Skip to content
Licensed Unlicensed Requires Authentication Published by De Gruyter March 10, 2017

Preliminary phytochemical analysis and evaluation of antioxidant, cytotoxic and inhibition of lipopolysaccaride - induced NOS (iNOS) expression in BALB/c mice liver by Ziziphus oenoplia Mill. fruit

  • Ramaraj Thirugnanasampandan EMAIL logo , Gunasekar Ramya , Gunasekaran Bhuvaneswari , Srinivasan Aravindh , Suresh Vaishnavi and Madhusudhanan Gogulramnath



Ziziphus oenoplia Mill. is an ethnomedicinal plant and its fruit has been traditionally used by Puliar tribes of Anamalai Hills, Tamil Nadu, India to treat various ailments. Phytochemical analysis, antioxidant, cytotoxic and inducible nitric oxide synthase (iNOS) gene downregulation activities of Z. oenoplia fruit (ZOF) were studied.


To explore bioactive compounds present in the ripened fruits, high-performance thin-layer chromatography (HPTLC) and gas chromatography/mass spectrometry (GC–MS) analysis were done. Free radical scavenging, hepatoprotective, inhibition of iNOS gene expression and cytotoxic activities of ethanol extract of fruit were also studied.


Total flavonoid content of ZOFwas estimated as 69 µg/mg catechin equivalent. HPTLC densitogram confirmed the presence of quercetin and GC–MS analysis showed a total of 16 compounds of 87.66 % with quinic acid as a major compound which accounted for 22.29 %. Free radical-scavenging activity of ethanolic fruit extract was ranged from 160.12 to 650.23 µg/mL. An amount of 1.5 µg lipopolysaccharide (LPS)- induced severe inflammation in BALB/c mice liver, followed by treatment with ethanolic fruit extract of 100 µg concentration, exhibited significant hepatoprotection and reverse transcriptase polymerase (RT-PCR) analysis showed downregulation of iNOS gene expression in hepatocytes at transcriptional level. ZOF also showed significant cytotoxicity and propidium iodide staining confirmed the induction of apoptosis in cervical cancer cells (HeLa).


Findings of the present study prove that ZOF is a rich source of bioactive compounds with a wide range of pharmacological activities. Hence, consumption of this wild edible fruit will be a cost-effective and easily available natural nutritional source for health protection.

  1. Author contributions: All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission.

  2. Research funding: We thank our college management for financial support.

  3. Employment or leadership: None declared.

  4. Honorarium: None declared.

  5. Competing interests: The funding organization(s) played no role in the study design; in the collection, analysis and interpretation of data; in the writing of the report or in the decision to submit the report for publication.


1. Jordana M, Sarnstrand B, Sime PJ, Ramis I. Immune inflammatory functions of fibroblasts. Eur Respir J. 1994;7:2212–2222.10.1183/09031936.94.07122212Search in Google Scholar

2. Ladecola C, Zhang F, Casey R, Nagayama M, Ross ME. Delayed reduction of ischemic brain injury and neurological deficits in mice lacking the inducible nitric oxide synthase gene. J Neurosci. 1997;17:9157–9164.10.1523/JNEUROSCI.17-23-09157.1997Search in Google Scholar

3. Yu HH, Wu FL, Lin SE, Shen LJ. Recombinant arginine deiminase reduces inducible nitric oxide synthase iNOS-mediated neurotoxicity in a coculture of neurons and microglia. J Neurosci Res. 2008;86:2963–2972.10.1002/jnr.21740Search in Google Scholar

4. Clifford GM, Smith JS, Plummer M, Munoz N, Franceschi S. Human papillomavirus types in invasive cervical cancer worldwide: A meta analysis. Br J Cancer. 2003;88:63–73.10.1038/sj.bjc.6600688Search in Google Scholar

5. Munger K, Basile JR, Duensing S, Eichten A, Gonzalez SL, Grace M,et al. Biological activities and molecular targets of the human papillomavirus E7 oncoprotein. Oncogene. 2001;20:7888–7898.10.1038/sj.onc.1204860Search in Google Scholar

6. Heald SV Rhamnaceae. In: Smith N, Mori SA, Henderson A, Stevenson DW, Heald SV,et al., editors. Flowering plants of the neotropics. New Jersey:Princeton University Press, 2004:323–324.Search in Google Scholar

7. Devi S, Pandey VB, Singh JP, Shah AH. Peptide alkaloids from Zizyphus species. Phytochem. 1987;26:3374–3375.10.1016/S0031-9422(00)82518-2Search in Google Scholar

