菟丝子不同炮制品化学成分和补肾阳作用的比较研究

【作者】 张颖；

【导师】 李向日；

【作者基本信息】 北京中医药大学 ， 中药炮制学， 2018， 硕士

【摘要】 菟丝子为旋花科植物南方菟丝子Cuscuta australis R.Br.或菟丝子Cuscuta chinensis Lam.的干燥成熟种子。始载于《神农本草经》,被列为上品。味甘、辛,性平,是常用的补益中药,具有补肝肾、固精缩尿、益精明目、止泻、安胎等功效。菟丝子炮制方法首载于晋代葛洪著的《肘后备急方》:“酒浸服”,菟丝子盐炙始见于宋代《圣济总录》:“入盐少许炒,乘热捣末”。菟丝子还有许多其他炮制方法,如:清炒、盐水拌蒸、黄精汁制、米泔制等。《中国药典》(2015版)收载的菟丝子炮制品规格为生菟丝子和盐菟丝子,福建、黑龙江、重庆、云南、陕西等省的炮制规范收载了酒菟丝子。中医理论认为:肾主生殖,菟丝子生品、酒炙品、盐炙品皆具有补肾作用,但其补肾作用的异同以及其作用机制尚不明确,酒炙和盐炙对菟丝子化学成分的影响也不清楚,因此限制了菟丝子的进一步开发利用。故本课题探讨菟丝子炮制前后的化学成分变化,以揭示菟丝子炮制理论,建立菟丝子炮制品的质量标准,比较研究菟丝子生品、盐炙品和酒炙品补肾作用的异同并探讨其增效机制,为临床应用合理选用炮制品规格提供科学依据。目的:1.分别建立菟丝子生品、盐炙品和酒炙品的特征图谱,确定盐炙品和酒炙品的差异性成分,以期为更好地控制菟丝子炮制品质量提供科学依据。2.探讨菟丝子生品、盐炙品和酒炙品化学成分的变化,对化学成分进行定性和定量分析研究,以找出其中的差异,明确炮制后化学成分发生量变还是质变。3.研究菟丝子生品、盐炙品和酒炙品补肾阳作用的药效差异并探讨其增效机制。方法:1.采用高效液相色谱法建立菟丝子生品、盐炙品、酒炙品的特征图谱。色谱柱:ZORBAXSB-C18(4.6×250mm,5μm);流动相:甲醇-0.4%磷酸梯度洗脱;紫外检测器:检测波长360nm;流速为1.0 mL/min,进样量:15μL,柱温:35℃。2.采用液质联用技术对菟丝子炮制前后的化学成分进行定性及定量研究,以找出其中的差异。a)定性分析:应用LTQ-Orbitrap XL静电场轨道阱组合式高分辨质谱仪,ACQUITY UPLC BEH C18色谱柱(2.1 ×100 mm,1.7μm),乙腈-0.05%甲酸水梯度洗脱,时间为33min,流速为0.2mL/min,柱温:35℃,进样量:2μl,质谱检测器采用电喷雾离子源负离子模式(ESI-),毛细管温度350℃,源电压4kV,分辨率30 000。b)定量分析:应用 Agilent 1290 液相色谱仪(Agilent,美国),Agilent 6460 Triple Quad LC/MS三重串联四级杆质谱仪,Agilent Eclipse Plus C18 色谱柱(2.1 × 100 mm,1.8μm)。流动相乙腈-0.05%甲酸水,流速为0.3mL/min,梯度洗脱。柱温室温。进样量:2μL。采用外标法对金丝桃苷、槲皮素、紫云英苷、山柰酚、异鼠李素、异槲皮苷、木犀草苷、绿原酸、隐绿原酸、异绿原酸A、异绿原酸B、异绿原酸C、阿魏酸、咖啡酸、对香豆酸定量。3.菟丝子生品、盐炙品、酒炙品补肾阳药效的比较研究。以氢化可的松制备肾阳虚大鼠模型,灌胃给药菟丝子生品、盐炙品、酒炙品醇提物和水提物,为期30天。实验结束后进行大鼠脏器系数、肾功指标、性激素指标、精浆生化指标和睾丸组织形态学检测。结果:1.采用高效液相色谱法建立菟丝子生品、盐炙品、酒炙品的特征图谱。生品、盐炙品、酒炙品间有13个共有峰,其中生品中未见峰1、3,盐炙品中未见峰3、12,酒炙品中未见峰4、10。与盐炙品比较,酒炙品消失了 2个成分,同时又增加了 2个成分,分别是消失了峰4、10,增加了峰3、12。由PCA score图可以看出,菟丝子生品、盐炙品和酒炙品能够明显聚为三类,由Loading Bi plot图可找到了盐炙品、酒炙品的差异成分,峰3、12是盐炙品的差异成分,峰4、10是酒炙品的差异成分。2.采用液质联用技术对菟丝子炮制前后的化学成分进行比较研究。结果:共鉴定出45个化学成分,其中包含绿原酸、新绿原酸等20个有机酸类,金丝桃苷、紫云英苷等23个黄酮类,Cuscutoside A、Cuscutoside D等2个木脂素类,其中有6-O-caffeoyl-β-glucose、3-O-(4’-O-Caffeoylglucosyl)quinicacid 等 19 个成分为首次发现,结果显示,菟丝子生品、盐炙品和酒炙品间化学成分未见差别。3.采用液质联用技术对菟丝子生品、盐炙品、酒炙品的化学成分进行含量测定。结果显示:与生品比较,菟丝子盐炙后除阿魏酸外,其余14种成分含量均显著升高;菟丝子酒炙后除绿原酸、异绿原酸B、异绿原酸C、槲皮素含量升高外,其余成分含量均降低,其中绿原酸含量升高但无统计学差异,阿魏酸含量无显著性差异。与酒炙品比较,除槲皮素、阿魏酸外盐炙品中各成分含量均高于酒炙品中各成分的含量。4.菟丝子生品、盐炙品和酒炙品对肾阳虚大鼠生殖机能作用的比较研究:检测结果显示,和正常对照组比较,模型组各指标均具有显著性差异,说明肾阳虚大鼠造模成功;与模型组比较,菟丝子生品、盐炙品和酒炙品组的各项指标均具有显著性差异。药效结果显示:菟丝子生品、盐炙品和酒炙品均能改善肾阳虚大鼠的生殖机能,说明其具有补肾作用。结论:1.建立了菟丝子生品、盐炙品和酒炙品的特征图谱,并得出结论:生品、盐炙品和酒炙品间存在化学成分差异,找到了盐炙品、酒炙品之间的差异成分但差异性成分较少,说明二者间的区别主要不在于成分的种类,而在于含量,这也可能是菟丝子盐炙品和酒炙品功效不同的原因。2.采用液质联用技术对菟丝子炮制前后的化学成分进行比较研究,定性和定量结果表明,菟丝子盐炙能增加黄酮类和有机酸类成分的含量,酒炙菟丝子成分呈降低趋势,而化学成分的种类未见发生质变。3.药效结果显示:菟丝子生品、盐炙品和酒炙品均能改善肾阳虚大鼠的生殖机能,说明其具有补肾、改善生殖机能作用。从药效学结果来看,菟丝子生品、盐炙品改善肾阳虚大鼠的生殖机能主要通过改善肾功能、提高精浆生化指标,酒炙品主要是通过调节性激素水平,且功效为盐炙品>生品>酒炙品。通过对生品、盐炙品和酒炙品中黄酮类、有机酸类及提取物中成分的含量测定可以得知,盐炙品>生品>酒炙品,因此我们推测菟丝子生品、盐炙品和酒炙品中化学成分含量的差异可能是其药效作用不同的主要原因。更多还原

