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International Journal of Natural Products Research

Universal Research Publications. All rights reserved

ISSN: 2249-0353 Original Article Qualitative analysis of phenolic constituents from leaves of Anaphalis contorta
Rajesh Kumar Joshi Department of Phytochemistry, Regional Medical Research Centre (ICMR), Nehru Nagar, Belgaum, Karnataka-590010, India Corresponding author: E-Mail: joshirk_natprod@yahoo.com Phone: +91-0831-2475477; Fax: +91-0831-2475479 Received 26 June 2011; accepted 08 July 2011 Abstract A reversed phase high performance liquid chromatography (RP-HPLC) was used for the screening of phenolic constituents in Anaphalis contorta Hook f. leaves. The phenolic compounds were identified by comparison with authentic standards. The phenolic compounds were identified as syringic acid, vanillic acid, p-hydroxybenzoic acid, ferulic acid, protocatechuic acid, gallic acid, p-coumaric acid, and 3,5-dihydroxybenzoic acid in the leaves extract of A. contorta. 2011 Universal Research Publications. All rights reserved Key words: Anaphalis contorta Hook f., phenolic compounds Introduction Phenolic compounds are secondary metabolites which synthesize by the plants and posses various biological properties such as: antioxidant, antiapoptosis, anti-aging, anticarcinogen, anti-inflammation, anti-artherosclerosis, cardiovascular protection, improvement of the endothelialfunction, as well as inhibition of angiogenesis and cell proliferation activity. Most of these biological actions have been attributed to their intrinsic reducing capabilities [1]. Crude extracts of fruits, herbs, vegetables, cereals and other plant materials rich in phenolics are increasingly of interest in the food industry because they retard oxidative degradation of lipids and thereby improve the quality and nutritional value of food [2]. Fruits, vegetables, and other plant foods, contain scores of functional phytochemicals and their consumption has long been associated with physical wellbeing. Among phytochemicals with health benefit are phenolic acids, flavonoids, and other polyphenols, which have been demonstrated to exhibit positive effects on certain types of cancer [3], coronary heart disease [4], and various inflammatory disorders [5]. Aromatic plants have been widely used to extend the self life of foods and in folk medicines. Anaphalis contorta Hook. f. of the family Asteraceae is widely distributed in temperate regions from Kashmir to Sikkim and Afghanistan to South West China at a height of 15004500 m. In Nainital it is found in Cheena (Naina) peak above 2000 m [6,7]. The fresh leaves of A. contorta and some other Anaphalis species are applied to cuts and wounds under a cloth bandage [8]. When taken before meals, the leaves of A. contorta stimulate the appetite. It is administered to convalescent for its sedative and tonic properties [9]. The aim of the present study was to identify the phenolic constituents from the leaves of A. contorta. To the best of my knowledge this is the first report on the phenolic constituents of the leaves of A. contorta. Materials and Methods Plant material The plant was collected from the pine forest of Nainital in the month of July 2007. The plant was identified by Prof. Y. P. S. Pangtey, Department of Botany, Kumaun University, Nainital. A voucher specimen (No. 28/100991) was

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Figure 1. Structure of phenolics identified in the leaves of A. contorta confirmed and deposited in the Herbarium of the Department of Botany, Forest Research Institute, Dehradun. Extraction of phenolics The extraction of the phenolics from the leaves of A. contorta was determined according to Andlauer et al., 2000 [10], with some modification. The fresh leaves (200 g) of A. contorta were shade dried and crushed to coarse powder. The powder (20 g) was macerated with 10 ml distilled water of 2 N-HCl and heated in water bath for 1 h at 100 oC using air condenser and filtered. The filtrate was extracted with diethyl ether using separating funnel. The diethyl ether layer was washed with distilled water, dried over anhydrous sodium sulphate and evaporated using thin film rotary vacuum evaporator at temperature range 2025 C to obtained diethyl ether extract. The obtained extract was collected and dissolved in known amount of (10 ml) HPLC grade methanol prior to the analysis. The sample was filtered through organic filter (Millipore) before injection to HPLC column. Analysis of phenolics The qualitative analysis of eleven phenolic compounds viz., caffeic acid, syringic acid, vanillic acid, p-hydroxybenzoic acid, ferulic acid, chlorgenic acid, protocatechuic acid, gallic acid, cinnamic acid, p-coumaric acid, 3,5-dihydroxybenzoic acid were used for study and performed by reverse phase high performance liquid chromatography (RP-HPLC) under following conditions: Apparatus: HPLC-Beckman model-322 equipped with 100 A model pump, 420 controller, mixer, 210 injector and BD-40 recorder Column: C18 reverse phase (Highchrome) stainless steel 12.5 cm x 1.4 inch (o.d) into 4.6 mm (i.d)

