Evaluation of cytotoxic effects and acute and chronic toxicity of aqueous extract of the seeds of Calycotome villosa (Poiret) Link (subsp. intermedia) in rodents

Document Type : Original Research Article


1 Laboratory of Physiology-Pharmacology and Environmental Health, University Sidi Mohamed Ben Abdallah, Fez, Morocco

2 Institute of Neuroscience, Université catholique de Louvain, Brussels, Belgium

3 GNOS research group, Louvain Drug Research Inistitute, Université catholique de Louvain, Brussels, Belgium


Objective: The present investigation was carried out to evaluate the safety of an aqueous extract of the seeds of Calycotome villosa (Poiret) Link (subsp. intermedia) by determining its cytotoxicity and potential toxicity after acute and sub-chronic administration in rodents.
Materials and Methods: Cytotoxic activity was tested in cancer and non-cancer cell lines HeLa, Mel-5, HL-60 and 3T3. Acute toxicity tests were carried out in mice by a single oral administration of Calycotome seed-extract (0 - 12 g/kg) as well as intraperitoneal doses of 0 - 5 g/kg.  Sub-chronic studies were conducted in Wistar rats by administration of oral daily doses for up to 90 days. Changes in body and vital organ weights, mortality, haematology, clinical biochemistry and histologic morphology were evaluated.
Results: The lyophilized aqueous extract of C. villosa exhibited a low cytotoxicity in all cell lines tested with an IC50 > 100 µg/ml. In the acute study in mice, intra-peritoneal administration caused dose-dependent adverse effects and mortality with an LD50 of 4.06 ± 0.01 g/kg.In the chronic tests, neither mortality nor visible signs of lethality was seen in rats. Even AST and ALT were not affected while a significant decrease in serum glucose levels, at 300 and 600 mg/kg was detected.  Histopathological examination of the kidney and liver did not show any alteration or inflammation at the end of treatment.
Conclusion: In conclusion, the aqueous extract of C. villosa seed appeared to be non-toxic and did not produce mortality or clinically significant changes in the haematological and biochemical parameters in rats.


