摘要
目的建立双波长高效液相法(High performance liquid chromatography,HPLC)比较研究山萸肉酒制前后配伍入六味地黄汤(以下分别简称六(山),六(酒))中7种药效成分:莫诺苷、马钱苷、獐牙菜苷、芍药苷、没食子酸、5-羟甲基糠醛以及丹皮酚的含量变化;探究六(山)、六(酒)干预绝经后骨质疏松症模型大鼠去卵巢术后90天的作用及其机制。方法①采用双波长HPLC法测定山萸肉酒制前后配伍入六味地黄汤中七种药效成分的含量。色谱条件:Hypersil C18色谱(4.6 mm×250 mm,5μm),流动相乙腈-0.3%磷酸,梯度洗脱(0-7 min,0%-8%乙腈;7-15 min,8%-10%;15-20 min,10%-15%;20-30 min,15%-23%;30-50 min,23%-45%),流速0.6 mL·min^(-1),检测波长:240 nm(莫诺苷、马钱苷、獐芽菜苷、芍药苷),274 nm(没食子酸、5-羟甲基糠醛、丹皮酚),柱温30℃,进样量20μL。②6-8月龄SD大鼠,分假手术组、模型组、阳性药物组、山萸肉配伍入六味地黄汤组(六(山)组)和酒萸肉配伍入六味地黄汤组(六(酒)组),去卵巢术后第5天开始给药,每周称重,并于术后90天取各组大鼠5只,称重后麻醉,取血清,双侧股骨,并取脏器(子宫、脾、肝、双肾以及心脏)称重。分别采用全自动生化仪以及化学发光微粒子免疫检测法检测大鼠血清钙(Calcium,Ca)、磷(Phosphorus,P)水平和雌二醇(Estradiol,E2)水平;双能X线骨密度仪测定大鼠骨密度(Bone mineral density,BMD)值;HE染色法观察大鼠骨组织形态学变化。结果①山萸肉酒制前后配伍入六味地黄汤中均含有上述7种药效成分,且六(酒)中7种药效成分总含量高于六(山)(P<0.01)。其中,六(酒)中没食子酸、5-羟甲基糠醛高于六(山)(P<0.01),而莫诺苷含量则较六(山)低(P<0.01)。②与假手术组比较:模型组大鼠血清中Ca、P、E2水平及子宫指数降低(P<0.01),BMD值降低(P<0.01),骨组织结构损害程度加深,表现为骨髓腔增大、脂肪细胞逐渐增多,骨小梁排列稀释,骨小梁数量变少甚至出现断裂。③与模型组比较,六(山)组及六(酒)组大鼠血清中的E2、Ca、P水平及子宫指数均升高(P<0.05),骨组织结构有明显改善,BMD值明显升高(P<0.05);六(酒)组肝脏、脾脏指数升高(P<0.05)。结论山萸肉酒制前后配伍入方六味地黄汤后均含有莫诺苷,马钱苷,獐牙菜苷,芍药苷,没食子酸,5-羟甲基糠醛(5-hydroxymethyl furfural,5-HMF)以及丹皮酚七种药效成分,可有效干预PMOP;其中,酒萸肉入方的六味地黄汤抗骨质疏松作用优于山茱萸入方的六味地黄汤,其机制可能与六(酒)中七种有效成分的总含量高于六(山),该7种成分与成骨细胞、破骨细胞增殖分化密切相关,从而可调节骨代谢失调,提高骨密度,改善PMOP有关。可为2020版药典六味地黄丸选用酒萸肉入方及临床上应用六味地黄汤防治PMOP提供一定的实验依据。
Objective To establish a dual wavelength HPLC method to compare the content changes of seven active components:monoglycoside,loganin,swertin,paeoniflorin,gallic acid,5-hydroxymethyl furfural and paeonol in Liuwei Dihuang Decoction(hereinafter referred to as"Liuweidihuang Decoction")before and after the preparation of Cornus wine;To explore the effect and mechanism of Liu(Shan)and Liu(Jiu)on 90 days after ovariectomy in postmenopausal osteoporosis model rats.Methods①The contents of seven active ingredients in the Cornus wine mixed with Liuwei Dihuang Decoction were determined by dual wavelength HPLC.Chromatographic conditions:hypersil C18 chromatography(4.6 mm×250 mm,5μm),mobile phase acetonitrile-0.3%phosphoric acid,gradient elution(0-7 min,0%-8%acetonitrile;7-15 min,8%-10%;15-20 min,10%-15%;20-30 min,15%-23%;30-50 min,23%-45%),flow rate 0.6 mL·min^(-1),detection wavelength:240 nm(monoglycoside,loganin,swertin,paeoniflorin),274 nm(gallic acid,5-hydroxymethyl furfural,paeonol),column temperature 30℃,injection volume 20μL。②6-8 month old SD rats,divided into sham-operated group,model group,positive drug group,shanshuang flesh with Liuwei Dihuang Decoction group(liu(shan)group)and wine cornelian flesh withLiuwei Dihuang Decoction group(liu(wine)group),dosing started on the 5 d after ovariectomy,weighed weekly,and 5 rats from each group were taken at 90 d after surgery,weighed,anaesthetized,serum taken,bilateral femurs,and organs(uterus,spleen,liver,both kidneys and heart)were weighed.Serum calcium(Ca),phosphorus(P)and estradiol(E2)levels were measured using a fully automated biochemical instrument and chemiluminescent