Ethnobotanical Leaflets 12: 446-451. 2008.
Free Radical Scavenging Activity of
Folklore: Pithecellobium dulce Benth. Leaves
M.
Sugumaran1*, T.
Vetrichelvan1 and
S. Darlin Quine2
1Adhiparasakthi College of
Pharmacy, Melmaruvathur, Tamilnadu-603319, 2School of Chemical and
Biotechnology, SASTRA University, Thanjavur, Tamilnadu-613402, *Corresponding Author: Tel: mobile-9443154362 E-mail: murugesansugumaran@yahoo.com Issued 25 June 2008 Abstract
In
the present study, the aqueous and alcoholic extract of Pithecellobium
dulce leaves were evaluated for radical scavenging activity using
reducing power assay method. Aqueous extract showed potent free radical
scavenging activity, than alcohol extract. The observed activity could be due
to higher phenolic content in the extracts (0.2171& 0.2042 mg/g in
aqueous and alcohol extract respectively). HPTLC fingerprint profile of the
ethanol and aqueous extracts were developed which would serve as reference
standard for quality control of these extracts. Introduction Pithecellobium dulce Benth. (Leguminosae)1 is a small to medium sized, evergreen, spiny tree
up to 18 m height, native of tropical America and cultivated throughout the
plains of India and in the Andamans. It is known as ‘Vilayati babul’ in Hindi
and ‘Kodukkapuli’ in Tamil. The bark of the plant is reported to be used as
astringent in dysentery, febrifuge and it is also useful in dermatitis and
eye inflammation. The leaves have been reported to possess astringent,
emollient, abortifiacient and antidiabetic properties. The presence of
steroids, saponins, lipids,
phospholipids, glycosides, glycolipids and polysaccharides have been reported
in the seeds. 2-5 The bark contains 37% of tannins of catechol
type. Quericitin, kaempferol, dulcitol and afezilin have been reported from
the leaves. 6, 7 Roots have been reported to possess estrogenic
activity.8 Studies on alkylated resins from seed oil have
been reported recently .9 Table 1. Ethanomedical uses of P. dulce.
It is evident that the plant has great potentials in treating a number of ailments where the free radicals have been reported to be the major factors contributing to the disorders.10 In continuation of our work on evaluation of ethano pharmacological properties11-13 of pithecellobium dulce, the present investigation was aimed to evaluate the in vitro antioxidant activity of ethanolic and aqueous leaf extract of P. dulce by reducing power assay method based on the medicinal values in folk medicine of Mexico ( Table 1). Materials
and Methods Plant material Fresh leaves of Pithecellobium dulce were collected from Sembulam Village at Kancheepuram District, T.N. in the month of January 2005. The plant was identified by local people of that village and authenticated by Dr. P. Jayaraman, Director, Plant Anatomy Research Centre (PARC), Chennai. A herbarium specimen (APCP-3/ 2005) of the plant was preserved in the Department of Pharmacognosy of our institute for further reference. All the reagents used were of analytical grade obtained from S.D. Fine chemicals Ltd., Mumbai and Qualigens Fine Chemicals, Mumbai. Preparation of Aqueous and
Alcoholic Extracts The fresh leaves of P. dulce were washed with water, air- dried at room temperature and then reduced to coarse powder. The powdered mass of leaf was defatted with petroleum ether (60-800 c) followed by extraction with alcohol (95% v/v) and then water for about 18 hr by using soxhlet apparatus. The extracts were filtered and the filtrates were concentrated under reduced pressure using rotary evaporator to obtain the extracts as solid residues. Extractive value (%w/w) of alcohol and aqueous extracts were 17.93 and 18.58 respectively. Preliminary
Phytochemical Screening The freshly
prepared extracts were chemically tested for the presence of different
constituents using standard methods.14 Reducing
Power Assay Method Reducing power of 70% ethanolic extract of P.
dulce was carried out as per Oyaizu.15 Different doses
of P. dulce were prepared and 1ml of each solution was mixed with
Phosphate buffer (2.5 ml, 0.2M, pH 6.60) and potassium ferricyanide 9 (2.5
ml, 1%). The mixture was incubated at 50˚C for 20 min. To
this mixture, 2.5 ml of 10% trichloro acetic acid ( (A control - A sample)/ A control ×100, where A control= Absorbance of blank and A sample= Absorbance with different dilutions of drug. The results are shown in Table-2. Table 2. Reducing
power activity of P .dulce
leaf extracts.
