IJCRR

IJCRR - 5(7), April, 2013

Pages: 17-24

Date of Publication: 18-Apr-2013

INVESTIGATION OF ANTI BACTERIAL AND ANTI FUGAL POTENTIALS OF MACARANGA PELTATA

Author: Meenakshi Verma, Narayanan K., Mitali Thakar B., Subrahmanyam V.M., Venkata Rao J., Dhanaraj S.A., Vasanth Raj P.

Category: General Sciences

Abstract:Macaranga peltata belonging to Euphorbiaceae family. Leaves and stem bark were separated, shade dried and extracted separately using methanol. The concentrated methanolic extracts of Macaranga peltata were subjected to antimicrobial studies. The percentage yield of the leaves and stem bark were found to be 47% and 30% respectively. The anti-bacterial activity of Leaf and Stem Bark extracts of Macaranga peltata was determined by Cup-plate method and Zone of Inhibition was measured. Zone of Inhibition was found to be 16mm for Leaf extract and 13mm and 14mm for Stem Bark extract in two different sets against Escherichia coli. Out of two Gram-positive bacteria Bacillus subtilis and Staphylococcus aureus Zone of Inhibition was obtained only for Staphylococcus aureus that was 16mm for Leaf extract and 12mm and 13mm for Stem Bark. Leaf extracts showed better anti-bacterial activity than the Stem Bark extract against both Gram-positive and gram-negative bacteria. Minimum Inhibitory concentration (MIC) for Leaf extract was between 62.5\?g/ml to 125\?g/ml for Escherichia coli, 125\?g/ml to 250\?g/ml for Pseudomonas aeruginosa, 62.5\?g/ml to 125\?g/ml for Bacillus subtilis and 31.25\?g/ml to 62.5\?g/ml for Staphylococcus aureus. Whereas MIC for Stem Bark extract was between 500\?g/ml to 1000\?g/ml for Escherichia coli, 250\?g/ml to 500\?g/ml for Pseudomonas aeruginosa, 62.5\?g/ml to 125\?g/ml for Bacillus subtilis and 62.5\?g/ml to 125\?g/ml for Staphylococcus aureus. Ciprofloxacin used as standard antibiotic showed MIC between 1.953 \?g/ml to7.813 \?g/ml for all four bacterial strains.

Keywords: Macaranga peltata, Euphorbiaceae, anti-bacterial and Cup-plate method

Full Text:

INTRODUCTION

The plant is a biosynthetic laboratory, not only for chemical compounds, but also a multitude of compounds like glycosides, alkaloids etc. These exert physiological and therapeutic effect. The compounds that are responsible for medicinal property of the drug are usually secondary metabolites. A systematic study of a crude drug embraces through consideration of primary and secondary metabolites derived as a result of plant metabolism. The plant material is subjected to phytochemical screening for the detection of various plant constituents [1]. With onset of scientific research in herbals, it is becoming clearer that the medicinal herbs have a potential in today’s synthetic era, as numbers of medicines are becoming resistant. According to one estimate only 20% of the plant flora has been studied and 60%of synthetic medicines owe their origin to plants. Ancient knowledge coupled with scientific principles can come to the forefront and provide us with powerful remedies to eradicate the diseases.
According to World Health Organization (WHO) more than 80% of the world's population relies on traditional medicine for their primary healthcare needs. Plants used for traditional medicine contain a wide range of substances that can be used to treat chronic as well as infectious diseases [2]. The 30th World Health Assembly adopted a resolution urging interested governments to utilize their traditional systems of medicine with regulations suited to their national health care systems [3]. Utilization of plants for medicinal purposes has been documented long back in ancient literature. However, organized studies in this direction were initiated in 1956 and off late such studies are gaining recognition and popularity due to loss of traditional knowledge and declining plant population [4]. Macaranga peltata is one of important plants widely found in Western Ghats in India. Macaranga peltata belonging to Euphorbiaceae family is a genus of important pioneer trees widely distributed in south east Asia. Macaranga is a dicot plant with more than 350 species. Genus Macaranga is native to Africa, Australia, Asia and the South Pacific. There are evidences that show that bergenin derivatives and polyphenols have been recorded from Macanga peltata and other flavonoids as well as diterpinoids are suspected to be present. A previous study on Macaranga genus includes study of Macaranga alnifolia for cytotoxic natural products from tropical rainforests in Madagascar through the International Cooperative Biodiversity Group (ICBG) program. Ethanolic extract of the fruit of Macaranga alnifolia Baker (Euphorbiaceae) was obtained for phytochemical investigation. This extract was found to be active in the A2780 ovarian cancer cytotoxicity assay. Bioassayguided fractionation led to the isolation of the five new compounds [5]. Although several traditional plant extracts have historically been known to have antimicrobial activity, to date, there has been relatively little or in some cases, no reports examining the activity against several medically important bacterial and fungal pathogens [6]. Considering the vast of potentiality of plants as sources for antimicrobial drugs with reference to antibacterial and antifungal agents, a systematic investigation was undertaken to screen the local flora for antibacterial and antifungal activity from Macanga peltata.