microparticle immunoassay,respectively;bone mineral density(BMD)values were measured using a dual-energy X-ray bone densitometer.HE staining was used to observe the morphological changes of bone tissue in rats.Results①All the seven potent ingredients mentioned above were contained in the Liuwei Dihuang Decoction before and after the wine preparation of Cornu Cervi Pantotrichum,and the total content of the seven potent ingredients in liu(wine)was higher than that in liu(shan)(P<0.01).Among them,gallic acid and 5-hydroxymethylfurfural were higher in Liu(Jiu)than in Liu(Shan)(P<0.01),while the content of monosidine was lower than in Liu(Shan)(P<0.01).②Comparison with the sham-operated group:90 d after surgery,the serum Ca,P,E2 levels and uterine index of rats in the model group decreased(P<0.01),the BMD value decreased(P<0.01),and the degree of structural damage to bone tissue The degree of damage to the bone tissue structure was deepened,manifested by enlargement of the bone marrow cavity,gradual increase of adipocytes,dilution of the bone trabeculae arrangement,reduction of the number of bone trabeculae or even fracture.③Compared with the model group,90 d after surgery,the serum E2,Ca,P levels and uterine index of the rats in the liu(shan)and liu(wine)groups increased(P<0.05),the bone tissue structure of the liu(shan)and liu(wine)groups improved significantly,and the BMD value increased significantly(The liver and spleen indices of the liu(wine)groups increased(P<0.05).Conclusion The combination of the wine preparation of Cornus officinalis and its use in the formula of LiuWei Di Huang Decoction both contain monosidine,strychnine,sweretin,paeoniflorin,gallic acid,5-hydroxymethyl furfural(5-HMF)and tannin,and can effectively interfere with PMOP.Among them,the antiosteoporotic effect of Liu Wei Di Huang Tang,which was formulated with wine and dogwood,was better than that of Liu Wei Di Huang Tang,which was formulated with Cornus officinalis,and the former contained more 5-HMF and gallic acid than the latter,which may provide some experimental basis for the selection of wine and dogwood in the 2020 edition of the Pharmacopoeia and the clinical application of Liu Wei Di Huang Tang in the prevention and treatment of PMOP.
作者
文枝
于慧
龙琼
谭佳颖
黄莉
肖望重
戴冰
Wen Zhi;Yu Hui;Long Qiong;Tan Jiaying;Huang Li;Xiao Wangzhong;Dai Bing(The First Hospital of Hunan University of Chinese Medicine,Changsha 410007,China;Qi Yang Hospital of Traditional Chinese Medicine,Yongzhou 426100,China)
出处
《世界科学技术-中医药现代化》
CSCD
北大核心
2023年第8期2717-2725,共9页
Modernization of Traditional Chinese Medicine and Materia Medica-World Science and Technology
基金
湖南省中医药管理局指导课题(E2022008):基于“肾藏精”理论探讨骨组织中“GPER介导的雌激素效应”削弱所致的PMOP发病机制及六味地黄汤的干预研究,负责人:戴冰
湖南省教育厅重点课题(21A0227):“肾藏精”理论探讨骨组织中“GPER介导的雌激素效应”削弱所致的PMOP发病机制及六味地黄汤的干预研究,负责人:戴冰
关键词
六味地黄汤
绝经后骨质疏松症
山萸肉
酒萸肉
HPLC
Liuwei Dihuang decoction
Postmenopausal osteoporosis
Cornus officinalis
Wine cornel meat
HPLC
作者简介
通讯作者:戴冰,主任药师,博士生导师,主要研究方向:临床中药学相关研究
