In vitro Evaluation of the Antimicrobial activity of Methanolic and Aqueous extract of Mucuna pruriens seed
Amita Shobha Rao1, Shobha K L1*, Preethi Lavina Concessao2, Gowrish Rao S1, Sreedhara R Pai K3
1Dept. of Microbiology, Melaka Manipal Medical College, Manipal Academy of Higher Education,
Manipal, Karnataka, India.
2Dept. of Physiology, Melaka Manipal Medical College, Manipal Academy of Higher Education,
Manipal, Karnataka, India.
3Department of Pharmacology, Manipal College of Pharmaceutical Sciences,
Manipal Academy of Higher Education, Manipal, Karnataka, India.
*Corresponding Author E-mail: shobha.kl@manipal.edu
ABSTRACT:
The present investigation was to find out efficacy of Mucuna pruriens (M. pruriens) seeds. The antimicrobial activity of methanol and aqueous extract of M. pruriens seeds was determined by well diffusion method with ATCC strains of gram positive and gram negative bacteria clinical strains of multidrug resistant (MDR) Klebsiella pneumoniae, and clinical strains of Candida albicans. It was observed that both the aqueous and the methanol extract of M. pruriens seeds had antibacterial activity against all the ATCC bacterial strains. The MDR strain of Klebsiella pneumoniae was resistant to both aqueous and methanol extract of M. pruriens seed. Antifungal activity against Candida albicans was exhibited by both aqueous and methanol extracts of M. pruriens seed.
KEYWORDS: Mucuna pruriens, Antibacterial, Antifungal, Agar well diffusion test, Methanol extract.
INTRODUCTION:
Now a days researchers are trying to find out substitutes for antibiotics as they have side effects. These are known to disturb the normal intestinal microflora2.
Plants of medicinal value are the local heritage with global importance. Herbal products today are being highly used for treatment of different types of diseases. The curative property is mainly because of presence of various chemical substances present in the plants and are of great interest to discover and develop a new drug3.
Medicinal plants exhibit antibacterial activities have high amount and variety of phytochemicals like flavonoids, alkaloids, polyphenols etc. This served as a rich source of therapeutic agents. Medicinal plants are now becoming the bases for production of synthetic drug4. The plant products studied has to be properly examined phytochemically and also in terms of their pharmacological properties to be used in alternative therapies. Several studies also showed that seed possesses powerful antimicrobial activities5,6.
Mucuna pruriens [M. pruriens] plant is tropical legume, belonging to the genus Mucuna and comes under the family Leguminosae7. It is commonly known as velvet bean or cowitch. Seeds are 12 mm long, ovoid and its color is shiny black or brown. M. pruriens is extensively used as cover crop for enhancement of water infiltration, softening the soil, improvement of soil fertility and to control the weeds8,9. It has been reported that the seed extract of M. pruriens showed antivenom activities. There are reports which describes the use of M. pruriens seed powder against stress10,11.
In the present investigation efforts have been made to study the antimicrobial activity of methanol and aqueous extract of M. pruriens seeds against various bacterial including the multidrug resistant strain and fungal species.
MATERIALS AND METHODS:
Method:
Methanol extract preparation:
Dry seed powder of M. pruriens was continuously refluxed with methanol using soxhlet apparatus at 40ºC-50ºC for 72 hours. The solvent extract was then stored in air tight container at 4ºC till further use.
Antimicrobial activity:
Agar well diffusion method was employed to study the antibacterial and antifungal susceptibility12.
Antimicrobial susceptibility was determined against following strains. The American Type Culture Collection (ATCC) strains of bacteria used were Staphylococcus aureus (ATCC 25923), Klebsiella pneumoniae (ATCC 700603), Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853). Clinical strains of MDR Klebsiella pneumoniae and clinical strains of Candida albicans were also taken for the study.
Sabouraud’s dextrose agar, Muller-Hinton agar was procured from Hi-media, Mumbai. The above mentioned bacterial and fungal strains were revived by plating on nutrient agar, and Sabouraud’s dextrose agar (SDA), respectively. Isolated colonies were selected after overnight incubation at 37°C. Identification of the organisms was done by standard procedure. Isolated bacterial colonies were then transferred to sterile Mueller- Hinton broth, and Candida albicans was transferred to Sabouraud’s dextrose broth and incubated overnight. 0.5 McFarland’s turbidity standard was used to adjust the concentration of growth of microorganisms to 105CFU/ml.
Ampicillin disc (10µg), Oxacillin disc (1µg), Gentamicin disc (30µg) and Ketoconazole disc (10µg), obtained from Hi-Media, Mumbai were used as controls.
