Antibacterial Activity of Aqueous, Ethanol and Acetone Extracts of Ocimum sanctum Linn

Aqueous, ethanol and acetone leaf and root extracts of Ocimum sanctum Linn were investigated for their antibacterial activities at various concentrations against Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa using standard methods. Antimicrobial studies indicated that both the acetone leaf and root extracts of O. sanctum were found comparatively more effective against these bacteria than any other extract tested while aqueous extract being the least effective against the tested microbes. The highest mean zone of inhibition of acetone leaf extract against P. aeruginosa was 20.74± 0.68 followed by 19.36± 0.29 at concentration 150mg/ml and 100 mg/ml respectively. The aqueous and ethanol root extracts of O. sanctum for had no inhibitory effect against the test microorganisms. Generally the acetone crude extracts showed activity against the three bacteria species with highest average zone of inhibition compared to other extracts. The antimicrobial activity of the leaf extract was more pronounced against test microbes than root extract. The study revealed that the plant possessed antimicrobial properties and could be a potential source of antibacterial agent in the treatment of bacterial infections.


Introduction
Ocimum sanctum Linn known as Holy Basil is an aromatic plant native to the tropics of Asia and Africa being medicinally important plant in the family Lamiaceae. The family Lamiaceae is one of the most exploited medicinal plant family worldwide not only as a source of medicinal plants but also with its valuable essential oils being used as spices and flavours for various food products [1,2]. Morphologically O. sanctum is an erect about 75 cm tall, much branched with hairy stems and simple opposite green leaves that are strongly scented. Leaves have petioles, and are ovate, up to 5 cm long, usually slightly toothed [3]. Ocimum sanctum is known as a consolidated source of extracts with many applications in folk medicine ranging from strong antibacterial and antioxidant properties to particularly in Asia and Africa [4,5]. The presence of essential oils in O. sanctum has been reported to exhibit repellence activity against insects [2,6].
The World Health Organization (WHO) estimates that up to 80% of the world's population in developing countries depends on locally available plant resources for their primary healthcare because they are easily accessible and less expensive compared to western pharmaceuticals which are often expensive or inaccessible [7,8,9]. Several modern drugs have been isolated or derived from natural sources based on their use in traditional medicine as herbal remedies or crude drugs, approved various regulatory agencies [10,11]. With consideration of O. sanctum as reservoir plant for many therapeutic applications, various studies have reported its usages in complementary and alternative medicine particularly as anticancer [12][13][14][15]. As an aromatic medicinal herb, O. sanctum is used customarily in the treatment of headaches, coughs, diarrhea, constipation, antifertility, worms, kidney malfunctions and dysentery [16,17].
Furthermore, studies have revealed O. sanctum to possess anti-inflammatory, analgesic, antipyretic, antidiabetic, hepatoprotective, hypolipidemic and antistress activities [13]. In the latest assessment for its clinical efficacy of herbal medicines, studies have shown its potential for the treatment of oral submucous fibrosis [18]. Traditionally as medicinal plant O. sanctum is taken in a variety of forms being hot or dried leaf tea, powdered leaf as well as seed, root, stem formulations, both systemically and topically [19]. Although several studies have been conducted for ethnomedicinal potential of O. sanctum, only scarce information is available for its different plant parts being extracted and screened for antimicrobial activity. The aim of this study was to evaluate the antimicrobial effects of aqueous, ethanol and acetone leaf and root extracts of O. sanctum against pathogenic bacteria to determine their potentials as antibacterial agent.

Plant Sample Collection
The plant samples of O. sanctum were collected from Ukonga ward, Dar es Salaam at the latitude 6° 38' 39"°S, longitude 39° 10' 29" °E. The identification of the plant was further confirmed at the Herbarium of the Botany Department, University of Dar es Salaam. The leaves and roots of O. sanctum were washed with distilled water to remove any impurities and then cut into pieces and oven dried at 70°C for 12 hrs to remove all moisture. The dried samples were then ground into fine powder with warring commercial laboratory blender and further milled (mesh size 850 um).

Extraction of Plant Material
Crude extracts were obtained by using aqueous (distilled water), acetone and ethanol. The aqueous extract was also prepared by mixing 20g of each powdered leaf and root samples with 100 mL boiling distilled water for 5 minutes. For ethanol and acetone extracts the powdered leaf and root samples (20g) each were extracted individually using 100mL of extraction solvent of 95% ethanol and 95% acetone for 24 hrs with intermittent shaking at room temperature. The extracts were filtered by using Whatman filter paper number 1 and air dried at 28 °C. The extracts were then concentrated by evaporating the solvents using a water bath at 40°C. After evaporation for 45 min to 1hr depending on the type of the solvents, the weight of crude extract was determined by subtracting the flask's weight from total weight of the flask after evaporation. The dried extracts were then labeled and kept in airtight bottles in the refrigerator at 4 °C prior to determination of antibacterial activity.

