CHEMICAL COMPOSITION AND ANTIMICROBIAL PROPERTIES OF DIFFERENT BASIL OILS

CHEMICAL COMPOSITION AND ANTIMICROBIAL PROPERTIES OF DIFFERENT BASIL OILS

H.C. Srivastava, Pankaj Shukla, Ajay Singh Maurya and Sonia Tripathi*

Department of Chemistry, Research lab, D.B.S. College (R.L.D.B.S.C.), Kanpur- 208 006, Uttar Pradesh, India

ABSTRACT:The aerial parts essential oils of Ocimum basilicum (Lamiaceae) from Togo were steam-distilled and investigated for their percentage composition (GC and GC/MS) and in vitro antimicrobial activities. Five oil chemotypes were identified and classified as follows in line with their principal components: estragole type; linalool/estragole type; methyleugenol type; methyleugenol/t-anethole type; tanethole type. The in vitro microbiological experiments revealed that only the methyleugenol and methyleugenol/t-anethole chemotypes were active against tested fungi and bacteria. Their minimum inhibitory concentrations (MIC) ranged from 80-150 μL/L and from 200-500 μL/L respectively. Likewise, on tested bacteria the MIC varied from 200-400 μL/L and from 250-500 μL/L respectively. These findings are supportive of the potential of both basil oil chemotypes for use as active ingredients in natural antibiotic drugs.

Ocimum basilicum, Lamiaceae, GC and GC/MS, In vitro Antimicrobial activities

INTRODUCTION: The genus Ocimum collectively called basil (in English), includes around 30 plant species from tropical and subtropical areas ^{1, 2}. Ocimum is widely cultivated and extensively used for food, perfumery, cosmetics, pesticides, medicine, and traditional rituals because of their natural aroma and flavour and other properties ^{3, 4}. Literature reports that O. basilicum leaf essential oils or leaf powder have effective insecticidal and pesticidal actvities against Vigna unguiculata pests Callosobruchus maculatus (F) ^{5, 6}.

O. basilicum commonly grows semi wild and is cultivated in Togo at small scale in vegetable gardens. Current domestic uses are only for food and folk medicine ⁷. Nevertheless, a large scale production of the basil essential for well-known value-added applications ^{8, 9} is quite viable under local circumstances. The chemical composition of O. basilicum essential oils has been intensively investigated throughout the world ^{10, 11, 12}, indicating that the estragole chemotype and the linaloolestragole one are the most widely distributed.

At the same time, very little work has been done on the chemical composition and the antimicrobial activities of basil oils from plants growing in India. In addition, there are very few data available either for practical use or for basic research needs about antimicrobial properties of the essential oils of O. basilicum growing in India.

In order to help fill this deficiency, partly at least, the aim of the present work was to investigate basil oils extract of plants sampled in different localities belonging to different ecological areas of India. Specifically, oils percentage composition was determined along with their bacteriostatic and fungistatic activity using 14 bacterial and 14 fungal strains.

The principal finding expected from this applied research is the considered basil oil(s) that could be produced in India for use as source(s) of active ingredients in natural antibiotics or cosmetics.

 MATERIALS AND METHODS:

Plant material sampling and Volatile oils isolation: Leaves and inflorescences of O. basilicum used in this work were harvested from plants at full flowering stage in vegetable gardens in five various locations of India from May to October 2011. Plant specimen was identified by Prof. J.P. Shukla, Department of Botany, Faculty of Science at the D.B.S. (P.G.) College, Kanpur, where voucher specimens were deposited in the herbarium. A sample (50 g) of air-dried plant material was extracted by the hydro distillation technique during 2hours in a modified Clevenger-type glass apparatus ¹³.

The extracted crude essential oils were stored in hermetically sealed dark glass flasks with rubber lids, covered with aluminium foil to protect the contents from light and kept under refrigeration at 4°C until use without any prior purification.