MATERIALS AND METHODS MATERIALS

All solvents and reagents purchased form Merck Pvt. Ltd, Mumbai, India. All media components purchased from Himedia (India). All the microorganisms were procured from National Collection of Industrial Microorganisms (NCIM), Pune and subcultured in the laboratory.

METHODS

Collection and Authentication of Plant
The plant Macaranga peltata was collected from the Syndicate circle, Manipal in the month of January 2009. The plant was authenticated by Dr. Chandrakant Bhat, Professor, Muniyal Ayurvedic College, Manipal, Karnataka India.

Preparation of Extracts:
The collected plant parts (leaves and stem bark) were separated and shade dried. Then the dried parts were chopped and coarsely powdered; the powdered plant materials were extracted separately using methanol by soxhlet extraction. The amount of methanol was taken as methanol : dry powder (100:15) The extracts were then concentrated to dryness under reduced pressure and controlled temperature to yield a deep brown – dark brown semisolids, the percentage yields was calculated.. All the extracts were stored in refrigerator till further use.

Phytochemical Studies: Qualitative Phytochemical Analysis:
The concentrated methanolic extracts of Macaranga peltata were subjected to chemical test as per the standard methods for the identification of the various constituents. 2-3 mL of the extract solution was taken for all the tests for viz., carbohydrates, Alkaloids, Steroids and Sterols, Glycosides, Saponins, Flavanoids, Tannins, Triterpenoids, Protein and Amino acid:[7] (results not shown).

Antimicrobial activity [8]:
(a) Growth and Maintenance of Test Microorganism for Antimicrobial Studies:
Bacillus subtilis – NCIM 2063 Escherichia coli – NCIM 5011 Pseudomonas aeruginosa – NCIM 7312 Staphylococcus aureus – NCIM 2079 Aspergillus niger – NCIM 545 Candida albicans – NCIM 3100 All the microorganisms were procured from National Collection of Industrial Microorganisms (NCIM), Pune and subcultured in the laboratory. The bacterial cultures were maintained in slants till further use and fungus cultures were maintained on Sabraud’s dextrose agar (SDA).
(b) Cup Plate method for determination of Zone of Inhibition: Nutrient agar (20ml) was prepared according to the manufacturers’ recommendation and sterilized. Plates containing 20 ml of sterile nutrient agar each were inoculated with standardized innocula using sterile Pasteur pipette. Six wells each of 5 mm diameter were made on each plate-1 for antibiotic (Ciprofloxacin for antibacterial), 1 for DMSO (Solvent control), 2 for Leaf Extract and 2 for Stem Bark Extract. 50µl of the plant extracts, antibiotic and DMSO were dispensed into subsequent wells. The extracts were allowed to diffuse into the medium for 1hr at room temperature. This was then incubated at for 24 h at 37ºC after which the zones of growth inhibition were measured and recorded in millimeter [9].
(c) Determination of Minimum Inhibitory Concentration by Tube Dilution method: MIC is the lowest concentration of the drug in which turbidity/growth is not observed. A series of test tubes were prepared containing the same volume of medium inoculated with the test organism to obtain about 104 CFU/ml. Two fold serial dilutions were carried out using 1.8 ml of the above inoculum and 0.2 ml test solution. Positive control containing antibiotic, negative control containing uninoculated broth and solvent control containing DMSO were also used. The tubes were incubated at 37º C for 18 hours (Bacteria) or 28º C for 40 hours (Fungi) [10]. The tubes are inspected visually to determine whether the microorganism has grown, as indicated by turbidity. Tubes in which the extract/ antibiotic is present in a concentration sufficient to inhibit bacterial growth remained clear.

Yield of plant extract:
Percentage yield obtained from leaf was 47% and from stem bark powder was 30% that is very good yield (Table no.1).

Antimicrobial activity:
Standardization of Colonies:
Two Gram-positive and two Gram-negative bacterial colonies were standardized and results were obtained. The numbers of bacterial colonies were represented in terms of Colony Forming Unit (Table no.2).

Antimicrobial Activity (Zone of Inhibition):
Anti-microbial activity of the plant extracts were determined by Cup-Plate Method and activity was measured by measuring Zone of Inhibition in millimeters. Results were obtained for each bacterial strain tested against Macaranga peltata Leaf and Stem Bark extract. Anti-bacterial activity was obtained only for one Gram-positive and one Gram-negative bacteria out of two gram-positive and two Gram-negative bacterial strains. Leaf extract showed better activity against both Gram-positive and Gramnegative bacteria. DMSO used as solvent control, did not inhibit growth of bacteria. Activity of the extracts was found to be less than the standard antibiotic Ciprofloxacin (Table no.3).

