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* For correspondence.

Oxidation Communications 36, No 4, 1067–1078 (2013)

Biological systems – antioxidants, inhibitors, antagonists

antioxidant, antimiCroBial studies and CharaCterisation of essential oil, fixed oil of Clematis graveolens By gC-ms

A. MUSHTAQa, N. RASOOLa*, M. RIAZa, R. B. TAREENb, M. ZUBAIRa, U. RASHIDc, M. AKMAL KHANd, Y. H. TAUFIQ-YAPe

aDepartment of Chemistry, Government College University, 38 000 Faisalabad, Pakistan E-mail: nasirhej@yahoo.co.uk bDepartment of Botany, University of Balochistan, Quetta, Pakistan cInstitute of Advanced Technology, University Putra Malaysia, 43 400 UPM Serdang, Selangor, Malaysia E-mail: umer.rashid@yahoo.com dDepartment of Chemistry, Government College University, 54 000 Lahore, Pakistan eCentre of Excellence for Catalysis Science and Technology Faculty of Science, University Putra Malaysia, 43 400 UPM Serdang, Selangor, Malaysia E-mail: taufiq@upm.edu.my

aBStRaCt

The GC-MS analysis of antimicrobial and antioxidant activity of Clematis graveolens was assessed to explore its medicinal importance. Medicinal importance of its genus plants encourages us to undertake the comprehensive investigation of the essential oil and fixed oil of the leaves and stem. GC-MS analysis of essential and fixed oils showed the presence of many compounds in the leaves and stem parts of the plant like 2,2 dimethoxy butane (15.16%), flouroethane (45.14%), undecane (5.16%), 1,2-benzenedicarboxylic acid (18.35%), 3,8,12-tri-O-acetoxy-7-desoxyingol-7-one (12.74%), propanoic acid, 2-(3-acetoxy-4,4,14-trimethylandrost-8-en-17-yl)- (9.14%) and vitamin E acetate (4.38%). The antimicrobial activity of the essential and fixed oil was resoluted by disc diffusion and minimum inhibitory concentration (MIC) assay and the plant showed potent activity. Furthermore the antioxidant potential of essential and fixed oil was assessed by the DPPH, reducing power and by percentage inhibition in linoleic acid system.

Keywords: Clematis graveolens, antioxidant, GC-MS, essential oil, fixed oil.

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AIMS AND BACKGROUND

The naturally occurrence and biological activity of essential oil and herbal extracts have paved sound path to the research1 as their functions are versatile and save2. the plant of under investigation, Clematis graveolens, belonged to Ranunculaceae family frequently known as toatal, is poisonous antiseptic which causes blistering or scorch-ing and loyal in ailment medication, used in neck and sore throat, hemorrhage and wounds healing3. Some authors have studied chemical and biological properties of various plants extracts in Pakistan4–7, but more research work is needed. Clematis is a Greek word meaning vine branch, a number of species are cultivated for attractive flowers in North America and 70 species are used in Chinese traditions8. Clematis genus is virtually ornamentals in folk therapeutic species9,10 mainly used to cure wound healing, headache, viral fever, dermatology, leprosy, cardiac disorders11, to lower down blood pressure12, for cartilage injuries13, promote lactation, stimulate menstrual discharge, persuade urination, human breast cancer8, HIV, influenza A, influenza B, tumor, for diuretic remedy14 . There is no report to the best of the knowledge on the GC-MS study, antioxidant and antimicrobial activities of the Clematis graveolens essential and fixed oil. So this research work was carried out in order to emphasise the significance of this plant in pharmaceutical field.

EXPERIMENTAL

General. The plant Clematis graveolens was selected because negligible work was done on this plant but intense ethno pharmacological information was available for its genus species and its family plants. Plant was collected in the Quetta and Ziarat valley and was further identified by Dr. Tareen from the Department of Botany, University of Balochistan Quetta, Pakistan. Plant was in the dried form and the whole plant collected was separated into different parts in stem and leaves. A voucher specimen (CG-RBT-07) was deposited in the herbarium/collection of the Department of Botany, University of Baluchistan Quetta, Pakistan. After collection the plant was screened into leaves and stem, dried in shade and grinded into powdered form.

