| 1Objective In this study,the determination of key saponins and HPLC fingerprints were used to provide the experimental basis for the quality assurance of crude materials which is from different origin and grades of Panax notoginseng(Burk.).PNR was prepared according to Chinese Pharmacopoeia with the raw material,and then according to the pharmacological actions with few reports in the literature but with some relevant applications of Panax notoginseng in the Clinical Chinese Medicine,through the establishment of the rat models separately with chronic liver injury induced by CCl4,researching on hepatic microsomal drug-metabolizing enzyme activity,antioxidant enzyme activity and inhibition of inflammatory factors by PNR,and observing the Pathological changes,in order to confirm the protective effects of PNR on chronic liver injury;through gastrointestinal intestinal water loaded,regulating the metabolism of urine,in order to explore the diuretic action of PNR and the primary mechanism;and with the methods of intestinal motility and pepsin activity test,further studying on gastrointestinal function.Furthermore,to evaluate the mechanism of PNR in the light of the treatment of hyperlipidemia and the health protection function of reducing blood lipids,through a hyperlipidemia model established by using high fat diet.To provide scientific basis for clinical application and preventive treatment of disease of PNR.2Methods 2.1 Study on quality evaluation of PNR The determination of key saponins:In accordance with the content determination method from the Chinese Pharmacopoeia(Part one),the choice of the real estate areas of 20 heads,30 heads,40 heads,60 heads and 80 heads from Wenshan,Qujing and Honghe in Yunnan,120 heads and numerous heads from Wuzhou,Jingxi and Napo in Guangxi,using the PNR processed by them as the research object,carrying on the preliminary comparison,not only the content of total saponins was analysized in PNR notoginsenoside R1,ginsenoside Rg1 and ginsenoside Rb1 determined,what's more,the content of R1 which as one of the main monomer saponins in roots of Panax notoginseng was also evaluated as the key component.The determination of PNR fingerprint:Samples of PNR were made by different origins and different grades were selected about 20 heads,30 heads,40 heads,60 heads and 80 heads separately from Wenshan,Honghe and Qujing in Yunnan Province,and 80 heads,120 heads and numerous heads separately from Wuzhou,Jingxi in Guangxi Province.The fingerprint detection conditions were determined through method of exploration about the detector(UVD and ELSD),column(C8 and C18 column)and gradient elution conditions,and the methodology study.Study on the quality of PNR made by the raw materials from different origins and grades,the common mode of fingerprint in 20 batches of samples were established,not only in order to campare overall differences,but also the characteristic peaks of our common peak identification and the differences of peak area;Furthermore,through the analysis of HPLC/MS,according to the reference samples and the literature of common peak,to do the identification of the common peak components.2.2 Study on pharmacological effects of PNR on protecting liver,diuresis and regulating gastrointestinal tract function The rat model of chronic liver injury was adopted.The rats are given high,medium and low doses of PNR by gavage administration q.d.in the course of modeling.These indexes were determined on the last day of administration including ALT,AST,IL-1,IL-6,IL-8,and TNF-? in the serum of rats in each group,MDA,SOD,GSH,and GSH-Px in liver,and liver enzyme Cyt.P450 and Cyt.b5.The pathological changes of rats' livers were observed by routine paraffin sections and HE staining.Additionally,the pre-stomach water loaded rat model was established,after 20 mins the rats were given with high,medium and low doses of PNR by gavage administration.And then recording the urine volume of rats for 6 h,the urine p H and the concentration of Na+,K+ and Cl-in the methods of metabolic cage and weighing.Furthermore,in order to observe the effect of PNR on promoting digestion in Chinese medicine,through testing intestinal peristalsis and the activity of pepsin.The appropriate volume of carbon powder were given by gavage to the ICR mices with high,medium and low doses of PNR by gavage administration after 0.5 h.To record the time interval about the defecation of the first black stool of each mouse,the numbers of black stool within 24 h and the propellant rates of carbon powder.At the same time,to study on the effect of PNR on pepsin activity in ICR mice by testing the pepsins of gastric juice from ICR mice with high,medium and low doses of PNR by gavage administration and normal group with isometric saline after 7 d.2.3 Study on the mechanism of reducing blood lipid by PNR Hyperlipidemia rats,as the experimental model,given high,medium and low doses of PNR with intragastric administration for 8 weeks.Then ALT,AST,TC,TG and LDL-C levels in the serum of them were detected separately;Paraffin sections and HE staining were carried out to the liver tissues of each group to observe its histopathological changes;Gene expressions of LDLR,SIRT1 and LXR-? in