Main Subjects

Akdogan M, Kilinc I, Oncu M, Karaoz E, Delibas N. 2003. Investigation of Biochemical and Histopathological Effects of Mentha piperita L and Mentha spicata L on Kidney tissue in Rats. Hum Exp Toxicol, 22: 213-219.
Alexeeff GV Broadwin R Liaw J Dawson SV .2002.  Characterization of the LOAEL-to-NOAEL uncertainty factor for mild adverse effects from acute inhalation exposures. Regul Toxicol Pharmacol, 36:96-105.
Aniagu SO, Nwinyi FC, AkumkaDD, AjokuGA, DzarmaS, IzebeKS, DitseM, Nwaneri,        PC, WambebeC, GamanielK . 2005 . Toxicity studies in rats fed nature cure bitters.  Afr J Biotechnol, 4: 72-78.
Avula BWang YHPawar RSShukla YJSchaneberg BKhan IA. 2006. Determination of the appetite suppressant P57 in Hoodia gordonii plant extracts and dietary supplements by liquid chromatography/electrospray ionization mass spectrometry (LC-MSD-TOF) and LC-UV methods. J Aoac Int, 89 :606-611.
Baliga MS ,  Jagetia GC ,  Ulloor JN ,  Baliga MP ,  Venkatesh P , Reddy R ,  Rao KV,  Baliga BS , Devi S ,  Raju SK ,  Veeresh V, Reddy TK, Bairy KL. 2004.  The evaluation of the acute toxicity and long term safety of hydroalcoholic extract of Sapthaparna (Alstonia scholaris) in mice and rats. Toxicol Lett, 151:317-326.
Cherkaoui TK, Lachkar M, Wibo M, Morel N, Gilani AH, Lyoussi B. 2008. Pharmacological studies on hypotensive, diuretic and vasodilator activities of chrysin glucoside from Calycotome villosa in rats. Phytother Res, 22: 356-361.
Cherkaoui TK., El-Hilaly J, Israili ZH, Lyoussi B. 2009. Ethnobotanical survey and   pharmacological screening of medicinal plants used as antihypertensive in Sefrou province (Middle North of Morocco): Benefits and challenges. Submitted in Journal of Ethnopharmacology
Dioka C, Orisakwe O.E, Afonne O.J, Agbasi P.U, Akumka DD, Okonkwo CJ, Ilondu N. 2002. Investigation into the haematologic and hepatoxic effects of rinbacin in rats. J Health Sci, 48: 393–398.
Drici MD, Clement N. 2001. Is gender a risk factor for adverse drug reactions? The example of drug-induced long QT Syndrome. Drug Saf, 24: 575–585.
Ebert, SN, Liu XK, Woosley, RL. 1998. Female gender as a risk factor for drug-induced cardiac arrhythmias: evaluation of clinical and experimental evidence. J Womens Health, 7: 547–557.
El Antri, A, Messouri, I, Bouktaib, M, El Alami R, Bolte, M, El Bali, B, Lachkar, M. 2004a. Isolation and X-ray crystal structure of tetrahydroisoquinoline alkaloids from Calycotome villosa Subsp. Intermedia. Molecules, 9: 650–657.
El Antri, A, Messouri I, Bouktaib, M,  El Alami R, El Bali, B, Lachkar, M. 2004b. Isolation and x-ray crystal structure of a new isoquinoline-N-oxide alkaloid from Calycotome villosa Subsp. Intermedia. Fitoterapia, 75: 774–778.
El Antri, A, Messouri I, Chendid Tlemcani R , Bouktaib, M,  El Alami R, El Bali, B, Lachkar, M. 2004c. Flavone glycosides from Calycotome villosa Subsp. Intermedia. Molecules 9: 568–573.
Gazda, V.E., Gomes-Carneiro, M.R., Barbi, N.S., Paumgartten FJR. 2006. Toxicological evaluation of an ethanolic extract from Chiococca alba roots. J Ethnopharmacol, 105: 187-195.
Greuter W, Burdet, HM, Long, G (eds). 1989. Med-Checklist 4: A Critical Inventory of  Vascular Plants of the Circum Mediterranean Countries; Dicotyledones (LauraceaeRhamnaceae). C.B. de Genève: Geneva. Grossel SS, Crowl AD. 1994.  editors. Marcel Dekker, Inc., New York.Handbook of Highly Toxic    Materials Handling and Management. 
Huang, RL, Chen, CC, Huang, YL., OU JC, Hu CP, Chen CF, Chang C. 1998. Anti- Tumor effects of d-dicentrine from the root of lindera megaphylla. Planta Med, 64: 212-215.
(IFCC) International Federation of Clinical Chemistry).1986. On IFCC methods for the measurement of catalytic concentration of enzymes. Part 2. IFCC method for aspartate aminotransferase (L-aspartate: 2-oxoglutarate aminotransferase, EC J Clin Chem Clin Biochem, 24:497-510.
IFCC (International Federation of Clinical Chemistry), 1980. IFCC methods for the   measurement of catalytic concentration of enzymes. Part 3. IFCC method for alanine aminotransferase (l-alanine: 2-oxoglutarate aminotrasferase, EC Clinica Chimica Acta, 105: 147F–154F.
Kallner A, Tryding N. 1989. IFCC Guidelines to the Evaluation of Drug Effects in Clinical Chemistry. Scandinavian Journal of Clinical and Laboratory Investigation, 49, 1–29.
