Abstract: The presence study was carried out to investigate on the possible antibacterial activity of methanol (MECO) and chloroform (CECO) extracts of Corchorus olitorius (Senaung betina) using the in vitro disc diffusion methods. The sterilized blank discs (6 mm diameter) was impregnated with 20 μL of the respective extract (in the concentration of 10,000; 20,000; 40,000 and 50,000 ppm) and tested against Corneybacterium diphtheria, Staphylococcus aureus (ATCC 25923), Bacillus cereus, Proteus vulgaris, Staphylococcus epidermidis, Kosuria rhizophila, Shigella flexneri, Escherichia coli (O 157), Aeromonas hydrophila and Listeria monocytogenes. The MECO and CECO, at all concentrations, were effective against C. diptheria and K. rhozophila with the latter also effective against S. aureus and B. cereus. At the concentration of 40,000 ppm and above, the MECO was effective only against S. aureus, B. cereus and S. epidermidis while the CECO was effective against S. epidermidis, S. flexneri and A. hydrophila. Based on this study, it was concluded that C. olitorius possesses antibacterial activity that is comparable to some of the standard antibiotics.
INTRODUCTION
Corchorus olitorius L., locally known to the Malaysia’s Sabahan as Senaung betina, is an annual herb that belongs to the family Tiliaceae. Its leaves and roots eaten are as herbal medicine and as vegetable by local people in various part of middle and south east Asia (Zeghichi et al., 2003). Traditionally, its leaves are used in the treatment of gonorrhea, chronic cystitis, pain, fever and tumours (Zeghichi et al., 2003; Abu-Hadid et al., 1994).
Recent study has demonstrated that the aqueous extract of C. olitorius possesses peripheral and central antinociceptive activities that might be mediated by the opioid receptor and enhanced at high temperature (Zakaria et al., 2005a). Further studies have also proven that C. olitorius possess anti-inflammatory and antipyretic activities (Zakaria et al., 2005b) and that its antinociceptive activity is influenced by pH and α-amylase (Zakaria et al., 2005c).
Based on the fact that there are no scientific research reporting on the antibacterial activity of this plant, we decided to take this opportunity to screen for its potential antibacterial activity. The aim of this study was to elucidate C. olitorius potential antibacterial activity using its methanol and chloroform extracts against a group of selected gram positive and gram negative bacteria.
MATERIALS AND METHODS
C. olitorius plants were collected from Shah Alam, Selangor, Malaysia and a voucher specimen (SK 963/04) was deposited at the Herbarium of Institute of Bioscience, Universiti Putra Malaysia, Malaysia.
Microorganisms tested in this study were Corneybacterium diphtheria, Staphylococcus aureus, Bacillus cereus, Proteus vulgaris, Staphylococcus epidermidis, Kosuria rhizophila, Shigella flexneri, Escherichia coli, Aeromonas hydrophila and Listeria monocytogenes.
Fresh leaves of C. olitorius were oven-dried for 24 h at 40°C according to the methods described by Somchit et al. (2003) but with slight modifications. It was then ground into small pieces under sterilized condition and extracted with methanol or chloroform in the ratio of 1: 20 (w/v) for 24 h by using Soxhlet apparatus. The resultant extraction of both solvents was completely evaporated by using rotary evaporator machine (Somchit et al., 2003). The obtained crude extracts of methanol (MECO) and chloroform (CECO) were prepared into various concentrations (10,000, 20,000, 40,000 and 50,000 ppm) by dissolving the respective extract in dimethyl sulfoxide (DMSO). Twenty microliter of the respective MECO or CECO were then loaded into empty sterilized blank discs (6 mm diameter, Oxoid, UK). In addition, commercial antibiotic discs (Chloramphenicol; 30 μg μL-1) were used for comparison.
Preparation of microorganism culture: The above-mentioned bacteria were incubated at 37°C±0.5 for 24 h after injection into nutrient broth. Mueller Hinton Agar (MHA) (Oxoid, UK), sterilized in a flask and cooled to 40-50°C was poured, in the volume of 15 mL, into sterilized Petri dishes (diameter of 9 cm) and allowed to harden under room temperature. This is followed by homogenous distribution of 0.1 mL bacteria cultures (106 bacteria per mL) onto medium in Petri dishes. Discs loaded with extracts were then positioned on the solid agar medium by pressing slightly (Sundar, 1996). Petri dishes were placed in incubator according to their respective growth temperature and condition for 18 to 24 h. At the end of the period, inhibition zones formed was measured in mm. The study was performed in triplicate and the formation of the inhibition zones were compared with those of antibiotic discs.
