Abstract: The aim of the present study was to evaluate antibacterial and antioxidant activity of different solvent extracts of Mangifera indica L. seeds. The seeds of Mangifera indica L. were successively extracted in petroleum ether, chloroform, ethyl acetate, acetone and methanol. All obtained extracts were evaluated for antimicrobial and free radical scavenging (DPPH) activity. The in vitro antimicrobial activity was done by agar disc diffusion method at a concentration of 600 μg disc-1 against 5 Gram-positive bacteria, 7 Gram-negative bacteria and 3 fungal strains. The antioxidant potential was evaluated using scavenging of DPPH radical. Maximum antibacterial activity was shown by methanol extract followed by acetone extract. Acetone (IC50 = 11 μg mL-1) and methanol (IC50 = 12 μg mL-1) extract also showed DPPH scavenging activity which was comparable with that of standard ascorbic acid (IC50 = 11.4 μg mL-1). This study has highlighted the potentiality of antibacterial and antioxidant properties of M. indica seeds.
INTRODUCTION
There is continuous and urgent need to discover new antimicrobial compounds with diverse chemical structures and novel mechanisms of action because there has been an alarming increase in the incidence of new and re-emerging infectious diseases. In addition, bacterial adaptation to antibiotics and increase in antibiotic resistance over the past decade has generated a considerable worldwide public health problem (Andersson, 2003). Plants contain numerous biologically active compounds, many of which have antimicrobial properties (Cowan, 1999). Numerous studies have been conducted with the extracts of various plants, screening antimicrobial activity as well as for the discovery of new antimicrobial compounds (Chah et al., 2006; Nair and Chanda, 2006; Parekh and Chanda, 2007a).
Free radicals are often generated as by products of biological reactions or from exogenous factors. The involvement of free radicals in the pathogenesis of a large number of diseases is well documented. A potent scavenger of free radicals may serve as a possible preventive intervention for the diseases (Gyamfi et al., 1999). A large number of naturally occurring compounds such as flavonoids, catechins, lignans and phenolic acids contained in edible plants and medicinal herbs have antioxidant properties. Dietary antioxidant intake has been associated for example with reduced risk of cardiovascular diseases, cancer and neurodegenerative diseases (Nair et al., 2007).
Mangifera indica L. is commonly grown in many parts of the world belongs to the family Anacardiaceae. Mangifera indica L., a plant widely used in the traditional medicinal systems of India, has been reported to possess antiviral, antibacterial and anti-inflammatory activities (Makare et al., 2001). The seeds have been used for anti-diarrhoeal activity in Indian traditional medicine (Sairma et al., 2003). The young and the unripe fruits of mango are acidic in taste and utilized for various culinary purposes. The ripe fruits are used in preparing various processed products such as squash, nectar, jam, cereal flakes, custard powder, baby food and toffee etc. (CSIR, 1962). The leaves showed antibacterial activity (Bbosa et al., 2007) however there are hardly any reports on the antimicrobial and antioxidant activity of mango seed. The aim of this study was to determine the antimicrobial and antioxidant activity of the seed extracts of M. indica.
MATERIALS AND METHODS
Plant Material
Fresh seeds of Mangifera indica L. (kesar) were collected in the
month of June 2008, from Talala (Gir), Junagadh, Gujarat, India. The taxonomic
identity of the plant was confirmed by Dr. N.K. Thakrar, Department of Biosciences,
Saurashtra University, Rajkot, India. Fresh seeds were washed, air dried and
then homogenized to fine powder and stored in airtight bottles.
Extraction
The dried powder of M. indica was extracted in a series of solvents
of increasing polarity. Petroleum ether, chloroform, ethyl acetate, acetone
and methanol were used for the extraction. Ten grams of dried powder of M.
indica was taken in 100 mL of solvent in a conical flask, plugged with cotton
wool and then kept on a rotary shaker for 24 h. After 24 h, the extract was
filtered with eight layers of muslin cloth. Remaining residue was dried and
used for extraction in another solvent. Filtrate was centrifuged at 5000 g for
10 min. The supernatant was collected and the solvent was evaporated. The dried
extract was stored at 4°C in airtight bottles.