Isolation of essential oil by hydro-distillation method. Isolation of essential oils was done on dried leaves and stem of the plant by using clevenger type apparatus according to the method given by Riaz et al.15 The extract of essential oil was collected, dried under Na2SO4 (anhydrous), filtered and kept at –4ºC till analysis.

Isolation of fixed oil with soxhlet. Fixed oil of the plant was prepared by using a soxhlet extractor following the method cided in Ref. 16 with some modifications. 20 g of shaded dried powder of leaves and stem of Clematis graveolens were extracted separately in 250 ml n-hexane solvent (99.9%) in a soxhlet extractor by hot extraction. The extracted oil was concentrated and stored in vial for further analysis at –4ºC.

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GC-MS analysis. Using a GCMS-QP2010 (Shimadzu, Japan) device the sample was subjected for GCMS investigation. With capillary column (DB-5) the gas chromato-graph was prepared, 30 m long, 0.25 μm film width and 0.25 μm inner diameter. In a 3-step gradient program oven temperature was set, initial temperature was set at 45ºC for 5 min, secondly slopped at 10ºC rise/min till 150ºC, till 280ºC followed by 5ºC/min increase. Flow rate for helium gas was 1 ml min. In scanning mode ions or fragments were monitored through 40 to 550 m/z. With the help of mass spectra the identification of components was done by using mass spectral library.

Evaluation of antioxidant activity of essential and fixed oil of Clematis graveolens. Antioxidant activity of essential and fixed oils of the plant was assessed by using subsequent parameters. The linoleic acid system was used to determine the antioxidant activity of essential oil and fixed oil of the plant in terms of percentage inhibition per oxidation in the linoleic acid system17. 1,1-diphenyl-2-picrylhydrazyl radical scaveng-ing activity of essential oil and that of fixed oil sample was checked by following the method given in Ref. 18. By using the method cited in Ref. 19 reducing power of the plant extracts was resoluted with little adjustments.

Antibacterial and antifungal activity. In order to analyse the antimicrobial activity of selected bacterial strains Escherichia coli, Staphylococcus aureus, S. epidermidis, Nitrosapira and fungal strains Aspergillus niger, C. albicans and Aspergillus flavus disc diffusion method20 and minimum inhibitory concentration method21 with little modification were used.

RESULTS AND DISCUSSION

Percentage yields of essential oil and fixed oil of Clematis graveolens leaves and stem parts are given in Table 1. Highest amount of fixed oil in the Clematis graveolens was obtained (0.95%) from the leaves of the plant and that of essential oil was found (0.67%) from the leaves also. Percentage yield of essential oil was in the range of 0.53–0.67% and that of fixed oil was 0.72–0.95%. No literature has been available regarding percentage yield of essential and fixed oils of this plant. The colour of the oil was yellow.

table 1. Percentage yield of essential and fixed oil of Clematis graveolens Plant parts Essential oil (%) Fixed oil (%)Stem 0.53 0.72Leaves 0.67 0.95

GC-MS analysis. GC-MS analysis revealed that 35 compounds were analysed from the fixed oil stem and 28 compounds were analysed from fixed oil leaves of Clema-tis graveolens. GC-MS analysis of essential oil found 24 compounds from essential oil of leaves and 22 compounds from essential oil stem of Clematis graveolens.