8. Kirtikar KR, Basu BR. Indian medicinal plant, 2nd ed. New Delhi:Periodical Experts Book Agency, 1991:595.Search in Google Scholar

9. Suksamrarn S, Suwannapoch N, Aunchai N, Kuno M, Ratananukul P, Haritakun R,et al. Ziziphine N, O, P and Q, new antiplasmodial cyclopeptide alkaloids from Ziziphus oenoplia var. brunoniana. Tetrahedron. 2005;61:1175–1180.10.1016/j.tet.2004.11.053Search in Google Scholar

10. Singh MP, Panda H. Medicinal herbs with their formulations Vol. 1. Delhi:Daya Publishing House, 2005:97–100.Search in Google Scholar

11. Jadhav SA, Prassanna SM. Evaluation of antiulcer activity of Ziziphus oenoplia Mill. roots in rats. Asian J Pharm Clin Res. 2011;1:92–95.Search in Google Scholar

12. Rao CV, Rawat AKS, Singh AP, Singh A, Verma N. Hepatoprotective potential of ethanolic extract of Ziziphus oenoplia (L.). Mill roots against antitubercular drugs induced hepatotoxicity in experimental models. Asian Pac J Trop Med. 2012;5:283–288.10.1016/S1995-7645(12)60040-6Search in Google Scholar

13. Zhishen J, Mengcheng T, Jianming W. The determination of flavonoid contents in mulberry and their scavenging effects on superoxide radicals. Food Chem. 1999;64:555–559.10.1016/S0308-8146(98)00102-2Search in Google Scholar

14. Mensor LI, Menezes FS, Leitao GG, Reis AS, Dos Santos T, Coube CS,et al. Screening of Brazilian plants extracts for antioxidants activity by the use of DPPH free radical method. Phytother Res. 2001;15:127–130.10.1002/ptr.687Search in Google Scholar PubMed

15. Zhao GR, Xiang ZJ, Ye TX, Yuan YJ, Guo ZX. Antioxidant activities of Salvia miltiorrhiza and Panax notoginseng. Food Chem. 2006;99:767–774.10.1016/j.foodchem.2005.09.002Search in Google Scholar

16. Singh N, Rajini PS. Free radical scavenging activity of an aqueous extract of potato peel. Food Chem. 2004;85:611–616.10.1016/j.foodchem.2003.07.003Search in Google Scholar

17. Choi BD, Kang SJ, Ha YL, Park GB, Ackman RG. Conjugated linoleic acid as a supplemental nutrient for common carp (Cyprinus carpio). Food Sci Biotechnol. 2002;11:457–461.Search in Google Scholar

18. Halliwell B, Gutteridge JMC, Aruoma OI. The deoxyribose method: A simple test-tube assay for determination of rate constants for reactions hydroxyl radical. Anal Biochem. 1987;165:215–219.10.1016/0003-2697(87)90222-3Search in Google Scholar

19. Chan MM, Huang HI, Fenton MR, Fong D. In vivo inhibition of nitric oxide synthase gene expression by curcumin, a cancer preventive natural product with antiinflammatory properties. Biochem Pharmacol. 1998;55:1955–1962.10.1016/S0006-2952(98)00114-2Search in Google Scholar

20. Yamasaki K, Edington HD, McClosky C, Lizonova A, Kovesdi I,et al. Reversal of impaired wound repair in iNOS deficient mice by topical adenoviral mediated iNOS gene transfer. J Clin Invest. 1998;101:967–971 . .10.1172/JCI2067Search in Google Scholar

21. Kim HY, Kim JK, Choi JH, Jung JY, Oh WY, Kim DC,et al. Hepatoprotective effect of pinoresinol on carbon tetrachloride induced hepatic damage in mice. J Pharmacol Sci. 2010;112:105–112.10.1254/jphs.09234FPSearch in Google Scholar

22. Yu JQ, Lei JC, Zhang XQ, Yu HD, Tian DZ, Liao ZX,et al. Anticancer, antioxidant and antimicrobial activities of the essential oil of Lycopus lucidus Turcz.var.hirtus Regel. Food Chem. 2011;126:1593–1598.10.1016/j.foodchem.2010.12.027Search in Google Scholar

23. Brana C, Benham C, Sundstorm L. A method for characterising cell death in vitro by combining propidium iodide staining with immunohistochemistry. Brain Res Protocol. 2002;10:109114.10.1016/S1385-299X(02)00201-5Search in Google Scholar

24. Wang S, Zhang Z, Gao W, Yan Y, Li X. Identification of chemical constituents in the extract and rat serum from Ziziphus jujuba Mill by HPLC-PDA-ESI-MSn. Iranian J Pharm Res. 2014;13:1055–1063.Search in Google Scholar

