Abstract: In this present study, it is tried to find out the antimicrobial effect and Minimum Inhibitory Concentration (MIC) of Oxalis corniculata leaf and Ocimum sanctum leaf extract against clinical isolates from urine, stool and sputum and their successive comparison with commercially, available antibiotic discs. Powdered leaves were prepared and used for extraction with various solvents, viz., methanol, ethanol and chloroform in case of Oxalis corniculata and ethanol, methanol, n-hexane and petroleum spirit in case of Ocimum santum. All the solvent extracts were evaporated to dryness in air. Using the disc diffusion method, the bacterial growth were inhibited. Among the solvent extracts tested, methanol extract of Oxalis corniculata leaf showed higher antibacterial activity compared to Erythromycin and Nalidixic acid against Staphylococci sp. whereas the methanol extract of Ocimum sanctum leaf showed higher antibacterial activity compared to CIP-5 against Staphylococcus aureus. The best MIC values were recorded to be 256 μg mL-1 against Salmonella typhi for ethanol extract of Oxalis corniculata leaf and 128 μg mL-1 against Staphylococcus aureus for the methanol extract of Ocimum sanctum leaf.
INTRODUCTION
The use of higher plants and their extracts to treat infections is an age old practice in traditional medicine. Traditional medical practice has been known for centuries in many parts of the world (Onyeagba et al., 2004). About 80% of the world’s population depends wholly or partially on traditional medicine for its primary health care needs (Kunwar and Adhikari, 2005). Bangladesh possesses a rich flora of Medicinal plants. Out of 5000 species of phanerogams and pteridophytes growing in this country more than a thousand are regarded as having medicinal properties (Batugal et al., 2004). More than 500 of such medicinal plants have so far been enlisted as growing in Bangladesh. The use of herbs is the most ancient approach to healing known. The herbal medicines may be in form of powders, liquids, or mixtures, which may be raw or boiled, ointments, liniments and incisions (Akpata, 1979). Roots, barks and leaves of various plants are employed in ethnomedicine. Plant extracts are given singly or as concoctions for various ailments. Many investigators have demonstrated the antimicrobial activity of the constituents of some higher plants (El-Shouny and Magaam, 2009; Misra et al., 1992; Habtemarian et al., 1993) and quite a number of chemical compounds of plant origin have been shown to possess antimicrobial activities (Prabuseenivasan et al., 2006; De Billerbeck et al., 2001; Corthout et al., 1992). In diseases of microbial origin, the plants function as a result of antimicrobial activity against the causative agents (Sofowora, 1993).
Oxalis corniculata Linn. is a small procumbent herb, with stems rooting and pubescent with appressed hairs, leaves palmately 3-foliolate. This plant is well known for its medicinal value as a good appetiser and as a remover of kapha, vata and piles. It is also known to cure dysentery, diarrhea and skin diseases (Raghavendra et al., 2006). Ocimum sanctum L. (Labiatae) is a strongly scented small annual herb, up to 18 inches tall and grows into a low bush and is commonly known as holy basil, Tulsi’ or Tulasi (Mahmood et al., 2008). It is being used as a tonic for the treatment of nervous disorders, stress related headaches, migraines and allergies (Bargava and Singh, 1981). Due to peculiar essence of O. sanctum oil, used to clear the mind and relieve the intellectual fatigue, while giving clarity and mental strength. The oil is also administered for asthma, bronchitis, sinus infections, constipation, nausea, vomiting and cramp (Gupta et al., 2002).
In this present study it is so tried to find out the antimicrobial effect and Minimum Inhibitory Concentration (MIC) of Oxalis corniculata leaf and Ocimum sanctum leaf extract against clinical isolates from urine, stool and sputum and their successive comparison with commercially available antibiotic discs.
MATERIALS AND METHODS
This research was conducted from July, 2007 to December 2007 at Department of Biotechnology and Genetic Engineering, Islamic University, Kushtia-7003, Bangladesh. The bacterial strains used in this study were Pseudomonas aeruginosa, Staphylococci sp., Staphylococcus aureus, Salmonella typhi, Klebsiella pneumonia, Shigella sp., Enterobacteria sp., Escherichia coli which were isolated from clinical specimens like stool, urine and sputum collected from infected patients by standard methods. The plants were taken from local garden in Kushtia district. Then leaves were collected, washed and cut into small pieces and dried in the shade, away from the sun (this will avoid the denaturing of the enzyme and prevent excessive dehydration). Once the leaves were dried, it was grounded into finely divided powder. Ocimum sanctum leaf was dissolved in selected solvents like n-hexane, ethanol, petroleum spirit and methanol to produce their respective extracts for their antibacterial activity. Similarly, for Oxalis corniculata leaf, chloroform, ethanol and methanol extracts were used as solvents. Twenty gram of the ground samples were soaked in 100 mL of different solvents in conical flasks sealed with foil. The flasks were placed in the shaker for 24 h for Oxalis corniculata leaf and 36 h for Ocimum sanctum leaf so that the oil in the leaf powder extracts out into the solvent. After shaking, the debris of the leaves was removed by filtration. The solutions were filtered twice to ensure no leaf particle gets retained in the crude extract. Into this, a pinch of charcoal was added to absorb the color of the extract. The crude extract was filtered again to remove the undissolved charcoal. The filters were finally transferred into the beaker and left in the air for drying till the solvents evaporates leaving a concentrated crude extract. All extracts were stored at 4°C when not in use. Sterile filter paper disks of 5 mm diameter were impregnated with different crude extracts and dried in a hot air oven at 60°C for 5 min.
