摘要
目的研究补肾活血方中马钱苷、莫诺苷、芍药苷含量测定方法及其对免疫性卵巢早衰小鼠激素血清卵泡刺激素(follicle-stimulating hormone,FSH)、黄体生成素(luteinizing hormone,LH)、雌二醇(estradiol,E2)水平的影响。方法 (1)采用HPLC,色谱柱Hypersil ODS C18柱(200 mm×4.6 mm,5μm),流动相:乙腈(A)-0.1%磷酸溶液(B)梯度洗脱,柱温30℃,流速为1.0 mL/min,进样量10μL,测定补肾活血方中马钱苷、莫诺苷、芍药苷含量;(2)以小鼠透明带3为抗原,皮下多点注射BALB/c雌性小鼠,建立免疫性卵巢早衰模型,分别用补肾活血方、马钱苷、莫诺苷、芍药苷进行干预,并设空白组、模型组为对照,酶联免疫吸附法测定各组FSH、LH、E2含量。结果 (1)马钱苷、莫诺苷、芍药苷的线性范围分别在58~1 160μg(r=1.000 0)、203~4 070μg(r=0.999 8)及117~2 340μg(r=0.999 4)内,线性关系良好;这3种成分的平均加样回收率分别为98.6%、98.0%、98.8%,RSD分别为2.18%、1.28%、1.77%;在补肾活血方中按生药量计算,莫诺苷、马钱苷、芍药苷的平均含量分别为(4.612±0.057) mg/g、(1.286±0.014) mg/g、(3.065±0.022) mg/g。(2)与空白组比较,模型组E2水平显著降低(P<0.01),FSH和LH的水平均明显升高(P<0.01);与模型组比较,补肾活血方组、马钱苷组、莫诺苷组E2水平均显著升高(P<0.01),FSH和LH的水平均显著降低(P<0.01),芍药苷组E2水平显著升高(P<0.01),LH的水平显著降低(P<0.01),FSH水平降低(P<0.05)。结论所建立的HPLC简便、灵敏、准确,可用于补肾活血方中莫诺苷、马钱苷、芍药苷的含量测定;马钱苷、莫诺苷、芍药苷为补肾活血方中可能的药效物质。
Objective To study a quantitative determination method of loganin, morroniside and paeoniflorin, and its effect on the level of follicle-stimulating hormone(FSH), luteinizing hormone(LH), estradiol(E2) in mice with immune premature ovarian failure. Methods HPLC procedure was performed on the chromatographic column of Hypersil ODS C18(200 mm × 4.6 mm, 5 μm)at 30 ℃. The mobile phase was acetonitrile(A)-0.1% phosphoric acid solution(B) in gradient elution at flow rate of 1.0 mL/min and sample size was 10 μL to determine the content of loganin, morroniside and paeoniflorin in Bushen Huoxue Formula;Mice ZP3 as antigen was injected subcutaneously into Balb/c female mice to establish a model of immune premature ovarian failure. Bushen Huoxue Formula, loganin, morroniside and paeoniflorin were used for the intervention, and the blank group and the model group were set as the control group. Enzyme-linked immunosorbent assay was used to determine the contents of FSH, LH, E2. Results The linear ranges of loganin, morroniside and paeoniflorin were 58~1 160 μg(r=1.000 0), 203~4 070 μg(r=0.999 8) and 117~2 340 μg(r=0.999 4), respectively, and the linear relationship was good.. The average recoveries of the 3 components were 98.6%(RSD=2.18%),98.0%(RSD=1.28%), 98.8%(RSD=1.77%);the average contents of morroniside, loganin and paeoniflorin were(4.612±0.057),(1.286±0.014) and(3.065 ±0.022) mg/g, respectively based on the crude drug quantity in Bushen Huoxue Formula;Compared with the blank group, the E2 level in the model group was significantly decreased(P<0.01), while the FSH and LH levels were significantly increased(P<0.01). Compared with the model group, the E2 level of the Bushen Huoxue Formula group was significantly increased(P<0.01), and the levels of FSH and LH were significantly decreased(P<0.01). The E2 level of the paeoniflorin group was significantly increased(P<0.01). The level of LH was significantly decreased(P<0.01), and the level of FSH was significantly decreased(P<0.05).Conclusion The method is simple, sensitive and accurate, which can be used for the quantitative determination of morroniside,loganin and paeoniflorin in Bushen Huoxue Formula. Loganin, morroniside and paeoniflorin are possible pharmacodynamic substance in Bushen Huoxue Formula.
作者
张韫玉
张思敏
刘平安
黄姗姗
张楚洁
胡金芳
刘慧萍
肖小芹
ZHANG Yunyu;ZHANG Simin;LIU Ping’an;HUANG Shanshan;ZHANG Chujie;HU Jinfang;LIU Huiping;XIAO Xiaoqin(Hunan University of Chinese Medicine,Changsha,Hunan 410208,China;The First Affiliated Hospital of Hunan University of Chinese Medicine,Changsha,Hunan 410007,China)
出处
《湖南中医药大学学报》
CAS
2020年第5期540-545,共6页
Journal of Hunan University of Chinese Medicine
基金
国家自然科学基金项目(81303123)
湖南省自然科学基金项目(2018JJ2299)
湖南省中医药科研计划项目重点项目(201801,201815)
湖南省教育厅创新平台开放基金项目(18K069)
国家级大学生创新项目(201810541001)
湖南省大学生研究性学习与创新性实验项目(S201910541013)
湖南中医药大学中西结合一流学科开放基金(2018ZXYJH24)
湖南中医药大学校级研究生创新课题(2018CX30)
中医方证研究转化医学湖南省重点实验室资助。
关键词
补肾活血方
马钱苷
莫诺苷
芍药苷
高效液相色谱法
含量测定
卵巢早衰
激素水平
Bushen Huoxue Formula
loganin
morroniside
paeoniflorin
HPLC
content determination
premature ovarian failure
hormone level
