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, India
2School of Chemical and Biotechnology, SASTRA University, Thanjavur,
*Corresponding Author: Tel: mobile-9443154362
E-mail: [email protected]
Issued 25 June 2008
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.
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
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 50C for 20 min. To this mixture, 2.5 ml of 10% trichloro acetic acid (TCA) was added and then centrifuged at 3000 rpm for 10 min. The upper layer of the solution (2.5 ml) was mixed with distilled water (2.5ml) and Fecl3 (0.5 ml, 0.1%) was added and the absorbance was measured at 700 nm. Increased absorbance of the reaction mixture indicated increased reducing power. The experiment was performed in triplicate. The percentage scavenging was calculated by using the formula,
(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 g/ 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 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 20-250c. 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 CATS 3.20 version software. 17
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, TCA and Fecl3. The polyphenolic content of ethanolic and aqueous leaf extract of P. dulce was estimated by Folin- Denis method. The method is based on the oxidation of the molecules containing a phenolic hydroxyl group. The tannin and tannin-like compounds reduce phospho tungusto molybdic acid in alkaline solution to produce a highly colored blue solution. The intensity of which is proportional to the amount of polyphenolic compounds and can be estimated against standard tannic acid solution at wavelength of 700 nm. The total phenolic compounds of two successive extracts were expressed as tannic acid equivalent in mg/ g of extracts. Phenolic compounds were reported18 to be potent free radical terminators and thus, the results are further supported by the varying amounts of total phenolic content in different fractions of leaves. HPTLC fingerprint profile of the ethanol and aqueous extracts were developed which would serve as reference standard for quality control of these extracts. Further work is, therefore, under progress to identify and isolate the anti oxidative constituents and to establish the activity in animal models.
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.
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