone 1620 1623 t MS, RI, CO-I 5 Germacrene A 1723 1723 2.0 MS, RI, 6 p-Guaiacol 1752 1755 t MS, RI, 7 Dodecanol 1965 1964 1.4 MS, RI, 8 NI 1977 - 1.8 MS 9 Methyl eugenol 2001 2011 t MS, RI, CO-I 10 2-Pentadecanone 2008 2027 1.5 MS, RI, 11 4-Ethyl guaiacol 2010 2031 1.6 MS, RI 12 2-Dodecenol 2018 2040 1.6 MS, RI 13 NI 2021 - 1.7 MS 14 Hexahydrofarnesyl acetone 2121 2118 1.6 MS, RI, 15 Eugenol 2147 2141 4.0 MS, RI, CO-I 16 m-Ethyl phenol 2162 2181 t MS, RI 17 Tetradecanol 2172 2173 1.4 MS, RI, 18 4-Vinyl guaiacol 2176 2178 4.5 MS, RI, 19 Methyl palmitate 2209 2217 1.5 MS, RI, CO-I 20 Ethyl palmitate 2256 2250 2.8 MS, RI, CO-I 21 Pentadecanol 2278 2287 1.3 MS, RI, CO-I 22 NI 2287 - 2.4 MS 23 Tetradecadiene 2299 - 1.4 MS (t) 24 9,12,15-Octadecatrienal 2365 - 2.0 MS (t) 25 1-Hexadecanol 2379 2385 2.1 MS, RI, 26 NI 2398 - 1.5 MS 27 Indole 2403 2405 t MS, RI 28 Tetracosane 2412 2400 1.4 MS, RI, CO-I 29 Ethyl octadec-9-enoate 2471 2477 1.6 MS, RI 30 Methyl-11,14-eicosadienoate 2480 - 1.4 MS (t) 31 Pentacosane 2514 2500 1.5 MS, RI, CO-I 32 Methyl linolenate 2547 2583 1.7 MS, RI 33 8,11,14-Ecosatrienoic acid 2580 - 1.6 MS (t) 34 Octadecanol 2585 2586 1.4 MS, RI 35 Phytol 2618 2611 6.9 MS, RI 36 Myristic acid 2663 2663 2.0 MS, RI, CO-I 37 Heptacosane 2686 2700 1.6 MS, RI, CO-I 38 Pentadecanoic acid 2800 2794 6.8 MS, RI 39 Palmitic acid 2952 2946 17.8 MS, RI, CO-I 40 Hydroquinone 3615 - 13.9 MS, CO-I Fatty acids 28.2 Long chain alcohols 16.1 Hydroquinone 13.9 Oxygenated monoterpenes 10.1 Esters 9.0 Higher alkanes 4.5 Ketones 3.1 Aldehyde 2.0 Other identified Constituents 4.7 Total identified constituents 91.6 RI a : Retention indices relative to C 10  – C 40    n - alkanes on Supelcowax -10 Column, RI b : Retention indices reported in the literature (26-28), Concentration c : Quantification was done using the calibration curves generated from the analyses of following standards i) undecanone (Sr. No. 10, 14) , ii) Pentadecanol (Sr. No. 7, 12, 17, 21, 25, 34, 35) iii) Eugenol (Sr. No. 11, 15, 18) iv) Methyl palmitate (Sr. No. 19, 20, 29, 30, 32) v) Palmitic acid (Sr. No. 33, 36, 38, 39) vi) Heptadecane (Sr. No. 28, 31, 37) vii) Hydroquinone (Sr. No. 40) viii) Limonene (Sr. No. 3, 5, 23). Concentration  d -These results were validated using a standard mixture of available authentic compounds.Method of identification e : MS – mass spectrum of the respective compounds from the Library, RI –Reported retention indices, Co-I – Co-injection with the authentic sample, MS (t): Tentative identification detector voltage: 1.3 kV; mass range: 45-450 u with an ionizing  voltage of 70 eV; scan speed: 1000 u/s; interval 0.50 s. Component identification: The chemical constituents of the essential oil were identified by comparison of their mass spectra to those reported in NIST and Wiley GC/MS libraries. The identification was further supported by cal- Naik et al.   Vol. 23, May/June 2011 Journal of Essential Oil Research/  15  where, A A(105) is the absorbance of antioxidants at 105 min, A C(105)  is the absorbance of control at 105 min, A c(0)  is the absorbance of control at 0 min.    ABTS radical cation decolourisation assay:   ABTS radical scavenging assay (32) was used to evaluate the ability of essential oil to scavenge the ABTS . +  radical cations. ABTS . + radical cations were generated by reacting ABTS solution (7 mM, 3 mL) with ammonium persulfate (2.45 mM, 15 mL). The reaction mixture was allowed to stand at room temperature for 16 h before use. The test solutions of different concentra-tions (100 m g/mL, 250 m g/mL, 500 m g/mL, 750 m g/mL and 1000 m g/mL) and the standard solutions of BHA and BHT (100 m g/mL) in methanol were prepared. The ABTS solution (0.6 mL) was added to each test tube containing test solution and standards (1 mL each) and the final volume was made 2 mL with methanol. The control was prepared by adding methanol (1.4 mL) to ABTS solution (0.6 mL) while blank  was prepared in the identical manner as the test solution but  without ABTS solution. Absorbance was read at 745 nm. All the tests were performed in triplicate.  