Chemical composition and antibacterial activity of essential oil from two Ocimum spp / Asian Journal of Traditional Medicines,

2011, 6 (5)

Regular articles

Chemical composition and antibacterial activity of essential oil from two Ocimum spp grown in sub-tropical India during spring-summer cropping season
Ram Swaroop Verma a*, Pawan Singh Bisht a, Rajendra Chandra Padalia a, Dharmendra Saikia b, Amit Chauhan a a. Central Institute of Medicinal and Aromatic Plants (CSIR-CIMAP), Research Centre, Pantnagar, P.O. - Dairy farm Nagla, Udham Singh Nagar, Uttarakhand 263149, India b. Central Institute of Medicinal and Aromatic Plants (CSIR-CIMAP), P.O. CIMAP, Lucknow 226015, India

Abstract Chemical composition and antibacterial activity of the essential oils of Ocimum gratissimum L. and Ocimum kilimandscharicum Guerke grown in sub-tropical region of northern India during spring/summer cropping season were investigated. The essential oil content in the fresh herb of O. gratissimum and O. kilimandscharicum was 0.45% and 0.48%, respectively. The essential oils of both Ocimum species was analyzed by capillary gas chromatography (GC-FID) and gas chromatography-mass spectrometry (GC-MS). Major components of the O. gratissimum essential oil were eugenol (63.7%), (Z)--ocimene (19.6%), and germacrene D (7.3%), while the principal components identified in the O. kilimandscharicum essential oil were camphor (63.4%), limonene (7.9%), camphene (5.8%), and -terpinene (4.7%). These oils were evaluated against four pathogenic bacteria. Interestingly, both oils showed good activity against tested strains. The activity index was noticed higher for O. gratissimum than O. kilimandscharicum essential oil. Key words: Ocimum gratissimum L.; Ocimum kilimandscharicum Guerke; essential oil, composition; eugenol; camphor; antibacterial activity

1. Introduction
Among the plants known for medicinal value, the members of genus Ocimum (family Lamiaceae) are very important for their therapeutic potentials. Ocimum sanctum L . , Ocimum gratissimum L.,
* Author to whom correspondence should be addressed. Address: Central Institute of Medicinal and Aromatic Plants (CSIR-CIMAP), Research Centre, Pantnagar, P.O. -Nagla Dairy farm, Udham Singh Nagar, Uttarakhand 263149, India; Tel.: +91-5944 234445; Fax: +91-5944 234712; E-mail: rswaroop1979@yahoo.com; rs.verma@ cimap.res.in Received: 2010-11-18 Accepted: 2011--08-23

Ocimum canum Sims, Ocimum basilicum L., Ocimum kilimandscharicum Guerke , Ocimum americanum L., and Ocimum micranthum Willd are examples of important species of genus Ocimum which grow in different parts of the world and are known to have medicinal properties [1]. The plant has been used for medicinal purposes as a digestive stimulant and for treatment of insomnia and constipation. The oils of basil have been used principally in the food and cosmetic industries [2]. The genus Ocimum is characterized by a great morphological and chemical variability, the ease of crosspollination leads to a large number

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of subspecies, varieties and forms, which differ in essential oil composition and morphological characters [3, 4]. O. gratissimum which is also known as Ram Tulsi or Bun Tulsi is a tall, much branched perennial aromatic shrub growing 1-2.5 meter height and it has wide distribution in the tropical areas of Africa and Brazil. In Asia, it is found distributed all over peninsular India, West Bengal, Assam and also in neighboring countries like Bangladesh and Indonesia. The essential oil of O. gratissimum possessed insecticidal [5], antibacterial [6-9], and antifungal properties [10]. The essential oil of O. gratissimum and its main component eugenol were efficient in inhibiting eclosion of Haemonchus contortus, a gastrointestinal parasite of small ruminants [11]. Camphor yielding O. kilimandscharicum , also known as Kapur Tulsi originating from the Kilimandscharo hill in Africa, has been domesticated in India. Major components found in the essential oil of O. kilimandscharicum are linalool, camphor, and 1, 8 cineole [12]. This oil is used in confectionary and pharmaceutical industries especially in Europe [13]. In India, Ocimum is generally cultivated in rainautumn cropping season (June-October) for the production of dry herb or essential oil therefore; most of the studies on Ocimum spp have been conducted in same cropping season. Literature survey revealed the potential of Ocimum spp has not been explored adequately in spring-summer cropping season. The aim of present study was to investigate the chemical composition and antibacterial potential of the essential oils of O. gratissimum and O. kilimandscharicum from sub-tropical India during spring-summer cropping season.

