American Journal of Food Technology

Year: 2011 | Volume: 6 | Issue: 4 | Page No.: 336-341
DOI: 10.3923/ajft.2011.336.341

Study of Antibacterial Activity of Ocimum sanctum Extract Against Gram Positive and Gram Negative Bacteria

Poonam Mishra and Sanjay Mishra

Abstract: This study was carried out to observe the antibacterial activity of aqueous extract, chloroform extract, alcohol extract and oil obtained from leaves of Ocimum sanctum against the selected bacteria i.e., E. coli, P. aeruginosa, S. typhimurium and S. aureus. The antibacterial activity of Ocimum was evaluated by liquid inhibition test. E. coli, P. aeruginosa, Staphylococcus aureus and Salmonella typhimurium were not found resistant against the Ocimum extract since reduction in optical density were observed from 0.20 to 0.85. Chloroform extract were found most effective against P. aeruginosa where 0.85 reductions in O.D were observed. Extract obtained from Ocimum sanctum were observed equally effective against the gram negative and gram positive bacteria. Present investigation reveals that Ocimum sanctum may be a better alternative as a preservative in Food Industries since it is equally effective against pathogenic gram positive and gram negative bacteria.

Keywords: S. aureus, Ocimum sanctum, antibacterial and E. coli

INTRODUCTION

Ocimum is a grassy and annual plant. The leaves of this plant are oval with sharp tip. It is a native of Iran, Afghanistan and India (Mann et al., 2000; Volak and Jiri, 1997; Zargari, 1990; Mirheidar, 1990). Ocimum respectively, named basil is an aromatic herb that has been used traditionally as medicinal herbs in the treatment of headaches, coughs, diarrhea, constipation, warts, worms and kidney malfunctions (Sikmon et al., 1990). Antimicrobials of plant origin have enormous therapeutic potential. They are effective in the treatment of infectious diseases while simultaneously mitigating many of the side effects that are often associated with synthetic antimicrobials. The beneficial medicinal effects of plants materials typically result from the combination of secondary metabolites such as alkaloids, steroids, tannins and phenol compounds, flavonoids, steroids, resins, fatty acids, gums, which are capable of producing definite physiological action on body (Joshi et al., 2009). These plants also have therapeutic effects for nasal polyps (Mann et al., 2000), an upper respiratory tract diseases and it has been used as a bathing solution for treatment of ulcers (Volak and Jiri, 1997). Recent interest on Ocimum has resulted from its inhibitory activity against HIV-1 reverse transcriptase and platelets aggregation induced by collagen and ADP (adenosine 5’-disphosphate) (Yamasaki et al., 1998; Okazaki et al., 1998). It is also a source of aroma compounds and essential oils containing biological active constituents that posses insecticidal (Deshpande and Tipnis, 1997), nematicidal (Chaterjee et al., 1982), fungistatic (Reuveni et al., 1984) and antimicrobial properties (Yamasaki et al., 1998; Wannissorn et al., 2005). Ocimum species contain a wide range of essential oils rich in phenolic compounds and a wide array of other natural products including polyphenols such as flavonoids and anthocyanins (Deshpande and Tipnis, 1997). These Phenolic compounds and flavonoids are potent antioxidants, free radicals scavengers and metal chelator (Cook and Samman, 1996). In previous studies the essential oil of Ocimum species reported to have antibacterial activities. Very few data have been reported regarding the antibacterial activity of leaf. Also no such review is available related with the preservative action of leaves. The purpose of this study is to identify the antibacterial activity of the different extract of leaf against the growth of Gram positive and Gram negative bacteria (Gram positive bacteria are encased in a plasma membrane covered with a thick wall of peptidoglycan while Gram negative bacteria are encased in a triple layer. The outermost layer contains lipopolysaccarides LPS) in detail.

MATERIALS AND METHODS

The study has been carried out in year 2008-2009. The fresh leaves of Ocimum sanctum were collected from local market. The collected leaves were macerated. Macerated leaves were divided in two parts; first part was subjected to cold aqueous extraction and second part was subjected to essential oil extraction.

Cold aqueous extract of Ocimum: Macerated leaves of 200 g were mixed with 500 mL of water and kept for 8 h. After 8 h the whole mixture was filtered by using cheese cloth and obtained extract was centrifuged. Supernatant obtained by centrifugation was used for antibacterial activity assay whereas residue obtained from both filtration and centrifugation was mixed thoroughly and were further used for CHCl₃ extraction.

Isolation of essential oils: The 200 g of leaves and 500 mL of water have been placed in a Clevenger type apparatus. The essential oil was isolated by hydrodistilltaion for 3 h. The obtained essential was separated, dried over anhydrous sodium sulphate and stored at -20°C before storage (Ezekwesili et al., 2004). Residue obtained from oil extraction was further used for Chloroform extract.