Determination of total phenolic content:   The total phenolic content was determined by the reported method using Folin-Ciocalteu reagent (33). A solution of the sample in methanol (100 µg/mL) was prepared. To this solution (1 mL), Folin-Ciocalteu reagent (1/10 dilution, 1 mL) was added. After 5 min, sodium carbonate (10 mL, 7 %), was added to the mixture. This solution was diluted to 25 mL using distilled water. The absorbance was measured at 750 nm after incubation for 90 min at room temperature. Total phenolic content of the sample is expressed as mg gallic acid equivalent (GAE)/g fresh leaves. The sample was analyzed in triplicate.  Determination of antimicrobial activity:   Antimicrobial activity was examined by well-diffusion method (31) using bacterial cultures, S. aureus, P. aeruginosa, P. asplenii, B.  subtilis, B. cereus, P. mirabilis, E. coli and   fungus  C. albicans.  The antimicrobial activity was studied using Mueller-Hinton medium with 2% agar for bacterial strains and Sabouraud-dextrose agar for fungal strain. Gentamycin was used as the positive control. The cultures were incubated at 37 ° C for 24 h at 100 rpm in respective medium to reach the stationary phase of growth before assays. A microbial concentration having 13 x 10 8 colony forming units per mL   (cfu/mL) was used for assays. Culture (100 m l) was sprayed on each separate sterile Petri plate with a spreader. The wells were prepared by sterile cork borer with 6 mm diameter. Each well was loaded with 40 m l sample (50 mg/mL) in methanol. The solvent blank was incorporated in each assay. All the plates were kept at 5 ° C for 30 min for diffusion. The plates were then incubated for 24 h at 37 ° C and the diameters of growth inhibition zones were measured. Each assay was performed in triplicate. The minimum inhibitory concentration (MIC) of essential oil indicating clear inhibition was determined (31). Statistical analysis : All the statistical analyses were performed using SPSS version 11.0. Values are presented as a means ± standard deviation. One way analysis of variance  was carried out and differences between variables were tested for significance by post hoc Tukey’s HSD multiple G. sylvestre palmitic acid, heptadecane, hydroquinone, limonene and methyl palmitate was generated using five concentration points. The response factor of the respective standard and the percentage of each chemical constituent in the essential oil  was determined by following the reported procedure (29). Determination of Antioxidant Activity  DPPH radical scavenging assay:  The standard protocol of DPPH assay (30) was followed with slight modifications. Solutions of different concentrations of essential oil and standard, BHT, (20, 40, 60 and 100 µg/mL) in methanol were prepared. To the test solution (1 mL), DPPH solution (0.1 mM, 1 mL) in methanol was added. The total volume was made 4 mL using methanol. After 30 min incubation in the dark, absorbance was recorded at 515 nm. The percentage inhibition activity was calculated by the following formula: where, A 0 is the absorbance of control, A t is the absor-bance of test solutions/standard, A b is the absorbance of blank solution. Antioxidant activity of the essential oil is expressed as IC 50  values. The IC 50  value is defined as that concentration of essential oil which inhibits 50% of DPPH radicals. All the experiments were performed in triplicate. b -Carotene bleaching assay : Antioxidant activity was measured by the standard b -carotene-linoleic acid assay (31)  with slight modifications. To the chloroform solution (1 mL), b -carotene (3.34 mg), linoleic acid (40 mg) and Tween-20 (400 mg) were added. Chloroform was then removed at 40°C under vacuum using a rotatory evaporator. The resulting mixture was diluted with distilled water (10 mL). The volume of resulting emulsion was further made up to 100 mL with hydrogen peroxide (0.01 M). The test solutions of increasing concentrations (1 mg/mL, 2.5 mg/mL, 5 mg/mL, 7.5 mg/mL and 10 mg/mL) of each sample