Anti-bacterial Evaluation and Minimum Inhibitory Concentration Analysis of Oxalis corniculata and Ocimum santum against Bacterial Pathogens
Md. Shahedur Rahman,
Md. Mahboob Hossain Khan
Md. Abu Hena Mostofa Jamal
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.
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 worlds 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
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,
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.
||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
|| Determination of Minimum Inhibition Concentration (MIC) of
Oxalis corniculata and Ocimum sanctum leaf in different solvents
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;
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
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.,
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.
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