Microorganisms
The tested microorganisms were obtained from National Chemical Laboratory,
Pune, India. Amongst fifteen microorganisms investigated five were Gram positive
bacteria (Staphylococcus aureus ATCC25923, Staphylococcus epidermidis
ATCC12228, Bacillus cereus ATCC11778, Bacillus subtilis TCC6633,
Micrococcus flavus ATCC10240) while seven were Gram negative bacteria
(Pseudomonas aeruginosa ATCC27853, Escherichia coli ATCC25922,
Klebsiella pneumoniae NCIM2719, Proteus mirabilis NCIM2241, Proteus
vulgaris NCTC8313, Salmonella typhimurium ATCC23564, Citrobacter
freundii ATCC10787) and three fungus strains were Candida albicans ATCC2091,
Candida tropicalis ATCC4563 and Cryptococcus luteolus ATCC32044.
The bacteria were grown in the nutrient broth and maintained on nutrient agar
slants at 4oC, while fugal strains were grown in sabourad broth and
maintained on MGYP slants at 4oC.
Antimicrobial Assay
The antimicrobial assay was performed by agar disc diffusion method (Bauer
et al., 1996; Parekh and Chanda, 2007b). The
molten Mueller Hinton Agar (HiMedia) was inoculated with 200 μL of the
inoculum (1x108 cfu) and poured into the sterile Petri plates (Hi-media).
The disc (0.7 cm) (Hi-Media) was saturated with 20 μL of the extract (600
μg disc-1) in 100% dimethyl sulphoxide (DMSO) and air dried.
Thereafter the discs were introduced on the upper layer of the seeded agar plate.
Piperacillin (100 μg disc-1), Amikacin (30 μg disc-1),
Fluconazole (10 μg disc-1) and Amphotericin-B (100 units disc-1)
were used as positive controls. Paper discs loaded with 20 μL of DMSO served
as negative control. The plates were incubated at 37°C for all the bacterial
strains while that of fungal strains were incubated at 28°C for 48 h. The
experiment was done three times and the mean values are presented. The antimicrobial
activity was evaluated by measuring the inhibition zones.
DPPH Free Radical Scavenging Assay
The free radical scavenging of the extracts of M. indica was measured
using the modified method of McCune and Johns (2002)
was used. The extracts were dissolved in methanol. One milliliter of the extract
was added to 1 mL of a solution of DPPH radicals (0.3 mM) dissolved in methanol.
The mixture was shaken vigorously and allowed to stand for 10 min at room temperature.
The absorbance of the resulting solution was measured at 517 nm. The radical
scavenger activity was expressed in terms of the amount of antioxidants necessary
to decrease the initial DPPH absorbance by 50% (IC50). The IC50
value for each sample was determined graphically by plotting the percentage
disappearance of DPPH as a function of the sample concentration.
RESULTS AND DISCUSSION
Disc diffusion methods are extensively used to investigate the antibacterial activity of natural substances and plant extracts. These assays are based on the use of discs as reservoirs containing solutions of substances to be examined (Sambath-Kumar et al., 2006). In this study, 5 Gram-positive, 7 Gram-negative and three fungal strains were used to screen the possible antimicrobial activities of different solvent extracts of M. indica seeds.
The extractive yield of petroleum ether, chloroform, ethyl acetate, acetone and methanol was 13, 2.9, 0.8, 1.8 and 5%, respectively. The maximum yield was in petroleum ether while minimum was in ethyl acetate. None of the extracts, showed activity against the fungal strains studied. Antifungal activity is not common in medicinal plants. For example 23 extracts of 12 Cuban plants, used in traditional medicine failed to inhibit the growth of yeast (Marting et al., 1996). Petroleum ether extract also did not show any activity against Gram positive and Gram negative bacterial strains investigated.