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Ethylene glycol monopropyl ether (21.41%), fluoroethane (45.14%), 1-phenyl-3-,3-diethyltriazene (6.62%), undecane (5.16%), dichloroacetyl chloride (4.79%) are the major compounds of essential oil stem and 1,2-benzenedicarboxylic acid (18.35%), 3,8,12-tri-O-acetoxy-7-desoxyingol-7-one (12.74%), propanoic acid, 2-(3-acetoxy-4,4,14-trimethylandrost-8-en-17-yl) (9.14%), arbutin, p-3,4,6-tetraacetate 2-(3,4-dihydroxycinnamate) diacetate (5.57%), 2,2-dimethoxy butane (7.32%) are the major compounds of fixed oil stem of Clematis graveolens. 3-octanone,8-(6-tricosyl-2-methyl-1,3-dioxan-4-yl) (12.56%), uridine, 5-tridecafluorohexyl- (8.18%), phosphonic acid, [1-(acetylamino)-2-[4-(acetyloxy)phenyl]ethyl]-, bis(trimethylsilyl)ester (7.88%), di-O-benzoyl methyl 4,6-benzylidene-alpha-d-galactoside (5.95%), 1-[bis-(2-hydroxyethyl)amino]-3-(3,6-diiodocarbazol-9-yl)-propan-2-ol (5.83%), are the major compounds of essential oil from leaves and 2,2-dimethoxybutane (15.16%), cholestano[3,2-c]isoquinolin-1′(2′H)-one, 3′,4′-dihydro-6′,7′-dimethoxy- (6.68%), 2-amino-N-methylpropanamide (5.95%), kopsinyl alcohol (4.47%), hexamethylcy-clotrisiloxane (4.69%) are the major compounds of the fixed oil leaves of Clematis graveolens and all these aspects are summarised in Tables 2–5. Some of the compounds were not detected in essential and fixed oils of Clematis graveolens. The phenolic compounds in plants have vital consequence for the antioxidant potential by self-protected mechanism against oxidising compounds due to the presence of hydroxyl group associated to their chemical moiety22 and work as hydrogen donor23. Therefore, the presence of these valuable and effectual compounds in Clematis graveolens may play crucial role in the biological and pharmaceutical field.

table 2. GC-MS analysis of essential oil leaves of Clematis graveolensSr. no

Retention time (min)

Name of compound area (%)

1 2 3 4 1 3.52 heptyl hydroperoxide 2.34 2 3.78 2,2-dimethoxybutane 7.74 3 15.94 5-ethyl-5-methyldecane 3.03 4 31.12 trans,trans-1,1′-(P-phenylene)bis(3-(P-(methylthio)phenyl)-2-

propen-1-one)3.02

5 31.19 bis[phenylsulfonyl]-4-trichloromethyphenyl chloromethane 1.68 6 31.28 roridine a 2.60 7 31.42 4-(p-methoxyphenyl)-2-(4-phenyl-2-pyridyl)-6-(2-pyridyl)-

pyridine4.02

8 31.48 not identified 2.08 9 31.57 di-O-benzoyl methyl 4,6-benzylidene-alpha-d-galactoside 5.9510 31.82 p-bis(trimethylsilyl)benzene 5.7011 31.96 hexa(methoxymethyl)melamine 4.7712 31.99 vitamine E acetate 3.5213 32.08 not identified 2.65

to be continued

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1 2 3 414 32.13 not identified 1.5315 32.19 1-[bis-(2-hydroxyethyl)amino]-3-(3,6-diiodocarbazol-9-yl)-

propan-2-ol5.83

16 32.49 3-octanone,8-(6-tricosyl-2-methyl-1,3-dioxan-4-yl) 12.5617 32.67 phosphonic acid, [1-(acetylamino)-2-[4-(acetyloxy)phenyl]

ethyl]-, bis(trimethylsilyl)ester7.88

18 32.93 fluphenazine enanthate 1.6819 33.74 rhodoviolascin 2.6320 33.86 silicic acid, diethyl bis(trimethylsilyl)ester 1.7121 33.91 N,N-dimethyl-4-nitroso-3-(trimethylsilyl) aniline 1.9122 34.05 1,3-bis(trimethylsilyl)benzene 4.0923 34.10 uridine, 5-tridecafluorohexyl- 8.1824 34.28 not identified 2.90

table 3. GC-MS analysis of essential oils of stem of Clematis graveolens Sr. no

Retention time (min)

Name of compound Area (%)

1 3.13 dichloroacetyl chloride 4.792 3.65 fluoroethane 45.143 3.77 ethylene glycol monopropyl ether 21.414 3.89 1-phenyl-3,3-diethyltriazene 6.625 4.28 thiomorpholine 0.236 5.95 furfuryl alcohol 0.737 8.62 dipropylene glycol 0.708 8.68 2-(2-hydroxypropoxy)-1-propanol 0.839 9.15 2-butanol, 3,3′-oxy-bis- 0.31

10 9.46 undecane 5.1611 9.79 larixic acid 0.4212 11.05 (S)-(+)-2′,3′-dideoxyribonolactone 0.7613 11.51 dianhydromannitol 1.3414 15.91 5-ethyl-5-methyldecane 0.5015 16.28 2,4-di-tert-butylphenol 0.2816 20.58 1-nonadecene 0.2417 22.39 palmitic acid, methyl ester 0.4018 22.83 1-(+)-ascorbic acid 2,6-dihexadecanoate 3.7219 23.29 1-nonadecanol 0.2620 25.65 lorazepam 0.2421 25.75 chloroacetic acid, octadecyl ester 0.1822 29.06 isooctyl phthalate 5.74