Essential oil analysis by Gas Chromatography: Gas chromatographic analysis was carried out on a Varian 3300 type gas chromatograph equipped with FID detector. An apolar capillary column DB-5 (30 m x 0.25 mm i.d.; film thickness 0.25 μm) and on a polar column supelcowax 10 with the same characteristics as above mentioned were used. DB-5 column operating conditions were as follows: from 50°C (5 min), 50°C to 250°C at the rate of 2°C/min and supelcowax 10 from 50°C (5 min), 50°C to 200°C at 2°C/min. The injector and detector temperatures were respectively 250°C and 300°C. The carrier gas was helium at a flow rate of 1.5 mL/min. Samples (0.2 μL) of undiluted essential oil were injected manually.

Gas Chromatography-Mass Spectrometry Analysis: The GC/MS analysis was carried out on a Hewlett Packard 5890 series II chromatograph, coupled with a mass spectrometer of the Hewlett Packard 5971 series typeoperating in the EI mode at 70 eV. The capillary column type was DB5-MS (30 m x 0.25 mm i.d.; film thickness 0.25 μm). The amount of sample injected and GC/MS parameters were the same as above.

Identification of Components:The components of oils samples were identified by their retention time, retention indices relative to C₈-C₂₄ n-alkanes, computer matching with Wiley 275L library and as well as by comparison of their mass spectra with the authentic samples or with data already available in the literature ^{14, 15}. The percentage of composition of the identified compounds was computed from the GC peak area without any correction factor and was calculated relatively.

In vitro Antifungal Testing:Fungal strains used are listed in Table 2. The fungi were cultivated on a sabouraud agar medium in which was added chloramphenicol 1%, all purchased from BioMerieux Co. (Paris, France). Pure estragole, linalool and methyleugenol commercial standards were also purchased. The antimicrobial activities of the essential oils were assessed according to agar dilution method ^{16, 17}. The tested essential oil and its pure major components from commercial origin were diluted in a minimal quantity of ethanol 95% 1/10 v/v to which was added an aqueous solution of Tween 80 (final concentration of 1% v/v) in order to obtain a homogeneous mixture. The later was incorporated as appropriate to the microbiological culture medium under solidification to obtain final concentrations of the active ingredient that ranged from 10 to 500 μL/L. The mixture was then poured into 3 cm diameter petri dishes.

After solidification fungal strains were respectively sealed as described below:

1. Dermatophytes were seeded with a disc of approximately 2 mm, from a mycelia carpet of preculture, laid in the middle area of a new petri dishes, upper side against the new culture media
2. 1 mL of a suspension of 105 conidia per mL of Aspergillus fumigatus or 105 blastospores per mL of yeast was poured on the surface of the culture medium.

Incubation time and temperatures depend on the fungal strains: 24 hours at 37°C for Candida albicans and Aspergillus, 48 hours at 37°C for Cryptococcus, 14 days at 24°C for the dermatophytes and Scopulariopsis brevicaulis.

Antibacterial testing: Bacterial strains were cultivated on 1.3% (m/v) nutrient broth- conventional antibiotics, are usually reported in human dermatomycosis in tropical and subtropical countries ²².

The African Trichophyton soudanense, a parasite frequent in school environment ^{23, 24} was interestingly also sensitive to the tested essential oils (MICs 100 and 200 μL/L). It was also the case for Cryptococcus neoformans, a hazardous opportunist yeast, which is a resistant germ usually infecting humans affected by HIV/AIDS, which group of patients is known to be generally at high risk with regard to mycosis opportune affections.

TABLE 1: CHEMICAL COMPOSITION OF O. BASILICUM VOLATILE OILS FROM INDIA

  TABLE 2: ANTIFUNGAL ACTIVITIES OF THE DIFFERENT O BASILICUM ESSENTIAL OILS CHEMOTYPES FROM INDIA

* MIC: Minimum inhibition concentration; Ob1: Estragole chemotype; Ob2: Linalol/estragole chemotype ; Ob3 Methyleugenol : chemotype; Ob4 : Methyleugenol/t-anethole chemotype ; Ob5 : t-anethole chemotype; B : Hôpital Saint Jacques de Besançon, France; CIP: Collection Institut Pasteur de Paris Fungal strains

Culture media with or without ethanol 95% and Tween 80 were used as controls. The antimicrobial activities were evaluated by the determination of the minimum inhibitory concentration (MIC). The MIC of a tested active ingredient was determined as the lowest concentration of the test antimicrobial ingredient sample that resulted in a complete inhibition of visible growth of the microorganisms. All tests were carried out in triplicate.

