In vitro evaluation of the antibacterial activities of the methanol, aqueous and n-hexane extracts of Ocimum lamiifolium from Ethiopia

Ocimum lamiifolium (local name Dama Kesse, Amharic) is a medicinal plant in Ethiopia. Its leaves are squeezed and sniffed to treat coughs and colds. They are also used to treat eye infections and to stop nose bleedings. In the present study, leaves of O. lamiifolium were collected from their growing habitats. Dried leaf powders were extracted using methanol, distilled water and n-hexane. 25, 50, and 100 mg/ml doses of the extracts made in Tween 80 (2%) were screened for their antimicrobial activities against Staphylococcus aureus, Escherchia coli, Pseudomonas aeruginosa and Shigella boydii using disk diffusion assay. The inhibition zones due to the methanolic extract ranged from 0 (in S. aureus due to 25 mg/ml) to 12 mm (in E. coli due to 100 mg/ml). Inhibition zones due to the aqueous extract ranged from 8 mm in S. aureus and S. boydii to 12 mm in S. boydii at concentrations of 25 and 100 mg/ml, respectively. The n-hexane extract at 25 mg/ml resulted in inhibition zone that ranges from 7 mm (against S. aureus) to 11 mm (against E. coli) at 50 and 100 mg/ml doses. The minimum inhibitory concentration of S. boydii and E. coli was 10 mg/ml due to all the extracts. The minimum inhibitory concentrations on S. aureus were 10, 20 and 50 mg/ml due to the aqueous, n-hexane and methanolic extracts, respectively. P. aeruginosa was minimally inhibited at 10 mg/ml due to the methanol and aqueous extracts and 15 mg/ml due to the n-hexane extract. The methanol, aqueous, and n-hexane extracts of O. lamiifolium leaf extracts inhibited the test bacteria with significantly higher levels of inhibition zones than the negative control (T80). The positive controls (Tetracycline and Chloramphenicol) also showed significantly higher inhibition zones than the 100 mg/ml concentration of the extracts and T80 except that Chloramphenicol failed to inhibit S. aureus and P. aeruginosa. However, combination of Chloramphenicol with plant extracts raised their inhibition zones from zero to 23 and 25 mm in S. aureus and P. aeruginosa, respectively.


Plant collection and identification
O. lamiifolium leaves were collected from their natural habitats Central and North East Ethiopia.The plants were not flowering during the period of collection.The collected specimens were authenticated by botanists from the National Herbarium of Addis Ababa University and voucher specimens were deposited at the same herbarium of Addis Ababa University.

Extraction of plant
Collected leaves of O. lamiifolium were washed by distilled water and subjected to shade drying at 25°C.Then the dried leaves were pulverized to get course powder.100 g of the powder was added to 1 L (1:10, w:v) of three solvent types, namely, methanol (absolute), n-hexane (absolute), and distilled water and each mixture was shaked for 48 h at 120 rotations/min.The solutions were filtered by Whatman No. 1 filter paper.Finally, the methanol and hexane extracts were concentrated under vacuum in a rotary evaporator (Büchi Laboratoriums-Tchnik AG CH-9230 Flawil/Schweiz) to give gummy residues and the aqueous extracts using a lyophylizer (Bioblock Scientific, Illkirch Cedex, France).The crude extracts were then weighed and the yield of the each extract was calculated as 17.8, 12 and 6.7% (methanol, aqueous and n-hexane extract, respectively).

Bacterial strains
Clinical isolates of Staphilococcus aureus, Shigella boydii, Escherichia coli, and Pseudomonas aeruginosa were obtained from the Ethiopian Public Health Institute (EPHI).These isolates were screened for their susceptibility towards different doses of the different extracts of O. lamiifolium as well as two standard antibiotics [Tetracycline (30 µg/disk) and Chloramphenicol (30 µg/disk)].In order to perform the antimicrobial screening, the bacterial isolates were cultured overnight at 37°C on Nutrient Agar medium.Colonies collected from each 24 h old bacterial culture were diluted in sterile saline and the optical density was adjusted in comparison with 0.5 McFarland' scale to prepare a standardized inoculum (1.5 × 10 8 cfu/ml).The bacteria from saline solutions were spread on Müller Hinton Agar plates using sterile cotton swabs.
The paper disc diffusion technique was applied to determine the antimicrobial activities of the tested plant extracts.Sterile paper discs (5 mm in diameter) immersed in stock solutions containing 25, 50 and 100 mg/ml prepared in 2% Tween 80 of plant extracts were placed on the surface of inoculated Nutrient Agar plates.Plates were then incubated for 24 h at 37°C, and diameters of the inhibition zones were recorded.All assays were applied in triplicates and the results are given as means ± standard error of the mean.