【Abstract】 Semen cuscutae is the dry and mature seed of Cuscuta australis R.Br.or Cuscuta chinensis Lam.which blongs to the Convolvulaceae family.It was first recorded in "Sheng Nong’s Herbal Classic" and listed as top grade.Semen cuscutae is sweet and pungent in taste,which is commonly used in traditional Chinese medicine.It is widely applied to nourishing liver and kiney,treat impotence and seminal emission,prevent miscarriage,and improve eyesight.Wine-processed product(WPP)was the initial processed product of Semen Cuscutae,which was first recorded in Ge Hong’s Handbook of Formulas for Emergencies"(Zhou Hou Bei Ji Fang)in Jin Dynasty of China which was described as "take it after soaking with wine".Salt-processed product(SPP)was first documented in "Comprehensive Recording of Sage-Like Benefit"(Sheng Ji Zong Lu)in North Song Dynasty which was described as"stir-fry with salt,pound into powder while it is hot".There were many other processed methods of semen cuscutae such as plain-frying,braizing after stirring with salt solution,and processing with juice of rhizoma polygonati or rice-water and so on.The crude products(CP)and salt-processed product(SPP)of semen cuscutae was recorded in "Pharmacopoeia of the People’s Republic of China(Edition 2015)".Wine processed product(WPP)was recorded in processing specifications of Fujian,Heilongjiang,Chongqing,Yunnan and Shanxi provinces.In traditional Chinese medicine,it is believed that kidneys control the reproduction.CP,SPP and WPP of semen cuscutae can tonify kidney to protect the reproductive system,however,the mechanism of protection of reproductive system is not clear which restrict the further utilization of semen cuscutae.Therefore,this task is to establish the quality standard of processed products of semen cuscutae.This study will explore the chemical composition changes before and after the processing of semen cuscutae,and reveal the processing theory of semen cuscutae.Moreover this study will take the comprartive study of differences and synergy mechanism of CP,SPP and WPP of semen cuscutae on nourishing kiney fuctions.This task will provide scientific basis for the reasonable specification selection of different processed products of semen cuscutae for clinical application.Objective:1.The purpose of this study was to establish the characteristic chromatogram of CP,SPP and WPP,respectively,and determine the difference ingredients of SPP and WPP.So as to provide a scientific basis for better controling the quality of different processed products of semen cuscutae.2.This study is to explore the chemical composition changes of CP,SPP and WPP.In order to find out the change in quality and quantity,qualitative and quantitative analysis of chemical compositions such as flavonoids and chlorogenic acids will be studied.3.To comprartively study the differences and synergy mechanism of CP,SPP and WPP on nourishing kiney fuctions.Methods:The main research contents and achieved results are as follow:1.To establish the characteristic chromatogram of CP,SPP and WPP with HPLC.The samples were separated on a ZORBAX SB-C18 column(4.6 mm×250 mm ID,5 μm particle size)with a mobile phase that consisted of methanol(A)and 0.4%phosphoric acid solution(B)at a flow rate of 1.0 mL/min.The injection volume is 15 μL and monitored at 360 nm.The column temperature maintained at 35℃.2.Qualitative and quantitative research on the chemical composition of CP,SPP and WPP