model rats were measured with RT-PCR analysis,and protein expressions levels of SREBP2 and SCAP were detected by Western-blotting assay.3Results 3.1 Study on quality evaluation of PNR Results on contents of saponins:(1)Key component R1: The other grades from Yunnan real estate areas of 20 heads and the same level of different regions and different grades of the same origin were determined,in accordance with the method on the determination of notoginsenoside R1 content,which is shown that the content of R1 from Yunnan 20 heads was the highest,30 heads the second,40 and 60 heads similar results;the content of R1 from the 20~60 heads samples was more than 1.15 %.But the 80 heads less than 0.95 %,and 120 and numerous heads much less.The content of R1 in 20 heads was about twice as much as 80 heads,5 times as 120 head and more than 8 times as many heads.The content of R1 in 60 samples was about 4 times as much as 120 heads and 7 times as many heads.Preliminary estimating,samples of Yunnan 20~60 heads PNR were superior to 80~numerous heads.This may be related to the different grades of the source of the medicinal herbs,leading to differences in content.(2)Comparing to the content of total saponins,the content of 20~60 heads was more than 9.95 %,and the content of 30 and 40 heads was similar,but the content of total saponins in 80 heads was less than 9.95 %.Particularly,PNR of 120 and numerous heads were unqualified,due to the content of total saponins less than 5 % from the requirement of Ch.P.It also shows that the quality of the PNR processed by 20~60 heads was superior.The determination results of characteristic spectra: From the overall evaluation of Yunnan region,between the sample similarity is relatively close,with 17 common peaks by visual analysis,including 5 common peaks which existed in the sample from Yunnan but none in Guangxi,at tR = 7.70 min,37.95 min,40.45 min,43.25 min and 47.95 min,thus the samples from Yunnan is high consistent between each other.But the samples of Guangxi with 13 common peaks,including one peak at tR = 33.0~35.0 min which existed in the sample from Guangxi but none in Yunnan by visual analysis.So that it's more different between the two origins;At the same time,in the form of the 20 batches of PNR samples of 12 common peaks in the analysis,it was most among the samples of chromatographic peak areas of Yunnan,and 20~60 heads of them has similar quality in spectrum characteristics;Furthermore,through the control of known peaks it was identified 3 common peaks of R1,ginsenoside Rg1 and Rb1.According to the peak area of common peak,the area of 120~numerous heads was less than 20~80 heads,and much less than 20~60 heads,furthermore the former was ten times as the latter.It's the same conclusion as the foregoing content.Additionally,based on the analysis of the relative retention time,the technical parameters of the characteristic spectrum of 12 common peaks are determined,the common peak number(relative retention time)as follows: 1(0.142);2(0.353);3(0.438);4(0.473);5(0.534);6(0.546);7(0.909);8(0.925);9(0.945);10(1.000);11(1.162);12(1.251).In further HPLC-ESI-MS analysis,it was obtained 8 chromatographic peaks in the spectra information.The remaining 4 peaks cannot obtain mass spectra information,which might be related to their ionization characteristics.Among them,in addition to identify 3 saponins which were written on the standard of Ch.P collection determination about P.notoginseng,No.4 means R1(tR = 26.58 min,[M+HCOO]-,m/z = 977),No.5 means Rg1(tR = 29.99 min,[M+HCOO]-,m/z = 845),No.10 means Rb1(tR = 56.14 min,[M-H]-,m/z = 1107),comparison with the standard fragmentation information,No.6 peak was also identified on ginsenoside Re(tR = 30.59 min,m/z: 991 [M+HCOO]-;945 [M-H]-;799 [M-H-rha]-;637 [M-H-rha-glu]-),and another common peak was preliminary judgment as ginsenoside Rd by literature(tR = 65.12 min,[m/z: 945;783;765;621]).On the whole,compared with other producing areas and grade samples,the samples from Yunnan(20~60 heads)were deemed to be the advantages of the most peaks and the best separation effect.3.2 Study on pharmacological effects of PNR on protecting liver,diuresis and regulating gastrointestinal tract function The result shows that: Compared with the model group,HG could significantly reduce the level of ALT and AST in model rats(P<0.01);MG could only reduce the level of serum ALT in model rats(P<0.05);HG,MG and LG could promote the viability of Cyt.P450 and Cyt.b5 enzymes and enhance the levels of the liver SOD and GSH-Px(P<0.01),and decrease the level of MDA(P<0.01),and have significant inhibition effect on the levels serum IL-1,IL-6,IL-8 and TNF-?