RESULTS

Minimum Inhibitory concentration (MIC): The results for MIC of Macaranga peltata observed against different bacterial species were recorded.

MIC for E.coli:
Very low concentration i.e. between 3.906 µg/ml to7.813 µg/ml of ciprofloxacin (used as standard anti-biotic) was able to inhibit the bacterial growth. Leaf extract inhibited the bacterial growth between 62.5µg/ml to 125µg/ml concentration while Stem Bark extract inhibited growth of bacteria between 500µg/ml to 1000µg/ml of concentration. DMSO at its higher concentrations was found to inhibit bacterial growth. Although activity showed by plant extracts was lesser as compared to standard, still Leaf extract showed better activity than Stem Bark extracts (Table no.4).

MIC for Pseudomonas:
Very low concentration i.e. between 1.953 µg/ml to 3.906 µg/ml of ciprofloxacin (used as standard anti-biotic) was able to inhibit the bacterial growth. Leaf extract inhibited the bacterial growth between 125µg/ml to 250µg/ml concentration while Stem Bark extract inhibited growth of bacteria between 250µg/ml to 500µg/ml of concentration. DMSO at its higher concentrations was found to inhibit bacterial growth. Although activity showed by plant extracts was lesser as compared to standard, still Leaf extract showed better activity than Stem Bark extracts (Table no.5).

MIC for Bacillus:
Very low concentration i.e. between 1.953 µg/ml to 3.906 µg/ml of ciprofloxacin (used as standard anti-biotic) was able to inhibit the bacterial growth. Both Leaf extract and Stem Bark extracts inhibited the bacterial growth between 62.5µg/ml to 125µg/ml concentration (Table no.6). DMSO at its higher concentrations was found to inhibit bacterial growth. Although activity showed by plant extracts was less, still it was comparable with standard.

MIC for Staphylococcus:
Very low concentration i.e. between 1.953 µg/ml to 3.906 µg/ml of ciprofloxacin (used as standard antibiotic) was able to inhibit the bacterial growth. Leaf extract inhibited the bacterial growth between 31.25µg/ml to 62.5µg/ml concentration while Stem Bark extract inhibited growth of bacteria between 62.5µg/ml to 125µg/ml of concentration. DMSO at its higher concentrations was found to inhibit bacterial growth. Although activity showed by plant extracts was lesser as compared to standard, still Leaf extract showed better activity than Stem Bark extracts (Table no.7).

DISCUSSION

Macaranga peltata belonging to Euphorbiaceae family is one of the important plants found in Western ghats specially in Udupi district, which contains potential for active compounds is very less studied. Since different species of Macaranga i.e. Macaranga alnifolia has been already studied for its anti-proliferative activity, Macaranga peltata is studied for its antimicrobial activity. Leaf and Stem Bark samples were collected from Macaranga peltata and shade dried and powdered. Methanolic extracts of these samples were obtained by Soxhlet extraction method. The yield obtained from leaf was 47% and from Stem Bark was 30%. Both plant extracts were subjected to phytochemical analysis. Leaf extract showed the presence of carbohydrates, steroids and sterols, glycisides, flavonoids, tannins, proteins and amino acids while Stem Bark extract showed positive results for carbohydrates, glycosides, saponins, flavonoids, tannins, proteins and amino acids. Biological studies of plant extracts indicate anti-microbial activity. Anti-microbial activity was performed in two steps, one for anti-bacterial and another for antifungal. To screen the plant Macaranga peltata for anti-bacterial activity four bacterial strains were selected; two Gram-positive (Bacillus subtilis and staphylococcus aureus) and two Gram-negative (Escherichia coli and Pseudomonas aeruginosa). These bacterial strains were maintained in slants containing Nutrient Agar and sub-cultured in nutrient broth during use. The standardization of each strain was done and Colony Forming Unit (CFU) for each was determined. The anti-bacterial activity of Leaf and Stem Bark extracts of Macaranga peltata was determined by Cup-plate method and Zone of Inhibition was measured. Zone of Inhibition was found to be 16mm for Leaf extract and 13mm and 14mm for Stem Bark extract in two different sets against Escherichia coli while there was no inhibition observed against another Gram-negative strain Pseudomonas aeruginosa. Out of two Gram-positive bacteria Bacillus subtilis and Staphylococcus aureus Zone of Inhibition was obtained only for Staphylococcus aureus that was 16mm for Leaf extract and 12mm and 13mm for Stem Bark extract in two different sets of the experiment. Leaf extracts showed better anti-bacterial activity than the Stem Bark extract against both Gram-positive and gram-negative bacteria. The extracts that were tested positive were estimated for Minimum inhibitory concentration (MIC). MIC of the extracts was determined against all bacterial strains selected for the experiment. MIC for Leaf extract was between 62.5µg/ml to 125µg/ml for Escherichia coli, 125µg/ml to 250µg/ml for Pseudomonas aeruginosa, 62.5µg/ml to 125µg/ml for Bacillus subtilis and 31.25µg/ml to 62.5µg/ml for Staphylococcus aureus. Whereas MIC for Stem Bark extract was between 500µg/ml to 1000µg/ml for Escherichia coli, 250µg/ml to 500µg/ml for Pseudomonas aeruginosa, 62.5µg/ml to 125µg/ml for Bacillus subtilis and 62.5µg/ml to 125µg/ml for Staphylococcus aureus. Ciprofloxacin used as standard antibiotic showed MIC between 1.953 µg/ml to7.813 µg/ml for all four bacterial strains. For screening of anti-fungal activity of plant Macaranga peltata two fungal strains Aspergillus niger and Candida albicans were selected. There was no antifungal activity observed by the extracts at the concentrations tested.

