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- v.33(3); Jan-Mar 2014
- PMC4264302
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Anc Sci Life. 2014 Jan-Mar; 33(3): 151–156.
PMCID: PMC4264302
PMID: 25538349
Rajesh K. Joshi
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Abstract
Context:
Ocimum basilicum L. (Lamiaceae) commonly known as sweet basil, has been used as a traditional medicinal plant for the treatment of headaches, coughs, diarrhea, constipation, warts, worms, and kidney malfunctions.
Materials and Methods:
The essential oil of the flowering aerial parts of O. basilicum growing in the Western Ghats region of North West Karnataka, India, was obtained by hydro-distillation and analyzed by gas chromatography equipped with flame ionization detector and gas chromatography coupled to mass spectrometry (GC–MS). The oil was tested against six Gram-positive, eight Gram-negative bacteria, and three fungi by the tube-dilution method at a concentration range of 5.00-0.009 mg/mL.
Results:
Twenty-five constituents were identified in the essential oil of O. basilicum. The major constituents were identified as methyl eugenol (39.3%) and methyl chavicol (38.3%), accounting for 98.6% of the total oil. The oil was found to be active against Gram-positive, Gram-negative bacteria, and fungi with minimal bactericidal concentration values in the range of 0.143 ± 0.031 to 0.572 ± 0.127 mg/mL, 0.781 ± 0.382 to 1.875 ± 0.684 mg/mL, and 0.312 ± 0.171 to 0.442 ± 0.207 mg/mL, respectively.
Conclusion:
The essential oil of O. basilicum of this region contains methyl eugenol/methyl chavicol chemotype and has bactericidal properties.
KEY WORDS: Bactericidal property, essential oil composition, gas chromatography-mass spectrometry, Lamiaceae, methyl chavicol, methyl eugenol, Ocimum basilicum L.
INTRODUCTION
Ocimum basilicum L. (sweet basil) belongs to the family Lamiaceae, which includes about 200 species occur in various botanic varieties and forms.[1] Traditionally, sweet basil has been used as a medicinal plant in the treatment of headaches, coughs, diarrhea, constipation, warts, worms, and kidney malfunctions.[2] O. basilicum is a popular culinary herb and a source of essential oils extracted by steam distillation from the leaves and the flowering tops which are used to flavor foods, in dental and oral products, and in fragrances.[3,4,5] The aromatic character of each type of basil is determined by genotype and depends on the major chemical compounds of essential oils primarily consisting of monoterpenes and phenylpropanoids.[6,7] The essential oil has antimicrobial,[8] antifungal, and insect-repelling,[9] anticonvulsant, hypnotic,[10] and antioxidant[11] activities. Various workers have reported chemical composition of the essential oil of O. basilicum from different parts of the world is summarized in Table 1.[12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27] In a study, on 270 sweet basil accessions, the major constituents were found to be linalool, methyl chavicol, or citral and 1,8-cineole, camphor, thymol, methyl cinnamate, eugenol, methyl eugenol, methyl isoeugenol, and elemicine.[28] According to Marotti et al.,[6] the European basil type has linalool and methyl chavicol as the major oil constituents. The reunion basils, another chemotype have methyl chavicol as a major constituent, whereas tropical chemotypes of basil have methyl cinnamate as the major constituent. Another basil chemotype grown in North Africa, Russia, Eastern Europe, and parts of Asia has eugenol as the major constituent.[6] Reports on the chemical composition of the essential oil of O. basilicum from Western Ghats region of Karnataka are very inadequate, and there is no report on the terpenoid composition of this plant from this region. Hence, this study was carried out to describe detailed chemical investigation and antimicrobial property of the essential oil of O. basilicum from Western Ghats region (one of the 34 global biodiversity hotspots[29]).
Table 1
Major constituents of the essential oil of Ocimum basilicum from different countries
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MATERIALS AND METHODS
Plant material
The flowering aerial parts of O. basilicum were collected in May 2011, at the height of 800 m from district Belgaum (N 15.88668; E 74.52353), Karnataka, India. The plant was identified by Dr. Harsha Hegde, Research Scientist, Regional Medical Research Centre (Indian Council of Medical Research), Belgaum (voucher specimen No. RMRC-532).
