| Chuanxiong(Szechwan Lovage Rhizome,the rhizome of Ligusticum chuanxiong Hort)is a widely used Chinese herb to activate blood and dissolve stasis.In the traditional identification experience,"a large,full,solid solid,yellow section of the yellow,oily,aroma thick" is better.The quality of Chuanxiong was assessed by the ethanol extract(12%)and ferulic acid content(0.1%)in the Chinese Pharmacopoeia(2015).Obviously,these two indicators are difficult to effectively assess its quality.Modern studies have shown that antiplatelet aggregation can reflect the efficacy of Chuanxiong blood circulation.The main efficacy components of Chuanxiong(coniferyl ferulate,Z-ligustilide,Senkyunolide A,etc.)is most of the oil,unstable,difficult to standardize.In this paper,we developed a quantitative analysis of multi-components by single-marker(QAMS)method by Levistolide A as the internal reference and a bioassay method for the antiplatelet aggregation effect,and compared the pharmacological compotents and antiplatelet aggregation activities of Ligusticum chuanxiong.provided reference for quality assessment research.This paper includes the following three parts:First,the quantifitive determination of anti-platelet aggregation activity in chuanxiong:(1)Developed the quantitative determination of anti-platelet aggregation activity of Chuanxiong by study various factors influencing the antiplatelet aggregation activity in vitro,namely blood after 800 rpm two centrifugation(10 min each time)by platelet rich plasma(PRP),3500 rpm centrifugal platelet poor plasma(PPP),adding 300 ?mol/L ADP induced platelet aggregation,the sample dissolved into 15% solvent two dimethyl sulfoxide(DMSO),the whole experiment from taking blood to the end was completed within 3 h,selection of sodium ferulate as positive drug,in 15 ~ 60 mg/m L concentration range and platelet inhibition the rate showed a good linear relationship(r = 0.9934,n = 4).The water extract of the test solution,extracting 4 times,according to the quantity response parallel line method(2.2)method,to determine the number of experiments at least 4 times,the reliability testing(regression P < 0.01,P > 0.05 from parallel)to meet the requirements of Pharmacopoeia,reproducibility of the experimental value of RSD was 3.43%(n = 6)the precision of the experiment,the RSD value was 6.98%(n = 6),the rate of confidence limit is 23.90%(n = 6)< 30%,indicating that the method is accurate,good repeatability.(2)From different cultivation and the medicine market,pieces of companies and pharmacies respectively collected 13 samples of chuanxiong material,2 samples of chuanxiong decoction pieces,2 samples of chuanxiong related Chinese medicine(Suxiaojiuxin Pills and Le Mai Granule).The results show that chuanxiong material,decoction pieces and related Chinese medicine were 0.957 U/g(n = 13),0.713 U/g(n = 2),0.646 U/g(n = 2),respectively.That method can be used for the determination of chuanxiong materials,pieces and the relevant Chinese medicine established quantitative determination method of antiplatelet aggregation activity and platelet aggregation activity.(3)According to the section of Chuanxiong smell,color,10 copies of 13 samples of chuanxiong in three grade.One grade is rich smell,yellow white section;two grade is rich smell,and yellow section;three grade light smell and brown section.And assays its antiplatelet aggregation activity.One,two,three grade such as anti platelet aggregation activity were 1.306 U/g(n = 4),0.914 U/g(n = 3),0.722 U/g(n = 3),respectively.The results were consistent with the classification of the odor and the color of the cross section,indicating that the anti platelet aggregation activity was strongly correlated with the odor and color of chuanxiong.In second,a determination of 7 bioactive components of chuanxiong by QAMS:(1)Through the optimization of chromatographic conditions and QAMS method related influence factors,to establish a method for determination of ferulic acid,senkyunolide I,senkyunolide H,coniferyl ferulate,senkyunolide A,Z-ligustilide and Levistolide A a total of 7 components by Levistolide A as the internal reference,relative correction factor of levisticum lactone A and the other 6 components were 1.14,1.46,1.44,0.54,0.30,0.81,respectively.The relative correction factors of 3 chromatographic systems,2 chromatographic columns,3 kinds of flow rates and 3 column temperatures were determined,and the RSD values were less than 5%.42 samples of chuanxiong by determination of 6 active components of external standard method and internal standard method(ferulic acid,senkyunolide I,senkyunolide H,coniferylferulate,senkyunolide A,Z-ligustilide content),RSD values were less than 5%,show that the content of a test can accurately determine the 7 for active components in chuanxiong.