*Corresponding Author:
D. K. Pal
Division of Pharmaceutical Chemistry, Seemanta Institute of Pharmaceutical Sciences, Jharpokharia, Mayurbhanj 757 086, India.
E-mail: [email protected]
Date of Submission 18 February 2005
Date of Revision 4 June 2005
Date of Acceptance 20 March 2006
Indian J Pharm Sci, 2006, 68 (2): 256-258  

Abstract

The in vitro antioxidant activity of the roots and rhizomes of Cyperus rotundus L. has been investigated by estimating degree of non-enzymatic haemoglobin glycosylation, measured colorimetrically at 520 nm. The ethanol extract of the roots and rhizomes of C. rotundus showed higher activity, than other extracts of it. The antioxidant activity of the extracts are close and identical in magnitude, and comparable to that of standard antioxidant compounds used.

Cyperus rotundus L., (Muthaghas (Bengali), Motha (Hindi), Nutgrass (English); family: Cyperaceae) is a perennial sedge distributed throughout India. The roots and rhizomes of this plant are used in different diseases like chronic diarrhoea, inflammation, skin rashes, and excess bleeding. It has also antiestrogenic, antimicrobial, anthelmintic, antihistaminic, antiemetic, antipyretic, and antidiabetic activities [1-5]. The roots and rhizomes of C. rotundus on preliminary chemical analysis, is found to contain β-sitosterol, cyperene, cyperol, flavonoids, sesquiterpenoids, ascorbic acid, and polyphenols [6,7]. It is of timely interest, to search for new antioxidants from plant sources. Recently, a great deal of interest has been directed towards the bioactivity of flavonoids, ascorbic acid, and polyphenols, as dietary sources of antioxidant [8]. Hence, the present communication deals with the evaluation of the antioxidant activity of the roots and rhizomes of C. rotundus L.

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 is carried out by in vitro non-enzymatic glycosylation of haemoglobin method. Since nonenzymatic 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.

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

The roots and rhizomes of C. rotundus L. were collected from Panua, in the district of Bankura, West Bengal in the month of June, and were authenticated at the Central National Herbarium, Botanical Survey of India, Howrah, West Bengal. A voucher specimen has been preserved in our laboratory for future reference (DPS 1). Shade-dried, powdered, sieved (40 mesh size) plant materials were exhaustively extracted successively with petroleum ether (40-60 C), chloroform, ethanol, and distilled water, using a soxhlet extractor. The extracts were concentrated to dryness in vacuum. The yield of petroleum ether, chloroform, ethanol, and water extracts, were 1.5%, 2.4%, 12.3% and 9.2%, respectively. The ethanol extract was subjected to silica gel preparative TLC, where two compounds were isolated using chloroform : ethanol (9:1) as solvent system.

Compound A (mp: 210°, Rf value: 0.59, λmax: 224.2 nm) having characteristic IR (Perkin Elmer, IR-297) peaks at 3350 cm-1, 2930 cm-1, 2850 cm-1, 1715 cm-1, 1690 cm-1, 1640 cm-1, 1600 cm-1, 1450 cm-1, 1380 cm-1 and 1300 cm-1 and compound B (mp: 255°, Rf : 0.64, λmax: 220 nm) having characteristic IR peaks at 2930 cm-1, 2850 cm-1, 1700 cm-1, 1600 cm-1, 1450 cm-1, 1380 cm-1 and 1300 cm-1 suggest the structural similarities with the polyphenolic type of compounds [9].

The antioxidant activity 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 [10-13].

Results of antioxidant activity of C. rotundus extracts are summarized in Table 1. The result obtained, indicates that ethanol extract has more antioxidant activity than the petroleum ether, chloroform, and aqueous extract. Ethanol extract showed 35.3%, aqueous extract 14.1%, chloroform extract 12%, petroleum ether extract 7.9% and inhibition of haemoglobin glycosylation with a concentration of 1.0 mg/ml of each. The antioxidant activity of the extracts are concentration dependent. The detailed chemical nature of the active principles responsible for antioxidant activity is not known. However, preliminary phytochemical screening has confirmed the presence of flavonoids (1.25%), ascorbic acid (0.01%) and polyphenols (1.62%) which might be responsible for this activity. D-α- tocopherol (vitamin E) and ascorbic acid (vitamin C) were used as standard antioxidant compounds.

Sample Final concentration of the tested compound (mg/ml)
0.5 1.0
PE 2.9 ± 0.16 7.9 ± 0.24
CE 6.1 ± 0.30 12.0 ± 0.32
EE 23.3 ± 0.25 35.3 ± 0.29
AE 9.2 ± 0.17 14.1 ± 0.21
D-α -tocopherol 11.4 ± 0.15 16.3 ± 0.14
Ascorbic acid 5.3 ± 0.11 9.4 ± 0.13

Table 1: The antioxidant activity of different extracts of C. Rotundus L.

Acknowledgements

The authors are grateful to the Principal, S. I. P. S. and Mr. P. C. Basa, President, S. I. P. S., Orissa for providing necessary facilities. The authors wish to express their sincere thanks to Dr H. J. Chowdhury, Joint Director, Central National Herbarium, Botanical Survey of India, West Bengal for his generous help in taxonomical identification.

References