Isolation of essential oil
The fresh plant material (500 g) was subjected to hydro - distillation using Clevenger type apparatus for 3 h. The oil was collected and dried over anhydrous sodium sulfate and stored in sealed vials at −4°C until analysis.[30] The oil yield was 0.21% v/w.
Gas chromatography
The gas chromatography (GC) analysis of the oil was carried out on Varian 450 gas chromatograph equipped with flame ionization detector (FID), using stationary phase CP Sil-8-CB (30 m × 0.25 mm i.d., 0.25 μm film thickness) column under the experimental conditions reported earlier.[31,32] Nitrogen was a carrier gas at 1.0 mL/min flow rate. Temperature was increased at the rate of 3°C/min between 60 and 220°C. Injector and detector temperatures were 230 and 250°C, respectively. The injection volume was 1.0 μL diluted in n-hexane; split ratio was 1:50.
Gas chromatography-mass spectrometry
The GC–mass spectrometry (MS) analysis of the oil was carried out on Thermo Scientific Trace Ultra GC interfaced with a Thermo Fisher Scientific SpA. Strada Rivoltana, 20090 Rodano-Milan, Italy. fitted with TG-5 (30 m × 0.25 mm i.d., 0.25 μm film thickness) column. The oven temperature was increased at the rate of 3°C/min between 60 and 220°C using helium as a carrier gas at 1.0 mL/min. The injector temperature was 230°C, injection size 0.1 μL prepared in n-hexane; split ratio 1:50. MS were taken at 70 eV with mass scan range of 40-450 amu.[33,34]
Identification of the components
Identification of constituents was done on the basis of retention index (RI) (determined with reference to the hom*ologous series of n-alkanes C8-C25, under identical experimental condition), MS library search (NIST and WILEY), and by comparison with MS literature data.[35] The relative amounts of individual components were calculated based on GC peak area (FID response) without using the correction factor.
Antimicrobial strains
The microorganisms screened for antimicrobial activity were obtained from the National Collection of Industrial Microorganisms (NCIM), National Chemical Laboratory, Pune. The microorganisms were Staphylococcus aureus (NCIM 2079), Staphylococcus epidermidis (NCIM 2493), Streptococcus faecalis (NCIM 2080), Micrococcus flavus (NCIM 2379), Micrococcus luteus (NCIM 2103), Bacillus subtilis (NCIM 2063) (Gram-positive); Escherichia coli (NCIM 2574), Enterobacter aerogenes (NCIM 2694), Klebsiella pneumoniae (NCIM 2957), Pseudomonas aeruginosa (NCIM5029), Proteus vulgaris (NCIM 2813), Proteus mirabilis (NCIM 2241), Serratia marcescens (NCIM 2078), Salmonella typhimurium (NCIM 2501) (Gram-negative bacteria), Aspergillus niger (NCIM 620), Aspergillus fumigatus (NCIM 902), and Penicillium chrysogenum (NCIM 733) (fungi).
Preparation of test sample
The essential oil of the flowering aerial parts of O. basilicum was dissolved in 10% dimethylsulfoxide (DMSO), which is reported to be nontoxic to microorganisms at this concentration,[36,37] with Tween 80 (1% v/v for easy diffusion). Erythromycin (Alembic Ltd., Solan, Himachal Pradesh, India), amikacin (Iskon Remedies, Sirmour, Himachal Pradesh, India), and amphotericin B (Chandra Bhagat Pharma Pvt. Ltd., Ankleshwar, India) were used as a positive reference standard for Gram-positive, Gram-negative bacteria and fungi, respectively.
