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The Natural Products Journal, 2013, 3, 66-70
In Vitro Antimicrobial Activity of Essential Oil of Swertia cordata Aerial Parts Shivani Joshia,*, Devendra Mishrab, Ganga Bishta and Khadga S. Khetwala a
Department of Chemistry, D.S.B. Campus, Kumaun University, Nainital, Uttarakhand, India; bDepartment of Applied Chemistry, Birla Institute of Applied Sciences, Bhimtal, Nainital, Uttarakhand, India Abstract: Essential oil from aerial parts of Swertia cordata (G. Don.) Clarke obtained by steam distillation was analysed through Gas chromatography and Gas chromatography-mass spectrometry. The main compounds found were palmitic acid (8.34), caryophyllene oxide (5.65), pentadecanal (4.77) and lachnophyllum ester (4.14%). Different concentrations of essential oil were tested for antimicrobial activity by disc diffusion method. The oil was found active against gram positive bacteria and fungi.
Keywords: Swertia cordata, essential oil, gas chromatography- mass spectrometry, zone of inhibition. INTRODUCTION Swertia Linn. is an annual or perennial herb comprising more than 170 species among which 79 and 40 species are distributed in China and India, respectively. The genus Swertia contains different bitter principles, iridoids and secoiridoids, for example amarogentin, swertiamarin, sweroside, amaroswerin and gentiopicroside, which are mainly responsible for its pharmacological activity. Approximately 20 species of this genus are used in Chinese traditional medicine to treat hepatic, choleric, and antiinflammatory diseases [1]. Swertia chirata attracts special attention because of its varied use as a bitter febrifuge and its antihelmintic, antimalarial and antidiarrhoeal activity [2]. The plant was once found growing abundantly in the wild and was exported from India, now it has the status of endangered species. Moreover, use of this herb by commercial houses on a large scale has resulted in extinction of the true S. chirata [3-5]. S. cordata is a branched herbaceous plant with yellowish white flowers. The plant has been used in medicines as an alternative febrifuge and antihelmintic and as a bitter tonic [1, 6, 7]. To the best of our knowledge there is no published report on essential oil composition of Swertia cordata so far, therefore the primary objective of this paper is the detailed analysis of the plant essential oil and its antimicrobial property. EXPERIMENTAL Plant Material The plant Swertia cordata (G. Don) Clarke was collected in the month of October, 2009 from village Vinayak near Pangot (29°25’25”N 79°25’37”E) 15 km away from Nainital, *Address correspondence to this author at the Department of Chemistry, D.S.B. Campus, Kumaun University, Nainital, Uttarakhand, India; Tel: +919568296342; E-mail:
[email protected] 2210-3163/13 $58.00+.00
Uttarakhand, India. The plant was authenticated by Botanical Survey of India (BSI), Dehradun. A voucher specimen (No.112968) was deposited in the Herbarium Section at BSI, Dehradun, India. Essential Oil Extraction The fresh aerial parts of Swertia cordata (5 kg) were chopped and steam- distilled using copper still fitted with spiral glass condensers. The distillate was saturated with NaCl and extracted with n-hexane. Anhydrous Na2SO4 was then added to dry the organic phase which was separated using separating funnel and finally the solvent was evaporated under reduced pressure. The percentage content of the oil was calculated on the basis of dry weight of plant material. The oil was then stored in screw-capped vials, under refrigeration until needed. GC-MS Analysis The oil was analysed using Agilent Technologies 6890 N Network system chromatography equipped with FID data handling system with manual injection/ injector with split ratio 1:50, N2 flow of 4.0 kg/cm2. HP-5MS 5% phenyl methyl siloxane capillary column (30x0.32 mm, 0.25 μm film coating, Model No. Agilent 19091J-413). The oven temperature was programmed from 60oC-210oC at 3o C/min using Helium as carrier gas at 1.0 ml/min. The injector temperature was 210oC, injection volume was 0.2μL prepared in hexane, with split ratio of 1:50. Mass spectra were taken at 70ev (EI) with mass scan range of m/z 40-450 amu with mass scan time 4 seconds. Identification of the constituents was done on the basis of retention index, library mass search database (NIST & WILEY) and Robert P. Adams [8]. Microorganisms Used The antimicrobial activity of S. cordata essential oil was conducted against three gram positive bacteria, © 2013 Bentham Science Publishers
Antimicrobial Activity of Swertia cordata Essential Oil
Table 1.
The Natural Products Journal, 2013, Vol. 3, No. 1
Essential oil Composition of Swertia cordata Aerial Parts
S.No.
