Comparative Antimicrobial Study of Different Parts of Ocimum americanum L., O. basilicum L. and O. sanctum L. in Comparison to Standard Antibiotics Collected from Dibrugarh, Assam

J. Chetia, L. R. Saikia, S. Upadhyaya, E. Khatiwora, A. Bawri Department of Life Sciences, Dibrugarh University, Dibrugarh, Assam-786004, India Present address: Silapathar College, Silapathar, Dhemaji, Assam, 787059, India Department of Botany, D. K. D. College, Dergaon, Assam, India-785614 Department of Chemistry, Y. M. College, Bharati Vidyapeeth, Deemed University, Pune, India411038 North Eastern Institute of Folk Medicines, Ministry of AYUSH, Government of India, Pasighat, East Siang District, AR, India


Introduction
Millions of people are dependent on various antibiotics to live a disease free life. But some multi-drug resistant pathogens are creating some problems to these existing might be due to the habitat and climatic condition of that place. The study was conducted to compare the differences in these properties with other literature which were done in other places.

Sample collection
Flowering branches of plants ( Fig. 1) were collected and brought to the laboratory. Different parts (young and mature leaves, inflorescence and stem) were separated and cleaned properly and washed under running water to remove dust and other debris. The materials were air dried at room temperature. The stems were sliced before allowed to dry. After removal of surface water, the materials were wrapped with brown paper and allowed sun drying for complete dryness (less than 1-2 % moisture content). The materials were grounded to fine powder using mortar and pestle and then in electric grinder. The fine powder was kept in air tight bottles for further analysis.

Preparation of extracts
Extracts were prepared in five solvents viz., water, methanol, ethanol, acetone and petroleum ether by cold maceration methods and are known as cold extracts. The solvents were selected on the basis of polarity level and their extraction ability. Extracts were collected by soaking 10 g of air dried powder in 500 mL of solvent (except water) for 72 h with intermittent shaking. The extracts were filtered through Whatman no. 1 filter paper into pre-weighed beakers. The filtrate was dried on water bath to obtain a dried mass. The water extract was prepared by soaking 10 g of powder in 500 mL distilled water for 48 h with intermittent shaking. The soaking for 72 h caused fungal growth. The solution was filtered through Whatman no. 1 filter paper. The filtrate was dried to sticky mass using a b c water bath. The extracts were kept in air tight glass bottles at 5 °C for further analysis. Hot petroleum ether extract was also prepared using soxhlet extractor and antimicrobial activity of the extract was done to observe the difference in activities of both cold and hot petroleum ether extract. The dried extracts were dissolved in DMSO (dimethyl sulfoxide) to obtain sample solution at 1 mg/mL of concentration. Aqueous extracts were dissolved in distilled water at 1 mg/mL of concentration.

Antimicrobial activity assay of the sample extracts
Antimicrobial activity of the bacterial strains was carried out by agar well diffusion method described by Nair et al. [47] using 6 mm borer. The intensity of the activity was determined by measuring the diameter of the zone of inhibition (ZOI) comparing with some standard antibiotics.
Gram positive and gram negative bacterial strains and fungal strains were used in this experiment to know the antimicrobial activity of the sample extracts. a) Gram positive bacterial strains-Bacillus subtilis ( (IMTECH), Chandigarh, India. The reference of bacterial strains were maintained on nutrient agar slants and fungal strains on PDA (Potato Dextrose Agar) slants and stored in freeze. Strains were regularly sub-cultured using nutrient broth for bacterial strains and Potato Dextrose Broth for fungal strains. The antibacterial activity was assayed by measuring the diameter of the ZOI formed around the well [48]. The resulting ZOI will be uniformly circular as there will be a confluent lawn of growth. The antifungal effect was seen as crescent shaped ZOI [49].

Statistical analysis
All the experiments were done in triplicate and mean and standard deviation (SD) was calculated and presented in '±' form.