was carried out by liquid mass combined technology to find out the difference.a)UPLC-ESI-MSn for qualitative analysisFor LC/MS analysis,an LTQ-Orbitrap mass spectrometer(Thermo Scientific,Bremen,Germany)was connected to the Ultra High Performance Liquid Chromatography instrument via an electrospray ionization(ESI)interface.Samples were analyzed in negative ion mode.The extracts were chromatographically separated on an ACQUITY UPLC BEH C18 column(2.1×100 mm i.d.,1.7μm).The mobile phase consisted of A(acetonitrile)and B(water containing 0.05%formic acid,v/v).The flow rate was 0.20 mL/min.The elution conditions applied with a linear gradient.Source voltage,4kV,capillary temperature of 350℃,resolution set at 30 000.The injection volume is 2 μL.b)UPLC-MRM-MS for quantitative analysisDetection was performed on an Agilent 1290 Agilent 6460 Triple Quad LC/MS(Agilent,American)with an ESI source operating in the negative ion mode.The extracts were chromatographically separated on an Agilent Eclipse Plus C18 column(2.1×100 mm i.d.,1.8μm).The mobile phase consisted of A(acetonitrile)and B(water containing 0.05%formic acid,v/v).The flow rate was 0.30 mL/min.The injection volume is 2μL The elution conditions for chemical content analysis applied with a linear gradient.Column temperature was at room temperature.The external standard method was used to quantify hyperoside,astragalin,quercetin,kaempferol,isorhamnetin,isoquercitrin and luteolin-7-O-glucoside,3-caffeoylquinic acid,4-caffeoylquinic acid,3,4-dicaffeoylquinic acid,3,5-dicaffeoylquinic acid,4,5-dicaffeoylquinic acid,p-hydroxycinnamic acid,caffeic acid and 5-O-feruloylquinic acid.3.Comparative study on efficacy in kidney-yang deficiency rats of CP,SPP and WPP.Kidney-Yang deficiency rats were administered by gastric perfusion with ehanol and water extract for thirty days.After the experiment,the viscera coefficient,renal function index,sex hormone index,seminal plasma biochemical index and tissue morphological detection of testicle were recorded.Results:1.Establish the characteristic chromatogram of CP,SPP and WPP with HPLC.There are thirteen common peaks in CP,SPP and WPP.Peak 1 and 3 were not seen in CP.Peak 3 and 12 were not seen in SPP.Peak 4 and 10 were not seen in WPP.Compared with SPP,WPP increased two components(peak 3 and peak 12)meanwhile decreased two components(peak 4 and peak 10).At PC A score plot,CP,SPP and WPP can divide into three categories because of significant difference.At Loading Bi plot,peak 3 and peak 12 were difference components of SPP.Peak 4 and peak 10 were difference components of WPP.2.Forty-five chemical components were identified,including twenty chlorogenic acids,3-caffeoylquinic acid and 3,4-dicaffeoylquinic acid for instance;twenty-three flavonoids,hyperoside,astragalin for example and two lignanoids such as Cuscutoside A、Cuscutoside D.Significantly,there were 19 of them reporting at the first time such as 6-O-caffeoyl-β-glucose,3-O-(4’-O-Caffeoylglucosyl)quinic acid.As a result,no diference between CP,SPP and WPP was seen.3.UPLC-MRM-MS for quantitative