Determination of antibacterial activity:
Mueller-Hinton Agar (MHA) and blood agar measuring 20ml each was poured into petri dishes. The bacterial culture was spread over the surface of the MHA plate and blood agar. 6mm diameter wells were punched into the agar and filled with 100μl solution of test compound. The inoculated plates were then kept in the incubator for 18 hrs at 37°C. Tests were done in triplicates and the average of the three was considered for the study.
Determination of antifungal activity:
20ml of SDA was poured into each petridishes. Culture of the Candida albicans was spread over the surface of the SDA plate. Wells were punched into the agar plate measuring 6mm in diameter and filled with 100μl solution of test compound. The plates were then kept in the incubator for 18 hrs at 37°C. Tests were done in triplicates and the average of the three was considered for the study.
RESULTS:
Diameter of the zone of inhibition was measured for the antimicrobial activity. It was observed that both the aqueous extract and the methanol extract of M. pruriens seeds had antibacterial activity against all the ATCC bacterial strains as given in Table 1 and Table 2.
The methanol extract of M. pruriens seeds had antibacterial activity better than the aqueous extract.
The MDR Klebsiella pneumoniae was resistant to both aqueous and alcoholic extract of M. pruriens seed
Both the aqueous and methanol extract of M. pruriens seeds were found to have antifungal activity towards Candida albicans.
Table: 1 Zone of inhibition of M. pruriens seed methanol extract against various organisms
|
Name of the organism |
200 μg/ml |
100μg/ml |
50μg/ml |
25μg/ml |
|
Staphylococcus aureus (ATCC 25923) |
25 |
21 |
18 |
12 |
|
Escherichia coli (ATCC 25922) |
12 |
10 |
08 |
- |
|
Pseudomonas aeruginosa (ATCC 27853) |
11 |
09 |
07 |
|
|
Klebsiella pneumoniae (ATCC 700603) |
17 |
09 |
6 |
- |
|
Klebsiella pneumoniae (MDR strain) |
- |
- |
- |
- |
|
Candida albicans |
18 |
15 |
9 |
- |
MDR: Multidrug resistant, (‑): Indicates no zone of inhibition
Table: 2 Zone of inhibition of M. pruriens seed aqueous extract against various organisms
|
Name of the organism |
200μg/ml |
100μg/ml |
50μg/ml |
25μg/ml |
|
Staphylococcus aureus (ATCC 25923) |
19 |
16 |
13 |
10 |
|
Escherichia coli (ATCC 25922) |
11 |
09 |
07 |
- |
|
Pseudomonas aeruginosa (ATCC 27853) |
10 |
08 |
- |
- |
|
Klebsiella pneumoniae (ATCC 700603) |
15 |
14 |
12 |
09 |
|
Klebsiella pneumoniae (MDR strain) |
- |
- |
- |
- |
|
Candida albicans |
15 |
10 |
8 |
- |
MDR: Multidrug resistant, (-) Indicates no zone of inhibition
DISCUSSION:
Now a days plants are the source of inspiration for preparation of novel drug compounds in developing countries. The drugs derived from plants contribute significantly towards human health. The antibacterial, antifungal, and antiviral activity is significant. The antibacterial effect against both gram positive and gram negative bacteria gives an indication that the plant can be a source of the bioactive substance which has broad spectrum of activity13-17
The extract and the biologically active compounds isolated from the plants have gained importance. Plant-derived drugs have been reported to be safe and without side effects; hence, much attention has been given to these natural products as new therapeutic agents18-24.
The present study observation was that the alcoholic extracts showed significant antibacterial activity when compared to aqueous extracts and this was supported by other investigators25-31. Antifungal activity against Candida albicans was exhibited by both aqueous and methanol extracts of M. pruriens seed in this study. This might be due to the fact that the antifungal bioactive components such as alkaloids, lectins, terpenes, and saponins.
In the present study it was observed that the methanol and aqueous extracts of M. pruriens seeds were effective in inhibiting the test pathogens. Based on the results obtained, it was seen that methanol extract of M. pruriens seed had better antibacterial activity and may be used in traditional medicine. Further work can be carried out on the isolation procedure for finding out the exact phytocompound responsible for the biological activity.
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Received on 14.07.2020 Modified on 24.09.2020
Accepted on 28.10.2020 © RJPT All right reserved
Research J. Pharm. and Tech. 2021; 14(8):4212-4214.
DOI: 10.52711/0974-360X.2021.00730