Results and Discussion
The results of antimicrobial activity study of the sample extract of O. americanum, O. basilicum and O. sanctum are presented in Tables 1 to 3 and standard antibiotics in Table  4. Extracts from the plant recorded good antimicrobial activity against B. subtilis, B. cereus, S. aureus and P. vulgaris. Acetone extract of young inflorescence of O. americanum showed the highest antibacterial activity against B. cereus (14 ± 1 mm) which was also higher (10 ± 1 mm) than the inhibition of standard of Clotrimazole (10 mcg). The sample extracts of O. basilicum did not recorded antifungal activity against C. albicans and P. chrysogenum. Out of twenty extracts from O. basilicum only four extracts recorded inhibition against E. faecalis. The more number of phytoconstituents present in oil of inflorescence might be the reason of higher activity. Hot petroleum ether extract of mature leaves of O. basilicum showed the highest activity against E. coli (16 ± 2 mm) which was also higher than the inhibition of Ampicillin (10 mcg) (10 ± 0 mm), Streptomycitin (10 mcg) (12 ± 0 mm), Erythromycin (15 mcg) (12 ± 2 mm). Twenty five extracts of O. sanctum did not record inhibition against P. chrysogenum. Petroleum ether extract of young leaves of O. sanctum recorded the highest inhibition against P. vulgaris (20 ± 2 mm) which is higher than the inhibition of standard Ampicillin (10 mcg) (12 ± 2 mm) and Erythromycin (15 mcg) (12 ± 2 mm).
In our study, only acetone extract of young inflorescence of O. americanum recorded inhibition against C. albicans (10 ± 0 mm). Thaweboon et al. [50] showed that essential oils from the plant recorded antimicrobial activity against three different micro-organisms including C. albicans using biofilm model. The essential oil from leaves also recorded antibacterial activity against oral bacteria related to periodontal disease [51]. In case of O. basilicum, hot petroleum ether extract of mature leaves recorded a significant inhibition against E. faecalis and all other extracts recorded no inhibition or negligible inhibition. Prasad et al. [52] observed that extracts from O. basilicum had no antibacterial activity against E. faecalis. In our study, ethanol extract of young leaves and methanol extract of mature leaves recorded inhibition against S. aureus. Ethanol, methanol, propanol, chloroform and isoamyl alcohol have activity as 9, 10, 9, 14 and 18 mm respectively against S. aureus as studied by Prasad et al. [52]. In our study, methanol extract of both young and mature leaves recorded inhibition against B. subtilis. Similarly, methanol, propanol, chloroform, petroleum ether and isoamyl alcohol extract recorded activity as 11, 11, 14 and 20 mm respectively against B. subtilis as observed by Prasad et al. [52]. In our study, various extracts recorded inhibition against E. coli and S. aureus in agar well diffusion method. Gebrehiwot et al. [53] showed that hydrodistilled oil of O. basilicum has antimicrobial activity against E. coli and S. aureus in paper disc diffusion method. But the crude extracts (chloroform:methanol = 1:1) at concentration of 10 and 20 µL, did not recorded activity against the tested microorganisms. Azam and Irshad [54] revealed that ethanol and methanol extract of O. basilicum has antibacterial activity against four tested strains. Methanolic, ethanolic and essential oil recorded ZOI as 5, 5 and 4 mm against S. aureus; 4, 6 and 8 mm against E. coli and 5, 6 and 4 mm against B.
subtilis. These results were compared to Ampicillin having ZOI as 6, 5 and 9 against S. aureus, E. coli and B. subtilis respectively using disc diffusion method. Similarly, in agar well diffusion method also the extracts recorded antibacterial activity against the tested organisms. Methanolic extract recorded ZOI as 7, 4 and 6 mm, ethanolic extract recorded 4, 7 and 4 and essential oils recorded 7, 6 and 7 mm and standard Ampicillin recorded 6, 7 and 9 mm against S. aureus, E. coli and B. subtilis respectively. They also recorded that there is no significant difference in ZOI in both disc diffusion and agar well diffusion method. In our study, the various extracts of different aerial parts recorded inhibition against E. coli, S. aureus and B. cereus. Moghaddam et al. [55] showed that the essential oil from aerial parts of O. basilicum have antimicrobial activity against E. coli, S. aureus and B. cereus. The Minimum Inhibitory Concentration (MIC) was determined against E. coli at concentration 9 µg/mL. In our study, ethanol extract of young leaves recorded inhibition against E. coli and S. aureus. Shweash et al. [36] showed that ethanolic extract of leaves of O. basilicum showed antibacterial activity against E. coli and ZOI was increased along with increase in concentration. It had lower MIC value (0.312 mg/mL) against E. coli. Adam and Omer [56] showed the ethanolic extract of leaves of O. basilicum have antibacterial activity against E. coli, S. aureus along with other tested bacterial strains. At 100 µg/disc concentration E. coli and S. aureus showed ZOI as 13.6 and 13.9 mm. Shafique et al. [57] also revealed that essential oil from O. basilicum have antibacterial activity against both Gram positive and Gram negative bacteria. Raghad et al. [58] revealed that ethanolic extract of seeds of O. basilicum have antimicrobial activity against E. coli (2 mm), S. aureus (5 mm), S. epidermis (2.5 mm), P. aeruginosa (5 mm) and fungi C. albicans (3 mm). Shweash et al. [59] showed that ethanolic extract of leaves of O. basilicum showed antibacterial activity against E. coli and ZOI was increased along with increase in concentration. It had lower MIC value (0.312 mg/mL) against E. coli. In the present study, antimicrobial activity of cold and hot petroleum ether extract recorded difference in their inhibition against bacterial strains.
In the present study, antimicrobial activity of cold and hot petroleum ether extract recorded difference in their inhibition against bacterial strains. Hot petroleum ether extracts of all the parts (except stem) recorded more inhibition than cold petroleum ether extracts of inflorescence. In our experiment, there is a variation in antimicrobial activities of cold petroleum ether and hot petroleum ether extract of the plants. Sharma et al. [60] carried out experiments on antimicrobial activity of ethanol and water extract of leaf and stem of three Lamiaceae members and recorded that ethanol extract have more activity than the water extracts, which may be due to the hot extraction of ethanol using soxhlet apparatus. Zalazare et al. [61] recorded that ethanol and hot water extracts of the mushrooms contained higher bioactive substances than cold water extracts. Variable antimicrobial activity was observed in cold and hot water extracts against tested bacterial pathogens [62][63][64][65]. According to some other workers, activity of samples also may vary with temperature [66][67][68][69]. Traub and Leonhard [70] showed that out of 62 types of antimicrobial material, 25 types were found stable after the heat treatment which is very essential for antimicrobial agents of foods. This kind of differences occurred may be due to the age of the plant, the time of harvest of the material, method of extraction or may be the thermo-sensitivity of the active compounds.