Preparation of inocula
The inocula of bacterial strains were prepared from 18 h old cultures using nutrient broth, and Sabourad's dextrose broth was used for fungi. The suspensions were adjusted to 0.5 of the McFarland standard turbidity -104 for bacteria and -103 for fungal colony forming units (CFU)/mL.[38]
Antimicrobial assay
The tube-dilution method was used to determine the minimum inhibitory concentration (MIC) of the essential oil of O. basilicum against the microorganisms under study. The oil was dissolved in 10% DMSO with Tween 80 (1% v/v for easy diffusion). The final concentration of the oil was 5.00 mg/mL. Serial two-fold dilutions were prepared from the stock solution to give concentrations ranging from 5.00 to 0.009 mg/mL of the essential oil for bacteria and fungi.[39] Erythromycin, amikacin, and amphotericin B were dissolved in sterile distilled water, and two-fold dilutions were prepared (1.0-0.002 mg/mL). About 1 mL of each concentration was mixed with 1.0 mL of sterile nutrient broth for bacteria at 104 CFU/mL concentrations, while Sabourad's dextrose broth for fungi at 103 CFU/mL concentrations obtained from McFarland turbidity (standard no. 0.5). Negative control was prepared with DMSO (10%) and Tween 80 (1% v/v), and blank control from virgin media. Tubes were incubated for 24 and 48 h at 37°C for bacteria and fungi, respectively. MIC was determined as the lowest concentration that inhibited the visible microbial growth.[40,41] The minimal bactericidal concentration (MBC) determination, 0.1 mL of the culture in each tube of MIC without visible growth was spread on nutrient agar plate and incubated for 24 and 48 h at 37°C for bacteria and fungi, respectively. The highest dilution at which 99.9% of the bacteria and fungi inoculum were killed was considered the MBC. The assays were replicated, and the mean value of six experiments was recorded (n = 6) with a standard error of the mean. The statistical analysis was performed using Graph Pad InStat software San Diego, California, USA.
RESULTS AND DISCUSSION
Twenty-five compounds were characterized and identified by GC–MS, comprising 98.6% of the total oil. The identified compounds are listed in Table 2 in elution order from the TG-5 column [Figure 1], along with the percentage composition of each component and its RI. The major constituents were methyl eugenol (39.3%) [Figure 2], and methyl chavicol (38.3%) [Figure 3]. Other minor constituents were terpinolene (7.7%), eugenol (4.5%), and cubenol (1.9%). On the basis of >200 analyses of essential oils isolated from O. basilicum classified four major essential oil chemotypes of basil: (1) Methyl chavicol-rich, (2) linalool-rich, (3) methyl eugenol-rich, (4) methyl cinnamate-rich, and also numerous subtypes.[42] The presence of essential oils and their composition determines the specific aroma of plants and the flavor of the condiment. Not only the type of cultivar but also the agronomical practices and environmental conditions affect the composition of sensory important compounds.[43,44] The presence of methyl eugenol and methyl chavicol has been reported in the high percentage in this region is contrary from the northern and rest of the southern part of India, suggested the methyl eugenol/methyl chavicol chemotype essential oil of O. basilicum was found from this region. This quantitative and qualitative divergence may be due to the geographical, climatic, and soil conditions in the southern part of India, which in turn may affect the composition and other secondary metabolites of the plant.
