Apr 28, 2012 - worms and leaf juice rubbed over the liver in remittent fever. (Kirtikar and ... was mixed with 0.5 ml of distilled water in a test tube followed ... their total phenolic content and flavonoid contents using commonly accepted methods. The plant ... as fruit and vegetables (Klimczak et al., 2007; Kiselova et al.,. 2006 ...
Drug Invention Today ISSN: 0975-7619 Research Article www.ditonline.info
Total phenolics, flavonoids and antioxidant activity of Tricosanthes cucumerena Linn Soniya Choudhary, Babeet Singh Tanwer* and Rekha Vijayvergia Plant Pathology and Plant Biochemistry Laboratory, Department of Botany, University of Rajasthan, Jaipur302004 Tricosanthes cucumerena Linn (Snake gourd) of Family: Cucurbitaceae was analyzed for their antioxidant activity as well as their total phenolic content and flavonoid contents using commonly accepted methods. The plant parts (stem, leaf and fruit) were extracted in methanol. The total level of phenolic contents (32.2±0.49mg GAE/gm DW) and flavonoids (7.82±0.67mg QE/gm DW) were found higher in leaves than other plant parts. The antioxidant activity was measured by DPPH radical scavenging activity and maximum activity was found in leaves (90.17±0.67%) in 100µg concentration and it is dose dependent. Key words: Trichosanthes Cucumerina L, Phenolics, Flavonoids, Antioxidant Power
INTRODUCTION Phenols comprising of phenolic acids, flavonoids, biflavonoids, anthocyanins and isoflavonoids posses a wide spectrum of biochemical activities such as antioxidant, antimutagenic, anticarcinogenic, as well as ability to modify the gene expression (Nakamura et al., 2003; Tapiero et al., 2002). The antioxidant capacity of phenolic compounds is determined by their structure, in particular the ease with which a hydrogen atom from an aromatic hydroxyl group can be donated to the free radicals. Flavonoids constitute a major group of phenolic compounds in plants. They provide pigmentation for fruits, flowers and seeds to attract pollinators and seeds dispersers. They assist in plant defense against pathogenic microorganism (Schijlen et al., 2004; Parr and Bowell, 2000). The number of flavonoids is constantly increasing due to the structural variation associated with these compounds. It is well known that antioxidant activity in higher plants has often been associated with phenolic compounds (Thabrew et al., 1998). Antioxidants are an important interacting system with different functions in higher plants, which ensures themselves a highly flexible organism (Borland et al., 2006; Shao et al, 2005). Antioxidants interfere with the production of free radicals and inactivate them (Dusinka et al., 1999). Antioxidants such as carotenoids, vitamins, phenols, flavonoids, and endogenous metabolites are naturally found in various parts of plants (Cao et al., 1997; Velioglu et al., 1998). Traditionally Trichosanthes cucumerina pacifies vitiated pitta, constipation, skin diseases, burning sensation, diabetes, anorexia, flatulence, constipation, worm infestation, fever and general weakness. The ripe fruit is highly bitter in taste, the bitter taste may suppose to contain medicinal properties (Choudhury, 1967) hence being used in various treatments as a cardiotonic, antipyretic, antiperiodic, useful for intestinal worms and leaf juice rubbed over the liver in remittent fever (Kirtikar and Basu, 2000) skin disease (Chopra et al., 1969) appetizer, laxative, aphrodisiac and blood purifier (Shivarajan and Indira, 1994). Root is used to cure bronchitis, headache and boils. Leaves for biliousness, emetic, externally applied
over bald patches alopecia to reduce congestion on congestive cardiac failure (Pullaih, 2006). Present work was carried out to estimate the total phenolic contents, flavonoid and to evaluate in vitro antioxidant activity of methanolic extracts of leaves, stem and fruits of Trichosanthes cucumerina Linn.