CONCLUSION

Macaranga peltata yield obtained from leaf was 47% and from Stem Bark was 30%. Both plant extracts were subjected to phytochemical analysis. Leaf extract showed the presence of carbohydrates, steroids and sterols, glycisides, flavonoids, tannins, proteins and amino acids while Stem Bark extract showed positive results for carbohydrates, glycosides, saponins, flavonoids, tannins, proteins and amino acids. Biological studies of plant extracts indicate antimicrobial activity. Leaf and Stem Bark samples extracts of Macaranga peltata were subjected to anti microbial studies. Both the extracts proved moderate anti bacterial activity among them leaf extracts showed better anti-bacterial activity than the Stem Bark extract against both Gram-positive and gram-negative bacteria.

ACKNOWLEDGEMENTS

We thank Manipal University for providing the facilities and support to conduct this study. Authors acknowledge the great help received from the scholars whose articles cited and included in references of this manuscript. The authors are also grateful to authors / editors / publishers of all those articles, journals and books from where the literature for this article has been reviewed and discussed. Authors are grateful to IJCRR editorial board members and IJCRR team of reviewers who have helped to bring quality to this manuscript.

Inference: Anti-bacterial activity was obtained only for one Gram-positive and one Gramnegative bacteria out of two gram-positive and two Gram-negative bacterial strains. Leaf extract showed better activity against both Gram-positive and Gram-negative bacteria. DMSO used as solvent control, did not inhibit growth of bacteria. Activity of the extracts was found to be less than the standard antibiotic Ciprofloxacin.

Inference: Very low concentration i.e. between 3.906 µg/ml to7.813 µg/ml of ciprofloxacin (used as standard anti-biotic) was able to inhibit the bacterial growth. Leaf extract inhibited the bacterial growth between 62.5µg/ml to 125µg/ml concentration while Stem Bark extract inhibited growth of bacteria between 500µg/ml to 1000µg/ml of concentration. DMSO at its higher concentrations was found to inhibit bacterial growth. Although activity showed by plant extracts was lesser as compared to standard, still Leaf extract showed better activity than Stem Bark extracts.

Very low concentration i.e. between 1.953 µg/ml to 3.906 µg/ml of ciprofloxacin (used as standard anti-biotic) was able to inhibit the bacterial growth. Leaf extract inhibited the bacterial growth between 125µg/ml to 250µg/ml concentration while Stem Bark extract inhibited growth of bacteria between 250µg/ml to 500µg/ml of concentration. DMSO at its higher concentrations was found to inhibit bacterial growth. Although activity showed by plant extracts was lesser as compared to standard, still Leaf extract showed better activity than Stem Bark extracts.

Inference: Very low concentration i.e. between 1.953 µg/ml to 3.906 µg/ml of ciprofloxacin (used as standard anti-biotic) was able to inhibit the bacterial growth. Both Leaf extract and Stem Bark extracts inhibited the bacterial growth between 62.5µg/ml to 125µg/ml concentration. DMSO at its higher concentrations was found to inhibit bacterial growth. Although activity showed by plant extracts was less, still it was comparable with standard.

Inference: Very low concentration i.e. between 1.953 µg/ml to 3.906 µg/ml of ciprofloxacin (used as standard anti-biotic) was able to inhibit the bacterial growth. Leaf extract inhibited the bacterial growth between 31.25µg/ml to 62.5µg/ml concentration while Stem Bark extract inhibited growth of bacteria between 62.5µg/ml to 125µg/ml of concentration. DMSO at its higher concentrations was found to inhibit bacterial growth. Although activity showed by plant extracts was lesser as compared to standard, still Leaf extract showed better activity than Stem Bark extracts.

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