Table 2
Chemical composition of the essential oil of Ocimum basilicum
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Figure 1
Gas chromatography-total ion current of the essential oil of Ocimum basilicum
Figure 3
Mass spectra and structure of methyl chavicol
The antimicrobial activity expressed as mg/mL, of the essential oil of O. basilicum against various strains of bacteria and fungi is summarized in Table 3. The organisms S. aureus, B. subtilis, A. fumigatus, S. faecalis, S. epidermidis, P. chrysogenum, and A. niger were found to be more susceptible to the oil with MBC values of 0.143 ± 0.031, 0.260 ± 0.080, 0.312 ± 0.171, 0.364 ± 0.127, 0.416 ± 0.415, 0.416 ± 0.161, and 0.442 ± 0.207 mg/mL, respectively. The organisms M. flavus, M. luteus, P. mirabilis, P. vulgaris, and P. aeruginosa were found moderately susceptible with the MBC values of 0.520 ± 0.161, 0.572 ± 0.127, 0.781 ± 0.382, 0.833 ± 0.322 and 0.937 ± 0.342, respectively. The microorganisms E. aerogenes, S. marcescens, S. typhimurium, E. coli, and K. pneumoniae were less susceptible and showed higher MBC values (MBC > 1.0 mg/mL). The observation of MBC assay suggested that the oil has bactericidal property. According to Wan et al.,[45] the majority of the essential oils assayed for their antibacterial properties showed a more pronounced effect against the Gram-positive bacteria. The resistance of Gram-negative bacteria to essential oil has been ascribed to their hydrophilic outer membrane which can block the penetration of hydrophobic compounds into target cell membrane.[46] The presence of phenolic components in the essential oil could be contributing for antimicrobial activity by causing leakage of intracellular ATP and potassium ions leading to cell death.[47,48]
Table 3
MBC values (mg/mL) of the essential oil of Ocimum basilicum
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ACKNOWLEDGMENT
The author is grateful to the Indian Council of Medical Research, New Delhi, India for providing necessary facilities. The author is thankful to Miss. Vijaylaxmi Badakar, Lab Assistant for her kind assistance for screening of antimicrobial activity of the oil.
Footnotes
Source of Support: Nil.
Conflict of Interest: None declared.
REFERENCES
1. Wierdak RN. Analiza zawartosci i składu chemicznego olejku dwoch form bazylii wonnej (Ocimum basilicum L.) Ann Univ Med Curie Sklodowska Sect EEE. 2001;10(Suppl):189–193. [Google Scholar]
2. Simon JE, Morales MR, Phippen WB, Vieira RF, Hao Z. Perspectives on New Crops and New Uses. Alexandria, VA: ASHS Press; 1999. A source of aroma compounds and a popular culinary and ornamental herb. [Google Scholar]
3. Akgul A. Volatile oil composition of sweet basil (Ocimum basilicum L.) cultivating in Turkey. Nahrung. 1989;33:87–8. [Google Scholar]
4. Guenther E. New York: Van Nostrand; 1952. The Essential Oils. [Google Scholar]
5. Heath HB. Westport: Avi Publishing; 1981. Source Book of Flavour. [Google Scholar]
6. Marotti M, Piccaglia R, Giovanelli E. Differences in essential oil composition of basil (Ocimum basilicum L.) Italian cultivars related to morphological characteristics. J Agric Food Chem. 1996;44:3926–9. [Google Scholar]
7. Tateo F. The composition of various oils of Ocimum basilicum L. J Essent Oil Res. 1989;1:137–8. [Google Scholar]
8. Lahariya AK, Rao JT. In vitro antimicrobial studies of the essential oil of Cyperus scariosus and Ocimum basilicum. Indian Drugs. 1979;16:150–2. [Google Scholar]
9. Dube S, Upadhyay PD, Tripathi SC. Antifungal, physicochemical and insect-repelling activity of the essential oil of Ocimum basilicum. Can J Bot. 1989;67:2085–7. [Google Scholar]
10. Ismail M. Anticonvulsant and hypnotic activities central properties and chemical composition of Ocimum basilicum L. essential oil. Pharm Biol. 2006;44:619–26. [Google Scholar]