RESULTS AND DISCUSSION
As can be seen from the Table 1, the MECO at the concentration between 10,000 and 50,000 ppm, was found to be effective against C. diphtheriae and K. rhizophila with the inhibition zone ranging from 14.0 to 18.0 mm, respectively. Other than the two bacteria, the CECO was also very effective against S. aureus. The inhibition zone of the CECO, at the concentration between 10,000 and 50,000 ppm, against C. diphtheriae, K. rhizophila, B. cereus and S. aureus ranged between 12.3 to 16.0, 12.0 to 17.0, 9.0 to 13.0 and 15.0 to 19.0 mm, respectively. The extracts high effectiveness is indicated by the presence of activity at the lowest concentration (10,000 ppm) used (Table 1).
The MECO was also found to show positive activity at the concentration of 20,000 or 40,000 ppm and above, against B. cereus and S. aureus and S. epidermidis, respectively. On the other hand the CECO was found to show positive inhibitory effect, at the concentration of 40,000 ppm, against S. epidermidis. However, the MECO and CECO extracts were found to give negative results when treated against P. vulgaris, S. flexneri, E. coli (O 157), A. hydrophila and L. monocytogenes (Table 1).
This study has demonstrated the potential used of C. olitorius as an antibacterial agent against the infection of C. diphtheria, S. aureus, B. cereus, S. epidermidis and K. rhizophila. The CECO was also found to be more effective against the said bacteria when compared to the MECO indicated by the presence of activity in the lowest concentration of CECO used (10,000 ppm) ( Table 1).
The traditional used of C. olitorius for treatment of various ailments by local people in India, Egypt and Philippines (Zeghichi et al., 2003) but lack of scientific papers to prove on those claims has lead to this study.
| Table 1: | The antibacterial activity of methanol and chloroform extracts of Corchorus olitorius determined by disc diffusion method |
| IZ = Inhibition zone, - = No inhibition zone, + = IZ ≤
9.0 mm, ++ = 9.0 mm < IZ ≤ 13.0 mm, +++ = 13.0 mm < IZ ≤ 16.0
mm ++++ = 16.0 mm < IZ ≤ 20.0 mm, Except for P. vulgaris (IZ = 9 mm), Chloramphenicol gave inhibition zone of ≤20 mm against all bacteria |
|
Present preliminary study on the aqueous extract of its leaves has demonstrated a potential antinociceptive (Zakaria et al., 2005a) and anti-inflammatory and anti-pyretic (Zakaria et al., 2005b) activities. The reason for not using the aqueous extract in this study is because the leaves were found to release sticky mucus once soaked in DH2O. Furthermore, the disc impregnated with the aqueous extract was found not to block but improved bacteria growth, which is observed even on the disc itself. This observation may be attributed to the extract sticky condition, which is suggested to take sometimes to dry and thus, provide enough moisture for bacteria growth. Even though we have dried the disc for one week under sterilized condition, the sticky appearance of the extract was still observed indicating the presence of moisture that might influence bacteria growth.
Even though the current study does not involved identification of the chemical constituents presence in the leaves extract, it is possible to relate the observed activity to the presence of tannins, flavonoids and glycosides (Ogunleye and Ibitoye, 2003), which have been known to occur abundantly in leaves of all plants.
Furthermore, present results also demonstrated that the polar, expected to be found in MECO and non-polar, expected to be found in CECO, compounds to be more effective in the treatment of Gram-positive bacteria rather than Gram-negative ones.
Finally, it was concluded that present results provided a basis for isolation of antibacterial compounds of interest from C. olitorius. Further study is being carried out to isolate and identify the antibacterial compounds present in both extracts of C. olitorius.
ACKNOWLEDGMENTS
The authors would like to thank Universiti Industri Selangor for the research grant (Project Code Number: 03018; Vote Number: 3090103018) and Universiti Putra Malaysia for the facilities.