2.1. Plant material

The samples of O. gratissimum and O. kilimandscharicum were collected in the month of April 2010 at flowering stage from Central Institute of Medicinal and Aromatic Plants, Research centre, Pantnagar. The experimental site is located between coordinates 29 N, 79.38 E and an altitude of 243.84 m in foot hills of Uttarakhand. The soil of foot hills was mollisol with neutral in reaction (pH 7.1).

2.2. Extraction of essential oil

Freshly harvested samples of the O. gratissimum and O. kilimandscharicum were hydrodistilled in a Clevenger type apparatus for 3 hrs to get essential oil [14]. Clevenger apparatus is an all glass apparatus consists of a round bottom flask, condenser and extraction burette. The plant material is loaded in to a round bottom flask with sufficient quantity of water. On heating the flask essential oil glands present in the plant material gets ruptured. The steam and essential oil vapours generated in the flask pass through a condenser to remove the energy which finally converts the vapours into liquid. The condensate (mixture of essential oil and water) is collected in an extraction burette. Since, the water and essential oil have different densities, essential oil floats on the surface of the water in the extraction burette. The essential oil measured directly in extraction burette, collected and dehydrated by anhydrous sodium sulphate and kept under refrigeration prior to analysis.

2.3. Gas chromatography (GC)

Analysis of the essential oils was carried out on Nucon gas chromatograph model 5765 and Perkin-Elmer Auto XL GC equipped with Flame Ionization Detector (FID) and two different stationary phases, CP-Wax 52 CB (30 m0.32 mm 0.25 m film thickness) and PE-5 (60 m0.32

2. Materials and methods

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Chemical composition and antibacterial activity of essential oil from two Ocimum spp / Asian Journal of Traditional Medicines, 2011, 6 (5)

mm; 0.25 m film coating) fused silica capillary columns, respectively. Hydrogen was the carrier gas at 1.0 ml/min. Temperature programming was done from 70C-230C at 4C /min with initial and final hold time of 2 minutes (for CP-Wax 52 CB) and from 70C-250 C at 3C/min (for PE-5). The injector and detector temperatures were 210C and 230C on CP-Wax 52 CB and 220C and 300C on PE-5 column, respectively. The injection volume was 0.02 l neat and Split ratio was 1: 30.

2.4. Gas chromatography-mass spectrometry (GCMS)

The GC-MS analysis of the oils was carried out on a Perkin-Elmer Turbomass Quadrupole Mass spectrometer fitted with Equity-5 (Perkin-Elmer) fused silica capillary column (60 m0.32 mm; 0.25 m film coating). The column temperature was programmed 70C, initial hold time of 2 min, to 250C at 3C/min with final hold time of 3 min, using helium as carrier gas at a flow rate of 1 ml/ min. The injector and source temperatures were 250C. The injection volume was 0.06 l neat with split ratio 1:30. MS were taken at 70 eV with an EI source with mass range of m/z 40-400.

435), Enterococcus faecalis (MTCC 439) , and Streptococcus mutans. Antibacterial activity testing was done using standard disc diffusion assay [17]. For preparing the discs, 5.0 l of the essential oil was pipetted on to 5.0 mm filter paper discs made from Whatman No. 3, which were carefully transferred on to the surface of seeded agar plate. The plates were incubated at 37C for 14-16 hours following which the diameter of the inhibition zone was measured. The net zone of inhibition was determined by subtracting the disc diameter (i.e. 5.0 mm) from the total zone of inhibition shown by the test disc in terms of clear zone around the disc.

3. Results and discussion

3.1. Essential oil composition
The essential oil content observed in fresh herbs of O. gratissimum and O. kilimandscharicum during spring-summer cropping season was 0.45% and 0.48%, respectively. The essential oil of the both Ocimum spp was analysed by GC-FID and GC-MS. The chromatograms of O. gratissimum and O. kilimandscharicum are shown in Fig. 1 and Fig. 2, respectively. The qualitative and quantitative results along with mode of identification of individual components are shown in Table 1. Major constituents in the essential oil of O. gratissimum were eugenol (63.7%), ( Z )--ocimene (19.6%), germacrene D (7.3%), -caryophyllene (1.7%), and -terpinene (1.0%). The essential oil composition of the different Ocimum spp has been investigated earlier in rain-autumn cropping season [18]. The comparison of present results with results from rain-autumn season showed sharp quantitative differences. The percentage of eugenol, methyl eugenol, caryophyllene oxide, and -terpineol was higher in rain-autumn cropping season as compared to the spring-summer season. On the other hand, the amount of ( Z )--ocimene, -terpinene,

2.5. Identification of compounds

The identification was done on the basis of retention time, retention indices (relative to n-alkane, C9-C24), MS Library search (NIST & WILEY) and by comparing mass spectra with the MS literature data [15, 16]. The relative amounts of individual components were calculated based on GC peak areas without using correction factors.