RESULTS AND DISCUSSION: Basil oil extraction yields were in the range of 1.4% to 2.2%, based on plant material dry weight. Identified oil constituents and percentage and their relative percentages are listed in Table 1. Using the relative importance of the oils major constituents as classification criteria permitted to determine five chemotypes as listed below:

i)        Estragole chemotype: It contained mainly estragole (85.50%), with a little amount of 1, 8-cineole (2.25%). The estragole content in this sample was similar to previous findings ^{18, 19, 30}.

ii)      Linalol-estragole chemotype: this type of basil oil contained linalool (41.21%), estragole (22.17%) and (E)-α-bergamotene (7.56%) as major constituents. This chemotype was very close to the European type previously reported by other workers in Nigeria ^{10, 31}.

iii)    Methyleugenol chemotype: This basil oil type contained mainly methyleugenol (74.45%), limonene (6.22%), γ-cadinene (4.07%) and (Z)-β-ocimene (3.16%).

iv)    Methyleugenol and t-anethole chemotype: This essential oil contained mthyleugenol (41.10%) and the t-anethole (32.56%) agar-granulated (NB-AG) culture medium (AES Labortories), in 9 cm diameter petri dishes. After cooling and solidification, the petri dishes were automatically inoculated with the bacterial suspensions 105 CFU/mL with STEERS apparatus. The petri dishes were incubated at 37°C for 48 hours under aerobic conditions. Major constituents: Others meaningful constituent were estragole (10.02%), (E)-α-bergamotene (3.64%) and linalool (2.91%).

v)      t-Anethole chemotype: This sample contained mainly t-anethole (74.64%), linalool (17.30%) and carvacrol (2.69%). To our knowledge, the last three basil oil chemotypes have not yet been reported. O. basilicum has been thoroughly investigated with regards to volatile oil composition but some of our findings are supportive of the idea that continuing chemical inventory on this species is still of scientific interest to support action aimed at plant biodiversity knowledge, conservation and sustainable exploitation.

The findings on the chemical composition of basil oils in Togo were very instructive with regard to the possible applications. Hence, the estragole chemotype found here resembled that described ^{19, 32}, which is well-known for its valuable usage in perfumery and food ²⁰. In addition, this chemotype has been recently indicated as a possible pesticide against larva and adult of Callosobruchus maculatus known as a major postharvest pest of Vigna unguiculata in storage ^{5, 6}.

The pesticidal activity of the volatile oil of Clausena anisata, containing mainly estragole and t-anethole^{20, 21}.Such indications on the pesticidal properties of thisessential oil containing estragole and t-anethole iscertainly indicative of the biocide potential of thesenatural molecules which might be endowed with antimicrobial properties ^{22, 32}, our focus in this investigation.

The experimental data in Table II show that all fungal strains tested were sensitive to the metyleugenol type of basil oil, but dermatophytes and pathogenic yeasts were more particularly affected. Hence, markedly low MICs (80 μL/L) were recorded with T. Mentagrophytes var mentagrophytes. Along with dermatophytes, imperfect filamentous fungi were also highly sensitive to the test volatile oil; MICs were in the range of 100- 150 μL/L. Likewise, the methyleugenol oil chemotype appeared very toxic to pathogenic yeast strains including Candida, Cryptococcus, Aspergillus, and Scopulariopsis, MICs varying from 80 to 100 μL/L. Also noticeable was the antifungal effect (MIC: 150 μL/L and 200 μL/L) of this oil chemotype on both strains of Scytalidium sp. The later, which often resist?

TABLE 3: ANTIFUNGAL ACTIVITIES OF THE DIFFERENT BASIL ESSENTIAL OILS CHEMOTYPES FROM TONGO 

* MIC: Minimum inhibitory concentration; **Ob1 : Estragole chemotype ; Ob2 : Linalol/ estragole chemotype; Ob3 : Methyleugénol chimiotype; Ob4 :Methyleugenol/t-anethole chemotype;Ob5 : t-anéthole chemotype; *** : Bacteria strains responsible of bad odours; IP : Bacteria strains provided by Institut Pasteur de Paris; L, LH : Bacteria strains provided by Dr. Marshall,Leeds University Microbiology Laboratory (United Kingdom)

The high antifungal activity of this basil oil chemotype on those pathogenic fungi like dermatophytes, filamentous fungi and yeasts confirmed the excellent fungal growth inhibition properties previously reported as a characteristic of essential oils rich in methyleugenol and/or other phenol derivatives ²⁵.