Antibacterial Activity
In testing the antibacterial activity O. sanctum leaf and root extracts the following bacterial strains were used; Escherichia coli-ATCC 25923, Staphylococcus aureus-ATCC 25923, Pseudomonas aeruginosa-ATCC 27853. These microorganisms were obtained from the Microbiology Department of Muhimbili National Hospital and they were maintained at 4°C in the refrigerator. Three different concentrations (50, 100 and 150 mg/ml) of each prepared aqueous, ethanol and acetone extracts were used for assessing the presence of zone diameter of inhibition.
Antibacterial activity was determined by using the well diffusion method. Each Petri dish containing Multer-Hinton agar medium was inoculated with one bacterial strain by scattering the suspension of the organism with a bended tip sterile glass rod. In each plate wells were made at equal distances using sterile cork borer. Gentamycin was used as a positive control and was prepared at a concentration of 25 µg/ml. Plates were kept in the refrigerator for 40 minutes to allow the diffusion of sample to the surrounding agar medium. The petri dishes were incubated at 37°C for 24 hrs. Plates were examined for zone of inhibition of that different extracts and the diameter of the zone of inhibition measured using a digital venial caliper and the average diameter for each sample was calculated.

Statistical Analysis
All experiments were carried out in four replicates and means and standard mean errors were calculated for the zones of inhibition measured for the replicate sets of experiments in each case. Results analyzed using Duncan's multiple range test (DMRT) to test the difference among the treatments.

Results
Results for the aqueous leaf and root extract of O. sanctum at concentrations 50, 100 and 150 mg/ml is indicated ( Table  1). The antimicrobial activity of aqueous leaf extract against E. coli, S. aureus and P. aeruginosa was revealed only at concentration 150 mg/ml with mean zone of inhibition 4.28 ± 0.68 against E. coli, 7.42 ± 0.36 against S. aureus and 11.42 ± 0.76 mm against P. aeruginosa (Table 1). Generally in the aqueous extract, the leaf showed significantly high inhibition against P. aeruginosa compared to what was revealed against E.coli and S. aureus while the aqueous root extract had no inhibition against all three tested microbes (Table 1).   The result in Table 3 indicates that both the acetone leaf extract and acetone root extract of O. sanctum showed inhibitory effect against E. coli and P. aeruginosa. The mean zone of inhibition of acetone leaf extract on E. coli was 14.30± 0.36 and 17.53± 0.29 at concentration 100 and 150 mg/ml respectively. The acetone root extract at concentration 150 mg/ml was the only concentration which revealed zone of inhibition against E. coli as other concentrations had no inhibition against the bacteria ( Table 3). The mean zone of inhibition of acetone leaf extract against P. aeruginosa was revealed in all three tested concentrations at 50, 100, and 150 mg/ml and the effect of extracts was 3.2 ± 0.56, 19.36± 0.29 and 20.74± 0.68 respectively for the concentrations while 20.74± 0.68 showed significantly high inhibition against S. aureus compared to other concentrations ( Table 3). The mean zones of inhibition of acetone root extract against P. aeruginosa were 15.57 ± 0.24 and 16.83 ± 0.5815.57 at concentration 100 and 150 mg/ml only as the 50 mg/ml concentration had no inhibition (Table 3). While the acetone leaf and root extract showed inhibition against E. coli and P. aeruginosa, zone of inhibition for S. aureus was only through the acetone leaf extract ranging from 12.67± 0.12 to 17.12± 0.34 at 100 and 150 mg/ml respectively. The acetone root extract had no inhibition against S. aureus for all concentrations tested ( Table 3). The gentamycin used as a positive control at a concentration of 25 µg/ml showed significantly high inhibition against all tested microbes (Table 1-3). The average zone inhibition for the different extracts indicating antibacterial potential of O. sanctum is shown in Figure 1. The acetone extract was the most effective against all the tested bacterial strains with highest average zone of inhibition of 15.04 mm for P. aeruginosa followed by E. coli with average zone of inhibition 9.16. The aqueous extract had the least average zone of inhibition in against all tested microbes. However, in comparison with the control, the inhibition of the microbes was significantly higher in the control than in plant extract namely aqueous, ethanol and acetone (Figure 1).
The average zone inhibition based on concentrations is shown in Figure 2. For the three different extract concentrations used (50, 100 and 150 mg/ml), the concentration 150 mg/ml had the highest average zone of inhibition followed by 100 mg/ml. At concentration 100 mg/ml and 150 mg/ml the antimicrobial activity against P. aeruginosa high compared to other tested microbes.
Furthermore, the concentration 50 mg/ml showed antimicrobial activity only for P. aeruginosa as the concentration showed no inhibition against E. coli and S. aureus (Figure 2).

