In vitro Antioxidant and Sub-acute toxicity studies of Aqueous extract of White butterfly (Clerodendrum volubile) leaves


Research Article | Published:

Print ISSN : 0970-4078.
Online ISSN : 2229-4473.
Pub Email:
Doi: 10.5958/2229-4473.2018.00099.X
First Page: 92
Last Page: 101
Views: 1862

Keywords: Clerodendrum volubile, antioxidant activity, free radical, sub-acute toxicity, Histopathology


The purpose of the study is to investigate the potential antioxidant property and the level of safety of aqueous extract of white butterfly (Clerodendrum volubile P. Beauv) (C. volubile) leaves. The radical scavenging activity of C. volubile extract against DPPH and ABTS+ radicals, in vitro was evaluated. Its reducing power was also measured using FRAP and lipid peroxidation method. In addition, sub-acute toxicity study in rats was carried out to establish the safe doses. Data revealed that the extracts showed no signs of mortalities up to a dose level of 5000 mg/kg body weight indicating that the LD50 is higher than 5g/kg. In vitro antioxidant studies revealed that C. volubile extract is rich in phenolics as typified by its total phenol and flavonoid contents. C. volubile extract showed a promising scavenging effect in DPPH, ABTS and ferric-reducing power assays in a dose-dependent manner. Hematological and biochemical parameters showed that C. volubile extract improved protein and blood levels preserved the levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT) and alkaline phosphatase (ALP) when compared with the control group. The extract also demonstrated lipid lowering effect. The significant increases in urea and creatinine levels observed suggested disturbances of kidney functions. The study indicate that C. volubile extract is non-toxic, safe at low dose levels and possesses antioxidant capacity. High dose, however could have toxic potential as reflected in weight increase in spleen, liver and kidney.

Clerodendrum volubile, antioxidant activity, free radical, sub-acute toxicity, Histopathology