and the standard solutions of BHA and BHT (1 mg/mL) in methanol were prepared. Aliquots (2 mL) of the b -carotene-linoleic acid emulsion was transferred into test tubes containing test samples (0.1 mL) and standards (0.1 mL) in methanol. A solution containing methanol (0.1 mL) and b -carotene-linoleic acid emulsion (2 mL) was used as a control. The blank was prepared in the same manner as that of test solution without b -carotene-linoleic acid emulsion. The test tubes were placed in a water bath maintained at 40°C. Absorbance of all the samples at 470 nm  was measured at zero time and after every 15 min until the color of b -carotene disappeared (105 min) in the control. All the samples were analyzed in triplicate. The % inhibition was determined by the following formula: %  16  /Journal of Essential Oil Research Vol. 23, May/June 2011 ters (9.0%), higher alkanes (4.5%) and ketones (3.1%). The major compounds were found to be palmitic acid (17.8%), hydroquinone (13.9%), phytol (6.9%), pentadecanoic acid (6.8%), 4-vinyl guaiacol (4.5%), and eugenol (4.0%). The assignments were further confirmed by co-GC/MS of some of the compounds.  Antioxidant activity: Reactive oxygen species (ROS) such as free radicals are responsible for oxidative damage known to cause various chronic diseases like atherosclerosis, Parkinson’s disease, Alzheimer’s disease, stroke, arthritis, chronic inflammatory diseases, cancer and other degenera-tive diseases (34). Such ROS are usually scavenged by the antioxidants including glutathione peroxidase, catalase and superoxide dismutase, which are naturally present in the body (35). Many times, endogenous antioxidant defenses are not totally efficient. Therefore, antioxidant supplementa-tion is required to diminish the oxidative damage. There are efficient synthetic antioxidants such as BHT and BHA, but these compounds are associated with side effects (36, 37). As free radicals and ROS are considered to be the important factors responsible for many diseases including diabetes (38), the search for antioxidants to scavenge free radicals and ROS Table II. Antimicrobial activity of essential oil of G. sylvestre .Sr. no. Microbial strains (Gram.) Essential oil Gentamycin MIC Zone of inhibition (mm) Zone of inhibition (mm) (mg/mL) 1 Pseudomonas aeruginosa (- ve) 15 18 282 Pseudomonas asplenii (- ve) 15 20 263 Escherichia coli (- ve) 16 20 254 Bacillus subtilis (+ ve) 16 25 225 Proteus mirabilis (- ve) 16 25 256 Candida albicans 20 11 257 Bacillus cereus (+ ve) - - -8 Staphylococcus aureus (- ve) - - - Figure 2. Determination of IC 50 value of G. sylvestre  leaf essential oil by DPPH radical scavenging assay.Figure 3. Variation of antioxidant activity of G. sylvestre  leaf essential oil with respect to time in b -carotene bleaching assay. comparison test. Differences were considered statistically significant at p < 0.05. Results and Discussion The hydrodistillation of G. sylvestre  leaves (60 g) yielded fragrant, light yellow colored oil (0.123 g, 0.2%), which was carefully isolated using a long tipped dropper. The isolated essential oil was directly used for chromatographic analyses. This avoided involvement of any solvent during isolation. GC/ MS analyses of all the batches of essential oil were identical. A representative chromatogram of the essential oil of G.  sylvestre leaves is shown in Figure 1  and indicated presence of 40 components ( Table I ). The compounds are listed in the order of their elution on the Supelcowax-10 column. The mass spectra were interpreted with the help of NIST and  Wiley libraries. Identification of 36 components representing 91.6 % of the total oil was made by the library search. These assignments were supported by comparison of their linear retention indices calculated using standard formula with those reported in the literature (26-28). Essential oil was found to contain long chain alcohols (16.1%), hydroquinone (13.9%), fatty acids (28.2%), oxygenated monoterpenes (10.1%), es- Naik et al.
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