Diameter of the cork borer = 6 mm, '-' indicates no inhibition
In our study, acetone extract of young and mature leaves and inflorescence recorded good inhibition against against B. subtilis and S. aureus. Baskaran [71] carried out antibacterial activity of O. sanctum against E. coli, B. subtilis, S. aureus and Klebsiella pneumonia using various extracts of the plant and recorded good antibacterial activity. The results showed that benzene and chloroform extracts are effective against S. aureus, K. pneumonia and B. subtilis. There was no activity against E. coli. Acetone extract showed strong activity against K. pneumonia, but less against S. aureus and B. subtilis. Chhetri et al. [72] showed that 1 % ethanol extract solution of O. sanctum recorded ZOI as 2.2 and 2.1 cm against E. coli and S. aureus respectively. Singh et al. [73] revealed that the aqueous and methanol extracts of O. sanctum did not recorded activity against E. coli. The extracts showed the largest ZOI (20 and 60 mm respectively) at 200 mg/L against S. aureus followed by 100 mg/L and 50 mg/L concentrations (16 mm and 14 mm) respectively of methanol extract. In case of aqueous extract the ZOIs were 11 and 14 mm at 100 and 50 mg/L concentrations. Prasad et al. [52] showed that extracts from O. sanctum tested did not show any activity against E. faecalis. Isoamyl alcohol showed activity of 18 mm against S. aureus. Similarly ethanol, methanol, propanol, chloroform and isoamyl alcohol have activity as 10, 10, 11, 13 and 24 mm, respectively against B. subtilis. Methanolic leaf extract showed activity against B. subtilis, S. aureus and E. coli [74]. The antimicrobial activity recorded by the solvent extracts is may be due to the phytochemicals present in the plant.
Diameter of the cork borer = 6 mm, '-' indicates no inhibition      From the above study, it can be concluded that the different parts of these three plants from Lamiaceae family have anti-microbial properties against tested bacterial and fungal strains. Instead of using whole plant, different parts can be used in medicinal practices which will help in sustainable management of these medicinal plants. This will also save the plants extinction.

Conclusion
Different extracts of different parts of the plants recorded antimicrobial inhibition against bacteria and fungi. Extracts from the leaves recorded more inhibition than the extracts from inflorescence and stem. Methanol, ethanol and acetone extracts recorded good inhibition than water and petroleum ether extract. The study can be concluded that instead of using the whole plant, different parts can be used in medicinal practices. This will also helps in sustainable management of these medicinal plants.