In this work there is no doubt that the antifungal activity of O. Basilicum chemotype methyleugenol against tested fungi is a predictable consequence of its high content in methyleugenol known as one of the phenolic volatile molecules endowed with antimicrobial properties ^{25, 26}. In comparison, commercial linalool and estragole tested as standards in this study were found noneffective unlike pure methyleugenol standard.

The methyleugenol/t-anethole chemotype showed a moderate antifungal activity only on dermatophytes with MICs ranged from 250-300 μL/L but not filamentous fungi and yeasts were not affected. The estragole type, the linalool/estragole type, and the tanethole type were ineffective on all fungal strain tested in this work. A lesson learned from the findings

mentioned above was that estragole and t-anethole, which are reported as possible natural pesticides, could not serve as natural fungicidal ingredients unlike methyleugenol against the fungal strains tested in this work. This simply indicates the broader understanding of what is generically termed to as the biological activity of essential oils.

Table 3 shows that all feet microflora bacterial strains tested were only sensitive to methyleugenol and methyleugenol/t-anethole oil chemotypes.

The MICs, which were in the range of 200-500 μL/L, were slightly higher than those recorded on fungal strains in this study, indicating a lesser activity. The results recorded against common armpit microflora bacterial strains, were especially promising for a possible use against Corybacterium xerosis, the principal one of the bacteria responsible for bad odours. Its growth was interestingly inhibited with at a dose of 100 μL/L antibacterial test suspension. Note that this bacteria metabolizes human steroids produced via transpiration to yield small and bad-smelling volatile molecules ².

The findings in this study indicated bacteriostatic effects against feet microflora germs like Staphylococcus epidermidis and Staphylococcus hominis (MIC: 200 and 300 μL/L) partly responsible for feet bad odours ²⁷. Methyleugenol and methyleugenol/t-anethole oil chemotypes samples were also very active against the Coryneform (Corynebacterium gr B, C and D2) (MIC: from 100-300 μL/L) which produce proteinase and lipase, both enzymes that cause feet surface skin bad odours ²⁷. Both oil chemotypes also showed a moderate antibacterial activity against Gram-negative bacteria (Acinetobacter sp, Moraxella sp.: MIC from 250- 500 μL/L) and Grampositve bacteria (Micrococcus luteus, Staphylococcus epidermidis: MIC: from 200-500 μL/L). The later are involved in nosocomial infections ^{28, 29}.

The proven antibacterial activities of the methyleugenol and metyleugenol/t-anethole types of basil oil found in this study are quite indicative of their potential as possible active ingredients for use in the formulation of deodorants for armpits or feet. Developing sustainable large scale consumer goods like natural drugs and cosmetics based on basil oil as well as on other essential oils bearing aromatic plants growing in Togo is of an obvious economic interest while helping preserve plant biodiversity.

In conclusion, the prospective investigation of the percentage composition of O. basilicum essential oils along with the evaluation of their antimicrobial properties on human normal cutaneous microflora bacterial and fungal strains is quite a typical applied research. The ultimate goal of this type of investigationis to help protect plant biodiversity and validate scientific basis the potential of some valuable species whether for food or non-food applications.

The present study has shown that it is feasible to use some kinds of the leaf essential oil of O. basilicum growing in India as a natural powerful antimicrobial ingredient whether in traditional or in modern medicines as well as in cosmetics. Such oils are the methyleugenol and the methyleugenol/t-anethole types. Conversely, the estragole type, the linalool/ estragole type, and the tanethole type, which were also included in the investigated samples, did show any useful antimicrobial properties.

ACKNOWLEDGMENT: The lead author of this report wishes to thank University Grants Commission for financial assistance, authors thankful to Dr. Nagendra Swarup, Honourable Secretary of Board of Management of D.B.S. (P.G.) College Kanpur and Dr. A.K. Srivastava, Principal, D.B.S. College, Kanpur for providing research environment this   investigation.

