Antioxidant Activity of Extract from the Leaves of Tylophora asthmatica
R. Malathi1 S. Ahamed John2 and A. Cholarajan3
1Department of Biotechnology, Bharathidasan University College (W), Orathanadu, Thanjavur, Tamil Nadu, India.
2Department of Botany, Jamal Mohamed College, Trichy, Tamil Nadu, India.
3Department of Microbiology, Meenakshi Chandrasekaran College of Arts and Science, Pattukkottai – 614 626, Thanjavur, Tamil Nadu, India
*Corresponding Author E-mail: email@example.com
Extract from the leaves of Tylophora asthmatica were investigated for antioxidant activity. The methanolic extract of T.asthmatica had a DPPH scavenging activity of 84.6% at 250μg/ml and a reductive potential of 0.77 at 100μg/ml. These values were comparable with those of Gallic acid, 91.4% at 250μg/ml and Ascorbic acid, 0.79 at 60μg/ml as standards for DPPH scavenging activity and reductive potential, respectively. These findings suggest that the rich phytochemical content of T.asthmatica and its good antioxidant activity may be responsible for its popular and wide traditional use. The experiment was carried out with the leaves of the selected medicinal plants. The results are discussed with the available literature.
Plants show enormous versatility in synthesizing complex materials which have no immediate obvious growth or metabolic functions. These complex materials are retired to as secondary metabolites. Plants secondary metabolites have recently been referred to as phytochemicals. Phytochemicals are naturally, occurring and biologically active plant compounds that have potential disease inhibiting capabilities. It is believed that phytochemicals may be effective in combating or preventing disease due to their antioxidant effect (Malliwell and Gutte ridge, 1992). Antioxidant protect other molecules (in vivo) from oxidation when they are exposed to free radicals and reactive oxygen species which have been implicated in the etiology of many diseases and in food deterioration and spoilage (Kasaikna, 1997).
The most important of these phytochemicals are alkaloids, tannins, flavanoids and phenolic compounds (Hill, 1952). Tylophora asthmatica, a wild indigenous plant, belongs to the family asclepidaceae and is commonly known as Indian ipecac. The powdered leaves, stems and root of T.asthmatica contain 0.2-0.3% alkaloids, of these, tylophorine, tylophorinidine are important alkaloids (Gopalakrishnan et al.,1979) the various studies have confirmed the anti-inflammatory activity (Gopalakrishnan et al., 1979) direct stimulate of adrenal cortex (Udupa et al., 1991), anti-inflammatory activity, anti-asthmatic (Shivpuri et al.,1972) and the treatment of bronchitis, rheumatism and dermatitis (Nadukarni, 1976). The present work has been designed to evaluate the antioxidant potential of extracts from the leaves of T.asthmatica and explore the basis for its traditional use.
MATERIALS AND METHODS:
DPPH (2, 2 dipheny 1-1-picryl hydiazyl) radical, gallic acid, ascorbic acid and folin ciocalteau reagent were obtained from sigma Aldrich, USA. All other chemicals are reagents used were of analytical grade.
Collection and proceeding of plant material:
The fresh leaves of T. asthmatica were collected during the month of February, 2008 in the Banks of Cauvery River, Tiruchirappalli, South India. It was botanically identified and authenticated. A voucher specimen (TAL-12) has been kept in our laboratory for future references. The leaves were shade dried, powdered, sieved through 410 meshes and stored in a tightly closed container for future use.
Preparation on of plant extract:
The powdered plant material (500) was extracted with petroleum ether (60-80oC) using soxhlet apparatus to remove lipids. It was filtered and the filtrate was discarded. The residue was extracted with methanol by soxhlet apparatus. The extract was completely dried in vauco, stored in refrigerator at 4oC and protected from sunlight until the time for extract administration. The yield of methanolic dried extract was 8.63% (w/w).
Total phenolic content:
Total phenolic content was determined using Folin-Ciocalteau reagent as previously described (McDonald et al., 2001). Total phenol value was obtained from the regression equation; y=0.0055x+0.1139 and expressed as mg/g gallic acid equivalent using the formula, C=cV/M; where C=total content of phenolic compounds in mg/g GAE, c= the concentration of gallic acid (mg/ml) established from the calibration curve, V=volume of extract and m=the weight of pure plant methanolic extract (g).
DPPH Radical scavenging activity :
The ability of the extract to scavenge DPPH radical was determined according to the method described by (Mensor et al., 2001). Sample stock solutions (1.0 mg/ml) were diluted to final concentrations of 250, 125, 50, 25, 10 and 5 μg/ml in methanol. 1 ml of a 0.3 mM DPPH methanol solution was added to 2.5ml solution of the extract or standard and allowed to react at room temperature for 30 min. The absorbance of the resulting mixture was measured at 518 nm and converted to percentage antioxidant activity (AA %) using the formula:
AA% = 100 – [(Abs sample – Abs blank) x 100] / Abs control Methanol (1.0ml) plus extract solution (2.5ml) was used as a blank. 1ml of 0.3 mM DPPH plus methanol (2.5ml) was used as a negative control. Solution of gallic acid served as positive control.