Compound
KI
Oil %
Mode of Identification
1
Cumene
933
0.19
a,b
2
- pinene
937
0.74
a,b
3
-citronellene
945
0.16
a,b
4
-pinene
972
0.39
a,b
5
Myrcene
985
0.20
a,b
6
- phellandrene
999
0.20
a,b
7
Limonene
1008
0.20
a,b
8
p-cymene
1020
0.20
a,b
9
E-( )- ocimene
1050
0.12
a,b
10
Menthone
1152
0.17
a,b
11
Tetrahydro lavandulol
1161
1.11
a,b
12
cis- chrysanthelol
1164
0.62
a,b
13
Furfuryl acetone
1183
0.13
a,b
14
Prenyl cyclopentanone
1225
0.12
a,b
15
Linalool acetate
1230
0.13
a,b
16
cis- - terpineol
1242
1.22
a,b
17
Piperitone
1252
0.19
a,b
18
Neryl formate
1282
0.14
a,b
19
Undecanal
1306
0.17
a,b
20
- cubebene
1345
0.67
a,b
21
9- decenoic acid
1360
0.15
a,b
22
- copaene
1370
0.19
a,b
23
10-(acetylmethyl) -3-carene
1383
2.39
a,b
24
- funebrene
1402
0.89
a,b
25
z- caryophyllene
1408
0.75
a,b
26
– cis- bergamotene
1412
0.73
a,b
27
Funebrene
1413
0.16
a,b
28
- humulene
1434
0.17
a,b
29
– himachalene
1450
0.57
a,b
30
cis- - farnesene
1454
2.69
a,b
31
-acoradiene
1470
0.20
a,b
32
2,5,9- trimethyl decane
1480
0.63
a,b
33
Germacrene-D
1484
1.33
a,b
34
– selinene
1498
0.29
a,b
35
- curcumene
1512
2.72
a,b
36
Iso-germacrene -D
1528
2.25
a,b
37
Lachnophyllum ester
1550
4.14
a,b
67
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Joshi et al.
Table 1. contd….
S.No.
Compound
KI
Oil %
Mode of Identification
38
- amorphene
1564
0.91
a,b
39
Spathulenol
1572
1.72
a,b
40
Caryophyllene oxide
1582
5.65
a,b
41
Globulol
1590
3.23
a,b
42
T- cadinol
1630
0.86
a,b
43
Trans-z- –bisabolene epoxide
1652
0.67
a,b
44
Pentadecanal
1719
4.77
a,b
45
E- -santalol
1739
1.56
a,b
46
Nuciferol
1755
0.38
a,b
47
Tetradecanoic acid
1768
2.10
a,b
48
Hexahydrofarnesyl acetone
1818
2.47
a,b
49
(5-Z, 9-E) Farnesyl acetone
1889
0.21
a,b
50
n- nonadecane
1900
0.14
a,b
51
Ethyl hexadecanoate
1934
0.39
a,b
52
Geranyl benzoate
1959
0.35
a,b
53
Iso- phyllocladene
1965
0.37
a,b
54
Palmitic acid
1970
8.34
a,b
55
Sclarene
1972
0.59
a,b
56
Bifloratriene
1976
0.53
a,b
57
1-eicosene
1988
0.32
a,b
58
Kaurene
1997
0.25
a,b
59
Eicosane
2000
1.04
a,b
60
Phyllocladene
2017
0.25
a,b
61
2-methyl eicosane
2035
3.65
a,b
62
Canellal
2046
0.32
a,b
63
Manool
2057
0.31
a,b
64
Scalreolide
2066
0.16
a,b
65
n- heneicosane
2100
0.28
a,b
66
Oleic acid
2142
0.31
a,b
Total
Staphylococcus aureus (MTCC 3160), Bacillus subtilis (MTCC 441), Bacillus mycoides (MTCC 645); three gram negative bacteria, Escherichia coli (MTCC 406), Pseudomonas aeruginosa (MTCC 424), Proteus vulgaris (MTCC 426) and two fungi, Candida albicans (MTCC 227) and Aspergillus niger (MTCC 404). Required microorganisms were procured from Institute of Microbial Technology, Chandigarh, India. Antibacterial and Antifungal Assay Antimicrobial activity of S. cordata essential oil was tested by the paper disc diffusion method according to the
69.45
slightly modified National Committee for Clinical Laboratory Standards Guidelines [9]. The sterilized nutrient agar media was poured and distributed to sterilized petri dishes with a diameter of 9 cm and cooled to 45-50°C for bacterial growth while potato dextrose agar medium was used for the growth of fungi. The prepared inoculums of bacterial and fungal strains were then poured and spread on the surface of prepared petri dishes. The filter paper discs (5 mm in diameter, Whatmann No.1) were individually impregnated with 10 μl of the oil dissolved in dimethylsulfoxide (DMSO), which was subsequently placed on the surface of
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Antimicrobial Activity of Swertia cordata Essential Oil
the inoculated petri dishes. The various concentrations of oil used were 1000, 500, 250 and 125 μg/ml. The petri dishes were kept at 4o C for 2 h and then incubated at 37oC for 24 h for the growth of bacteria and at 27oC for 48 h for the growth of fungi. The diameters of the inhibition zones including the diameter of paper disc were measured in millimeters. Controls were set up with equivalent quantities of DMSO. Chloramphenicol (25 μg/ disc) and fluconazole (25 μg/ ml) were used as positive controls [10]. All the experiments were performed in triplicate and the results (mm of zone of inhibition) were expressed as their mean values. RESULTS AND DISCUSSION Essential Oil Composition The 0.02 % w/w of pale yellow colour oil of S. cordata was extracted by steam distillation method. The chemical composition of the essential oil was analyzed by GC and GC-MS. Total sixty six constituents were identified, representing 69.45% of the total oil. The major constituents of the oil were palmitic acid (8.34), caryophyllene oxide (5.65), pentadecanal (4.77), lachnophyllum ester (4.14%). Essential oil from plant species of family Gentianaceae were reported to contain oxygenated sesquiterpenes and sesquiterpene hydrocarbons as the characterstic constituents [11]. The present report of Swertia cordata oil also found to possess sesquiterpene rich compounds, comprising oxygenated sesquiterpenes (17.58%), sesquiterpene hydrocarbons (14.48%) in addition to monoterpene hydrocarbons (4.96%), oxygenated monoterpenes (3.88%) and diterpenes (2.30%). Swertia genus is not known to contain high yield of essential oil. Swertia chirayita was reported to yield o.23% w/w essential oil [12]. There are very few reports on essential oil composition of Swertia species and this is the first report of Swertia cordata essential oil composition and its antimicrobial activity. The compounds, together with their retention index and relative percentage concentration are presented in Table 1. Identification of the constituents was Table 2.
