Preparation Of Total Lignans From Vitex Negundo Seeds And Its Anti-Rheumatoid Arthritis Activity

Objective This study was designed to investigate preparation technics of total lignans from Vitex negundo seeds and evaluate its anti-rheumatoid arthritis activity and acute toxicity in vivo.Methods1. Taking the extraction capacity of total lignans as evaluation indicator, the extraction technics of Vitex negundo seeds was optimized by orthogonal experimental design based on the single factor test.2. Taking the purity and transfer rate of total lignans as evaluation indicator, the purification process of total lignans from Vitex negundo seeds based on the combination of polyamide and macroporous resin was studied.3. The mice were intragastricly administered with total lignans from Vitex negundo seeds in maximum dosage in acutetoxicity test. The clinical symptoms were recorded for 14 days after the mice were given the maximum dosage including body weight, activity, spirit, appetite, urine, excrement, hair color and death.4. The collagen type II-induced RA rat model was used to investigate the anti-RA activity of total lignans from Vitex negundo seeds.5. Xylene-induced ear oedema was used to assess anti-inflammatory activity of total lignans from Vitex negundo seeds and analgesic activity was evaluated by acetic acid-induced abdominal writhing test.6. HPLC was used to determinate the content of VNL in fruits of four vitex species.Results1. The optimum extraction technology was finally determined as follows: 70% ethanol concentration, 10 times of solvent usage, and extracted twice each for 60 min at the temperature of 75?.2. D101 macroporous resin combined with Polyamide can be used to purify total lignans from Vitex negundo seeds. The optimum technology conditions of polyamide were: 1.5:1 of extract solution to polyamide ratio(g/g) to be evenly blended and 2:1 of extract solution to polyamide ratio(g/g) as the resin dosage. eluting it with 7 BV distilled water,then eluting by 35% ethanol,collecting 6 BV as the eluent volume. The optimal technological conditions were found in D101 as follows: the concentration of sample solution 1.5 mg/m L, maximum sample quantity 11 m L to 1 m L resin, eluting it with 8BV distilled water, then eluting by 35% ethanol,collecting 8BV as the eluent volume. Then the eluent volume was extracted with ethyl acetate. The percentage content of lignans in the purified sample reached 51.4%, transfer rate was 63.4% and the yield of total lignans was 2.0‰.3. No animal died and the mice had not abnormal response and pathological changes in autopsy observation compared to the control group in the maximum dosage. The maximal tolerance dosage was 16 g?kg-1(equivalent of 8000 g material?kg-1), which was 6400-fold the dosage normally used for human body intake.4. 40 mg/kg and 20 mg/kg groups of total lignans from Vitex negundo seeds had an obvious inhibitory activity to collagen type II-induced arthritis in rats, which can significantly inhibite the paw edewa and decrease the arthritis score and also can decrease the levels of IL-1?, IL-6, L-8?IL-17 A and TNF-?. MMP-3, MMP-9, COX-2 and INOS protein expression and the phosphorylation of key enzymes Ik B and PDPK were inhibitied in total lignans gourps.While can increase the level of IL-10. Especially, the high dose(40 mg/kg) was well tolerated during animals experiment.5. Total lignans from Vitex negundo seeds at dosages of 40, 20 and 10 mg/kg significantly reduced xylene-induced ear oedema by 42.42%(p<0.01), 30.27%( p<0.01), 22.16%( p<0.05), respectively. Total lignans from Vitex negundo seeds significantly reduced acetic acid-induced writhing at the dose of 40 mg/kg by 35.63%(p<0.05) and 20 mg/kg by 32.23%(p<0.05).6. The variation of VNL content in four species was observed and its content in V. negundo and V. trifolia was higher than the other two species.Conclusion It was concluded that the preparation technics of total lignans from Vitex negundo seeds established in this thesis can successfully enrich lignans from Vitex negundo seeds and was stable, reliable, less loss of effective components, high impurity removal rate. Total lignans purified from Vitex negundo seeds had significant anti-RA activity and had notable analgesic and anti-inflammatory effects. The acute toxicity test proved the total lignans from Vitex negundo seeds were safer. The animal experiment results showed total lignans from Vitex negundo seeds possess a wide prospect of a safe and effective anti-RA drug.