Determination of minimum inhibitory concentration (MIC)
MIC is the lowest concentration of an antimicrobial that inhibits the visible growth of microorganisms after overnight incubation (Yilmaz, 2012).MICs were defined as the lowest concentration of the aqueous, methanol and n-hexane extracts of O. lamiifolium inhibiting visible growth of the bacteria.On the other hand, the MBC was defined as the lowest concentration of the extracts of O. lamiifolium required to kill all the test bacteria (Yilmaz, 2012).The MIC was determined using agar dilution method which is described by the European Committee for Antimicrobial Susceptibility Testing (EUCAST) of the European Society of Clinical Microbiology and Infectious Diseases (ESCMID, 2000).The following procedure was followed to determine the MIC; 20 ml agar was used in 9-cm Petri dishes for agar dilution.Nineteen-milliliters molten agar was added to 1 ml of each plant extract to make the total volume 20 ml.Müller Hinton agar was prepared as recommended by the manufacturer.The sterilized agar was set to cool to 50°C in a water-bath.Extracts of O. lamiifolium were prepared into doses of 5, 10, 15, 20, 25, 50 and 100 mg/ml in 25 to 30 ml containers.Nineteen-milliliters of molten agar was added to each container and mixed thoroughly, and finally poured into pre-labeled sterile Petri dishes on a level surface.The plates were allowed to dry at room temperature so that no drops of moisture remain on the surface of the agar.Bacterial suspensions were prepared in 0.85% normal saline and were standardized by 0.5 McFarland standards to 1.5 × 10 8 colony forming units (CFU)/ml.The inocula were inoculated on the dry plates.The inoculum spots were then allowed to dry at room temperature before inverting the plates for incubation.Finally, the plates were incubated at 37°C in air for 18 h.The MIC (the lowest concentration of the extracts that completely inhibited visible growth) was judged by the naked eye.

Determination of susceptibility test of bacteria towards standard antibiotics and their combinations with O. lamiifolium extracts
Susceptibility of the test bacteria towards Chloramphenicol, Tetracycline and their combinations with the leaf extracts of O. lamiifolium was determined according to the classification indicated by Bauer et al. (1966).Based on this literature, inhibition zones due to Chloramphenicol (30 µg) can be classified as resistant (≤12 mm), intermediate (13 to 17 mm), and sensitive (≥18 mm) and zones of inhibition for Tetracycline (30 µg) are interpreted as resistant (≤14 mm), intermediate (15 to 18 mm), and sensitive (≥19 mm).

Antibacterial activities of O. lamiifolium leaf extracts
The methanol extract inhibited the test bacteria in a dose dependent manner (Figure 1).At 25 mg/ml, it did not inhibit S. aureus while the rest bacteria were inhibited by this dose with inhibition zones just below 10 mm.The 50 mg/ml concentration of the methanol extract on the other hand inhibited all the bacteria with mean inhibition zones ranging from 6 mm (S. aureus) to over 10 mm (E.coli).At 100 mg/ml concentration, the methanol extract inhibited three of the test bacteria with mean inhibition zones above 10 mm and S. aureus with mean inhibition zone close to 10 mm.The antibacterial activity of the methanol extract was generally lower than that of Tetracycline and Chloramphenicol.However, it was better than Chloramphenicol in inhibiting S. aureus and S. boydii.The aqueous extract inhibited all the test bacteria at 25, 50 and 100 mg/ml doses minimally inhibiting S. aureus and E. coli each with mean inhibition zones of 8 mm at 25 mg/ml dose and maximally S. boydii (12 mm) at 100 mg/ml.The aqueous extracts too were generally less effective than Tetracycline and Chloramphenicol although Chloramphenicol resistant strains (S. aureus and P. aeruginosa) were sensitive to these extracts.Like that of the aqueous extract, the n-hexane extract inhibited all the test bacteria at the three dose levels, S. aureus being inhibited minimally (7 mm) at 25 mg/ml and E. coli being inhibited maximally (11 mm) at 100 mg/ml.In general, the trend of inhibition of the test bacteria by the three extracts of O. lamiifolium showed that the aqueous extract is the best followed by its methanol and n-hexane extracts, respectively.

Determination of the MIC
The MIC concentrations of O. lamiifolium leaf extracts ranged from 10 to 50 mg/ml (Table 1).The 50 mg/ml concentration of its methanol extract inhibited all the bacteria and its 10 mg/ml inhibited 75% of them.The aqueous extract, on the other hand, inhibited all the bacteria at a concentration of 10 mg/ml and the n-hexane extract inhibited the microorganisms with a range of MICs from 10 to 20 mg/ml of which the 20 mg/ml inhibited the entire, 15 mg/ml inhibited 75%, and 10 mg/ml inhibited 50 percent of them.

Antibacterial effects of the combinations of Tetracycline (30 µg/ml) and Chloramphenicol (30 µg/ml) with O. lamiifolium leaf extracts at 100 mg/ml
Chloramphenicol (30 µg) resulted in inhibition zones of 31 and 33 mms in S. boydii and E. coli, respectively (Figure 2).On the contrary, it did not inhibit S. aureus and P. aeruginosa.Tetracycline (30 µg) inhibited S. aureus with inhibition zone of 11 mm and the rest bacteria with inhibition zones above 19 mm.Combination of these antibiotics to O. lamiifolium extracts at a dose of 100 mg/ml, however, increased the inhibition zones of the test bacteria.Inhibition of S. boydii and E. coli due to Chloramphenicol surpassed inhibition due to the combination of Chloramphenicol and plant extracts (100 mg/ml) (Figure 2).On the contrary, inhibition of S. aureus and P. aeruginosa was found to be higher than either caused by Chloramphenicol or extracts.In the same manner, inhibition of S. boydii and E. coli by Tetracycline were higher than inhibition by combination of plant extracts (100 mg/ml) and Tetracycline while S. aureus and P. aeruginosa were more sensitive to the combinations than individual parts.Generally, combining standard drugs with plant extracts boosted the inhibition of S. aureus and P. aeruginosa than that of either the drugs or the extracts.