Continuation of Table 2

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table 4. GC-MS analysis of stem of fixed oil of Clematis graveolensSr. no

Retention time (min)

Name of compound Area (%)

1 3.03 2-amino-N-methylpropanamide 5.94 2 3.24 butanoic acid, methyl ester 1.58 3 3.78 2,2-dimethoxybutane 15.16 4 4.94 3,3-dimethoxy-2-butanone 0.84 5 5.29 L-5-propylthiomethylhydantoin 1.41 6 5.42 valeric acid, 3-oxo-, methyl ester 1.11 7 9.45 undecane 3.73 8 11.46 4,5-dimethylnonane 1.89 9 15.55 3,4-dimethyheptane 2.0710 15.87 3,7-dimethylnonane 1.0111 15.94 3-ethyl-3-methylheptane 3.0112 28.77 4-chloro-2-iodobenzoic acid 1.113 29.00 1,2-benzenedicarboxylic acid, mono(2-kethylhexyl) ester 2.5714 29.08 kopsinyl alcohol 4.4715 30.13 octadeamethyl-cyclononasiloxane 0.8116 30.67 not identified 0.8117 30.8 3,6-bis(N-dimethylamino)-9-ethylcarbazole 1.2118 30.88 cyclopenta[a]quinolizine-6,7,9,10-tetracarboxylic acid, 6,7-

dihydro-8-methyl-,tetramethyl ester1.13

19 30.96 p-bis(trimethylsilyl)benzene 2.2320 31.03 hexamethylcyclotrisiloxane 4.6921 31.21 bis[phenylsulphonyl]-4-trichloromethylphenyl chloromethane 1.6722 31.27 di-O-benzyl methyl 4,6-benzylidene-alpha-d-galactoside 2.2723 31.40 silicic acid, diethyl bis(trimethylsilyl) ester 0.7524 31.81 7,15-dihydroxydehydroabietic acid, methyl ester, di (trimeth-

ylsilyl) ether3.13

25 31.94 not identified 5.0726 32.13 butanedioic acid, 2,3-bis[(tert-butyldimethylsilyl)oxy]-, bis(tert-

butyldimethylsilyl) ester1.99

27 32.19 dl-alpha-tocopheryl acetate 3.9128 32.29 vitamin E acetate 4.3829 32.48 not identified 6.6830 32.58 not identified 2.6831 32.61 5-(p-aminophenyl)-4-(p-tolyl)-2-thiazolamine 1.2932 32.64 1,2,4-benzentricarboxylic acid, 1,2-dimethyl ester 2.7733 32.75 not identified 2.4234 32.79 not identified 3.135 32.97 roridine a 1.12

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table 5. GC-MS analysis of fixed oil of leaves of Clematis graveolens Sr. no

Retention time (min)

Name of compound Area (%)

1 3.79 2,2-dimethoxybutane 7.32 2 7.48 octamethyl cyclotetrasiloxane 0.72 3 18.71 3-ethyl-3-methyldecane 2.02 4 39.36 1,2-benzenedicarboxylic acid 18.35 5 39.52 not identified 1.00 6 40.83 tetradecamethylhexasiloxane 1.02 7 41.79 4-(1,1,3,3-tetramethylbutyl)phenyl trimethylsilyl ether 1.22 8 41.94 2,6-dimethyl-3,3-bis(trimethylsilyloxymethyl)pyridine 1.5 9 42.09 vitamin E acetate 1.0110 42.89 butanedioic acid,2,3-bis[(tert-butyldimethylsilyl)oxy]-,

bis(tert-cutyldimethylsilyl) ester1.63

11 43.29 not identified 1.5212 43.4 zirconium(IV) trifluoroacetylacetonate 4.2013 43.6 benzoic acid, 4-methyl-2-trimethysilyloxy-, trimethylsilyl ester 0.7214 43.63 di-O-benzoyl-methyl-4,6-benzylidene-alpha-galactoside 0.7415 43.67 2-amino-4,4,6,6-tetramethyl-4,6-dihydro-thieno[2,3-c]furan-3-