2.6. Antibacterial assay

The antibacterial activity of the essential oils of both Ocimum species was evaluated against following microorganisms: Staphylococcus aureus (MTCC 96), Staphylococcus epidermidis (MTCC

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Chemical composition and antibacterial activity of essential oil from two Ocimum spp / Asian Journal of Traditional Medicines, 2011, 6 (5)

Voltage (mv)

Voltage (mv) t (min) Fig. 1. Chromatogram of Ocimum gratissimum essential oil (for GC analysis conditions see materials and methods section)

t (min) Fig. 2. Chromatogram of Ocimum kilimandscharicum essential oil (first peak is solvent)

and germacrene D were higher during springsummer season. Further, the major constituents identified in the O. kilimandscharicum essential oil were camphor (63.4%), limonene (7.9%), camphene (5.8%), -terpinene (4.7%), and terpinen4-ol (2.5%). Interestingly, the amount of major compounds in O. kilimandscharicum grown in spring-summer was comparable with that observed in rain-autumn season [18]. The effect of harvesting time/season on chemical composition of essential oil in other members of family Lamiaceae has also been reported [19-23]. These variations indicated that the dynamics of essential composition in aromatic

plants is possibly associated with the expression of different genes in different season.

3.2. Antibacterial activity

The essential oils of O. gratissimum and O. kilimandscharicum were tested against four Gram positive bacterial stains. The results of antibacterial activity are summarized in Table 2. The O. gratissimum oil was found to be active against all tested strains. The maximum zone of inhibition was observed in Staphylococcus aureus (16.0 mm), followed by Streptococcus mutans (14.0 mm), Enterococcus faecalis (10.0 mm), and

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Table 1. Essential oil composition of Ocimum gratissimum and Ocimum kilimandscharicum grown in spring/summer cropping season S. No. Compound RI a RI b S/M 1. 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 -Pinene Camphene -Pinene -Myrcene -Terpinene Limonene 1, 8-Cineole (Z)--Ocimene -Terpinene (E)--Ocimene Terpinolene (E)-Sabinene hydrate -Copaene Bourbonene Camphor Linalool -Elemene -Caryophyllene Terpinen-4-ol (E)--Farnesene -Terpineol Borneol Germacrene D Caryophyllene oxide Methyl eugenol Eugenol Essential oil content (%) 1026 1065 1104 1158 1177 1195 1199 1230 1244 1245 1278 1463 1481 1508 1512 1543 1582 1590 1606 1673 1685 1689 1705 2005 2132 2192 931 997 976 986 1014 1030 1031 1042 1060 1051 1087 1065 1374 1145 1097 1394 1423 1175 1465 1183 1165 1483 1588 1400 1362 0.2 t 0.2 t 0.3 t t 19.6 1.0 t 0.7 0.3 0.2 t 1.7 0.2 0.1 t 7.3 t 0.1 63.7 0.45 Peak area (%) OG R/A [18] t t 0.2 t 0.1 7.6 t 0.4 t 0.7 0.3 0.1 0.1 1.7 0.2 0.1 0.2 2.7 0.2 0.2 77.2 S/M 2.0 5.8 0.2 1.2 0.2 7.9 0.1 4.7 1.1 t 0.8 t 63.4 0.9 t 1.5 2.5 t 0.8 1.5 t 0.48 OK R/A[18] 1.4 6.4 t 1.2 0.5 8.7 0.7 0.6 3.0 1.1 1.4 64.9 1.4 t 1.2 0.6 2.7 t 0.7 0.1 RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS, Co-I RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS, Co-I RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS, Co-I Identification

OG: Ocimum gratissimum; OK: Ocimum kilimandscharicum; S/M: spring-summer cropping season; R/A: rain-autumn cropping season; [18] Padalia and Verma, 2010 RIa: Retention index on CP-Wax 52 CB column (polar); RIb: Retention index on PE-5 column (non-polar); MS: comparison of mass spectra with NIST and WILEY libraries; Co-I: Co injection of standard compound; t: trace (<0.05%)

Staphylococcus epidermidis (8.0 mm). Eugenol has been demonstrated to have antibacterial property [7, 24]. Therefore, activity of the O. gratissimum

essential oil observed in present study may be due to the presence of eugenol in good percentage. Like O. gratissimum , the essential oil of O.