25. Cardozo ML, Ordonez RM, Alberto MR, Zampini IC, Isla MI. Antioxidant and antiinflammatory activity characterization and genotoxicity evaluation of Ziziphus mistol ripe berries, exotic Argentinean fruit. Food Res Int. 2011;44:2063–2071.10.1016/j.foodres.2011.02.040Search in Google Scholar

26. Memon AA, Memon N, Bhanger MI, Luthira DL. Assay of phenolic compounds from four species of ber (Ziziphus mauritiana L.) fruits: Comparison of three base hydrolysis procedure for quantification of total phenolic acids. Food Chem. 2013;139:496–502.10.1016/j.foodchem.2013.01.065Search in Google Scholar PubMed

27. Sousa CD, Silva M, Vieira JR, Ayres GM, Costa MCC, Araujo CD,et al. Total phenolics and antioxidant activity of five medicinal plants. Quimica Nova. 2007;30:351–355.10.1590/S0100-40422007000200021Search in Google Scholar

28. Sun YF, Liang ZS, Shan CJ, Viernstein H, Unger F. Comprehensive evaluation of natural antioxidants and antioxidant potentials in Ziziphus jujuba Mill var. spinosa (Bunge) Hu ex H. F. Chou fruits based on geographical origin by TOPSIS method. Food Chem. 2011;124:1612–1619.10.1016/j.foodchem.2010.08.026Search in Google Scholar

29. Brito SM, Coutinho DM, Talvani T, Coronel C, Barbosa GR, Vega C,et al. Analysis of bioactivities and chemical composition of Ziziphus joazeiro Mart. using HPLC-DAD. Food Chem. 2015;186:185–191.10.1016/j.foodchem.2014.10.031Search in Google Scholar PubMed

30. Arhoghro EM, Ekpo KE, Anosike EO, Ibeh GO. Effect of aqueous extract of bitter leaf (Vernonia amygdalina Del) on carbontetrachloride (CCl4) induced liver damage in albino wistar rats. Eur J Sci Res. 2009;26:122–130.Search in Google Scholar

31. Lowe SW, Lin AW. Apoptosis in cancer. Carcinog. 2000;21:485–495.10.1093/carcin/21.3.485Search in Google Scholar PubMed

32. Jana S, Paliwal J. Apoptosis: Potential therapeutic targets for new drug discovery. Curr Med Chem. 2007;14:2369–2379.10.2174/092986707781745569Search in Google Scholar PubMed

33. Okun I, Balakin KV, Tkachenko SE, Ivachtchenko AV. Caspase activity modulators as anticancer agents. Anticancer Agents Med Chem. 2008;8:322–341.10.2174/187152008783961914Search in Google Scholar PubMed

34. Huang XD, Kojima-Yuasa A, Norikura T, Kennedy DO, Hasuma T, Matsui-Yuasa I,et al. Mechanism of the anticancer activity of Zizyphus jujuba in HepG2 cells. American J Chinese Med. 2007;35:517–532.10.1142/S0192415X0700503XSearch in Google Scholar PubMed

35. Mishra T, Khullar M, Bhatia A. Anticancer potential of aqueous ethanol seed extract of Ziziphus mauritiana against cancer cell lines and Ehrlich ascites carcinoma. Evid Based Complement Alternat Med. 2010;765029 . .10.1155/2011/765029Search in Google Scholar PubMed PubMed Central

36. Taha H, Arya A, Paydar M, Looi CY, Wong WF, Vasudeva MC,et al. Upregulation of insulin secretion and downregulation of proinflammatory cytokines, oxidative stress and hyperglycemia in STZ nicotinamide induced type 2 diabetic rats by Pseuduvaria monticola bark extract. Food Chem Toxicol. 2014;66:295–306.10.1016/j.fct.2014.01.054Search in Google Scholar PubMed

37. Arya A, AlObaidi MM, Shahid N, Noordin MB, Looi CY, Won WF,et al. Synergistic effect of quercetin and quinic acid by alleviating structural degeneration in the liver, kidney and pancreas tissues of STZ-induced diabetic rats: A mechanistic study. Food Chem Toxicol. 2014;71:183–196.10.1016/j.fct.2014.06.010Search in Google Scholar PubMed

Received: 2016-1-21
Accepted: 2017-1-25
Published Online: 2017-3-10
Published in Print: 2017-3-9

© 2017 Walter de Gruyter GmbH, Berlin/Boston

Downloaded on 5.10.2023 from
Scroll to top button