(2)Chuanxiong material samples were collected from different cultivative areas and medicinal materials markets.The samples were divided into three grades according to the smell and color.First,section two aroma white;product aroma,section yellow;three,smell lighter section yellow brown or brown,appeared bleeding phenomenon.The contents of 7 components were determined.According to the comprehensive score of the results of principal component analysis,there is no significant difference between the one or two grades(P > 0.05),there was a significant difference between the one or two grades and three grade(P < 0.01),and one grade(-15.192,n = 7),two grade(-17.290,n = 25 > three grade(-7.062,n = 10);Cluster analysis can be three grades and one or two grades of one or two separate,but such samples cannot be separated;the content of phenolic acids,phthalide compounds,total phenolic acids and phthalide compounds as indicators,there is no significant difference between among the one or two products(P > 0.05),there was a significant difference between the one or two products and three products(P < 0.01).And one or two grades > three grades;the content of single medicinal components as index,the content of Z-ligustilide was positively correlated with different levels of chuanxiong,there was a significant difference between the one or two and three items(P < 0.01),the first grade(13.492 mg/g,n = 7)> two products(9.899 mg/g,n = 25)> three products(5.818 mg/g,n = 9).(3)Comparison of chuanxiong material in different cultivations: the samples of chuanxiong material in Dujiangyan(7),Pengzhou(13),Meishan(11)were respectively collected in Sichuan,and the contents of the 7 components were determined.According to the comprehensive score of the results of principal component analysis,Sichuan Pengzhou(6.11,n = 13)> Sichuan Meishan(5.44,n = 11)> Sichuan Dujiangyan(5.28,n = 7);the results of variance analysis showed that there is no difference between Sichuan and Meishan Sichuan Dujiangyan(P > 0.05),there are differences between Sichuan Meishan,Sichuan,Dujiangyan and Pengzhou(P < 0.01),namely Sichuan Pengzhou Sichuan Dujiangyan Meishan and Sichuan.There was no significant difference in the content of phenolic acids,phthalide compounds,total phenolic acids and phthalide compounds in the 3 areas(P > 0.05).As a single active ingredient content as the index,the content of Z-ligustilide,there is no significant difference between Sichuan and Dujiangyan Sichuan Pengzhou(P > 0.05),there was a significant difference between Sichuan Dujiangyan and Sichuan Pengzhou,Sichuan Meishan(P < 0.01);the content of senkyunolide A,there was no significant difference between Sichuan and Pengzhou Sichuan and Meishan(P > 0.05),there was a significant difference between Sichuan Pengzhou,Sichuan Meishan and Sichuan Dujiangyan(P < 0.01),no difference between the content of effective components of the remaining(P > 0.05).It indicates that the content of Z-ligustilide and senkyunolide A are different in chuanxiong material.Third,study on the correlation between chuanxiong bioactive components and antiplatelet aggregation activity: in order to study the correlation of chuanxiong bioactive components and antiplatelet aggregation activity,(1)The establishment fingerprint of different concentrations ethanol(100%,70%,50%,30%,0)of chuanxiong extract,according to a 9 peak retention time and UV absorption control the identification of ferulic acid,senkyunolide I,senkyunolide H,coniferylferulate,senkyunolide A,E-ligustilide,Z-ligustilide and butylidenephthalide,Levistolide A.The grey correlation analysis,the correlation coefficient of 9 active components and antiplatelet aggregation activity from large to small in turn is 0.6492(E-ligustilide),0.6316(senkyunolide H),0.6198(ferulic acid),0.6163(senkyunolide I),0.6101(coniferylferulate 0.6071(senkyunolide A),0.5588(butylidenephthalide),0.5703(Z-ligustilide),0.5230(Levistolide A),indicating that the 9 bioactive components and antiplatelet aggregation activity correlation are similar.Total phthalides and phenolic acids,phenolic acids,phthalide compounds with antiplatelet aggregation activity and correlation coefficient from big to small in turn 0.6105(phthalide compounds),0.6099(phenolic acids),0.6098(total phenolic acids and phthalide components),indicating phenolic acids,phthalide class composition,phenolic acids and phthalide compounds and antiplatelet aggregation activity correlation are also similar.(2)Determination of bioactive components of chuanxiong in water extracts(ferulic acid,senkyunolide I,senkyunolide H,senkyunolide A)in biological potency aggregation activities of medicinal components in water extract of different grades of Rhizoma Chuanxiong were estimated with antiplatelet curve estimation,only the ferulic acid and chuanxiong the water extract of the antiplatelet aggregation activity curve by t test shows a S shape change;the grey correlation analysis,the correlation coefficient of water extract of Chuanxiong medicinal ingredients and anti platelet aggregation activity in descending order of 0.6452(A Weisuan),0.6316(senkyunolide I),0.6246(senkyunolide H),0.5375,(senkyunolide A),shows that the water extract of chuanxiong and 4 bioactive constituents and antiplatelet aggregation activity correlation are similar.The above results suggest that the antiplatelet aggregation effect of chuanxiong is multi-effect by various compotents. |
PDF Full Text Request