| 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. |
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