ANTIOXIDANT ACTIVITY OF TAXIFOLIN OBTAINED FROM METHANOLIC EXTRACTS OF CORDIA DICHOTOMA LINN. SEEDS

Received on 30 January, 2014; received in revised form, 20 March, 2014; accepted, 03 May, 2014; published 01 July, 2014

ANTIOXIDANT ACTIVITY OF TAXIFOLIN OBTAINED FROM METHANOLIC EXTRACTS OF CORDIA DICHOTOMA LINN. SEEDS

Mahasweta Roy*, Bhupendra Kumar, Nishant Kumar, Amrit Patel and Babita Kumar

College of Pharmacy, Shree Ganpati Institute of Technology, Ghaziabad, Uttar Pradesh, India

Cordia dichotoma forst, Antioxidant activity, Taxifolin, DPPH, seeds extracts.

INTRODUCTION: Free radicals i.e. chemical species possessing an unpaired electron are produced continuously in cells either as accidental byproducts of metabolism or as deliberates. The most important free radicals in biological systems are radical derivatives of oxygen are antioxidant in nature.

The Middle East have revealed that a large number of indigenous plant species are being used as a source of herbal therapies show beneficial therapeutic potentials.Various herbs and spices have been reported to exhibit antioxidant activity, including Ocimum sanctum, Piper cubeba Linn., Allium sativum Linn., Terminalia bellerica ,Camellia sinensis Linn., Zingiber officinale Roscoe and several Indian and Chinese plants .

The majority of the antioxidant activity is due to the flavones, isoflavones, flavonoids, anthocyanin, coumarin lignans, catechins and isocatechins ¹. Antioxidant-based drug formulations are used for the prevention and treatment of complex diseases like atherosclerosis, stroke, diabetes, Alzheimer’s disease and cancer ².

Cordia dichotoma L. family Boraginaceae is a medium sized tree with short crooked trunk; leaves simple, entire and slightly dentate, elliptical-lanceolate to broad ovate with a round and cordate base; flower white, small in lax terminal or axillary cyme; fruits drupe, yellowish brown, pink or nearly black when ripe with  viscid sweetish transparent pulp surrounding a central stony part. The plant part used is bark, leaves and fruits ³.

It is also known by various other names; for example Bird Lime Tree (common name), Sebesten Plum, Indian cherry (English), Lasora, Lasura (Hindi) and Slesmatakah (Sanskrit) It is taken as food. The immature fruits are pickled and are also used as vegetable ⁴.

Cordia dichotoma seeds have disclosed the presence of α-amyrins, betulin, octacosanol, lupeol-3-rhamnoside, β-sitosterol, β-sitosterol-3-glucoside, hentricontanol, hentricontane, taxifolin-3, 5-dirhamnoside and hesperitin-7- rhamnoside. Cordia dichotoma is already reported to have anti-inflammatory, Antihelminthic and Wound healing activity ^{5, 6, 7}.

MATERIAL AND METHODS:

Collection of Plant Material: The fruits of Cordia dichotoma were collected from the local market of Lucknow  and were authenticated as C. dichotoma  (Boraginaceae) by pharmacognostic evaluation and a voucher specimen was deposited at National Botanical Research Institute Lucknow, for future reference (voucher no. NBRI/CIF/Re./08/2008/32)

Animals: Healthy male or female albino Wister rats each weighing 150-200 g were used for study. The rats were housed in polypropylene cages and maintained under standard conditions (12 h light and dark cycles, at 25 ±3^oC and 35-60% humidity). Standard palletized feed and tap water were provided ad libitum.

Successive Extraction with Various Solvents: gms of air-dried seed powder of Cordia dichotoma was extracted by successive extraction (exhaustive) with various solvents of different polarity like Petroleum ether (60-80^oC), Diethyl ether, Chloroform, Acetone and Methanol in a soxhlet. Each extract was concentrated to a small volume and allowed to dry.  After drying, the respective extracts were weighed and percentage extractive values were determined.

Isolation of Constituent Fractions: The methanolic extract was subjected to column chromatography. For this, the extract was chromatographed on a silica gel column (60-80 mesh) with methanol as a stationary phase. Proper time was allowed for seggregation and stabilization of fractions on the column. First elution was done by using chloroform (50 ml) after leaving the solvent for 10 minutes in the column for sufficient partitioning and then collection of 10 ml fractions in 5 test tubes at the rate of 20-25 drops per minute. All the fractions in the test tubes were subjected to chemical test and chromatography (TLC) for identification of components.

All fractions showing single spot TLC were analyzed by UV spectroscopy scanning consequent fractions with single spot TLC with similar R_f values and UV scan were combined and subjected to precipitation of components by using various solvents of opposite polarity. There after the IR spectroscopy of these isolated components were done to identify the type of components. After chloroform second elution was done by adding chloroform: methanol 70: 30 and fractions were collected, subjected to TLC, UV scanning and precipitation as above

Fractionation was continued by using chloroform: methanol (40: 60) and fractions similarly obtained and treated as above.

Lastly 50 ml methanol was used as eluent and fractions were collected.

Screening For Antioxidant Activity ⁸:

Reagents used for Antioxidant activity: Butylated hydroxyl toluene, DPPH, Phosphate Buffer saline (pH 7.4), Stock TBA-TCA-HCl reagent (Trichloroacetic acid (15 % w/v), thiobarbituric acid (0.375% w/v), hydrochloric acid (0.25 N). The solution was heated to assist in the dissolution of thiobarbituric acid)

DPPH Method: DPPH is stable nitrogen centered free radical and has been extensively used to characterize an antioxidant. The reduction of DPPH radical serves as a quick and simple method to detect the antioxidant potential of compounds especially those with phenol group. It is known that DPPH react rapidly with compound containing weak N–H or O–H bonds. Electron transport is also an important mechanism for its reduction. It is reversible, reduced and due to its unpaired electron, densely colored. This property makes it suitable for spectrophotometric studies.