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  1. Adefegha SA and Oboh G (2011) Enhancement of total phenolics and     antioxidant properties of some tropical green leafy vegetables   by steam cooking. J Food Process Preserv 35: 615–622.
  2. Adefegha SA and Oboh G (2011) Enhancement of total phenolics and     antioxidant properties of some tropical green leafy vegetables   by steam cooking. J Food Proc Preserv 35 (5): 615-622.
  3. Adefegha SA and Oboh G (2016) Antioxidant and inhibitory properties          of Clerodendrum volubile leaf extracts on key enzymes        relevant to non-insulin dependent diabetes mellitus and      hypertension. J Taibah Univ Sci 10: 521–533.
  4. Alia M, Horcajo C, Bravo L and Goya L (2003) Effect of grape        antioxidant dietary fiber on the total antioxidant capacityand      the activity of liver antioxidant enzymes in rats. Nutr Res 23: 1251–1267.
  5. Alvarez AM, Mukherjee D (2011) Liver Abnormalities in cardiac diseases and heart failure. Int J Angiol 20(3): 135–142.
  6. Atanas G, Waltenberger AB, Pferschy-Wenzig E, Linder T and Wawrosch C (2015). Discovery and resupply of pharmacologically active plant-derived natural products: a review Biotechnol Adv 33(8): 1582–1614.
  7. Belle NAV, Dalmolin GD, Fonini G, Rubim MA and Rocha JBT (2004) Polyamine reduces lipid peroxidation induced by different pro-oxidant agents. Brain Res 1008:245–251.
  8. Bent S (2008) Herbal medicine in the United States: Review of efficacy, safety, and regulation: Grand rounds at University of California, San Francisco Medical Center. J Gen Internal Med 23: 854-859.
  9. Brewer MS (2011) Natural Antioxidants: Sources, Compounds, Mechanisms of Action, and Potential Applications. Comprehen Rev Food Sci Food Safety 10: 221–247.
  10. Burkill HM (1985) The Useful Plants of West Tropical Africa. Royal Botanic Gardens, Kew. (1) : 319.
  11. Calixto BJ (2000) Efficacy, safety, quality control, marketing and regulatory guidelines for herbal medicines (phytotherapeutic agents). Braz J Med Biol Res 33(2): 179-189.
  12. Cruz RCB, Meurer CD, Silva EJ, Schaefer C and Santos ARS (2006) Toxicity evaluation of Cucurbita maxima seed extract in mice. Pharm Biol 44 (4): 301-303.
  13. Dabla PK (2010) Renal function in diabetic nephropathy World J Diabetes 1(2): 48–56.
  14. Dastmalchi K, Dorman HJD, Kosar M and Hiltunen R (2007) Chemical composition and in vitro antioxidant evaluation of a water soluble Moldavian balm (Dracocephalum moldavica L.) extract. Lebensm Wiss. Technol 40: 239–248.
  15. Dhama K, Latheef  SK, Mani S, Samad HA and Karthik K (2015) Multiple beneficial applications and modes of action of herbs in poultry health and production-a review. Int J Pharmacol 11: 152-176.
  16. Ekor M (2013). The growing use of herbal medicines: issues relating to adverse reactions and challenges in monitoring safety. Front Pharmacol 4: 177-180.
  17. El-Hilaly JE, Israili ZH and Lyoussi B (2004) Acute and chronic toxicological studies of Ajuga iva in experimental animals. J Ethnopharmacol 91:43–50.
  18. El-Hilaly JE and Lyoussi B (2002) Hypoglycaemic effect of the lyophilized aqueous extract of Ajugaiva in normal and streptozotocin diabetic rats. J Ethnopharmacol 80:109–113.
  19. Erukainure OL, Oke OV, Ajiboye AJ and Okafor OY(2011) Nutritional qualities and phytochemical constituents of Clerodendrum volubile, a tropical non-conventional vegetable. Intl Food Res J 18 (4): 1393–1399.
  20. Erukainure OL, Choudhary MI,  Adhikari A, Islam S and Hafizur RM (2017) Anti-diabetic effect of the ethyl acetate fraction of Clerodendrum volubile: protocatechuic acid suppresses phagocytic oxidative burst and modulates inflammatory cytokines. Biomed Pharmacoth 86 :307–315.
  21. Erukainure OL, Ebuehi OAT, Choudhary IM, Adhikari A and Hafizur RMS (2014)   Iridoid glycoside from the leaves of Clerodendrum volubile beauv shows potent antioxidant activity against oxidative stress in rat brain and hepatic tissues. J Diet Suppl  doi: http://
  22. Erukainure OL, Zaruwa MZ, Choudhary MI, Naqvi SA and Ashraf A  (2016) Dietary fatty acids from leaves of Clerodendrum volubile induce cell cycle arrest downregulates matrix metalloproteinase (MMP)-9 expressions, and modulate redox status in human breast cancer.  Nutr Canc  doi:http:// 01635581.2016.1156714
  23. Fleming D and Hunt D (2000) Biological Safety, Principles and Practices. ASM Press, Washington, DC, USA. 267
  24. Fred-Jaiyesimi A and Adekoya Y (2012) Pharmacognostic studies and anti-inflammatory activities of Clerodendrum volubile P. Beauv leaf. Int J Phytomed  4: 414–418.
  25. Gülçin, I, Oktay M, Kirecci E and Kufrevioglu OI (2003) Screening of antioxidant and antimicrobial activities of anise (Pimpinella anisum L.) seed extracts. Food Chem 83: 371–382
  26. Gyamfi MA, Yonamin EM and Aniya Y (1999) Free-radical scavenging action of medicinal herbs from Ghana: Thonningia sanguinea on experimentally-induced liver injuries. Gen Pharmacol 32: 661–667.Gautam MK and Goel RK (2014) Toxicological study of Ocinum sanctum Linn leaves: Heamatological, biochemical and histopathological studies. J Toxicol volume 2014, Article ID 135654, 9 pages. doi: 10.1155/2014/135654
  27. Halim SZ, Abdullah NR, Afzan A, Rashid BA and Jantan I (2011) Acute toxicity study of Carica papaya leaf extract in Sprague dawley rats. J Med Plant Res 5(10):1867-72.
  28. Janero DR (1990) Malondialdehyde and thiobarbituric acid-reactivity as diagnostic indices of lipid peroxidation and peroxidative tissue injury. Free Rad Biol Med 9: 515 – 540.