11. Politeo O, Jukic M, Milos M. Chemical composition and antioxidant capacity of free volatile aglycones from basil (Ocimum basilicum L.) compared with its essential oil. Food Chem. 2007;101:379–85. [Google Scholar]
12. Mohiuddin M, Chowdhury MJ, Alam MK, Hossain MK. Chemical composition of essential oil of four flavouring plants used by the tribal people of Bandarban hill district in Bangladesh. Int J Med Aromat Plants. 2012;2:106–13. [Google Scholar]
13. Oliveira JS, Porto LA, Estevam CS, Siqueira RS, Alves PB, Niculau ES, et al. Phytochemical screening and anticonvulsant property of Ocimum basilicum leaf essential oil. Bol. Latinoam. Caribe. Plant Med Aromat. 2009;8:195–202. [Google Scholar]
14. Klimankova E, Holadova K, Hajslova J, Cajka T, Poustka J, Koudela M. Aroma profiles of five basil (Ocimum basilicum L.) cultivars grown under conventional and organic conditions. Food Chem. 2008;107:464–72. [Google Scholar]
15. Keita SM, Vincent C, Schmit JP, Belanger A. Essential oil composition of Ocimum basilicum L., O. gratissimum L. and O. suave L. in the Republic of Guinea. Flavour Fragr J. 2000;15:339–41. [Google Scholar]
16. Vani SR, Cheng SF, Chuah CH. Comparative study of volatile compounds from genus Ocimum. Am J Appl Sci. 2009;6:523–8. [Google Scholar]
17. Zheljazkov VD, Cantrel CL, Evans WB, Ebelhar MW, co*ker C. Yield and composition of Ocimum basilicum L. and Ocimum sanctum L. grown at four locations. HortScience. 2008;43:737–41. [Google Scholar]
18. Purkayastha J, Nath SC. Composition of the camphor-rich essential oil of Ocimum basilicum L. native to Northeast India. J Essent Oil Res. 2006;18:332–4. [Google Scholar]
19. Hanif MA, Al-Maskari MY, Al-Maskari A, Al-Shukaili A, Al-Maskari AY, Al-Sabahi JN. Essential oil composition, antimicrobial and antioxidant activities of unexplored Omani basil. J Med Plant Res. 2011;5:751–7. [Google Scholar]
20. Katarzyna DK. Biological value and essential oil content in sweet basil (Ocimum basilicum L.) depending on calcium fertilization and cultivar. Acta Sci Pol. 2010;9:153–61. [Google Scholar]
21. Benedec D, Oniga I, Oprean R, Tamas M. Chemical composition of the essential oils of Ocimum basilicum L. cultivated in Romania. Farmacia. 2009;57:625–9. [Google Scholar]
22. Kathirvel P, Ravi S. Chemical composition of the essential oil from basil (Ocimum basilicum Linn.) and its in vitro cytotoxicity against HeLa and HEp-2 human cancer cell lines and NIH 3T3 mouse embryonic fibroblasts. Nat Prod Res. 2012;26:1112–8. [PubMed] [Google Scholar]
23. Bunrathep S, Palauvej C, Ruangrungsi N. Chemical compositions and antioxidative activities of essential oils from four Ocimum species endemic to Thailand. J Health Res. 2007;21:201–6. [Google Scholar]
24. Hassanpouraghdam MB, Gohari GR, Tabatabaei SJ, Dadpour MR. Inflorescence and leaves essential oil composition of hydroponically grown Ocimum basilicum L. J Serbe Chem Soc. 2010;75:1361–8. [Google Scholar]
25. Hassanpouraghdam MB, Hassani A, Shalamzari MS. Menthone and estragole rich essential oil of cultivated Ocimum basilicum L. from Northwest Iran. Chemija. 2010;21:59–62. [Google Scholar]
26. Ozcan M, Chalchat JC. Essential oil composition of Ocimum basilicum L. and Ocimum minimum L. in Turkey. Czech J Food Sci. 2002;20:223–8. [Google Scholar]
27. Lee SJ, Umano K, Shibamoto T, Lee KG. Identification of volatile components in basil (Ocimum basilicum L.) and thyme leaves (Thymus vulgaris L) and their antioxidant properties. Food Chem. 2005;91:131–7. [Google Scholar]
28. Kruger H, Wetzel SB, Zeiger B. The chemical variability of Ocimum species. J Herbs Spices Med Plants. 2002;9:335–44. [Google Scholar]
