六味地黄汤水提醇溶部位的成分分析及对小鼠的补肾作用机制研究

Component analysis of ethanol-soluble portion of Liuwei Dihuang Decoction and its function mechanism on kidney deficiency in mice

目的研究分析六味地黄汤水提醇溶部位的主要成分及其补肾作用机制。方法用Hypersile C18(4.6 mm×250 mm,5μm)色谱柱,以甲醇-水(27∶73)为流动相,检测波长为236 nm;流速为1.0 m L·min^(-1);柱温为25℃,用高效液相色谱法测定六味地黄汤水提醇溶部位的主要成分及含量。按照性别和体重将30只小鼠随机分为3组(n=10):正常组、模型组、实验组。实验组按9.75g·kg^(-1)体重灌胃六味地黄汤水提醇溶部位,连续给药9 d;在给药第6天,除正常组外,其他各组按50 mg·kg^(-1)体重灌胃氢化可的松,连续4 d,建立肾阴虚小鼠模型。第10天,用ELISA法测定小鼠血浆环磷酸腺苷(c AMP)、环磷酸鸟苷(c GMP)含量,用放射免疫分析法测定小鼠血清促卵泡素(FSH)、雌二醇(E2)及睾酮(T)含量。结果莫诺苷、獐牙菜苷、芍药苷和马钱苷的线性范围分别在0.24~2.40μg(r=0.999 6)、0.14~1.38μg(r=0.999 1)、0.12~1.20μg(r=0.999 1)及0.24~2.40μg(r=0.999 4)内,线性关系良好;这4种成分的回收率分别为(100.22±1.80)%,(100.83±1.94)%,(102.40±1.47)%,(101.40±1.50)%,RSD分别为1.80%,1.92%,1.44%,1.50%。在六味地黄汤水提醇溶部位中按生药量计算,莫诺苷、獐牙菜苷、芍药苷和马钱苷的平均含量分别为0.20%,0.02%,0.06%,0.13%。给药9 d后,模型组与实验组c AMP含量分别为(8.20±0.63),(6.90±0.15)nmol·L^(-1),差异有统计学意义(P<0.01);模型组与实验组FSH、E2、T含量分别为(0.54±0.10),(0.88±0.04)m U·m L^(-1);(13.93±0.29),(15.48±0.43)pg·m L^(-1);(2.23±0.14),(5.63±0.48)ng·m L^(-1),差异均有统计学意义(均P<0.01)。结论六味地黄汤水提醇溶部位主要含有马钱苷、莫诺苷、獐牙菜苷、芍药苷,其补肾作用机制可能与其调节c AMP含量及HPG轴中各环节激素水平有关。

Objective To analyze the chemical components of the ethanol-soluble portion of Liuwei Dihuang Decoction,and to explore its mechanism in kidney-Yin deficiency of mice. Methods Bioactive components were analyzed by HPLC,the method was used at Hypersile C18column（ 4. 6 mm × 250 mm,5 μm）,the mobile phase consisted of methanol-water（ 27 ∶ 73）,the detective wavelength was 236 nm,the flow rate was 1. 0 m L·min^-1,the column temperature was 25 ℃. The mice were divided randomly into three groups（ n = 10） ： blank group,model group, experimental group. The mice were gavaged with oraladministration of corresponding medicines（ 9. 75 g·kg^-1） for nine weeks. At time 6 d after administration,in addition to that the normal group was given distilled water,the other groups were gavaged with hydrocortisone injection per day according to 50 mg·kg^-1body weight for 4 d to establish the kidney Yin deficiency mice model. The contents of cyclic adenosine monophosphate（ c AMP）,cyclic guanosine monophosphate（ c GMP） were determined by ELISA and the follicle-stimulating hormone（ FSH）,estradiol（ E2）,testosterone（ T） were determined by radioimmunoassay after administration. Results The linear range of morroniside,sweroside,paeoniflorin and loganin were 0. 24-2. 40 μg（ r = 0. 999 6）,0. 14^-1. 38 μg（ r = 0. 999 1）,0. 12^-1. 20 μg（ r = 0. 999 1）,0. 24-2. 40 μg（ r = 0. 999 4）respectively. The recovery and relative standard deviation of them were（ 100. 22 ± 1. 80） %,（ 100. 83 ± 1. 94） %,（ 102. 40 ± 1. 47） %,（ 101. 40 ± 1. 50） %,and RSD were 1. 80%,1. 92%,1. 44%,1. 50%. The average content of morroniside,sweroside,paeoniflorin and loganin in Liuwei Dihuang Decoction water extract-alcohol soluble parts were calculate in terms of material were 0. 20%,0. 02%,0. 06%,0. 13%. The plasma level of c AMP in model group and experimental group were（ 8. 20 ± 0. 63）,（ 6. 90 ± 0. 15） nmol·L^-1with significant different（ P〈0. 01）. The serum contents of FSH,E2,T in model group and experimental group were（ 0. 54 ± 0. 10）,（ 0. 88 ± 0. 04） m U · m L^-1;（ 13. 93 ± 0. 29）,（ 15. 48 ± 0. 43） pg·m L^-1;（ 2. 23 ± 0. 14）,（ 5. 63 ± 0. 48） ng ·m L^-1with significant different（ P〈0. 01）. Conclusion It is found that ethanol-soluble portion might be one of the bioactive component of Liuwei Dihaung Decoction to improve kidney Yin deficiency,and it mainly contains loganin,morroniside,sweroside and paeoniflorin. Its function mechanism of tonifying kidney may be related to the regulation of c AMP content and the levels of hormones in the HPG axis.