  29. Johnston DE (1999) Special considerations in interpreting liver function tests. Am Fam Physician 59(8):2223–30.
  30. Kalonia H, Kumar P and Kumar A (2011) Comparative neuroprotective profile of statins in quinolinic acid induced neurotoxicity in rats. Behaviour Brain Res 216: 220 – 228.
  31. Lahlou S, Israili ZH and Lyoussi B (2008) Acute and chronic toxicity of a lyophilized aqueous extract of Tanacetum vulgare leaves in rodents. J Ethnopharmacol 117:221–227.
  32. Lameire N, Van Biesen W, Vanholder R (2005) Acute renal failure. Lancet 365(9457): 417–430.
  33. Marieb EN and Hoehn KN (1999) Human Anatomy and Physiology. 9th ed, USA
  34. Meda A, Lamien C, Romito M, Millogo J and Nacoulma OG (2005) Determination of the total phenolic, flavonoid and proline contents in Burkina Faso honey, as well as their radical scavenging activity. Food Chem 91: 571-577.
  35. Meir S, Kanner J, Akin B and Hadas SP(1995) Determination and involvement of aqueous reducing compounds in oxidative defense systems of various senescing leaves. J Agric Food Chem 43: 1813–1815.
  36. Molehin OR, Oloyede OI and Boligon AA (2017) Comparative study on the phenolic content, antioxidant properties and HPLC fingerprinting of the extracts of Clerodendrum volubile P. Beauv. J App Pharm Sci 7 (03): 135-140.
  37. Molehin OR, Adefegha SA, Oboh G, Saliu JA and Athayde Ml (2014) Comparative study on the phenolic content, antioxidant properties and HPLC fingerprinting of three varieties of Celosia species. J  Food Biochem 38: 575–583.
  38. Oboh G and ROcha JBT (2007) Antioxidant in Foods: A New Challenge for Food Processors. Leading Edge Antioxidants Research. Nova Science Publishers Inc., New York, NY 35-44.
  39. Oboh G, Raddatz H and Henle T (2008) Antioxidant properties of polar and non-polar extracts of some tropical green leafy vegetables. J Sci Food Agric  88: 2486–2492.
  40. Ohkawa H, Ohishi N and  Yagi K (1979) Assay for lipid peroxides in animal tissues by thiobarbituric acid reactions. Ann Biochem 95: 351–358.
  41. Olson H, Betton G, Robinson D,  Thomas K and Monro A (2000)  Concordance of the toxicity of pharmaceuticals in humans and in animals. Regulatory Toxicol Pharmacol 32(1): 56–67.
  42. Orisakwe OE, Afonne OJ, Chude MA and Obi E Dioka CE (2003) Sub- chronic Toxicity Studies of the Aqueous Extract of Boerhavia diffusa Leaves. J Health Sci 49(6): 444 – 447.
  43. Oviasogie PO, Okoro D and Ndiokwere CL (2009) Determination of total phenolic amount of some edible fruits and vegetables. Afr J Biotechnol 8: 2819–2820.
  44. Oyaizu M (1986) Studies on products of browning reaction: Antioxidative activity of products of browning reaction prepared from glucosamine. Japan J Nutr 44: 307–31.
  45. Paul S, Ali Y, Rumpa N, Tanvir EM and Hossen S (2017) Assessment of toxicity and beneficiary effects of Garcinia pedunculata on the hematological, biochemical and histological homeostasis in rats. Evidence-Based Compl Alter Med, Article ID 4686104, Pp 11. doi:10.1155/2017/4686104
  46. Ramaiah SK (2007) A toxicologist guide to the diagnostic interpretation of hepatic biochemical parameters. Food Chem Toxicol 45 (9): 1551–1557.
  47. Re R, Pellegrini N, Proteggente A, Pannala A and Yang M (1999) Antioxidant activity;  applying an improved ABTS radical cation decolorisation assay. Free Radic Bio Med 26: 1231– 1237.
  48. Saidu Y, Bilbis LS, Lawal M, Isezuo SA and Hassan SW (2007) Acute and Sub-chronic toxicity studies of crude aqueous extract of Albizzia Chevalieri. Asian J Biochem 2(4): 224-236.
  49. Satpal S, Jain K and Punia JS (2010) Studies on biochemical changes in sub-acute toxicity of thiodicarb in rats. Toxicol Int 17: 30-32.
  50. Scalbert, A, Manach C, Morand C and Remesy C (2005) Dietary polyphenols and the prevention of diseases. Crit Rev Food Sci Nutr 45:287–306.
  51. Silva EJR, Goncalves ES, Aguiar FJS, Evencio LB and Lyra MMA (2007) Toxicological studies on hydroalcohol extract of Calendula officinalis L. Phytother Res 21:332–336.
  52. Singleton VL, Orthofer R and Lamuela-Raventos RM. (1999) Analysis of total phenols and other oxidation substrates and antioxidants by means of Folin–Ciocalteau’s reagent. Methods Enzymol 299:152–178.
  53. Velioglu YS, Mazza G, Gao L and Oomah BD (1998) Antioxidant activity and total phenolics in selected fruits, vegetables and grain products.  J Agric Food Chem  46: 4113–4117.
  54. Whittaker BS, Berlett ER and Stadtman RR (1997) Protein Oxidation in aging disease and oxidative stress. J Biol Chem 272: 20313- 20316.
  55. Wintrobe MM and Greer JP (2009) Wintrobe’s Clinical Hematology, Lippincott Williams and Wilkins.
  56. Wolf PL (1999) Biochemical diagnosis of liver disease. Indian J Clin Biochem 14(1): 59–90.
  57. Zhang L, Li J, Hogan S, Chung H and Welbaum GE (2010) Inhibitory effect of raspberries on starch digestive enzyme and their antioxidant properties and phenolic composition. Food Chem 119: 592–599.



This study was supported by the Ekiti State University, Ado-Ekiti Needs Assessment Intervention Training Scholarship under the Implementation Monitoring Committee of the Federal Ministry of Education Nigeria [VC/ NADO/VOLII/04] to Olorunfemi R. Molehin].

Author Information

Olorunfemi Raphael Molehin*
Department of Biochemistry, Faculty of Science, Ekiti State University, Ado-Ekiti P.M.B.5363 Ado-Ekiti Nigeria
Omotade Ibidun Oloyede
Department of Biochemistry, Faculty of Science, Ekiti State University, Ado-Ekiti P.M.B.5363 Ado-Ekiti Nigeria