INTRODUCTION:
Naregamia alata Wight and Arn. belongs to the family Meliaceae. The present family consist of 51 genera and 575 species distributed in the tropics and subtropics of which 19 genera and 70 species were found in India. The plants belong to this family are under shrubs, branched with a woody root stock and indumentum of simple hairs. The genus Naregamia consists of two species. One species in India. In India they are seen in Maharashtra, Andra P radesh, Karnataka, Tamilnadu and Kerala[1].
N. alata is a small shrub, up to 50 cm high; young parts covered with an indumentums of golden brown, simple hairs, almost glabrous when old. Leaves alternate, 3-foliate, up to 10 cm long; rachis winged; leaflets sessile, cuneate-obovate or spathulate, tapering into a narrow base. Flowers are solitary, or few in axillary clusters, bisexual, white. Calyx of 5, oblong, lanceolate sepals with fused lower parts, hairy outside. Petals 5, distinct, linear spathulate, free from staminal tube; expanded at apex with 10 crenatures; apiculate, spreading[2,3].
The plant is reported to be used in south India in rheumatism and itch. In Konkan, the leaves and stem are given in a decoction with bitters and aromatics as a remedy for biliousness. They have a pungent aromatic odour and are considered emetic, cholagogue and expectorant [4,5]. The plant is used as an ingredient of a compound powder preparation which is given in malarial and chronic fevers[6,7].
An antioxidant is a molecule capable of slowing or preventing the oxidation of other molecules. Oxidation is a chemical reaction that transfers electrons from a substance to an oxidizing agent. Oxidation reactions can produce free radicals, which start chain reactions that damage cells. Antioxidants terminate these chain reactions by removing free radical intermediates, and inhibit other oxidation reactions by being oxidized themselves.
“Free radicals” are dangerous substances, chemical compounds, and toxins found within the body. Herbal antioxidants help rid the body of these toxins and help people live healthier lives. The body uses oxygen and nutrients to make energy. Oxygen also helps the immune system fight disease and harmful substances.
To protect the cells and organ systems of the body against reactive oxygen species, humans have evolved a highly sophisticated and complex antioxidant protection system, that functions interactively and synergistically to neutralize free radicals. Thus, antioxidants are capable of stabilizing or deactivating, free radicals before they attack cells[8].
Medicinal plants represent a rich source of antimicrobial agents. Plants are used different countries and are a source of many potent and powerful drugs. A wide range of medicinal plant parts are used for extract as raw drugs and they possess varied medicinal properties. The different parts used include root, stem, flower, fruit, twigs exudates and modified plant organs. While some of these raw drugs are collected in smaller quantities by the local communities and folk healers for local used, many other raw drugs are collected in larger quantities and traded in the market as the raw material for many herbal industries[9]. Although hundreds of plant species have been tested for antimicrobial properties, the vast majority of have not been adequately evaluated[10]. Considering the vast potentiality of plants as sources for antimicrobial drugs with reference to antibacterial and antifungal agents. There are many types of biological assays performed with micro organism. Many therapeutic agents which inhibit the growth of micro- organisms are essential for their growth, can be standardized by microbial assay. The inhibition of microbial growth under standard condition may be utilized for demonstrating therapeutic efficacy of antibiotics. The microbiological assay is based on the comparison of bacteria by measured concentration of antibiotics under test with that produced by known concentration of standard. Microbiological assays are generally based on either i) Serial dilution method or ii) Diffusion method. Microbial study was conducted with gram positive organisms like Bacillus cereus, Staphylococcus aureus, Bacillus subtilis, Proteus vulgaris and gram negative organisms like Escherichia coli, Klebseilla Pneumonia, Salmonella typhi, Serratia marcescens, Pseudomonas aureogenosa.
MATERIALS AND METHODS
Plant material
The whole plant of Naregamia alata Wight and Arn. were collected in February 2011 from Thiruvaniyoor, Ernakulam district, Kerala and identified by Dr. V. J. Dominic, Head, Dept. of Botany, Sacred Heart College, Thevera, Cochin.
A voucher specimen (No.50955) was deposited at the herbarium of Tropical Botanic Garden and Research Institute, Thiruvananthapuram, Kerala, India.
Extraction of plant materials
Naregamia alata (whole plant) was collected, shade dried and powdered. Powdered material (350 g) was subjected to successive extraction in a soxhlet extractor using petroleum ether (60-80oc), chloroform and methanol for18 h each. The extracts were filtered and concentrated by distillation[11]. Finally traces of solvents were removed using rotary vacuum evaporator and the extracts were preserved in a refrigerator until further analysis.
In vitro Antioxidant Assays
DPPH Radical Scavenging Activity
1, 1-Diphenyl-2-picrylhydrazyl (DPPH) reducing activity is widely used method for the screening of phytochemicals for their antioxidant activities. DPPH is characterized as a stable free radical due to delocalization of the spare electron over the molecule. This delocalization also gives rise to the deep violet color, characterized by an absorption band in ethanol solution centered at about 517 nm. When a solution of DPPH is mixed with that of a substance that can donate a hydrogen atom, then this gives rise to the reduced form with the loss of the violet color. Representing the DPPH radical by Z. And the donor molecule by AH, the primary reaction is,
Z+ AH Ű ZH + A.