29. Myers N, Mittermeier RA, Mittermeier CG, Gustavo AB, Fonseca DA, Kent J. Biodiversity hotspots for conservation priorities. Nature. 2000;403:853–8. [PubMed] [Google Scholar]
30. Joshi RK, Badakar V, Kholkute SD. Carvacrol rich essential oils of Coleus aromaticus (Benth.) from Western Ghats region of North West Karnataka, India. Adv Environ Biol. 2011;5:1307–10. [Google Scholar]
31. Joshi RK. Chemical composition of Senecio belgaumensis from India. Chem Nat Compds. 2012;47:1010–1. [Google Scholar]
32. Joshi RK, Pande C, Mujawar MH, Kholkute SD. Chemical composition and antimicrobial activity of the essential oil of Anaphalis nubigena var. monocephala. Nat Prod Commun. 2009;4:993–6. [PubMed] [Google Scholar]
33. Joshi RK. GC/MS analysis of the essential oil of Senecio belgaumensis flowers. Nat Prod Commun. 2011;6:1145–6. [PubMed] [Google Scholar]
34. Joshi RK. Pulegone and menthone chemotypes of Mentha spicata Linn. from Western Ghats region of North West Karnataka, India. Natl Acad Sci Lett. 2013;36:349–52. [Google Scholar]
35. Adams RP. Carol Stream, Illinois, USA: Allured Publication; 2007. Identification of Essential Oil Components by Gas Chromatography/Mass Spectroscopy. [Google Scholar]
36. Pujol V, Seux V, Villard J. Recherche de substances antifongiques secretes par les champignons superieurs en culture. Ann Pharm Fr. 1990;48:17–22. [PubMed] [Google Scholar]
37. Joshi RK, Badakar V. Chemical composition and in vitro antimicrobial activity of the essential oil of the flowers of Tridax procumbens. Nat Prod Commun. 2012;7:941–2. [PubMed] [Google Scholar]
38. McFarland J. Standardization of bacterial culture for the disc diffusion assay. J Am Med Assoc. 1987;49:1176–8. [Google Scholar]
39. Joshi RK. Volatile composition and antimicrobial activity of the essential oil of Artemisia absinthium growing in Western Ghats region of North West Karnataka, India. Pharm Biol. 2013;51:888–92. [PubMed] [Google Scholar]
40. Murthy MM, Subramanyam M, Giridhar KV, Jetty A. Antimicrobial activities of bharangin from Premna herbaceae Roxb. and bharangin monoacetate. J Ethnopharmacol. 2006;104:290–2. [PubMed] [Google Scholar]
41. Joshi RK. Chemical constituents and antibacterial property of the essential oil of the roots of Cyathocline purpurea. J Ethnopharmacol. 2013;145:621–5. [PubMed] [Google Scholar]
42. Lawrence BM. Amsterdam: Elsevier Science Publisher BV; 1988. A Further Examination of the Variation of Ocimum basilicum L. Flavors and Fragrances: A World Perspective. [Google Scholar]
43. Jirovetz L, Buchbauer G, Shafi MP, Kaniampady MM. Chemotaxonomical analysis of the essential aroma compounds of four different Ocimum species from southern India. Eur Food Res Technol. 2003;217:120–4. [Google Scholar]
44. Vina A, Murillo E. Essential oil composition from twelve varieties of basil (Ocimum spp.) grown in Columbia. J Braz Chem Soc. 2003;14:744–9. [Google Scholar]
45. Wan J, Wilco*ck A, Coventry MJ. The effect of essential oils of basil on the growth of Aeromonas hydrophila and Pseudomonas fluorescens. J Appl Microbiol. 1998;84:152–8. [PubMed] [Google Scholar]
46. Inouye S, Yamaguchi H, Takizawa T. Screening of the antibacterial effects of a variety of essential oils on respiratory tract pathogens, using a modified dilution assay method. J Infect Chemother. 2001;7:251–4. [PubMed] [Google Scholar]
47. Ultee A, Kets EP, Smid EJ. Mechanisms of action of carvacrol on the food-borne pathogen Bacillus cereus. Appl Environ Microbiol. 1999;65:4606–10. [PMC free article] [PubMed] [Google Scholar]
48. Juven BJ, Kanner J, Schved F, Weisslowicz H. Factors that interact with the antibacterial action of thyme essential oil and its active constituents. J Appl Bacteriol. 1994;76:626–31. [PubMed] [Google Scholar]
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