(P<0.01).Besides,the result shows that HG could significantly increase the urinary volume in rats from 2 to 5 h after administration.however,hardly any diuretic effects at 6 h.Among them,HG could increase the urine Na+,Cl-discharge,reduces K+ excretion,and inhibit renal tubular reabsorption of Na+ and K+ excretion,so as to increase rat urine p H.In addition,the result shows that HG could shorten the interval of 1st time black stool and raise the carbon powder propelling rates.Likewise,HG and MG could significantly enhance the activities of pepsin in mice.3.3 Study on the mechanism of reducing blood lipid by PNR The results demonstrates that the high(HG)and low dose groups(LG)of PNR significantly dropped the serum LDL-C levels in model rats(P <0.01);the HG and medium dose groups(MG)of PNR significantly reduced the serum TG levels in model rats,and the difference is statistically significant(P <0.01 or P <0.05);the HG,MG and LG could significantly lower the level of serum TC in model rats(P <0.01);serums AST in the medium and low dose groups of PNR are significantly lower than model groups(P <0.01),but HG can't lower serum AST(P >0.05).Histological studies have shown that HG,MG and LG could significantly reduce the liver injury and fatty liver in rats induced by the high-fat diet.The result of molecular level proves that HG,MG and LG could up-regulate the expression of LDLR and SIRT1 in the liver of hyperlipidemia rats and down-regulate the expression of LXR-? gene.At the same time,PNR significantly could reduce the expression of SREBP2 and SCAP protein in liver of hyperlipidemic rats.4Conclusion 4.1 Study on quality evaluation of PNR Through the determination of PNR saponins index components showed that it was relatively higher from 20~60 heads PNR in Yunnan origin than the others on the content of notoginseng R1 and total saponins;80 heads the second.But 120 and numerous heads PNR samples failed in quality.Therefore,80 heads and higher grades should be selected as the raw materials of PNR,and preferably 20~60 heads.What's more,through studying on the fingerprint of PNR by HPLC/MS,it was found that the similarity of all samples from Yunnan areas is closed.Additionally,it was with high quality consistency between different grades of the same origin and the same grade from different planting in Yunnan,but the peaks of all samples between Guangxi and Yunnan existed relatively large difference;especially,chromatographic peaks from Yunnan area sample were much more.And 4 saponins R1,Rg1,Re and Rb1 were identified by contrast of reference characteristics,and a possible monomer ginsenoside Rd by literature.Due to the relative retention time and peak area change trend,it can be analysis to determine that Yunnan 20~60 heads has the advantages of chromatogram and similar quality,80 heads the second,and better than 120~numerous heads.As a result,the whole quality of the PNR from 20~60 heads in Yunnan area was better,which could be selected as the study object of pharmacological effects on reducing blood lipids,protecting liver,diuresis and gastrointestinal tract function,so as to improve the accuracy and reliability of the results.In addition,because the product of PNR commonly using as clinical medicine for many kinds of diseases such as mainly cardiovascular and metabolic system,and adjuvant therapy digestive system,it's suggested that the selected raw material was not less than 80 heads PNR,particularly above the level of 60 heads more reliably.4.2 Study on pharmacological effects of PNR on protecting liver,diuresis and regulating gastrointestinal tract function In the study,it was found that PNR had the pharmacological effects on protecting liver(chronic liver injury),diuresis and regulating gastrointestinal tract function.Firstly,the mechanism of protecting liver might be related as follow,on the one hand,by affecting the metabolism of liver enzymes,enhancing the detoxification function of the liver;On the other hand,by improving the function of antioxidant system in the body,inhibiting the production of lipid peroxidation,apart from these,by inhibiting of excessive expression of inflammatory cytokines in liver diseases.Secondly,the diuretic mechanism might be related to the reabsorption of Na+ and the excretion of K+ in the renal tubule,and the balance of electrolyte and p H in the body.Further studies can judge PNR regulating gastrointestinal function such as promoting intestinal peristalsis and enhancing pepsin activity.4.3 Study on the mechanism of reducing blood lipid by PNR PNR has a significant hypolipidemic effect,and may have a role in improving liver steatosis caused by hyperlipidemia.The positive effect of PNR on reducing blood lipid might be related to the improvement of the liver uptaking LDL-C in the blood circulation through up-regulating the gene expression of hepatic LDLR;More importantly,its effects might be related to the inhibition of cholesterol synthesis,through up-regulating the gene expression of SIRT1,and then influencing the down-regulation of SCAP-SREBP2 signaling pathway;Equally,down-regulating the gene expression of LXR-?,thereby inhibiting the absorption and increasing the degradation and peripheral tissue transport of cholesterol.It's embodied in a dual mechanism of action on inhibition of exogenous lipid absorption and endogenous lipid synthesis.Last but not least,the results provide a scientific basis for the evaluation of PNR in the application of metabolic system,urinary system and digestive system,and also lay the foundation for the development and application of health food containing Panax notoginseng ingredients on the functions of axiliary of reducing blood fat,protecting liver and relaxing bowel. |
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