Discussion
The results of this study show that the acetone and ethanol extracts of Ocimum sanctum possess antimicrobial activities against the test bacteria. All the three extracts tested showed varying degree of antibacterial activity against the tested bacteria species. Effective extraction of biological compounds from plant material mostly is dependent on the type of solvent, in this study the plant extracts by acetone provided more reliable antibacterial activity followed by to ethanol extracts and the aqueous extract was ineffective to all tested microorganisms. This study is in agreement with other studies particularly for effectiveness of ethanol extracts and ineffective of aqueous extracts which revealed relatively lower antimicrobial activity compared to other extracts [20,21,22]. The effectiveness of acetone and ethanol extracts of O. sanctum against Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa respectively is an indication that the extracts from the selected parts of this plant have potential for use as an antimicrobial agent. This finding supports the ethnomedicinal use of O. sanctum in the management various microbes' diseases such as stomach aches and gastroenteritis [12,19,20].
Ocimum sanctum contain a wide range of essential oils rich in phenolic compounds and a comprehensive array of phytochemicals including flavonoids and anthocyanins, antioxidants and free radicals scavengers which could be the reason for many medicinal potentials of the plant [14,19,23,24]. Several studies have revealed that the presence of bioactive compounds in plant materials of O. sanctum were responsible for antibacterial activity, particularly the presence of high content of linoleic acid which is reported to contribute towards its antibacterial activities against staphylococcus bacillus and Pseudomonas aeruginosa [25,26].
In this study leaf extract had good result against all tested selected bacteria and this could be due to the presence of rich phytochemicals in leaves compared to roots. Although all parts of the plant might have the active compounds but in leaves the presence of phytochemicals such as eugenol, rosmarinic acid, apigenin, and carnosic acid in O. sanctum has previously been reported to be significant high which account for many medicinal activities of the plant [13,26]. Findings of this study is in agreement with other studies reported antimicrobial activity of crude extracts from leaves of O. sanctum revealed to be the most active extract to inhibit a good number of bacteria tested [27,28]. Other antimicrobial effects of leaf extracts of this plant are also reported in its use on cotton fabrics [22].
In relation to medicinal potential O. sanctum and the frequently utilized plant parts, several studies have reported that regularly utilized parts were leaves [17,29]. Consideration to use only specific parts particularly the leaves seems to be a sustainable means for harvesting and conservation of the species bearing in mind its medicinal values in traditional system. For conservation of medicinal plants several studies have recommended the leaf harvest and strongly discouraged unsustainable means such as root and bark harvesting [9,30]. Harvesting medicinal plants through root excavation and bark striping can be very devastating and pose a serious threat to the plant survival. The high utilization of roots has also been reported as putting many plant species at a risk of extinction because of the damages inflicted on them in the course of uprooting the plants [31].
Several drug resistances have developed due to haphazard use of commercial antimicrobial drugs frequently used in the treatment of communicable diseases [32,33]. Such an occurrence of drug resistance through the uses of commercial antibiotics has prompted numerous efforts in search for antimicrobial agents from plants with less vulnerable to many side effects but efficient for therapeutic potential to heal many infectious diseases [34,35]. The antimicrobial activity of O. sanctum revealed in this study further supports its uses as alternative medication of ailments mostly caused by bacteria such as stomach ache and diarrhea, thus could safely be used as topical antibacterial agents where commercial antimicrobial drugs may not be utilized or may not be easily accessible.

Conclusion
The study has revealed that O. sanctum has good antibacterial activity, hence supporting the use of this plant as traditional medicine. Based on plant parts tested, the leaf extracts of O. sanctum showed higher potential for antibacterial effects than the root extract, thus recommending the sustainable use of leaf for medicinal harvesting compared to the use of roots. The plant can further be explored for the proper phytochemical compounds present in the leaves since this part showed good results for the antibacterial activity. Furthermore, the present study provides an important basis for the use of the acetone extracts which has not only revealed more potential for antimicrobial activity compared to other extracts but also the crude extract could be valuable for the development of new antibacterial drugs.