• Kadiri Sunil Kumar
  • Avanapu Srinivasa Rao


Objective: This investigation involves the extraction, isolation, and characterization of flavonoid from a Euphorbiaceae family plant Chrozophora
plicata followed by evaluation of its antioxidant principles.

Methods: The dried leaves were subjected to sequential soxhlation with polar and nonpolar solvents. Methanolic extract reveals the presence of large
amount of flavonoids. Methanolic extract was subjected to isolation using column chromatographic analysis with solvents such as petroleum ether,
chloroform, hexane, ethyl acetate, methanol, and water. Further, the isolated compound was subjected to thin layer chromatography technique and
spectral analysis such as infrared, 1HNMR, 13CNMR, mass spectroscopy, and high performance thin layer chromatography (HPTLC) finger printing
techniques. The compound was evaluated for in vitro antioxidant studies using 2,2-diphenyl-1-picrylhydrazyl (DPPH), NO assay, reducing power assay,
H2O2 scavenging assay, superoxide anion scavenging assay and β-Carotene linoleate system and in vivo antioxidative studies using carbon tetrachloride
(CCl4), and acetaminophen intoxicated rats.

Results: The compound was isolated in methanol:water in the ratio of 80:20 using column chromatographic technique. On the basis of phytochemical,
chromatographic, and spectral analysis, the isolated compound was identified as kaempferol and finally with HPTLC finger printing technique it was
found that the Rf value of the isolated compound was found to be 0.58 which is nearly similar to the Rf value of standard kaempferol (0.55). Hence,
the isolated compound was confirmed as kaempferol and is structurally elucidated as 3,5,7-trihydroxy-2-(4-hydroxyphenyl)chromen-4-one. This
compound was isolated for the first time from the C. plicata leaves. The in vitro antioxidant assay of isolated flavonoid has shown a dose-dependent
increase in free radical scavenging activity using DPPH, no assay, reducing power assay, H2O2 scavenging assay, superoxide anion scavenging assay, and
β-carotene linoleate system. Further, the methanolic extract of C. plicata (MECP) leaves was subjected to single dose acute toxicity study for 14 days in
female rats on the basis of OECD guidelines 423 and the therapeutically selected doses were 200 mg/kg and 400 mg/kg. In vivo antioxidant studies in
CCl4 and acetaminophen intoxicated rats indicated that the MECP leaves have significantly decreased lipid peroxidation in a dose-dependent manner
and increased the levels of catalase, superoxide dismutase, and glutathione.

Conclusions: By the above results, it was concluded that the isolated compound from C. plicata leaves was confirmed as kaempferol and it possesses
significant antioxidative potentials.

Keywords: Chrozophora plicata leaves, Flavonoids, Extraction, Isolation, Characterization, Methanolic extract, Antioxidant activity, Carbon
tetrachloride, Acetaminophen.