This was determined according to the method of Oyaizu (1986). Different concentrations of the methanolic extract of (20, 40, 60 and 100μg/ml) in 1 ml of distilled water were mixed with phosphate buffer (2.5ml, 0.2M, pH 6.6) and potassium ferricyanide (2.5ml, 1%). The mixture was incubated at 50oC for 20 min. Trichloroacetic acid (10%, 2.5ml) was added to the mixture. A portion of the resulting mixture was mixed with FeCl3 (0.1%, 0.5ml) and the absorbance was measured at 700 nm in a spectrophotometer. Higher absorbance of the reaction mixture indicates higher reductive potential.
Data were expressed as mean ± SEM. A one-way analysis of variance was used to analyze data. P<0.5 represented significant difference between means (Duncan’s multiple range test).
RESULTS AND DISCUSSION:
The total phenolic content in the methanolic extract was 5.68 ± 0.06 mg/g GAE. Phenolics are the largest group of phytochemicals and have been touted as accounting for most of the antioxidant activity of plants (or) plant products. The result of the DPPH scavenging activity of T.asthmatica extract compared to that of gallic acid is shown in figure: l. both showed a dose-dependent antioxidant activity. The AA% of GA was remarkable higher than that of T.asthmatica at lower concentrations but significant differences between them seem to be less conspicuous at higher concentrations. The reductive potentials of T.asthmatica extract and ascorbic acid (AA) were also dose-dependent (Figure 2). The reductive potential of AA was clearly higher than that of T.asthmatica at all concentrations except the lease (20μg/ml). However, it should be noted that the reductive potential of T.asthmatica was still appreciable reported that methanolic extract of T.asthmatica is rich in phytochemicals specific biologically important compounds have been identified in extracts from the plant. The present works also reveal that the extract from the leaves of T.asthmatica possesses because of its phytochemical constituents (Thabrew et al., 1998). The DPPH scavenging activities of OG showed a good correlation with its reductive potentials. These facts justify the medicinal use of the plant for the treatment of various maladies (Dhawan et al., 1977; Oliver, 1980).
However further work is necessary to ascertain the clinical safety of the extracts from the plant (Effraim et al., 2001) and to determine appropriate concentration for therapy so as to safeguard the health of the teeming mass of traditional users who more often than not, do not take these factors into consideration.
1. Dhawan B.N., Patnik G.R., Rastogy RAT, Sing K.K., Tandol T.S., (1977). Screening of Indian plants for biological activity YL India Exp.B.15:108.
2. Gopalakrishnan, C.D., Shankaranarayan, L., Kamesuxuan and Natarajan, S., (1979). Pharmacological investigation of tylophorine, the major alkaloid of Tylophora asthmatica. Indian J. Med. Res., 69:513-520.
3. Effraim. K.D., Jacks, T.W., Sodipo, O.A., (2001) Histopathological studies on the toxicity of Ocimum gratissimum leave extract on some organs of rabbit. Afr. J. Biomed. 6:21-25.
4. Halliwell, B., Gutteridge, J.M.C., (1992). Free radicals, antioxidants and human diseases. Where are we now? J. Lab. Clin. Med. 119:598-620.
5. Hill, A.F., (1952). Economic Botany A text book of useful plants and plant products, 2nd edn. McGraw-Hill book company Inc, New York.
6. Kasaikina, O.T., Kortenska, V.D., Marinova, E.M., Rusina, I.F., Yarisbheva, N.V., (1997). Russa. Chem. Bull. 46:1070-1073.
7. McDonald, S., Prenzler, P.D., Autolovich, M., Robards, K., (2001). Phenolic content and antioxidant activity by the use of DPPH free radical method. Phytotherapy research, 15:127-130.
8. Mensor, L.I., Menezes, F.S., Leitao. G.G., Resis, A.S., Dos Santos, T., Coube, C.S., Leitao, S.G., (2001). Screening of Brazillian plant extracts for antioxidant activity by the use of DPPH free radical method. Phytotherapy research 15:127-130.
9. Oyaizu, M., (1986). Studies on product of browning reaction prepared from glucose amine, Japan Journal of Nutrition, 44: 307-315.
10. Thabrew, M.I., Hugher, R.D., Fartane, I.G., (1998). Antioxidant activity of Osbeckia aspera. Phytother. Res. 12:288-290.
Received on 02.05.2012 Accepted on 24.05.2012
© Asian Pharma Press All Right Reserved