*Each value is an average of three
determinations. . Estimation
of Phenolic Content Clear aqueous and alcoholic leaf extracts of P.dulce were prepared and each 1.0 ml of the test solution was withdrawn in 10 ml volumetric flask separately. The blue colored complex was developed in the similar manner as in calibration curve studies, replacing the tannic acid with extracts and absorbance for aliquots of each extract was noted at 700 nm. The corresponding concentrations of total phenol against respective absorbances were determined as tannic acid using the calibration curve. All determinations were performed in triplicate. Total phenolic content of P. dulce was calculated in terms of tannic acid equivalent (TAE) by the following formula, C= c.v/ m, Where C is total content of phenolic compounds in mg/ g of plant extract, c is the con of polyphenol established from the calibration curve in µg/ ml, v is the volume of extract in ml and m is the weight of pure plant extract in g.16 The results are shown in Table 3. Table
3. Amount of phenolic compounds in p.dulce benth leaves.
HPTLC
Analysis HPTLC
fingerprinting was performed on 20x 20 cm aluminum sheets precoated with
silica gel 60 F254 Merck plates of 0.2 mm thickness. CAMAG HPTLC
linear thin trough (20x20 cm) was saturated with the developing solvent, for
2 hrs at 20-250°c.
The sample 2 or 5 ml
was applied in a 5 mm width band through LINOMAT IV in a space of 5 mm.
Detection was carried at scanning wavelength 254 nm using Camag scanner II
equipped with Fig 1. HPTLC Finger printing of alcohol extract of Pithecellobium dulce
Benth.
Fig.
2. HPTLC Finger printing of water extract of Pithecellobium dulce Benth Results and Discussion Preliminary
phytochemical investigation showed the presence of phenolics including
flavonoids as a major class of compounds. It was observed that absorbance of
the test sample was increased with increase in concentration of test. So, P.
dulce showed concentration dependant reducing capacity. Aqueous extract
showed potent free radical scavenging activity, than ethanol extract. The
aqueous extract has the highest phenolic content (0.2171 mg/g), followed by water
extract (0.2042mg/g). HPTLC fingerprint profile of the ethanol and aqueous
extract were developed in n-butanol: water: acetic acid (9.0: 0.5:0.5) at 254
nm and shown in Figs.1 and 2. Reducing
power of P. dulce was determined based on the ability of antioxidant
to form coloured complex with potassium ferricyanide, Acknowledgement The authors are grateful to Director, NISCAIR, New Delhi for assistance in literature collection of this plant. They are also thankful to Dr. P. Jayaraman, Director, Plant Anatomy Research Center (PARC), Chennai for his help in authentication of plant. The authors are further thankful to Director, Asthagiri Herbal Research Foundation, Chennai for providing facilities to take HPTLC finger printing. References
1. Anonymous. The Wealth of India: Raw materials, Vol.VIII. New Delhi: Publications and Information Directorate, CSIR, 1969: 140. 2. Nigam Shyam K, Mitra . and Chittranjan R. Fette 1971; 73(2): 75.
3. Bhargva Krishna P, Gupta M B.and Chittranjan R. Indian J Med Res 1970; 58:724. 4. Nigam S K, Misra G, Uddin R, Yoshikawa K, Kawamoto M.and Arihara S. Phytochemistry 1997; 44 (7): 1329. 5. Misra G, Nigam S K, Singh S, Nigam S G and Saxena R C. Indian Drugs 1979; 17(1):6. 6. Adinarayana D. and Ramachandraiah chetty P. Indian J Chem. 1985; 24B: 453. 7. Zapesochnaya G G, Yarosh E A, Syanidze N V.and Yarosh G I. Khim Prir Soedin, 1980; 2:252. 8. Saxena V K. and Singal M. Fitoterapia 1998; 69(4):305. 9. Anup Banarjee. J Indian Chem Soc 2005; 82: 186. 10. Aruoma O I. J Am Oil Chem Soc 1998; 75:199. 11. Sugumaran M, Vetrichelvan T, DarlinQuine S. and Dhandeep Singh. Pharma Buzz 2007 ;2(12): 48-52. 12. Sugumaran M, Vetrichelvan T. and Venkappaya K. The Antiseptic 2008; 105 (1): 45. 13. Sugumaran M, Vetrichelvan T. and Darlin Quine S. Indian J Nat Prod 2006; 23(1): 27-30. 14. Trease G E. and Evans W C. Pharmacognosy. London: Bailliere Tindall P, 1985: 539. 15.
Oyaizu M. Jap J Nutrition 1986; 44: 307. 16. Schanderl S H. Methods in Food Analysis. New York: Academic Press, 1970: 709. 17. Sethi P D. High Performance Thin Layer Chromatography- Quantitative analysis of Pharmaceutical Formulations. New Delhi: CBS Publishers and Distributors, 1996:10-68 18. Sawa T M, Nakao T, Akaike K. and Maeda H. J Agri Food Chem 1999; 47: 397. |