nonitrile0.99

16 43.74 not identified 4.4417 43.88 arbutin, p-3,4,6-tetraacetate 2-(3,4-dihydroxycinnamate) 5.5718 44.03 5-methyl-2-trimethylsilyloxy-acetophenone 0.7119 44.07 6-hydroxy-7-n-pentadecylmercapto-5,8-quinolinedione 1.3920 44.14 (4-tert-butylphenoxy)methyl acetate 1.7021 44.29 cholestane-3,6,7-triol 1.6622 44.76 not identified 1.0523 44.8 not identified 2.6224 44.98 3,8,12-tri-O-acetoxy-7-desoxyingol-7-one 12.7425 45.22 methyl hexatriacontanoate 4.0626 45.23 not identified 7.0527 45.42 not identified 9.1428 45.52 not identified 3.91

table 6. Percentage inhibition and IC50 analysis of Clematis graveolens essential and fixed oilPlant parts Extracts/fraction Inhibition (%) IC50 (μg/ml)Stem essential oil 69.69±0.12 31.93±0.01 fixed oil 59.59±0.14 47.82±0.02Leaves

essential oil 71.61±0.22 15.83±0.02fixed oil 79.92±0.45 14.06±0.03BHt 89.04±0.23 9.22±0.03

Antioxidant activity. Different assays were used to analyse antioxidant potential of the essential and fixed oils of Clematis graveolens. The percentage inhibition in

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linoleic acid system of essential oil ranges from 69.69–71.61% and that of fixed oil from 59.59–79.92. The results showed that maximum percentage inhibition in linoleic acid system was observed by the fixed oil of leaves and minimum percent-age inhibition in linoleic acid system was shown by the fixed oil of stem (Table 6). Essential oil and fixed oil of stem and leaves also showed antioxidant activity when analysed by reducing power and a linear increase in absorbance was observed when concentration of test sample increased (Fig. 1). With the increase in concentration, absorbance also increases, this aspect was observed in reducing power analysis of Clematis graveolens.

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

2.5 5 7.5 10

concentration (mg/ml)

abso

rban

ce

essential oil (leaves)

essential oil (stem)

fixed oil (leaves)

fixed oil (stem)

fig. 1. Reducing potential of essential and fixed oils of Clematis graveolens

The fixed oil of stem showed lower free radical scavenging activity and highest IC50 values (47.82 μg/ml) and fixed oil of leaves showed lower IC50 values (14.06 μg/ml) and highest free radical scavenging activity as described in Table 2. On comparing the fixed and essential oil of stem and leaves maximum IC50 values were shown by fixed oil of stem (47.82 μg/ml) and there results are given in Table 6. The essential oils posses antioxidant activities1 and this potential is might be due to the presence of phytoconstituents in essential oils24 chiefly due to the presence of mono- and oxy-genated terpenes, furthermore in literature is also highlighted the fact that some non-phenolic rich essential oils also showed these types of activities25. To secure human fitness the side effects of the artificial compounds enhanced the reimbursement of use of the natural plant as antimicrobial and antioxidant agents against microbial growth and oxidation, respectively26, and ability of oils is the main reason for extensive use of oils in pharmaceutical field, natural therapies, medicines and to preserve fresh and processed food1.

Antimicrobial activity. Results showed that essential oil of leaves showed good in-hibitory activity against E. coli (15.66 mm) and minimum activity against B. cereus (9.44 mm) and for fungal strains good activity was shown against C. albicans (11.44 mm) and minimum activity was shown against A. niger (9.86 mm). Regard-