The antibacterial activity was different with different solvent extracts. Minimum antibacterial activity was with chloroform extract followed by ethyl acetate, acetone and methanol extracts (Table 1). Maximum activity was in methanol extract. This trend was observed in all the Gram positive and Gram negative bacterial strains investigated.
| Table 1: | Antimicrobial activity of mangifera indica L. seed extracts in different solvents |
| SA: Staphylococcus aureus SE: Staphylococcus epidermidis, BC: Bacillus cereus, BS: Bacillus subtilis, MF: Micrococcus flavus, PA: Pseudomonas aeruginosa, EC: Escherichia coli, KP: Klebsiella pneumoniae, PM: Proteus mirabilis, PV: Proteus vulgaris, ST: Salmonella typhimurium, CF: Citrobacter freundii, Zone of inhibition includes disc diameter (7 mm), n = 3, -: No activity, negative control did not show any activity | |
Ahmad et al. (1998), Ellof (1998) and Parekh et al. (2005) reported that methanol is a better solvent than ethanol or hexane for consistent extraction of antimicrobial substances from medicinal plants. The most susceptible Gram positive bacteria was M. flavus with inhibition zone of 22 mm (methanol extract) and 20 mm (acetone extract) while most susceptible Gram negative bacteria P. vulgaris with 17 mm inhibition zone with methanol and acetone extracts. The most resistant bacteria was Gram positive B. subtilis with almost no antibacterial activity.
The antibacterial activity of different extracts was comparable with standard antibiotics. Generally Gram-negative bacteria are more resistant than Gram-positive bacteria because of the complexity of the cell wall of Gram negative bacteria but in the present study, the M. indica seed extracts in different solvents showed activity against both Gram positive and Gram negative bacteria may be indicative of the presence of broad spectrum antibiotic compounds or simply general metabolic toxins in the plant. Some antibiotics have become almost obsolete because of the problem of drug resistance (Ekpendu et al., 1994) and the consequences of drug resistance implies that new drugs must be sought for and to treat diseases for which known drugs are no longer useful.
The effect of antioxidants on DPPH radical scavenging is thought to be due to their hydrogen-donating ability. DPPH is a stable free radical and accepts an electron or hydrogen radical to become a stable diamagnetic molecule (Soares et al., 1997). The extract of plant is allowed to react with the stable radical DPPH, in methanol solution. The reduction capability of DPPH radical is determined by the decrease in its absorbance at 517 nm, induced by an antioxidant (AH) 10 min, as follows:
DPPH• + AH→DPPH-AH + A• |
| Fig. 1: | DPPH radical scavenging activity of seed extracts of M. indica (A) ethyl acetate extract (B) chloroform extract (C) acetone extract and (D) methanol extract |
The concentration of antioxidant needed to decrease the initial DPPH concentration by 50% (IC50) is a parameter widely used to measure the antioxidant activity (Sanchez-Moreno et al., 1998). A lower IC50 value corresponds with a higher antioxidant activity.
All the four extracts of M. indica seeds exhibited different extent on antioxidant activity. The IC50 values of acetone (11 μg mL-1) and methanol (12 μg mL-1) extracts were almost equal to or better than standard ascorbic acid (11.4 μg mL-1) while that of ethyl acetate and chloroform were 25 and 120 μg mL-1, respectively. The IC50 values for all the four extracts are shown in Fig. 1A-D. DPPH radical scavenging activity of acetone and methanol extracts of M. indica seeds revealed very high potency considering the fact that the free radical quenching properties were only from the crude extracts. Such high free radical scavenging properties of the crude extracts are shared by few other plants (Gulcin et al., 2003; Harish and Shivanandappa, 2006). In the longer term, plant species or their active constituents identified as having high levels of antioxidant activity in vitro may be of value in the design of further studies to unravel novel treatment strategies for disorders associated with free radical induced tissue damage.
CONCLUSION
It is interesting to note that the extracts are not pure compounds and in spite of it good results were obtained which only suggests the potency of these extracts. Hence M. indica seed extract could be used as a guide in our continuing search for new natural products with potential medicinal properties. The results of the present study indicate that M. indica extracts can be used as easily accessible source of natural antioxidants and antimicrobics. The potential for developing antimicrobials from higher plants is rewarding as it will lead to the development of a phytomedicine to act against microbes. Plant based antimicrobials and antioxidants have enormous therapeutic potential as they can serve the purpose without any side effects that are often associated with synthetic compounds.
ACKNOWLEDGMENT
The authors are thankful to Prof. S.P. Singh, Head, Department of Biosciences, Saurashtra University, Rajkot, India for providing excellent research facilities. One of the author (Yogesh Vaghasiya) is thankful to Department of Special Assistance (DSA), [Ref. No. F. 4-9/2000 (SAP-2) May 8th 2001], UGC, New Delhi for financial assistance as junior research fellow.