                     REDUCTION OF 1, 1 DIPHENYL - 2 - PICRYL HYDRAZYL

Procedure: The free radical scavenging effect of fractions was assessed by the decoloration of a methanolic solution of DPPH with minor modifications. Samples were dissolved in 0.1 ml DMSO and added to 0.1 ml of 0.1 mM DPPH in methanol. The mixture was shaken vigorously and allowed to stand for 10 min at room temperature in the dark. The absorbance at 565 nm by DPPH was measured by a spectrophotometer. BHT was used as a positive control.

Statistical Analysis: One way analysis of variance (ANOVA) followed by Newmann Keul’s, was carried out & p<0.001 was considered as significant. Groups were compared with control group.

RESULT:

Column Fractionation and Chromatographic Profile of the Methanolic Extract ⁹: The methanolic extract on column chromatography gave the following fractions with the following characters.

Solvent system for TLC:

1)      Chloroform: Methanol (9: 1)

2)      Forestal

3)      Phenol

Spraying agent: Anisaldehyde reagent 0.5%

TABLE 1:

The fractions C₃ and C₄ (chloroform fractions) from methanolic extract of Cordia dichotoma seeds gave single component spot on TLC with R_f value 0.941with mobile phase Chloroform: Methanol:: 9:1 which gave typical positive reactions for steroidal glycosides and matched comparably with sitosterol. The melting point of the substance obtained was 84-85^o which was in confirmation to earlier reported data for hentricontanol, a substance earlier reported for the seeds ¹⁰. This fraction was designated as hentricontanol fraction (HTF).

The fractions CM₁ and CM₂ (Chloroform: Methanol:: 7:3) from methanolic extract of Cordia dichotoma seeds showed single component spot on TLC with R_f value 0.86 with mobile phase Chloroform : Methanol :: 9:1 which gave strong positive reactions for flavonoid glycosides. The fractions were combined, concentrated and precipitated to yield yellow component with melting point 148^o which on spectroscopic analysis showed UV (CH₃OH) λ_max (288 nm) and IR (KBr) peaks (3400, 2900, 2935, 1683, 1536, 1558, 1455, 1417, 1280, 1271, 1124, 867 and 822 cm^-1 which matched comparably to earlier reported data for Taxifolin which is reported as a constituent ¹¹ and to standard data for flavonoids.

This fraction was designated as Taxifolin fraction (TF). The fractions CMM₂ and CMM₃ (Chloroform: Methanol:: 4:6) from methanolic extract of Cordia dichotoma seeds showed single component spot on TLC with R_f value 0.83 with mobile phase Chloroform : Methanol :: 9:1 which gave strong positive reactions for flavanoid glycosides. This could not be further identified and was designated as flavanoid fraction (FF).

The fractions M₁ and M₂ (Methanol) from methanolic extract of Cordia dichotoma seeds showed single component spot on TLC with R_f value 0.12 with mobile phase Chloroform: Methanol:: 9:1 and BAW and matched comparably with hesperitin and rutin standards. Acid hydrolysis of the precipitate obtained after combining the fractions and  precipitation with Ethyl acetate: Petroleum ether, gave a compound with melting point 227-228^o which on spectroscopic analysis showed IR (KBr) peaks (3400, 3128, 2939, 2345, 1647, 1508, 1458, 1294, 1062 and 808 cm^-1) which matched comparably to earlier reported data for Hesperitin ¹². Thus, the fractions must be Hesperitin glycoside (HF).

Effect of Extracts and Fractions on Dpph Free Radical Scavenging Activity:

TABLE2:SHOWING THE RESULT OF ANTIOXIDANT ACTIVITY OF THE VARIOUS EXTRACTS AND FRACTIONS FROM OF THE SEEDS OF C. DICHOTOMA.

Sample I- Petroleum ether extract; Sample II- Chloroform extract; Sample III- Methanol extract; Sample IV- Hentricontanol (HTF) Fraction; Sample V- Taxifolin (TF) fraction ; Sample VI- Flavanoidal (FF) fraction; Sample VII- Hespitin (HF) fraction. Values are expressed as Mean ±SEM (n=6). P*<0.05, P**<0.01, P***<0.001

DISCUSSION AND CONCLUSION:

Effect of Extracts and Fractions on DPPH Free Radical Scavenging Activity: In the study of antioxidant by scavenging free DPPH radicals, the various extracts and fractions showed following responses. The methanolic extract (P<0.001) of seeds of Cordia dichotoma Linn. showed promising DPPH free radical scavenging activity at a concentration of 100 µg/ml whereas the petroleum ether extract and chloroform extract and the FF was non-significant at 100µg/ml and 10 µg/ml concentration. The fraction HTF showed (P<0.05) significance whereas HF and TF showed (P<0.001) significance at the 100 µg/ml concentration. At 10 µg/ml concentration the methanolic extract, TF and HF showed significance (p<0.01) (figure 1).

FIG. 1: GRAPH SHOWING THE EFFECT OF THE VARIOUS EXTRACTS AND FRACTION ON THE DPPH FREE RADICAL SCAVENGING ACTIVITY. Sample I- Petroleum ether extract; Sample II- Chloroform extract; Sample III- Methanol extract; Sample IV- Hentricontanol (HTF) Fraction; Sample V- Taxifolin (TF) fraction ; Sample VI- Flavanoidal (FF) fraction; Sample VII- Hesperitin (HF) fraction.

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