Agar plates were inoculated with 0.1 mL broth culture of test organisms and spread with an L-shaped glass rod. Then the commercially available antibiotic discs were placed on the inoculated Petri dishes by using sterile forcep in the laminar Airflow cabinet. Discs prepared from the extract from the leaves were also placed in the same way. Then the plates were left for incubation at 37°C for 48 h. The Minimum Inhibitory Concentration (MIC) of all the solvents were also determined by serially diluting the crude extract from 512 μg mL-1 to 2 μg mL-1 with distil water (Reiner, 1982).
RESULTS AND DISCUSSION
The extract of Oxalis corniculata is prepared by dissolving their powders into different solvents like methanol, ethanol and chloroform. From Table 1 it has been seen that methanol extract of Oxalis corniculata leaf showed the effective zone formation against all the micro-organisms, while chloroform shows the least amount of zone formation. When a comparative study was made, it was seen that the crude extract of the methanol showed a high activity than E-15, TS-25 and NA-30 against Staphylococci sp. The chloroform extract of Oxalis leaf also showed more activity then the commercial discs, i.e., E-15, NA-30 and TS-25 against the Staphylococci sp. The ethanol extracts showed less antibacterial activity when compared to the commercial discs. Table 1 also mentioned that methanol extract of Ocimum leaf showed a good response when compared with the other extracts. It was noticed that the Staphylococcus aureus showed higher sensitivity to methanol extract of Ocimum leaf than CIP-5 and NA-30.
| Table 1: | Antibacterial activity of Oxalis corniculata and Ocimum sanctum leaf extracts on pathogenic bacteria (zone of inhibition in cm) and their comparison with commercial antibiotics |
| E: Erythromycin, NA: Nalidixic acid, 1: Ethanol Extract of Oxalis leaf, 2: Methanol extract of Oxalis, 3: Chloroform Extract of Oxalis, 4: Methanol extract of Ocimum leaf, 5: N-Hexane extract of Ocimum leaf, 6: Petroleum spirit extract of Ocimum leaf, 7: Ethanol extract of Ocimum leaf, -: No inhibition zone | |
| Table 2: | Determination of Minimum Inhibition Concentration (MIC) of Oxalis corniculata and Ocimum sanctum leaf in different solvents (μg mL-1) |
These finding agrees with that of other researchers (Joshi et al., 2009; Unni et al., 2009; Mahmood et al., 2008; Cock, 2008; Raghavendra et al., 2006; Taranalli et al., 2004; Burt, 2004).
Due to variable diffusability in agar medium, the antibacterial property may not demonstrate as ZOI commensurate to its efficacy. Therefore Minimum Inhibitory Concentration (MIC) value has also been computed in this study. MIC is the lowest concentration of antibacterial substance required to produce a sterile culture (Cheesbrough, 1987).
From Table 2 it is observed that in case of Oxalis corniculata leaf extract the lowest MIC value in observed at 256 μg mL-1 of the ethanol extract against Salmonella typhi. Where Ocimum sanctum leaf extract lowest MIC value was observed at 128 μg mL-1 concentration of methanol extract against Shigella sp. In general, it can be stated as that the methanol (Kathiriya et al., 2010) and chloroform extract of Oxalis and ethanol extract of Ocimum leaves showed higher antibacterial activity then the commercial discs. Thus, these plants can be used as efficiently for curing common diseases like diarrhea, cough, fever, etc.
These findings support the traditional knowledge of local users and it is a preliminary, scientific, validation for the use of these plants for antibacterial activity to promote proper conservation and sustainable use of such plant resources. Awareness of local community should be enhanced incorporating the traditional knowledge with scientific findings. In conclusion, the results of the present study support the folkloric usage of the studied plants and suggest that some of the plant extracts possess compounds with antimicrobial properties that can be further explored for antimicrobial activity. This antibacterial study of the plant extracts demonstrated that folk medicine can be as effective as modern medicine to combat pathogenic microorganisms. The millenarian use of these plants in folk medicine suggests that they represent an economic and safe alternative to treat infectious diseases (Bylka et al., 2004; Kilani, 2006; Joshi et al., 2009).
CONCLUSION
The best MIC values were recorded to be 256 μg mL-1 against Salmonella typhi for ethanol extract of Oxalis corniculata leaf and 128 μg mL-1 against Staphylococcus aureus for the methanol extract of Ocimum sanctum leaf.