Chloroform extraction of Ocimum: The residue obtained after oil extraction and cold aqueous extraction was separately treated with CHCl₃, in both cases equal volume of chloroform and residue (v/w) were mixed thoroughly and kept for 24 h for CHCl₃ extraction. After 24 h the mixture was filtered by using cheese cloth. Extract obtained from residue of cold aqueous and oil extraction were termed as CHCl₃^# and CHCl₃^##, respectively whereas residue obtained from both cases were further used for alcoholic extraction.

Alcoholic extraction of Ocimum: The residue obtained after chloroform extract was separately treated with methanol. Method of alcoholic extraction of Ocimum was same as was described in section chloroform extraction of leaves. Extract obtained from residue of cold aqueous and oil extraction were termed as CH₃OH^## and CH₃OH^### respectively.

Microorganism used for assay

Liquid inhibition: For liquid inhibition test, Nutrient broths were prepared by boiling peptone (5 g), Beef extract (5 g) and 2.5 g of NaCl in 500 mL of distilled water. Boiled broth was autoclaved at 121 psi. Four test tubes having 9.8 mL of broth and 0.2 mL of bacteria i.e., E. coli, P. aeruginosa, S. aureus and S. typhimurium were prepared in duplicates. O.D of each tube was observed at 550 nm. Similarly the 24 test tubes having 9 mL of Nutrient broth and 1 mL of Ocimum extract (essential oil/cold aqueous extract/chloroform/methanol extract separately) in duplicates were prepared, O.D of each test tubes were estimated at the same wavelength. Another 24 test tubes in duplicates having 8.8 mL of broth, 1 mL of extract and 0.2 mL of bacteria were prepared. All test tubes were incubated at 37°C for 24 h and after that the O.D were taken. All readings were taken against the blank (9 mL of nutrient broth and 1 mL solvent i.e., water, chloroform or methanol correspondingly) (Shukla and Sunadaram, 2002).

Statistical analysis: The data obtained were analyzed statistically for Analysis of Variance (ANOVA) using completely randomized design with least significant difference (LSD) at p≤0.05 using Co.Stat 6.303, Co Hart Software USA.

RESULTS

Strains of E. coli, P. aeruginosa, Stphylococcus aureus and Salmonella typhimurium were selected to study the effect of antibacterial activity of Ocimum sanctum extract. The antibacterial activity of Ocimum extract was analyzed by using Liquid inhibition test. Ocimum sanctum extract was found significantly effective against the growth of Stphylococcus aureus since reduction in optical density for all extracts tested were observed from 0.40 to 0.78 (Table 1). CHCl₃^## extract was found comparatively more effective against S. aureus than any other extract tested (Table 1) while CH₃OH^### extract was found least effective against all selected strains. Similarly Ocimum sanctum extracts were found significantly effective against E. coli where reduction in OD were observed from 0.26 to 0.69 (Table 2).

Ocimum extract showed maximum activity against the growth of P. aeruginosa where total 0.85 reduction in OD was observed when treated with CHCl₃^### extracts (Table 3) while CHCl₃^## was found least effective for P. aeruginosa where only 0.34 reduction in OD was observed. The same trend was observed when tested against S. typhimurium (Table 4). The therapeutic potential of the essential oils extracted from fresh leaves of Ocimum sanctum L. has been found to be largely due to eugenol (major constituents of the essential oils) which is a phenolic compound (1-hydroxyl-2-methoxy-4-allylbenzene) (Sen, 1993; Rajeshwari, 1992; Mukherjii, 1987). Eugenol which was analyzed to be the major compounds present in the essential oil of this plant has been reported to present antibacterial activity (Jamine et al., 2005; Loughrin and Kasperbaucer, 2001; Iwalokun et al., 2003) insecticidal activity (Chavan and Nikam, 1982) and nematicidal (Chaterjee et al., 1982). Mann et al. (2000) reported that the essential oil of Ocimum had highest antibacterial activity against gram positive bacteria as opposed to gram negative bacteria while present findings were in contradiction with Mann et al. (2000) and essential oil obtained from Ocimum sanctum was found equally effective against gram positive and gram negative bacteria (Table 1-4). Nakamura et al. (1999) reported that essential oil obtained from Ocimum gratissium had no any bacterial activity against the growth of P. aeruginosa while in present investigation P. aeruginosa was not found resistant against the Ocimum sanctum extracts. The present finding supports the Joshi et al. (2009) that the crude extracts of Ocimum sanctum is effective against the S. aurues and other selected gram positive microorganisms. Cold aqueous extract of Ocimum also showed potent antibacterial activities against the selected strains. Hence it may be interpreted from the present findings that Ocimum extract may be a better alternative as natural food preservatives in Food Industries.

CONCLUSION

Ocimum respectively, named basil is an aromatic herb that has been used traditionally as medicinal herbs in the treatment of headaches, coughs, diarrhea, constipation, warts, worms and kidney malfunctions. Present investigation reveals that Ocimum sanctum may be a better alternative as a preservative in Food Industries since it is equally effective against pathogenic gram positive and gram negative bacteria. In present investigation the concentration O. sanctum was kept constant for all selected strains hence the same experiments may be carried out with varying concentration of O. sanctum in future.

REFERENCES