Bioassay-Guided Isolation and Antioxidant Evaluation of Rutin from Leaf of Polyalthia longifolia

Subramanion Lachumy Jothy, Tamio Saito, Jagat Rakesh Kanwar, Subramaniam Kavitha, Leow Chiuan Herng, Yeng Chen, Leong Yin-Hui, Sreenivasan Sasidharan


Medicinal plants are sources of antioxidant compounds that have been reported to protect the human body from the adverse effects of free radicals. Therefore, the present study was intended to identify the DPPH (2, 2-diphenyl-1-picrylhydrazyl) free-radical scavenging constituent from the methanol extract of Polyalthia longifolia leaf using bioassay-guided fractionation. On the basis of DPPH radical scavenging assay-guided isolation, the leaf extract of P. longifolia was separated by employing a solvent partition of methanol leaf extract followed by Medium Pressure Liquid Chromatography (MPLC) and High-Performance Liquid Chromatography (HPLC) fractionation. The ethyl acetate fraction (EtOAc) revealed a strong antioxidant activity, compared to other fractions through in vitro DPPH radical-scavenging assay. The repeated fractionation of active EtOAc by MPLC separation and elution procedure yielded a sub-fraction EtOAc_F007 with strong antioxidant potential. The results indicated that the ethyl acetate sub-fraction EtOAc_F007 has noticeable effects on DPPH radical compared with other sub fractions. The sub fraction EtOAc_F007 was further purified using a preparative HPLC system. Subsequent Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) analysis of the purified constituent from sub-fraction EtOAc-F007 led to the identification of rutin as the antioxidant agent in P. longifolia leaf extract. The results obtained suggested that extracts from P. longifolia leaf have potential use as a bioactive source of natural antioxidants by contributing valuable health effects.



natural antioxidants; Bioassay-Guided Isolation; P. longifolia; DPPH radical

Full Text:



Newman, D.J., Cragg, G.M., Snadder, K.M., Natural products as sources of new drugs over the period 1981-2002. J. Nat. Prod. 66, 1022-1037, 2006.

Gilani, A.H., Rahman, A.U., Trends in ethnopharmacology. J. Ethnopharmacol. 100, 43-49, 2005.

Pitchai, D., Manikkam, R., Rajendran, S.R., Pitchai, G., Database on pharmacophore analysis of active principles, from medicinal plants. Bioinformatic. 5, 43-45, 2010.

Sticher, O., Natural product isolation. Nat. Prod. Rep. 25, 517-554, 2008.

Jothy, S.L., Chen, Y., Kanwar, J.R., Sasidharan, S., 2013. Evaluation of the genotoxic potential against H2O2-radical-mediated DNA damage and acute oral toxicity of standardized extract of Polyalthia longifolia leaf. Evid. Based Complement Alternat. MedArticle ID 925380, 13 pages, 2013.

Jothy, S.L., Aziz, A., Chen, Y., Sasidharan, S., 2012. Antioxidant activity and hepatoprotective potential of Polyalthia longifolia and Cassia spectabilis leaves against paracetamol-induced liver injury. Evid. Based Complement Alternat. Med. Article ID 561284, 10 pages, 2012.

Bose, T.K., Das, P., Maiti, G.G., Trees of the world, Vol. 1. Regional Plant Resource Centre, Bhubaneswar, India, 1998.

The Wealth of India, Publication and information directorate, vol. 3, 1st ed. CSIR, New Delhi, India, 1969.

Palma, M., Pineiro, Z., Barroso, C.G, Stability of phenolic compounds during extraction with superheated solvents. J, Chromatogr. A. 921, 169-174, 2001.

Sasidharan, S., Darah, I., Mohd Jain Noordin, M.K., Free radical scavenging activity and total phenolic compounds of Gracilaria changii. Int, J. Nat. Eng. Sci. 1, 115-117, 2007.

Duan, X.J., Zhang, W.W., Li, X.M., Wang, B.G., Evaluation of antioxidant property of extract and fractions obtained from red alga Polysiphonia urceolata. Food Chem. 95, 37-43, 2006.

Sarker, S.D., Latif, Z., Gray, A.I., Natural products isolation: an overview. in: Sarker, S.D., Latif, Z., Gray, A.I. (Eds), Natural products isolation. 2nd ed. Humana Press, New Jersey, 2005.

Atta-ur-Rehman, Choudhary M.I., William, J.T., Manual of Bioassay Techniques for Natural Product Research. Harward Academic Press, Amsterdam, pp. 82-84., 1999.

Otsuka, H. Purification by solvent extraction using partition coefficient, in: Sarker, S., Latif, Z., Gray, A.L. (Eds), Natural products isolation. Humana Press, New Jersey, 2nd ed., pp. 269-273, 2006.

Bucar, F., Wube, A., Schmid, M., Natural product isolation- how to get from biological material to pure compounds. Nat. Prod. Rep. 30, 525-545, 2013.

Sarker, S.D., Zahid, L., Alexander, I.G., Natural Products Isolation. in: Sarker, S.D., Zahid, L., Alexander, I.G. (Eds), Methods in Biotechnology. 2nd edn. Humana Press, Totowa, NJ, 2006.

Yang, J., Guo, J., Yuan, J., In vitro antioxidant properties of rutin. LWT- Food Sci. Technol. 41, 1060–1066, 2008.

Rosane, W.I., Oliveira, Z.D., Fernandes, S.C., Vieira, I.C., Development of a biosensor based on gilo peroxidase immobilized on chitosan chemically crosslinked with epichlorohydrin for determination of rutin. J. Pharm. Biomed. Anal. 41, 366–372, 2006.

Calabro`, M.L., Tommasini, S., Donato, P., Stancanelli, R., Raneri, D., Catania, S., Costab, C., Villarid, V., Ficarraa, P., Ficarraa, R., The rutin/b-cyclodextrin interactions in fully aqueous solution: spectroscopic studies and biological assays. J. Pharm. Biomed. Anal. 36, 1019–1027, 2005.

Barnaulow, O.D., Machineva, O.A., Kamissarenk,o, N.F., Comparative evaluation of the effect of some flavonoids on change in the gastric wall of reserpine treated or inmobilized mice. Khim, Farm. Zh. 17, 946–951, 1983.

Patil, S.L., Mallaiah, S.H., Patil, R.K., Antioxidative and radioprotective potential of rutin and quercetin in Swiss albino mice exposed to gamma radiation. J. Med. Phys. 38, 87–92, 2013.

Zielińska, D., Szawara-Nowak, D., Zieliński, H., Determination of the antioxidant activity of rutin and its contribution to the antioxidant capacity of diversifed buckwheat origin material by updated analytical strategies. Pol. J. Food Nutr. Sci. 60, 315-321, 2010.


  • There are currently no refbacks.

Creative Commons License
This work is licensed under a Creative Commons Attribution-NoDerivatives 4.0 International License.