1. Prakash P, Gupta N. Therapeutic uses of Ocimum sanctum Linn (Tulsi) with a note on eugenol and its pharmacological actions: A short review. Indian J Physiol Pharmacol 2005;49(2):125-31.
2. Farnsworth NR. The role of ethnopharmacology in drug development. Ciba Foundation Symposium 154 - Bioactive Compounds from Plants. Baffins Lane, Chichester, England: John Wiley & Sons; 1990. p. 2-21.
3. Anonymous. Sectoral Study on Indian Medicinal Plants Status, Perspective and Strategy for Growth. New Delhi: Biotech Consortium India Ltd.; 1996.
4. World Health Organization. Guidelines for the Assessment of Herbal Medicines. Document No. WHO/TRM/91.4. Geneva: World Health Organization; 1991.
5. Kumar S, Mishra A, Pandey AK. Antioxidant mediated protective effect of Parthenium hysterophorus against oxidative damage using in vitro models. BMC Complement Altern Med 2013;13:120.
6. Kumar S, Pandey AK. Phenolic content, reducing power and membrane protective activities of Solanum xanthocarpum root extracts. Vegetos 2013;26:301-7.
7. Cook NC, Samman S. Review: Flavonoids-chemistry, metabolism, cardio protective effects and dietary sources. J Nutr Biochem 1996;7(2)66-76.
8. Rice-Evans CA, Miller NJ, Bolwell PG, Broamley PM, Pridham JB. The relative antioxidant activities of plant derived polyphenolic flavonoids. Free Radic Res 1995;22(4):375-83.
9. Pandey AK. Anti-staphylococcal activity of a pan-tropical aggressive and obnoxious weed Parihenium histerophorus: An in vitro study. Natl Acad Sci Lett 2007;30(11-12):383-6.
10. Kumar S, Gupta A, Pandey AK. Calotropis procera root extract has capability to combat free radical mediated damage. ISRN Pharmacol 2013;2013:8.
11. Abdel-Fattah MR. The Chemical Constituents and Economic Plants of the Euphorbraceae. Doha, Qatar: Chemistry Department, Faculty of Science, Qutar University; 1987.
12. Hashim OK, Abou-Zaid MM, Abdel-Galil FM, Saleh NA. The flavonoids of Egyptian Chrozophora species. Biochem Syst Ecol 1990;18:151-2.
13. Burkill HM. Families E-I. The Useful Plants of West Tropical Africa. 2nd ed., Vol. 2. Kew, Richmond, United Kingdom: Royal Botanical Gardens; 1994. p. 636.
14. Evans WC. Pharmacognosy. 16th ed. Edinburgh: Saunders Elsevier; 2009. p. 3-4.
15. Satinder A, Karen MA. Handbook of Isolation and Characterization of Impurities in Pharmaceuticals. California: Academic Press; 2003. p. 214-20.
16. Barrett M. The Hand Book of Clinically Tested Herbal Remedies. 1st ed. New Delhi: CBS Publishers and Distributors; 2007. p. 3-6.
17. Sharma PC, Bhatia V, Bansal N, Sharma A. A review in Bael tree. Nat Prod Radiance 2007;6(2):171-8.
18. Yadav NP, Chanotia CS. Phytochemical and pharmacological profile of leaves of Aegle marmelos Linn. Pharm Rev 2009;10(3):144-9.
19. Nugroho AE, Riyanto S, Sukari MA, Maeyama K. Effects of aegeline, a main alkaloid of Aegle marmelos Correa leaves, on the histamine release from mast cells. Pak J Pharm Sci 2011;24(3):359-67.
20. Blois MS. Antioxidant determination by the use of stable free radical. Nature 1958;29:1199-200.
21. Mallaiah P, Sudhakara G, Srinivasulu N, Rao BS, Vijayabharathi G,Kumari DS. Assessment of in vitro antioxidant potential and quantification of total phenols and flavonoids of aqueous extract of Phyllanthus amarus. Sri Krishna Devaraya University, Anantapur. Int J Pharm Pharm Sci 2015;7(9):439-45.
22. Ruch RJ, Cheng SJ, Klaunig JE. Prevention of cytotoxicity and inhibition of intercellular communication by antioxidant catechins isolated from Chinese green tea. Carcinogenesis 1989;10(6):1003-8.
23. Kiliçgün H, Altiner D. Correlation between antioxidant effect mechanisms and polyphenol content of Rosa canina. Pharmacogn Mag 2010;6(3):238-41.
24. Kumaran A, Karunakaran RJ. Antioxidant activities of the methanolic extract of Cardiospermum halicacabum. Pharm Biol 2006;44(2):146-51.
25. Jainu M, Shyamala Devi CS. In vitro and in vivo evaluation of free radical scavenging potential of Cissus quadrangularis. Pharm Biol 2005;43(9):773-9.
26. Khanam S, Prasad, S.H.N. Devi K. Antioxidant activities of the methanolic extract of Cardiospermum halicacabum. Indian J Pharm.Educ 2004;38(4):180-83.
27. Fatima Z, Abderrahmane B, Seddik K, Lekhmici A. Antioxidant activity assessment of Tamus communis L. roots. Int J Pharm Pharm Sci 2016;8(12):64-71.
28. OECD Guidelines. OECD Guidelines for Testing of Chemicals, Test No. 425. Acute Oral Toxicity; 1996.
29. Ballantyne B, Timothy M, Turner P. General and Applied Toxicology. Abridged Edition. London: The Macmillan Press Limited; 1995. p. 53-6.
30. Krishna KL, Mruthunjaya K, Patel JA. Antioxidant and hepatoprotective potential of stem methanolic extract of Justicia gendarussa Burm. Int J Pharmacol 2010;6(2):72-80.
31. Srivastava A, Shivanandappa T. Hepatoprotective effect of the root extract of Decalepis hamiltonii against carbon tetrachloride-induced oxidative stress in rat. Food Chem 2010;118(2):411-7.
32. Ramachandra Setty S, Quereshi AA, Viswanath Swamy AH, Patil T, Prakash T, Prabhu K, et al. Hepatoprotective activity of Calotropis procera flowers against paracetamol-induced hepatic injury in rats. Fitoterapia 2007;78(7-8):451-4.
33. Sivakrishna S, Kottaimuthu A. In vivo antioxidant activity of ethanolic extract of aerial parts of Albizia procera roxb (Benth) against paracetamol induced liver toxicity in wistar rats. J Pharm Sci Res 2013;5(9):174-7.
34. Sumanth M, Ahmed R. Antihepatotoxic and antioxidant activity of root of Taraxacum officinale in CCl4-intoxicated rat. Pharmacogn Mag 2008;4(16):188-94.
35. Catherine W, Hosni MH. Introduction and inactivation of catalase and superoxide dismutase of Escherichia coli by ozone. Arch Biochem Biophys 1987;259(2):464-71.
36. Kono Y. Generation of superoxide radical during autoxidation of hydroxylamine and an assay for superoxide dismutase. Arch Biochem Biophys 1978;186(1):189-95.
37. Paglia DE, Valentine WN. Studies on the quantitative and qualitative characterization of erythrocyte glutathione peroxidase. J Lab Clin Med 1967;70(1):158-69.
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How to Cite
Kumar, K. S., and A. S. Rao. “EXTRACTION, ISOLATION AND STRUCTURAL ELUCIDATION OF FLAVONOID FROM CHROZOPHORA PLICATA LEAVES AND EVALUATION OF ITS ANTIOXIDATIVE POTENTIALS”. Asian Journal of Pharmaceutical and Clinical Research, Vol. 10, no. 4, Apr. 2017, pp. 460-9, doi:10.22159/ajpcr.2017.v10i4.17106.
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