Acute And Sub-Acute Toxicity And Quality Control Method For The Extracts Of Cajanus Cajan Leaves

Cajanus cajan is a Fabaceae plant distributed in Asia, Africa, and the Americas. Its leaves are widely used as fodder and traditional medicine. However, there is no systematically toxicological information available for the safety evaluation of the leaf extracts.Objective1、Content determination study of active ingredients of water or 90% ethanol extract of Cajanus cajan leaves (WEC or EEC), dichloromethane or n-butanol fractionation of EEC (named as DEC or BEC), to provide quality assurance for toxicological studies of these extracts.2、Oral acute toxicity of WEC, EEC, DEC and BEC in Kunming (KM) mice, and sub-acute oral toxicity of WEC or EEC in Sprague Dawley (SD) rats were investigated. To provide the foundation for the safety evaluation of Cajanus cajan leaf or its extracts.Methods1、Content determination studyCharacteristic flavonoids and stilbenoids were used in quality analysis of the extracts by means of high performance liquid chromatography (HPLC). Three constituents genistein, vitexin and orientin were used as referent markers for the qualitative analysis of WEC or BEC. Three constituents pinostrobin, longistyline C and longistyline A were used as referent markers for the qualitative analysis of EEC or DEC.2、Toxicology researchIn the acute toxicity study, maximal single dose of WEC, EEC, DEC, or BEC were orally dosed in KM mice, mortality, bodyweight (b.wt.), general behavior, and signs of toxicity were observed for 14 days to afford maximum tolerance doses (MTDs).In the sub-acute toxicity study, repeated oral doses of WEC or EEC at 1.5 (low),3.0 (middle) and 6.0 (high dose) g·kg-1 in SD rats were administered for 4 weeks, bodyweight, clinical signs, hematological and biochemistry parameters, relative organ weight and organ pathology were evaluated in the extract-exposure period and 2 weeks after the treatment.Results1、HPLC analysis revealed that the characteristic compounds in water solube extract WEC or BEC were flavonoidal glycosides, among them genistein, vitexin and orientin presented, with a content range of 0.25%-0.78%(WEC) or 0.51%-2.98%(BEC), while characteristic compounds in EEC or its water insoluble fractionation DEC involved in lipophilic compounds longistyline A, longistyline C and pinostrobin, with a content range of 0.22%-1.06%(EEC) or 1.80%-7.34%(DEC).2、No mortality or abnormality in general behavior, respiratory pattern, motility, reflex or fur of mouse was observed in the acute toxicity test; oral MTDs in KM mice were 15.0 g·kg-1 for WEC, EEC, BEC, and 11.3 g·kg-1 bodyweight (b.wt.) for DEC respectively (According to the crude drug, the MTD of WEC, EEC, BEC, DEC in turn were 115.4 g·kg-1、65.2 g·kg-1、862.1 g·kg-1、283.2 g·kg-1). In both acute toxicity and sub-acute toxicity studies, ascending trend of the bodyweight gains were observed in all groups. In the sub-acute toxicity study, no significant variance in hematological and biochemical parameters as well as in pathologic examinations in the treated rats, at both ends of treatment period and recovery period, was observed when compared with the control. While notable increase of the relative organ weight in kidneys (P< 0.05) in female rats administered with middle dose of WEC or middle and high dose of EEC, and significant decrease of the relative organ weight in testes and epididymis (P< 0.01 and P< 0.001) in male rats dosed with three doses of EEC were observed, but these changes became not significant at the end of recovery period, and did not accompany with toxicological changes in hematological, blood biochemical parameters and pathological lesions.ConelusionFour of the tested extracts, either prepared by direct extraction from C. cajan leaves with water or ethanol, or prepared by fractionation of the ethanol extract of C. cajan leaves with dichloromethane or n-butanol, did not show oral acute toxicity in mice at the tested doses. Oral administration of WEC or EEC for 4 weeks at the tested doses did not cause remarkable abnormal changes in SD rats in general behavior, bodyweight, feed intake, hematological and blood biochemical parameters, and pathological lesions. Interestingly, significant increases in relative kidney weight in female rats dosed with WEC or EEC, and notable decrease in relative testes and epididymis weight in male rats dosed with EEC were observed. These alterations became not significant at the end of recovery period, and did not accompany with abnormal changes in hematological, clinical biochemical markers and pathological lesions. WEC or EEC did not show obvious oral sub-acute toxicity in SD rats under this experimental condition.