done on the basis of retention index, library mass search database (NIST &WILEY) and Robert P. Adams [8]. Antimicrobial Activity Antimicrobial activity of steam distilled essential oil of Swertia cordata carried out against six bacterial and two fungal strains is summarized in Table 2. The results revealed that aerial parts of the plant showed moderate antibacterial and antifungal activity. The zone of inhibition decreased on decreasing the concentration of the oil. It was observed that the oil showed good antibacterial activity against gram positive bacteria as compared to gram negative bacteria while both the fungal strains were found sensitive to the essential oil. Among the bacterial strains, essential oil showed good activity against S. aureus which was found even comparable to that with the standard Chloramphenicol while the highest zone of inhibition was showed against B. mycoides; E. coli was found to be less sensitive to the oil. The activity showed against C. albicans and A. niger was observed to be comparable with Fluconazole. The antimicrobial properties of the essential oil of S.cordata may be due to the presence of terpenes. There are reports on the antimicrobial activity of essential oils containing lachnophyllum ester [13], -citronellene, limonene, germacrene-D, caryophyllene oxide, spathulenol [14], hexahydrofarnesyl acetone, pentadecanal [15,16] and the essential oil of S. cordata contains the above said constituents in the significant amount. It has been reported that the constituents present at lower concentrations might be involved in the synergism with the other active constituents [17]. As no previous reports on the antimicrobial activity of this plant could be found in the literature, the present study claims some useful results. The results obtained for its inhibitory effect on various microorganisms used indicate that the essential oil of S. cordata aerial parts could be useful for treating diseases of plants and animals. Therefore, study on the essential oil of this plant can be recommended.
Antimicrobial Activity of Essential oil of Swertia cordata Aerial Parts Standard
Zone of Inhibition (mm)* Essential Oil (μg/ml)
Microbes 1000
500
250
125
CP
FZ
S. aureus
15 ± 0.6
12 ± 0.5
9 ± 0.3
8 ± 0.6
12 ± 0
-
B. mycoides
18 ± 0
16 ± 0.3
14 ± 0.3
10 ± 0.6
25 ± 0.6
-
B. subtilis
14 ± 0.3
12 ± 0.6
10 ± 1.0
7 ± 1.1
30 ± 0.6
-
P. vulgaris
8±1
7±0
6 ± 0.3
-
14 ± 0
-
E. coli
7 ± 0.6
-
-
-
23 ± 0.3
-
P. aeruginosa
9 ± 0.5
7 ± 0.6
-
-
10 ± 0
-
C. albicans
14 ± 0.8
13 ± 0.8
10 ± 0.5
8 ± 1.1
-
15 ± 0.6
A. niger
14 ± 1.2
12 ± 0.3
10 ± 0.3
9±0
-
13 ± 0.6
*All the values are mean ± SEM of three findings CP= Chloramphenicol (25 μg/ disc), FZ= Fluconazole (25 μg/ ml)
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Joshi et al. [6]
CONCLUSIONS The present study confirmed antimicrobial properties of essential oil from S. cordata that showed significant growth inhibition for B. mycoides and C. albicans. The encouraging results indicate that S. cordata might be exploited as natural antibiotic for the treatment of several infectious diseases caused by these two pathogens and could be useful in understanding the relations between traditional cures and current medicines.
[7] [8] [9]
[10]
CONFLICT OF INTEREST The author(s) confirm that this article content has no conflict of interest.
[11]
ACKNOWLEDGEMENTS The authors are highly thankful to Center for Aromatic Plants, Dehradun, Uttarakhand, India, for GC and GC/MS analysis of the essential oil. We are equally grateful to Department of Pharmaceutical Sciences, Bhimtal, Nainital, India, for providing necessary lab facilities for antimicrobial activity.
[12]
[13] [14]
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Revised: January 16, 2013
Accepted: January 22, 2013