Differences in zones of inhibition among the methanol, aqueous, and n-hexane extracts of Ocimum lamiifolium at concentrations of 100 mg/ml
Differences in inhibition zones due to the different extracts of O. lamiifolium against the test bacteria are presented in Table 2.

DISCUSSION
Inhibition was concentration dependent in all of the bacteria with S. boydii being the most sensitive bacterium followed by E. coli, P. aeruginosa and S. aureus in decreasing order of sensitivity.Similar findings were demonstrated in a study by Gebrehiwot and Unakal (2013) where E. coli was the most sensitive followed by S. aureus and P. aeruginosa, respectively to the aqueous and ethanol extracts of O. lamiifolium.This antibacterial activity may be due to the occurrence of antibacterial active components like eugenol and sabinene in the extracts (Adebolu and Oladimeji, 2005;Wiart, 2006;Uinoiseau et al., 2010).In the present study, S. aureus and P. aeruginosa were found to be resistant to Chloramphenicol may be due to the ability of these bacteria to inactivate Chloramphenicol by enzymes coded by the cat genes or the ability of P. aeruginosa to inactivate Chloramphenicol by Chloramphenicol acetyltransferase enzyme and decreased outer membrane permeability or active efflux of this drug (Byarugaba, 2009).S. aureus was resistant to tetracycline may be due to its abilities like active efflux of the antibiotic and ribosome protection or modification of   the antibiotic (Byarugaba, 2009).All the extracts of O. lamiifolium showed better inhibition than the negative control (Tween 80) and were less effective than Tetracycline and Chloramphenicol.However, the leaf extracts were more effective than Chloramphenicol against S. aureus and P. aeruginosa.On the other hand, these extracts were less effective than Chloramphenicol against S. boydii and E. coli.Application of the aqueous extract inhibited even the most resistant bacterium (S. aureus) at the lowest concentration (25 mg/ml).
E. coli and S. boydii were the most sensitive to the extracts followed by P. aeruginosa and the least sensitive of all was S. aureus showing that the Gram negative bacteria were more sensitive to the plant extracts than the Gram positive one (S.aureus).The results confirmed that O. lamiifolium extracts are important to inhibit S. boydii and E. coli, followed by P. aeruginosa, and least effective against S. aureus.The aqueous extract of O. lamiifolium seem to be effective than its methanol and nhexane extracts against these bacteria.This result clearly distinguishes the importance of the aqueous extract which contains the most effective components to inhibit bacterial growth contradicting to the finding by Goyal and Kaushik (2011) where the methanolic extract of Ocimum sanctum L. showed comparatively higher activity than other organic and aqueous extracts.On the other hand, this result agrees with the work of Gebrehiwot and Unakal (2013) where the aqueous extract was found to be more effective than its ethanol extract against S. aureus, E. coli and P. aeruginosa.Generally, differences in activities of O. lamiifolium extracts may be due to the differences in their chemical compositions which are determined by different factors such as climate, plant nutrition, stress (Carson and Hammer, 2011), fertilizer application (Duke, 2009), plant organs used, plant developmental stage, plant origin, chemotypes, and methods used (Zuzarte et al., 2011).
Sometimes, the effectiveness of antibiotics can be increased by coupling them with plant extracts (Kekuda, 2012).In the present study, combination of Tetracycline (30 µg/disc) to the methanol extract of O. lamiifolium (100 mg/ml) increased the sensitivity of S. aureus.On the other hand, combination of Chloramphenicol (30 µg/disc) with all the extracts increased the sensitivity of S. aureus and P. aeruginosa.The implication of this finding is that the use of plant extracts in combination with less effective antibiotics can increase the susceptibility of bacteria to these antibiotics and can be solutions to bacterial resistance to antibiotics.
The majority of the dosages of all the extracts of O. lamiifolium inhibited the test bacteria with inhibition zones significantly higher than that of tween 80.The positive control (Tetracycline) on the other hand inhibited all the bacteria with inhibition zones significantly higher than all the extracts except the 50 mg/ml and the 100 mg/ml concentrations of the methanol, aqueous, and n-hexane extracts of O. lamiifolium which resulted in inhibition zones on S. aureus which were not significantly different from that exerted by Tetracycline.This leads to the conclusion that the aqueous, methanol and n-hexane extracts of O. lamiifolium have comparable activities with Tetracycline against S. aureus.

Table 2 .
Inhibition zones of Ocimum lamiifolium extracts at different concentrations.