Special regards also to Dr. R.K. Khare (H.O.D), Dr. Ashok Mishra, Dr. Sunil Prasad, Dr C.P. Singh, for taking time out of their busy schedules, during their sabbatical periods, to edit this manuscript.

REFERENCES:

1. Paton A. A synopsis of Ocimum L. (Labiatae) in Africa. Kew Bull. 1992; 47: 403-36.
2.  Rennie PJ, Gower DB, Holland KT. In vitro studies of human axillary odour and cutaneous microflora. Br J Dermatol. 1991; 124: 596-602.
3.  Albuquerque UP. de Taxonomia e ethnobotânica do gênero Ocimum L. (Lamiaceae) no Nordeste do Brasil-renfêrencia especial para Pernambuco. Recife. Dissertação (Mestradoem Biologia Vegetal)- Centro de Ciencias Biológicas, Universidade Federal de Pernambuco, 1996, p 125
4. Darrah H. Investigation on cultivars of Basils (Ocimum). Econ Bota. 1974; 28: 63-67.
5. Keita SM, Vincent C, Schmit J, Arnason JT, Belanger A. Efficacy of oil of Ocimum basilicum L. and O. gratissimum L. applied as an insecticidal fumigant and powder to control Callosobruchus maculatus (Fab). J Stored Prod Res. 2001; 37: 339-49.
6. Ketoh KG, Glitoh AI, Huignard J. Susceptibility of the bruchus Callosobruchus maculatus (Coleoptera: Bruchidae) and its parasitoid Dinarmus basalis (Hymenopterae: Pteromatidae)to three essential oils. J Econ Entomol. 2002; 95: 174-82.
7. Adjanohoun E, Ahyi MRA, Aké Assi L, Akpagana K, Chibon P, El-Hadj A, Eymen I, Goutote E, Ginko S, Hodouto KK, Hougnon P, Keita A, Kéoula Y, Klouga-Ocloo WP, Lo I, Siamevi K, Taffame KK, Garba M, Gassita JN, Gbeassor M. Médecine traditionnelle et Pharmacopée: Contribution aux études ethno-botaniques et floristiques du Togo. ACCT, Paris, 1986.
8. Berrada M, Ait Igri M, Il Idrissi A, Bellakdar. Contribution à l’analyse de l’huile essentielle d’un cultivar d’Ocimum basilicum L. du Maroc. Al Buruniya, Rev Mar Pharm. 1987; 3: 137-44.
9. Archtander S. Perfume and flavour material of natural originCarol Steam, Allured Publishing Corporation, 1994, p 81.
10. Ekundayo O, Laakso I, Oguntimein B, Okogun JL, Elujoba A, Hulbinen A. Essential oil of Ocimum basilicum from Nigeria. Acta Pharm Fen. 1987; 96: 101-06.
11. Sanda K, Koba K, Nambo P, Gaset A. Chemical investigation of Ocimum species growing in Togo. Flav Fragr J. 1998; 13: 226-32
12. Yayi E, Moudachirou M, Chalchat JC. Chemotyping of three Ocimum species from Benin: O. basilicum, O. canum and O. gratissimum . J Essent Oil Res. 2001; 13: 13-17.
13. Craveiro AA, Matos FJ, Alencar JW. A simple and inexpensive steam generator for essential oils extraction. J Chem Ed. 1976; 53: 652.
14. Kondjoyan N, Berdagué JL. A compilation of relative retention indices for the analysis of aromatic compounds. Ed. Laboratoire Flaveur, INRA de Theix, France, 1996.Koumaglo K, Dotse K, Akpagana K, Garneau FX, Gagnon H, Jean IF, Moudachirou M, Addae-Mensah I. Analyse des huiles essentielles de deux plantes aromatiques du Togo. Riv Ital EPPOS. 1996; 7: 680-91.microbiology of normal human feet. J Appl Bacteriol. 1987; 62: 39-46.
15. Adams RP. Identification of essential oil components by gas chromatography/ quadrupole mass spectroscopy. Carol Stream, Ilinois, Allured Publishing Corporation, 2001.
16. Griffin SG, Markham JL, Leach DN. An agar dilution method for the determination of the minimum inhibitory concentration of essential oils. J Essent Oil Res. 2000; 12: 249- 55.
