钩藤总碱的提取工艺和急性毒性及其对人脐静脉内皮细胞的保护作用

Extraction Process of Total Alkaloids of Uncaria Rhynchophylla, and Its Acute Toxicity and Protective Effect on HUVEC

关键词:  钩藤  钩藤总碱  正交试验  主成分分析  提取工艺优化  急性毒性  人脐静脉内皮细胞

Key words:  Uncaria rhynchophylla  Uncaria rhynchophylla total alkaloids  orthogonal test  principal component analysis  optimization of extraction process  acute toxicity  HUVEC

摘要:

目的 利用正交试验优选钩藤最佳提取工艺,研究钩藤总碱对小鼠急性毒性和对过氧化氢(H₂O₂)诱导损伤的人脐静脉内皮细胞(human umbilical vein endothelial cells,HUVEC)的保护作用。方法 采用L₉(3⁴)正交试验法,选择乙醇浓度、提取次数、提取时间、物料比为考察因素,用HPLC测定异钩藤碱、钩藤碱、去氢毛钩藤碱和毛钩藤碱含量并计算转移率,以主成分分析综合得分为指标,优选钩藤总碱的最佳提取工艺。选用KM小鼠,灌胃钩藤总碱溶液,进行最大耐受量(maximum tolerated dose,MTD)的测定,观察14 d内小鼠情况变化,后摘眼球取血,测定小鼠血清中的尿素氮(blood urea nitrogen,BUN)、谷丙转氨酶(glutamic-pyruvic transaminase,GPT)、谷草转氨酶(glutamic-oxaloacetic transaminase,GOT)含量变化。测定钩藤总碱对1.0 mmol·L^-1 H₂O₂诱导损伤的HUVEC存活率和上清液乳酸脱氢酶(lactate dehydrogenase,LDH)、内皮素(endothelin-1,ET-1)、一氧化氮(nitric oxide,NO)含量的影响。结果 优选的钩藤总碱最佳提取工艺为用70%乙醇加热回流提取2次,每次2 h,物料比1:8。急性毒性试验表明,给药后14 d内,各组小鼠活动正常、无明显不良反应、无死亡;钩藤总碱溶液对小鼠的MTD为21.0 g·kg^-1·d^-1,相当于临床成人日用量的1 377倍;与空白组比较,钩藤总碱组GPT显著降低(P<0.01)。与空白组比较,模型组HUVEC存活率及上清液中NO含量显著降低(P<0.01或P<0.05),LDH、ET-1含量显著升高(P<0.01);与模型组比较,钩藤总碱5 μg·mL^-1细胞存活率显著升高(P<0.01),LDH、ET-1含量显著降低(P<0.01或P<0.05)。结论 优选的钩藤总碱提取工艺简单可行,钩藤总碱对小鼠急性毒性较小,对HUVEC损伤有保护作用。

Abstract:

OBJECTIVE To optimize the optimal extraction process of Uncaria rhynchophylla by orthogonal experiment, to study the acute toxicity of Uncaria rhynchophylla total alkaloids to mice and protective effect on H₂O₂-induced injury of human umbilical vein endothelial cells(HUVEC). METHODS The L₉(3⁴) orthogonal test method was used, ethanol concentration, extraction times, extraction time, and material ratio were selected as the factors to be investigated, the contents of isorhynchophylline, rhynchophylline, hirsuteine and hirsutine were determined by HPLC, the transfer ratios were calculated, and the comprehensive score of principal component analysis was taken as the index to optimize the extraction process of Uncaria rhynchophylla total alkaloids. KM mice were selected. After intragastric administration of total alkaloids solution of Uncaria rhynchophylla for twice, maximum tolerated dose(MTD) was calculated, the changes of mice within 14 d were observed. The eyeballs were removed to collect blood. The contents of urea nitrogen(BUN), glutamic-pyruvic transaminase(GPT) and glutamic- oxaloacetic transaminase(GOT) in the serum of mice were determined. HUVECs were selected and damaged by 1.0 mmol·L^-1 H₂O₂ to observe the protective effects of total alkaloids of Uncaria rhynchophylla. The survival rate of HUVECs and the contents of lactate dehydrogenase(LDH), endothelin-1(ET-1), nitric oxide(NO) in the supernatant were measured. RESULTS The optimum paratemeters of Uncaria rhynchophylla total alkaloids extraction process were as following:Uncaria rhynchophylla was extracted by 70% ethanol for twice, each time for 2 h, and the material ratio was 1:8. Within 14 d of the acute toxicity test, the mice's activity was normal, no obvious adverse reactions were observed, and no death after administration; the MTD was 21.0 g·kg^-1·d^-1, which was equivalent to the daily dose of clinical adults 1 377 times. Compared with blank group, GPT in Uncaria rhynchophylla total alkaloids group was significantly decreased(P<0.01). Compared with the blank group, the survival rate of HUVEC and the NO content in the supernatant of the model group were significantly decreased(P<0.01 or P<0.05), while the LDH and ET-1 contents were significantly increased(P<0.01); compared with the model group, the cell viability of Uncaria rhynchophylla total alkaloids(5 μg·mL^-1) was significantly increased(P<0.01), and the content of LDH and ET-1 in the supernatant was significantly decreased(P<0.01 or P<0.05). CONCLUSION The optimized extraction process of total alkaloids of Uncaria rhynchophylla is simple and feasible, total alkaloids of Uncaria rhynchophylla has less acute toxicity in mice, and has a protective effect on HUVECs damage.