analysis.Compared with CP,the contents in SPP were increased except for 5-O-feruloylquinic.Contents in WPP were decreased except for 3-caffeoylquinic acid,3,4-dicaffeoylquinic acid,4,5-dicaffeoylquinic acid,and quercetin.The content of 3-caffeoylquinic acid increased,however there was no statistical difference.There was no significant difference in 5-O-feruloylquinic acid content.Compared with WPP,the contents in SPP were higher than that in WPP except for quercetin and 5-O-feruloylquinic acid4.Comparative study on efficacy in kidney-yang deficiency rats of CP,SPP and WPP of semen cusuctae.The results showed that,Compared with the control group,each index was increased or decreased significantly in model group which indicate that kidney deficiency rats were successfully made.Compared with the model group,the indexes of CP group,SPP group and WPP group were different significantly.The results showed that CP,SPP and WPP could improve the reproductive fuctions of kidney-yang deficiency rats which indicate that CP,SPP and WPP can tonic kidney.Conclusion:In this study,the characteristic chromatogram of CP,SPP and WPP of semen cuscutae were established,and concluded that there were differences in chemical composition among CP,SPP and WPP.The different contents between the salt-processed products and the wine-processed products were found,however there were no many different contents.It showed that the chemical species were not the main difference between SPP and WPP,but the contents were.This may also be the reason for different efficacy of SPP and WPP of semen cuscutae.Qualitative and quantitative research on the chemical composition of SPP and WPP was carried out by UPLC combined with mass to find out the difference among CP,SPP and WPP.The category of chemical components among CP,SPP and WPP were indistinguishable.At the same time,the contents of chemical components were considerable different.After salt processed,the contents on flavonoids and chlorogenic acid were increased.There is a decreasing trend in WPP.The results of pharmacodynamics showed that the CP,SPP and WPP could improve reproductive fuction of kidney-yang deficiency rats,indicating that they had the function of tonifying the kidney and improving the reproductive function.According to the results of pharmacodynamics,CP and SPP improve the reproductive function of kidney yang deficiency rats were mainly by improving the function of kidney and biochemical index of seminal plasma.WPP was mainly improve the function of the reproductive function of kidney yang deficiency rats through the regulation of sex hormone level.The effect of SPP is stronger than CP.CP is stronger than that of WPP.Through the determination of the contents of flavonoids,chlorogenic acids,and extracts in CP,SPP and WPP,it can be known that the contents of each component in SPP are higher than those in CP,while the contents in CP are higher than those in WPP.Therefore,we speculated that the differences on chemical composition in CP,SPP and WPP may be the main reasons for the different effects of pharmacodynamics.更多还原