Where, ZH is the reduced form and A. is free radical produced in this first step. This A. radical will then undergo further reactions which control the overall stoichiometry. The absorbance of the resultant solution is measured at 517 nm. The important parameter in this method is the IC50 value. IC50 is defined as the concentration of substrate that causes 50% loss of the DPPH activity [12].
Superoxide Scavenging Activity
Superoxide scavenging activity was determined by the method of Mc-Cord and Fridovich. This method involves the light induced super oxide generation by riboflavin and the corresponding reduction of nitro blue tetrazolium (NBT). Super oxide anions (O2-) act as an electron reducing agent in several reactions including reduction of quinines, tetra nitro methane, cytochrome C and nitro substituted aromatics such as NBT. The reduction of NBT to blue formazan has been widely used as a probe of O2- generation in chemical and biological systems. NBT in oxidized form is a yellow compound soluble in aqueous mixture. Its reduction to formazan is accompanied by disappearance of positive charges resulting in substantial decrease in solubility, disruption of the tetrazole ring with marked modification of the absorption spectrum in the visible region and the appearance of intense blue colour.
The reaction mixture(s) contained EDTA (6 mM), NaCN (3 µg), riboflavin (2 µg), nitro blue tetrazolium (50 µg), KH2PO4 buffer (67 mM, pH 7.8) and various concentrations of the extract in a final volume of 3 ml. The tubes were illuminated in bright sunlight for 15 min. The optical density at 560 nm was measured before and after illumination. The inhibition of superoxide radical generation was determined by comparing the absorbance values of the control with those of treatments [13].
Reducing Power assay method
In this assay the yellow colour of the test solution changes to various shades of green and blue depending on the reducing power of the extract. Presence of reducers causes the conversion of Fe3+ / ferricyanide used in this method to ferrous form. By measuring the formation of persion blue colour at 700nm, It is possible to determine the Fe2+ concentration, a higher absorption at 700nm indicate a higher reducing power.
Various concentrations of AVLS fractions (1 ml) were mixed with 1 ml of 200 mmol/l sodium phosphate buffers (pH 6.6) and 1 ml of 1% potassium ferricyanide. The mixture was incubated at 50oC for 20min. Then 1 ml of 10% trichloroacetic acid (w/v) was added. From that 1 ml was mixed with 1 ml of deionised water and 250μl of fresh ferric chloride (0.1%). The absorbance was measured at 700 nm: a higher absorbance indicates a higher reducing power [14].
Antimicrobial assay:
Antibacterial assay
Bacteria grown in nutrient broth for 24 h (pH 7.2-7.4) were used as inoculum. Mueller-Hinton agar medium were poured into petri plates to a uniform depth of 5 mm and allowed to solidify. Microbial suspensions at 5 x 106 cfu/ml were spread over the surface of the media using a sterile cotton swab to ensure the confluent growth of the organism. Aliquots (10 μl) of each plant extract 5mg/0.5ml dilutions in the inert solvent dimethyl sulphoxide (DMSO) were impregnated on Whatman No.1 filter paper discs (6 mm diameter). These discs were then aseptically applied to the surface of the agar plates at well-spaced intervals. These plates were incubated at 37°C for 24 h and observed inhibition zones including the diameter of the discs were measured. Control discs impregnated with 10 μl of the solvent (DMSO) and streptomycin (2 μg/disc, reference for bacteria) were used alongside the test discs in each experiment [15,16,17].
Antifungal assay
Fungus grown in nutrient broth for 24 hr (pH 7.2-7.4) was used as inoculum. Mueller-Hinton agar medium were poured into petri plates to a uniform depth of 5 mm and allowed to solidify. Microbial suspensions at 5 x 106 cfu/ml were spread over the surface of the media using a sterile cotton swab to ensure the confluent growth of the organism. Aliquots (10 μl) of each plant extractat4mg/10μl dilutions in the inert solvent Dimethyl sulphoxide were impregnated on Whatman No.1 filter paper discs (6 mm diameter). These discs were then aseptically applied to the surface of the agar plates at well-spaced intervals. These plates were incubated at 37°C for 24 hr and observed inhibition zones including the diameter of the discs were measured. Control discs impregnated with 10 μl of the solvent (DMSO) and Nystatin (2 μg/disc, reference for fungus) were used alongside the test discs in each experiment[17,18,19,].
RESULTS AND DISCUSSION:
Free Radical Scavenging Assay
DPPH Radical Scavenging activity
In this method the percentage inhibition of methanolic extract of Naregamia alata (NA) is compared with standard ascorbic acid. The percentage inhibition increases with concentration. The results are shown in the table 1.