MATERIALS AND METHODS Plant material: The stem, leaves and fruits of selected plant was collected from Jaipur, Rajasthan, India during the month of December, 20011 and authenticated as RUBL as 21070 by Herbarium, department of Botany, University of Rajasthan, Jaipur. The samples were dried at room temperature, crushed in grinder and the powder was extracted with methanol for 48h, filtered through Whatman no. 1 filter paper and appropriately diluted with methanol. Determination of total phenolic content: Total phenolics were analyzed spectrophotometrically using a modified Folin-Ciocalteu colorimetric method (Dewanto et al., 2002). 125µl of the standard Gallic acid solution or sample extract was mixed with 0.5 ml of distilled water in a test tube followed by 125 µl of Folin- Ciocalteu reagent. The samples were mixed well and allowed to stand for 6 min before 1.25 ml of a 7% of sodium carbonate was added. Water was added to adjust the final volume to 3ml. After incubation at room temperature for 90 min, the absorbance was recorded at 760 nm. Reference curve was prepared using 10- 400 µg/ml of Gallic acid (linear regression r2 = 0.9975) and the results are presented as amount of phenolic content (Gallic acid equivalent, GAE) per g dry weight. Determination of total flavonoid contents: Flavonoid quantification was done using aluminium chloride colorimetric method (Chang et al., 2002). Plant extracts (0.5 ml) were mixed with 1.5 ml of methanol, 0.1 ml of 10 % aluminium chloride, 0.1 ml of 1 M potassium acetate and 2.8 ml of distilled water and kept at room temperature for 30 min. The absorbance of the reaction mixture was measured at 415nm. The calibration curve was prepared using 12.5 to 100µg/ml of Quercetin in methanol (linear regression r2 =
Corresponding Author: Babeet Singh Tanwer, Plant Pathology and Plant Biochemistry Laboratory, Department of Botany, University of Rajasthan, Jaipur- 302004 Received 18-12-2011; Accepted 28-04-2012 May, 2012
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Soniya Choudhary, et al.: Total phenolics, flavonoids and antioxidant activity of Tricosanthes cucumerena Linn
0.9991) and the results are expressed as amount of flavonoid content (Quercetin equivalent, QE) per g dry weight. DPPH radical scavenging capacity: The antioxidative activity of the extracts was elucidated by 2, 2-diphenyl-1picryhydrazyl (DPPH) free radical scavenging capacity of the extracts (Hasan et al., 2006). DPPH solution (0.004% w/v) was prepared in methanol. Two ml of this solution was added to a sample solution (0.1ml, 1mg/ml in methanol). After 30 min, absorbance at 515nm was measured and the percentage of radical scavenging activity was calculated from the following equation:
Table 2 DPPH radical scavenging activity (%) of Tricosanthes cucumerena Linn. Concentration (µg) 25 50 75 100
Leaf 75.75±0.23 88.09±0.47 89.00±1.10 90.17±0.67
Stem 40.50±1.14 59.50±0.49 68.84±1.02 86.25±0.03
Fruit 48.00±0.67 70.34±0.43 75.42±0.41 81.59±1.14
Graph.1: DPPH radical scavenging activity (%) of Tricosanthes cucumerena Linn 100 90
DPPH radical scavenging (%)
80
% Radical scavenging = (1-Abs. sample/Abs. control) ×100 Abs. control is the absorbance of the DPPH solution without sample and Abs. sample is the absorbance of the tested sample.
RESULTS AND DISCUSSION
70 60 Leaf
50
Stem 40
Fruit
30 20 10
Phenolic compounds could be a major determinant of antioxidant potentials of food plants and could therefore be a natural source of antioxidants and because phenolic compounds have been associated with the health benefits derived from consuming high levels of fruits and vegetables, The correlation between total phenol contents and antioxidant activity has been widely studied in different foodstuffs such as fruit and vegetables (Klimczak et al., 2007; Kiselova et al., 2006; Jayaprakasha et al., 2008; Kedage et al., 2007). Flavonoid and total phenol contents of the extracts: The flavonoid contents of the extracts in terms of quercetin equivalent (the standard curve equation: y = 0.0067x + 0.0132, r2 = 0. 0.9991) were maximum in leaves 7.82±0.67mg g-1 and minimum in fruits 2.05±0.49 mg g-1. (Table-1) Table 1 Total phenolic contents, flavonoids and antioxidant activity in Tricosanthes cucumerena Linn Plant part Stem Leaf Fruit
Total Phenolic content (mg GAE/gm DW) 27.3±1.14 32.2±0.49 17.8±1.67
Total Flavonoid content (mg QE/gm DW) 4.75±1.14 7.82±0.67 2.05±0.49
Table 1 also show the contents of total phenols that were measured by Folin Ciocalteu reagent in terms of gallic acid equivalent (standard curve equation: y = 0.05x + 0.0545, r2= 0.9975). The leaves with total phenol contents of 32.2±0.49 mg g-1 had the highest amount among the other plant parts plants in this study. The compounds such as flavonoids, which contain hydroxyls, are responsible for the radical scavenging effect in the plants (Das and Pereira, 1990; Younes, 1981). According to our study, the high contents of these phytochemicals in leaf extract can explain its high radical scavenging activity. DPPH radical scavenging activity of the standard compound BHT was 68.2%. The highest radical scavenging activity was showed by leaf extract (90.17±0.67%). The DPPH radical scavenging activity in the leaf extracts decreased as Leaf> stem> fruit. (Table 2 and graph 1).
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0 25
50
75
100
Concentration (µg)
CONCLUSION The result of the present study showed that the extract of leaves, which contain highest amount of flavonoid and phenolic compounds, exhibited the greatest antioxidant activity. The high scavenging property of leaves may be due to hydroxyl groups existing in the phenolic compounds’ chemical structure that can provide the necessary component as a radical scavenger. Free radicals are often generated as byproducts of biological reactions or from exogenous factors. The involvements of free radicals in the pathogenesis of a large number of diseases are well documented. A potent scavenger of free radicals may serve as a possible preventative intervention for the diseases (Gyamfi et al., 1999). All of the extracts in this research exhibited different extent of antioxidant activity. In conclusion, the results of the present study suggest that tested plant materials have moderate to potent antioxidant activity and/or free radical scavenging activity. However, we do not know what components in the plant extracts show these activities. More detailed studies on chemical composition of the plant extracts, as well as other in vivo assays are essential to characterize them as biological antioxidants which are beyond the scope of this study. It should also be kept in mind that antioxidant activity measured by in vitro methods may not reflect in vivo effects of antioxidants (Wu et al., 2004). Many other factors such as absorption/metabolism are also important. The findings of this study support this view that some medicinal plants are promising sources of potential antioxidant and may be efficient as preventive agents in some diseases. The providing data can just enrich the existing comprehensive data of antioxidant activity of plant materials.
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Source of support: Nil, Conflict of interest: None Declared
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