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ing fixed oil of this plant maximum activity for bacterial strains was shown by fixed oil of stem against B. cereus (13.87 mm) and minimum activity was shown against E. coli (8.91 mm) and for fungal strains maximum activity was shown by the fixed oil leaves against A. flavus (10.34 mm) and minimum activity was shown by the stem against C. albicans (8.02 mm). Fungone and Novidate standards were used and these drugs showed good activity against tested strains than the under observation oils because of their purity. Mminimum inhibitory concentration (MIC) activities of the oils were inverse of the antimicrobial activity. Summary of these results is given in Tables 7–10. Antioxidant potential might be due to the presence of phytochemicals but in the case of essential oils it is very difficult to correlate the antioxidant activity to specific compounds due to their complexity and variability27. However, alcohols, ester, aldehydes and terpenes of all types, fatty acids and methyl esters contribute towards antimicrobial activity of essential oils28. Antimicrobial activity of essential oils is thought to be due to the monoterpenes components which are phenolic in nature and cause damage of membrane of microbial strains and potassium ions to leake from the membrane. This is the accountable for lethal action of cytoplasmic damage29. Me-dicinal plants are the rich source of antimicrobial agent. According to some authors30, antimicrobial activity of the essential oils of many plants might be due to the presence of fatty acids and alkanes constituents of the plants. Thus, compounds like undecane, 3-ethyl, 3-methyl decane, palmitic acid methyl ester, 1,2-benzenedicarboxylic acid, and 1,2-benzenedicarboxylic acid mono(2-ethylhexyl) ester may be responsible for antimicrobial activity of the Clematis graveolens. As semi-synthetic and synthetic drugs are available in market, it is the need of the hour to search antimicrobial resist-ant agents31. For this purpose Clematis graveolens is used as this type of activity is not already reported for essential oil and fixed oil of this plant.

table 7. Determination of antibacterial activity of essential and fixed oils of Clematis graveolens by zone of inhibition (mm)

Samples S. aureus E. coli B. cereus N. spiraStem essential oil 12.98±0.01 14.65±0.01 na 14.77±0.04 fixed oil 9.15±0.01 8.91±0.01 13.87±0.01 13.14±0.01 essential oil na 15.66±0.01 9.44±0.24 14.55±0.03 fixed oil 10.32±0.01 11.22±0.02 na 10.00±0.01Leaves novidat 17.77±0.49 18.26±0.36 19.77±0.49 20.26±0.36Note: NA – not active.

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table 8. Antifungal activity of the essential and fixed oils of Clematis graveolens by zone of inhibition (mm)

Sample C. albicans A. niger A. flavusLeaves essential oil 11.44±0.03 10.65±0.01 9.86±0.01

fixed oil na na 10.34±0.01Stem essential oil na 10.12±0.02 10.12±0.01

fixed oil 8.02±0.03 9.34±0.01 naStandard fungone 20.23±0.45 19.23±0.21 18.21±0.43Note: NA – not active.

table 9. Minimum inhibitory concentration (MIC) in mg/ml by essential and fixed oils of Clematis graveolens

Sample A. flavus A. niger C. albicansStem essential oil 0.89±0.003 0.69±0.003 0.76±0.001

fixed oil 0.82±0.001 0.89±0.003 naLeaves essential oil 0.85±0.003 na na

fixed oil na 0.79±0.003 0.99±0.002Standard fungone 0.43±0.003 0.36±0.002 0.29±0.002Note: NA – not active.

table 10. Minimum inhibitory concentration (MIC) in mg/ml against bacterial strains by essential and fixed oils of Clematis graveolens Sample S. aureus E. coli B. cereus N. spiraLeaves essential oil na 0.52±0.003 0.90±0.003 0.59±0.003 fixed oil 0.84±0.003 0.72±0.001 na 0.72±0.004Stem essential oil 0.60±0.002 0.62±0.004 0.65±0.003 0.59±0.002 fixed oil 0.92±0.001 0.94±0.002 na 0.80±0.005Standard novidate 0.48±0.002 0.41±0.004 0.32±0.005 0.22±0.001Note: NA – not active.

CONCLUSIONS

This study has been carried out in order to highlight the medicinal importance of Clematis graveolens. The work was focused to determine the chemical composition, antibacterial and antioxidant activities of fixed and essential oils of the plant stem and leaves. The GC-MS analysis showed the presence of various effective chemical moie-ties. Essential and fixed oils reveal excellent antibacterial and antioxidant potential. All the results showed that the plant may be used as a good therapeutic agent.

ACKNOWLEDGEMENTS

The authors are highly thankful to Higher Education Commission under Access to Scientific Instrumentation Project scheme for providing funds to GC-MS analysis. We are also thankful to Dr. Muhammad Athar Abbasi, the Department of Chemistry,

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Government College University Lahore, Pakistan, for providing the facility regarding the analysis by GC-MS.

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Received 12 April 2013 Revised 7 May 2013