17. Benjilali B, Tantaoui-Elaraki A, Ismaïlou-Alaoui M, Ayadi A. Méthode d'étude des propriétés antiseptiques des huiles essentielles par contact direct en milieu gélosé. Plant Méd Phytother. 1986; 20: 155–67.
18. Guenther E. The essential oils. Vol. 2. New York, Van Nostrand, 1949, 16-18, 503-05.
19. Baritaux O, Richard H, Touche J, Dersbesy M. Effects of drying and storage of herbs and spices on the essential oil. Part I. Basil, O. basilicum L. Flav Fragr J. 1992; 7: 227-71.
20. Moudachirou M, Ayedou MA, Gbenou J, Garneau FX, Koumaglo KH, Mensah-Addae I. Composition chimique des huiles essentielles des feuilles de Clausena anisata récoltées dans la sous-region Bénin-Togo-Ghana. J Soachim. 1997; 3: 49-54.
21. Okunde AL, Olaifa JL. Estragole: An acute toxic principle from the volatile oil of the leaves of Clausena anisata. J Nat Prod. 1987; 50: 990 91.
22. Alvarez P, Enriquez AM, Toro C, Martinez I, Buhigas I, de Miguel S, Lago M, Puente S, del Palacio A, Baquero M. Dermatomiucosis de importación por Scytalidium dimidiatum: A propósito de tres casos. Rev Iberoam Micol. 2000; 17: 102–06.
23. Ouaffak L, Gati A, Lyagoubi M. Les teignes du cuir chevelu dans les écoles Primaires de Khermisset (Maroc). J Mycol Méd. 2001; 114: 181-84.
24. Vandemeulebroucke BT, Mounkassa B, Deloye J, Jousserand P, Poujade F, Petithory JC. Teignes du cuir chevelu en milieu scolaire rural au Mali. J Mycol Med. 1999; 92: 11-13.
25. Friedman M, Henika PR, Mandrell RE. Bactericidal activities of plant essential oils and some of their isolated constituents against Campylobacter jejuni, Escherichia coli, Listeriamonocytogenes, and Salmonella enterica. J Food Prot. 2002; 65: 1545 60
26. Viollon C, Chaumont JP. Antifungal properties of essential oils and their main components upon Cryptococcus neoformans. Mycopathologia 1994; 128: 151-53
27. Marshall J, Holland KT, Gribbon EM. A comparative study of cutaneous microflora of normal feet with low and high levels of odour. J Appl Bacteriol. 1988; 65: 61-68.
28. Bergogne-Bérézin E. The increasing significance of outbreaks of Acinetobacter spp.: The need for control and new agents. J Hosp Infect. 1995; 30: 441-52.
29. Center for Disease Control and Prevention (CDC). National nosocomial infections surveillance system report. Data sumary from January 1990-May 1990. Am J Infect Control1999; 27: 520-32.
30. Chaumont JP, Mandin D, Sanda K, Koba K, De Souza C. Activités antimicrobiennes de cinq huiles essentielles de lamiacées togolaises vis à vis de germes représentatifs de la microflore cutanée. Acta Bot Gallica. 2001; 148: 93-101.
31. Chaumont JP, Leger D. Propriétés antifongiques de quelques phénols et de composés chimiquement très voisins. Relations structure-activité. Plant Med Phytother. 1989; 23: 124-28.
32. Baba-Moussa F, Koumaglo K, Ayédoun K, Akpagana K, Moudachirou M, Bouchet P. Activité antifongique d’huiles essentielles extraites au Bénin et au Togo. Cryptogamie, Mycol. 1997; 18: 165-68.
33. Kayibou G. Les Eucalyptus du Congo. Variations inter et intraspécifiques du rendements et de la composition de leurs huiles essentielles. Thèse de doctorat Université de Montpellier II, France, 1992, 112.
    

    ---

    How to cite this article:

    Srivastava HC, Shukla P, Maurya AS and Tripathi S: Chemical composition and Antimicrobial properties of different Basil oils. Int J Pharm Sci Res 2013; 4(3); 1210-1216.

    ---