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Table 2. Antibacterial activity of the essential oil of Ocimum gratissimum and Ocimum kilimandscharicum grown in spring/summer cropping season Pathogen Gram positive strain Staphylococcus aureus Staphylococcus epidermidis Enterococcus faecalis Streptococcus mutans Activity index 16.0 8.0 10.0 14.0 12.0 5.0 3.0 4.0 4.0 4.0 Essential oil / ZOI (mm) OG OK

kilimandscharicum could also be grow as essential oil crop in spring-summer season.

Acknowledgement
We are thankful to the Director, Central Institute of Medicinal and Aromatic Plants (CSIRCIMAP) for providing necessary facilities and encouragement.

References
[1] Prakash P, Gupta N. Therapeutic uses of Ocimum sanctum Linn (Tulsi) with a note on eugenol and its pharmacological actions: a short review. Indian J Physiol Pharmacol, 2005, 49(2): 125-131. [2] Vina A, Murillo E. Essential oil composition from twelve varieties of basil (Ocimum spp) grown in Colombia. J Braz Chem Soc, 2003, 14(5): 744-749. [3] Krishnan R. Natural outcrossing in sweet basil- Ocimum basilicum L. Indian Perfumer, 1981, 25(3&4): 74-77. [4] Simon JE, Quinn J, Murray RG. Basil: A source of essential oils. In Advances in New Crops; Janik, J; Simon, JE, Eds; Timber Press: Portland. O.R., 1990, pp: 484-489. [5] Keita SM, Vincent C, Schmit J P, Arnason JT, Blanger A. Efficacy of essential oil of Ocimum basilicum L. and O. gratissimum L. applied as an insecticidal fumigant and powder to control Callosobruchus maculatus (Fab.) (Coleoptera: Bruchidae). J Stored Prod Res, 2001, 37(4): 339-349. [6] Janssen AM, Scheffer JJC, Ntezurubanza L, Svendsen AB. Antimicrobial activities of some Ocimum species grown in Rwanda. J Ethnopharmacol, 1989, 26(1): 57-63. [7] Nakamura CV, Ueda-Nakamura T, Bando E, Melo AFN, Cortez DAG, Dias Filho B P. Antibacterial activity of Ocimum gratissimum L. essential oil. Mem Inst Oswaldo Cruz, 1999, 94(5): 675-678. [8] Cimanga K, Kambu K, Tona L, Apers S, de Bruyne T, Hermans N, Tott J, Pieters L, Vlietinck AJ. Correlation between chemical composition and antibacterial activity of essential oils of some aromatic medicinal plants growing in the Democratic Republic of Congo. J Ethnopharmacol, 2002, 79(2): 213-220. [9] Ngassoum MB, Essia-Ngang JJ, Tatsadjieu LN, Jirovetz L, Buchbauer G, Adjoudji, O. Antimicrobial study of essential oils of Ocimum gratissimum leaves and Zanthoxylum xanthoxyloides fruits from Cameroon.

ZOI: net zone of inhibition; OG: Ocimum gratissimum; OK: Ocimum kilimandscharicum

kilimandscharicum was also showed good activity towards tested bacterial strains. The maximum zone of inhibition was observed in Staphylococcus aureus (5.0mm) followed by Enterococcus faecalis and Streptococcus mutans (4.0mm each). Camphor is well-known chemical having antimicrobial activity [25] . Since, the O. kilimandscharicum essential oil was mainly constituted of camphor therefore; the antibacterial activity of the oil could be attributed mainly to this compound.

4. Conclusions
The aim of this study was to explore the potential of O. gratissimum and O. kilimandscharicum essential oils produced during spring-summer cropping season in subtropical region of India. It was noticed that the essential oil composition of O. gratissimum was quantitatively different when it compared with the composition from rain-autumn cropping season. However, essential oil composition of the O. kilimandscharicum was almost similar in both cropping seasons. The antimicrobial activity of these two essential oils is mainly due to the presence of eugenol and camphor which were notice in significant amount in spring-summer season. Thus, it was concluded that O. gratissimum and O.

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