Kennedy GL, Ferenz RL, Burgess BA. 1986. Estimation of acute oral toxicity in rats by the determination of the approximate lethal dose rather than the LD50. J Appl Toxicol, 6:145-148.
Kim JH, Hahm DH, Yang DC, Kim JH, Lee HJ, Shim I. 2005. Effect of crude saponin of Korean red ginseng on high-fat diet-induced obesity in the rat. J Pharmacol Sci, 97:124-31.
King DJ, Kelton JG.1984. Heparin – Associated Thrombocytopaenia. Ann Intern Med, 100:535-40.
Kouitcheu Mabeku LB, Penlap Beng V, Kouam J, Essame, O, Etoa, FX. 2007. Toxicological evaluation of ethyl acetate extract of Cylicodiscus gabunensis stem bark (Mimosaceae). J Ethnopharmacol, 111: 598–606.
Lee TY, Wu ML, Deng, JF, Hwang DF. 2002. High-performance liquid chromatographic determination for aristolochic acid in medicinal plants and slimming products. J Chromatograph B: Analytical Technol Biomed  Life Sci, 766:169-174.
Liechti  ME, Gamma A, Vollenweider FX. 2001. Gender differences in the subjective effects of MDMA. Psychopharmacology, 154: 161–168.
Lin TJ, Su CC, Lan CK, Jiang DD, Tsai JL, Tsai MS. 2003. Acute Poisonings with Breyniaofficinalis – An Outbreak of Hepatoxicity. J Clin Toxicol, 41: 591–594.
Litchfield JT, Wilcoxon F.1949. A simplified method of evaluating dose effect experiments. J Pharmacol Exp Ther,  96: 99–113.   
Lopez TA,  Campero CM, Chayer R, Cosentino B, Caracino M. 1996. Experimental toxicity of verbesina encelioides in sheep and isolation of galegine. Veterin  Human Toxicol, 38: 417-419.
Loy G, Cottiglia F, Garau D, Deidda D, Pompei R, Bonsignore L. 2001. Chemical composition and cytotoxic and antimicrobial activity of Calycotome villosa (Poiret) link  leaves. Farmaco , 56: 433-436.
Mosmann T. 1983. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J immunol method, 65: 55-63
Mukinda JT, Syce JA. 2007. Acute and chronic toxicity of the aqueous extract of Artemisia afra in rodents. J Ethnopharmacol, 112: 138–144.
Neese JM, Duncan P, Bayse D, Robinson M, Cooper T, Stewart C. 1976. Development and evaluation of Hexokinase/Glucose-6-Phosphat Dehydrogenase Procedure for Use as a National Glucose Reference Method. U.S. Department of HEW No. (CDC) 77-8330, 1–7.
Olson H, Betton G, Robinson D, Thomas K, Monro A, Kolaja G, Lilly P, Sanders J, Sipes G, Bracken W, Dorato M, Deun KV, Smith P, Berger B, Heller A. 2000. Concordance of toxicity of pharmaceuticals in humans and in animals.  Regul Toxicol Pharmacol,32: 56–67.
Raza M, Al-Shabanah OA, El-Hadiyah TM, Al-Majed AA. 2002. Effects of prolonged vigabatrin treatment on hematological and biochemical parameters in plasma, liver and kidney of Swisss albino mice. Scientia Pharm, 70: 135-145.
Rebecca MA, Ishii-Iwamoto EL, Grespan R, Cuman RKN, Caparroz-Assef SM, Mello JCP de, Bersani-Amado CA. 2002. Toxicological studies on Stryphnodendron adstringens. J Ethnopharmacol, 83: 101-104.
Ross MH, Reith EJ, Romrell IJ. 1989. A text an atlas. Histology 1-2. Silva EJR, Goncalves ES, Aguiar F, Evencio LB, Lyra MMA, Coelho MCOC, Fraga  MCCA, Wanderley AL. 2007. Toxicological studies on hydroalcohol extract of Calendula officinalis L. Phytother Res, 21: 332–336.
Stévigny C, Block S, De Pauw-Gillet MC, de Hoffmann E, Liabrès G, Adjakidjé V, Quetin-Leclercq J. 2002. Cytotoxic aporphine alkaloids from Cassytha filiformis. Planta Med, 68:1042-1044.
Tabacco A, Meiattini F, Moda E, Tarli P. 1979. Simplified enzymic/ colorimetric serum urea nitrogen determination. Clin Chem, 25: 336–337.
Teo S, Stirling D, Thomas S, Hoberman A, Kiorpes A, Khetani V. 2002.  A 90-day oral gavage toxicity study of D-methylphenidate and D, L-methylphenidate in Sprague Dawley rats. Toxicology, 179: 183-196.
Twaij HAA, Kery A, Al Khazraji NK. 1983. Some pharmacological, toxicological and phytochemical investigations on Centaurea phyllocephala. J Ethnopharmacol, 9: 299–314. 
Van Miert AS. 1989. Extrapolation of pharmacological and toxicological data based on metabolic weight. Arch fur Exp Veterinarmed, 43: 481-488.    
Waynforth BH, Flecknell PA. 1980. Injection techniques. In: Experimental and Surgical Techniques in the Rat. Academic Press, London, N.Y., USA 30–61.
Zhu M, Lew KT, Leung, PL. 2002. Protective effects of Plant formula on ethanolic-induced gastric lesions in rats. Phytother Res, 16: 276–280.