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Mobile phase:

Methanol: Water (1% acetic acid in 30: 70 v/v); the mobile phase was degassed before use

produces a sedative effect and convulsant properties as reported the traditional use of A. contorta leaves. Acknowledgements Supported instrumental facility by Prof. N. K. Sand, Department of Chemistry, G. B. Pant University of Agriculture and Technology, Pantnagar is thankful acknowledged. References [1] X. Han, T. Shen and H. Lou, Dietary polyphenols and their biological significance. Int. J. Mol. Sci. 8 (2007) 950988. [2] M. P. Kahkonen, A. I. Hopia, H. J. Vuorela, J. P. Rauha, K. Pihlaja, T. S. Kujala, and M. Heinonen, Antioxidant activity of plant extracts containing phenolic compounds. J. Agric. Food Chem. 47 (1999) 3954-3962. [3] D. Birt, Phytochemicals and cancer prevention: from epidemiology to mechanism of action. J Am Diet Assoc. 106 (2006) 20-21. [4] M. G. L. Hertog, D. Kromhout, C. Aravinis, H. Blackburn, F. Fidanza, S. Giampaoli, A. Jansen, A. Menotti and S. Nedeljkovic, Flavonoid intake and long-term risk of coronary heart disease and cancer in Seven Countries Study. Arch Int. Med. 155 (1995) 381-386. [5] R. Andriantsitohaina, E. Andriambeloson and J. C. Stoclet, Pharmacological approaches of endothelial nitric oxidedependent vasorelaxation induced by polyphenols from plant extracts. Meth. Enzymol. 301 (1999) 522-532. [6] R. K. Gupta, Flora Nainitalensis. Navayug Traders, New Delhi, 1968. [7] O. Polunin and A. Stainton, Flowers of the Himalaya. Oxford University Press, New Delhi, 1984. [8] K. R. Kirtikar and B. D. Basu, Indian Medicinal Plants, Part I, Indian Press. Allahabad, 1918. [9] C. Pande, R. K. Joshi and S. S. Sammal, Chemical composition of the essential oil of Anaphalis contorta Hook f. J. Essent. Oil Res. 20 (2008) 444-445. [10] W. Andlauer, C. Stumpf and P. Furst, Influence of the acetification process on phenolic compounds. J Agric. Food Chem. 48 (2000) 3533-3536. [11] B. Halliwell, J. M. C. Gutteridge and C. E. Cross, Free radicals, antioxidants and human disease: Where are we now? J. Lab Clin. Med. 119 (1992) 598-620. [12] A. O. Ayoka, R. O. Akomolafe, E. O. Iwalewa, M. A. Akanmub and O. E. Ukponmwan, Sedative, antiepileptic and antipsychotic effects of Spondias mombin L. (Anacardiaceae) in mice and rats. J. Ethnopharm. 103 (2006) 166-175.

HPLC condition: Flow rate 1 ml min-1 Chart speed 1 cm min-1 Detector UV detector, max 254 nm(Beckman DU70) Attenuation 0.02 aufs Pressure 1570 psi Mode isocratic The detector response for individual phenolics was calibrated with varying amounts of authentic phenolic acids used for analysis. All the standard phenolics were procured from Sigma Chemical Company, USA. Table 1. Phenolics analyzed in the leaves of A. contorta S. Phenolics (Standards) A. contorta No. 1. Caffeic acid 2. Syringic acid + 3. Vanillic acid + 4. p-Hydroxybenzoic acid + 5. Ferulic acid + 6. Chlorgenic acid 7. Protocatechuic acid + 8. Gallic acid + 9. Cinnamic acid 10. p-Coumaric acid + 11. 3,5-Dihydroxybenzoic acid + + = Present, - = Absent Results and discussion The reverse phase HPLC analysis of the extract of the leaves of A. contorta showed the presence of eight phenolic compounds Table 1. Eight phenolic compounds viz., syringic acid, vanillic acid, p-hydroxybenzoic acid, ferulic acid, protocatechuic acid, gallic acid, p-coumaric acid, and 3,5dihydroxybenzoic acid were detected, Figure 1. Phenolic compounds of different origins and functions, most of them belongs to principal biologically highly active components of foodstuff of plant origin and play the very important role of protecting organisms against harmful effects [11]. Phenolics are the dietry constituents and are used as chemopreventive agents. Phenolic compound(s) were present in the ethanolic and methanolic extracts, which exhibited anticonvulsant properties in the picrotoxin-induced convulsions model [12]. Thus the phenolic compounds present in this study could be

Source of support: Nil; Conflict of interest: None declared

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