DPPH is a stable free radical at room temperature often used to evaluate the antioxidant activity. The reduction capacity of DPPH radical was determined by the decrease in its absorbance at 517nm, which induced by antioxidants. Radical scavenging activity increased with increasing percentage of free radical inhibition. Figure 1 shows DPPH Free radical Scavenging Activity of Methanol extract and Ascorbic acid.
Table 1: Percentage Inhibition of Methanol extract of NA and Ascorbic acid
|
S.NO |
Concentration((μg/ml) |
% Inhibition |
|
|
Extract |
Ascorbic acid |
||
|
1. |
5 |
4.02 |
15.92 |
|
2. |
10 |
7.27 |
21.32 |
|
3. |
15 |
11.29 |
28.44 |
|
4. |
20 |
16.80 |
33.9 |
|
5. |
25 |
22.14 |
41.70 |
Figure 1: DPPH Free radical Scavenging Activity of Methanol extract and Ascorbic acid
Super Oxide Scavenging Activity
In this method the percentage inhibition of methanolic extract of Naregamia alata(NA) is compared with standard ascorbic acid. The percentage inhibition increases with concentration. The results are shown in the table 2. The figure 2 representing Super oxide Scavenging Activity of Methanol extract and Ascorbic acid
In this method Nitro blue tetrazolium (NBT) is used as one of the reagent. The percentage inhibition of NBT reduction can be used to quantify the super oxide- scavenging. Percentage inhibition increases with concentration. Figure-2 shows Super oxide Scavenging Activity of Methanol extract and Ascorbic acid.
Table 2: Percentage Inhibition of Methanol extract of NA and Ascorbic acid
|
S.NO |
Concentration (μg/ml) |
% Inhibition |
|
|
Extract |
Ascorbic acid |
||
|
1. |
5 |
5.09 |
22.36 |
|
2. |
10 |
9.35 |
32.06 |
|
3. |
15 |
17.11 |
37.55 |
|
4. |
20 |
23.82 |
43.07 |
|
5. |
25 |
28.30 |
47.80 |
Table 3: IC50 value of DPPH and Superoxide scavenging activity
|
Antioxidant assay |
Extract |
Standard |
|
DPPH Radical scavenging activity |
56.4906 ± 4.14438 |
32.0673± 1.819121 |
|
Super oxide radical scavenging activity |
42.32493± 0.350927 |
25.88854± 0.658331 |
Figure 2: Super oxide Scavenging Activity of Methanol extract and Ascorbic acid
Comparison of IC50 Value of DPPH and Super Oxide scavenging activity
Table 3 shows the IC50 value of DPPH and Superoxide scavenging activities of extract and standard. Figure 3 representing the IC50 value of DPPH and Superoxide scavenging activity.
Reducing Power Assay Method
In this method Quercetin was used as standard. The results are shown in the table 4.
The figure 4 showed the comparison of methanol extract and standard Quercetin in the reducing power assay method.
In this method the concentration of methanolic extract of NA and quercetin taken was 25-100(μg/ml). The graph is plotted against absorbance vs concentration. When the concentration increases absorbance also increases because when the concentration increased, reduction of ferri form to ferrous form also increased. Figure-4 shows reducing power assay method of Methanol extract and Quercetin.
Table 4: Absorbance of Methanol Extract of NA and Quercetin
|
S.No
|
Concentration (μg/ml) |
Absorbance |
|
|
Methanol Extract |
Quercetin |
||
|
1. |
25 |
0.078 |
0.461 |
|
2. |
50 |
0.098 |
0.624 |
|
3. |
75 |
0.184 |
0.791 |
|
4. |
100 |
0.454 |
0.954 |
Figure 4: Reducing power assay method of Methanol extract and Quercetin.
ANTI MICROBIAL ASSAY:
Anti bacterial assay
MTCC (Microbial type culture collection)
In this method different gram positive and gram negative bacterias like Bacillus cereus, Staphylococcus aureus, Escherichia coli, Klebseilla pneumonia, Salmonella typhi, Serratia marcescens, Pseudomonas aureogenosa, Bacillus subtilis were used. The results are shown in table 5. Antibacterial activity of Pet.ether, Chloroform and Methanol extracts of NA were tested. Streptomycin used as the standard. In the case of Bacillus cereus, Salmonella typhi, Serratia marcescens, Pseudomonas aureogenosa and Bacillus subtilis, CHCl3 having highest activity than Pet.ether and Methanol. In Staphylococcus aureus, Pet.ether having highest activity than CHCl3 and Methanol. In Escherichia coli, CHCl3 and Methanol extracts having same and highest activity than Pet.ether. In Klebseilla pneumonia, Methanol extract having highest activity than CHCl3 and Pet.ether. So we can concluded that CHCl3 having highest antibacterial activity than Pet. ether and Methanol. The concentration of the extracts taken for disc diffusion method was 40mg dissolved in 100μl DMSO. From this stock solution 10μl/disc was taken. The results expressed along with the disc diameter i.e.; 6mm.
Disc diameter-6mm including disc diffusion
40mg 100µL 4mg (10
µL/disc)