A preliminary study on the in vitro antioxidant activity of the stems of opuntia vulgaris.

The in vitro antioxidant activity of stems of Opuntia vulgaris Mill had been investigated by estimating degree of non-enzymatic haemoglobin glycosylation measured colorimetrically at 520 urn. It was found that benzene and chloroform extract of O. vulgaris had better antioxidant activity than petroleum ether, ethyl acetate, ethanol and aqueous extract. The antioxidant activity of the extracts was concentration dependent and comparable to that of D-α- tocopherol (vitamin E) and ascorbic acid (vitamin C), standard antioxidant compounds used.

INTRODUCTION

Opuntia vulgaris Mill (Sappattukkalli in Tamil, Nagophema in Oriya, family: Cactaceae) is a large succulent shrub common throughout India, found widely in the Northern India[1]. Various parts of this plant were used in tribal medicine as bitter, laxative, stomachic, c native, antipyretic and cures biliousness, tumours, anemia, ulcers and enlargement of spleen. The juice of the plant was applied in the treatment of syphilis in Ayurveda[23]. The plant has been found also to posses’ anthelmintic, analgesic and anticonvulsant activities[45].

Recently, a great deal of interest has been directed towards the bioactivity of natural plants as sources of antioxidant[6-8]. As O. vulgaris posses anti-tumor and anti-inflammatory activities which are related to antioxidant property; the present communication deals with the in-v it m evaluation of the antioxidant activity of aerial parts of O. vulgaris.

Evaluation of the antioxidant activity of any drug sample or herbal extract can be carried out either by in vitro or in vivo models. Various procedures are available in each model to determine the antioxidant capacity. Here, the evaluation was carried out by in vitro non-enzymatic glycosylation of haemoglobin method. Since non-enzymatic glycosylation of haemoglobin is an oxidation reaction, an antioxidant is expected to inhibit the reaction. The degree of haemoglycosylation in vitro in the presence of different concentration of extracts can be measured colorimetrically.

MATERIAL AND METHODS

Chemicals

Haemoglobin was purchased from Nice Chemicals Pvt. Ltd., Cochin. Glucose, phosphate buffer and D-α-tocopherol were procured from Merck, Mumbai. Ascorbic acid and gentamycin were obtained from Biokem International Pvt. Ltd., Bangalore and Nicholas Piramol India Ltd., Pithampur, respectively. All other reagents and solvents used were of analytical grade.

Preparation of extracts

The fresh stems of O. vulgaris were collected from hill area near the Subarnarekha River in the District of Mayurbhanj, Orissa, India in the month of September. They were authenticated by Dr H. J. Chowdhury, Additional Director, Central National Herbarium, Botanical Survey of India, Howrah, and West Bengal, India. The voucher specimens have been preserved in our laboratory for future reference (DM1). Shade-dried, powdered, sieved (40 mesh size) plant materials were exhaustively extracted successively with petroleum ether (40-60° C), chloroform, ethyl acetate, ethanol and distilled water using a soxhlet extractor. The extracts were concentrated to complete dryness in vacuum. The extracts were subjected to antioxidant studies.

Phytochemieal Screening

The phytochemical examinations of stems of O. vulgaris were performed by the standard methods[4910].

Antioxidant studies

Non-enzymatic haemoglyeosylation method: The antioxidant activities of different extracts were investigated by estimating degree of non-enzymatic haemoglobin glycosylation measured colorimetrically. Haemoglobin, 60 mg/ 100 mL in 0.01 M phosphate buffer (pH 7.4) was incubated in presence of 2 g/ 100 mL concentration of glucose for 72 h in order to find out the best condition for haemoglobin glycosylation. The assay was performed by adding 1 mL of glucose solution, 1 mL of haemoglobin solution and 1 mL of gentamycin (20 mg/ 100 mL) in 0.01 M phosphate buffer (pH 7.4). The mixture was incubated in dark at room temperature for 72 h. The degree of glycosylation of hemoglobin in the presence of different concentration of extracts and their absence were measured colorimetrically at 520 nm[11-14].

RESULTS AND DISCUSSION

Results of antioxidant activity of stems of O. vulgaris Mill extracts are summarized in Table-1 and Figure 1, respectively. The results obtained indicated that benzene and chloroform extract of stems of O. vulgaris had better antioxidant activity than petroleum ether, ethyl acetate, ethanol and aqueous extract. Benzene extract showed 40.9%, Chloroform extract 36.3%, petroleum ether extract 6.2%, ethyl acetate extract 30.4%, ethanol 11.5% and aqueous extract 11.6% inhibition of haemoglobin glycosylation with a concentration of 1.0 mg/ml of each. It was also found that the antioxidant activities of the extracts were concentration dependent. The activities were compared with D-α--tocopherol (vitamin E) and ascorbic acid (vitamin C) that were used as standard antioxidant compounds.

Table 1

Antioxidant activity of different extracts of O. vulgaris Mill

Fig. 1

The antioxidant activity of different extracts of bark of O. vulgaris Mill.

Preliminary phytochemical investigations indicated the presence of saponin and alkaloid in the stems of O. vulgaris. The detailed chemical nature of the active principle(s) responsible for antioxidant activity and their mode of action are under investigation.

Percent inhibition of haemoglobin glycosylation was measured at two concentrations of petroleum ether extract, benzene extract, chloroform extract, ethyl acetate extract, ethanol extract and aqueous extract. The activities were compared with those of D-α-tocopherol and ascorbic acid. Values are mean ± S.E.M. of three replicates.

Percent inhibition of haemoglobin glycosylation was measured at two concentrations of petroleum ether extract, benzene extract, chloroform extract, ethyl acetate extract, ethanol extract and aqueous extract. The activities were compared with those of D-α-tocopherol and ascorbic acid. Values are mean ± S.E.M. of three replicates.

CONCLUSION

The in vitro antioxidant activity of stems of O. vulgaris Mill revealed that benzene and chloroform extract of it had excellent antioxidant property. Hence, the present study demonstrates the potential effectiveness of stems of O. vulgaris, which supports the claim by traditional medicine practitioners’ as an anti-tumor and anti-inflammatory remedy.