| ObjectivesCistanche is a traditional Chinese medicine.According to the 2015 edition of Chinese Pharmacopoeia,Cistanche comes from the dried scaly stem of Cistanche deserticola or Cistanche tubulosa,which has high medicinal value.Due to the shortage of Cistanche resources,Cistanche sinensis is used as the genuine Cistanche in the market.In order to improve the quality control level of Cistanche and its preparation to ensure the safety and effectiveness of clinical drugs,based on the existing literature reports,this study intends to use UPLC method with higher resolution and shorter analysis time to analyze and compare the quality of different species of Cistanche deserticola,Cistanche tubulosa and Cistanche sinensis,to establish the analysis and identification methods of different species,and to explore the rationality of Cistanche sinensis as a substitute of Cistanche deserticola,Cistanche tubulosa.On this basis,the standard decoction of Cistanche deserticola and Cistanche tubulosa decoction pieces were studied to provide reference for the quality control of Cistanche prescription granules.Methods1.UPLC method was used to establish the quantitative analysis methods of four kinds of phenylethanoid glycosides(echinacoside,cistanoside A,acteoside,isoacteoside)in Cistanche deserticola and Cistanche tubulosa,and the difference of the content of the components between Cistanche deserticola and Cistanche tubulosa was compared;2.UPLC method was used to establish the quantitative analysis method of three phenylethanoid glycosides(acteoside,acetylpoliumoside,2’-acetylpoliumoside)in Cistanche sinensis,and compare with the chemical composition of Cistanche deserticola and Cistanche tubulosa;3.The UPLC fingerprint analysis method of three species of Cistanche was established.The common model,similarity comparison and combination of cluster analysis and principal component analysis were used.Methods To systematically evaluate the quality differences of Cistanche deserticola,Cistanche tubulosa and Cistanche sinensis.4.Determine the preparation process of Cistanche standard decoction,prepare 14batches of Cistanche standard decoction,and evaluate the quality of the standard decoction by studying the quality control indexes such as the extract rate,index component transfer rate,fingerprint and so on.Results1.The UPLC quantitative analysis method of four phenylethanoid glycosides(echinacoside,cistanoside A,acteoside,isoacteoside)in Cistanche deserticola and Cistanche tubulosa was established.The standard curve of the four components has a good linear relationship(R~2≥0.9999).The LOD and LOQ were0.0000777-0.000709mg/m L and 0.000259-0.00236 mg/m L,respectively.The RSD of precision,stability and repeatability test is less than 4.30%.The recovery rate of sample addition is between 96.22%and 100.93%(RSD<2.0%).The contents of 4phenylethanoid glycosidesin 15 batches of Cistanche deserticola and 10 batches of Cistanche tubulosa were determined.The results showed that the average contents of the four components in Cistanche deserticola and Cistanche tubulosa were in the order of echinacoside>acteoside>cistanoside A>isoacteoside,and the contents of Cistanche deserticola and Cistanche tubulosa were different in different batches.The average value of the four components in Cistanche tubulosa is 5.13%,and the average value of the four components in Cistanche deserticola is 1.96%,indicating that the average content of the four components in Cistanche tubulosa is significantly higher than that of Cistanche deserticola.2.The UPLC quantitative analysis method of three phenylethanoid glycosides(acteoside,acetylpoliumoside,2’-acetylpoliumoside)in Cistanche sinensis was established.The standard curve of the three components has a good linear relationship(R~2≥0.9999).The LOD and LOQ were 0.000130～0.000319mg/m L and0.000434～0.00106 mg/m L,respectively.The RSD of the precision,stability and repeatability tests are all less than 2.67%,and the recovery rate of sample addition is between 97.21%and 101.13%(RSD<1.0%).The contents of three components in 13batches of Cistanche sinensis were determined.The results showed that the content of acetylpoliumoside was the highest in 13 batches of Cistanche sinensis,accounting for58.1-80.1%of the total amount of the three compounds,followed by 2’-acetylpoliumoside,while the content of acteoside was lower.There were obvious differences in the type and content of phenylethanoid glycosides between Cistanche deserticola,Cistanche tubulosa and Cistanche sinensis.Acetylpoliumoside and 2’-acetylpoliumoside were the specific components of Cistanche sinensis highest component content.The content of acteoside is the common component of Cistanche deserticola,Cistanche tubulosa and Cistanche sinensis,but there is great difference among the three varieties.The highest content is 0.79%in Cistanche tubulosa,0.52%in Cistanche deserticola,and only 0.19%in Cistanche deserticola.the content of the three species is different,the highest is 0.79%in Cistanche tubulosa,0.52%in Cistanche deserticola,the lowest is 0.19%in Cistanche sinensis.3.The UPLC fingerprint analysis method of three species of Cistanche was established.The UPLC fingerprints of 38 samples were analyzed,and the UPLC fingerprints and common patterns of three species of Cistanche were obtained.Nine common peaks were determined for Cistanche deserticola samples,and six common peaks were determined for Cistanche tubulosa samples,both of which identified four peaks of echinacoside,cistanoside A,acteoside,isoacteoside.Six common peaks were determined for Cistanche sinensis samples There are three peaks of acteoside,acetylpoliumoside,2’-acetylpoliumoside.The similarity evaluation results showed that:the similarity between Cistanche deserticola and Cistanche tubulosa was 0.932,while the similarity between Cistanche tubulosa and Cistanche sinensis was 0.070,and the similarity between Cistanche sinensis and Cistanche deserticola was only0.045 lower;the quality differences of Cistanche deserticola,Cistanche tubulosa and Cistanche sinensis were evaluated by combining chemometrics cluster analysis and principal component analysis.The results showed that 38 batches of samples were grouped into two groups,Cistanche deserticola,Cistanche tubulosa and three batches of Cistanche sinensis were clustered in the first category,and the rest of Cistanche sinensis were clustered in the second category.The principal component analysis could distinguish the three species of Cistanche.Among them,the sample point distribution of Cistanche tubulosa is relatively discrete,which shows that the quality of the samples is different between batches.4.14 batches of Cistanche standard decoction were prepared.The results showed that the extraction rate of Cistanche standard decoction ranged from 25.07%to 56.64%,the average transfer rate of echinacoside was 22.05%,the average transfer rate of acteoside was 15.07%,and the p H value ranged from 4.30 to 5.15.In addition,The fingerprint of the standard decoction of Cistanche was established,and 6 peaks were identified.Among them,No.1 peak was echinacoside,No.3 peak was cistanoside A,No.4 peak was acteoside,and No.5 peak was isoacteoside.The results showed that the prepared standard decoction had good homogeneity and controllable quality.ConclusionThis study provides a new reliable and rapid UPLC analysis method for the component analysis,species identification and quality evaluation of Herba cistanche and its pieces.Meanwhile,the core samples of its quality markers are established through the study of the standard decoction of Cistanche decoction pieces.It is found that there are obvious differences in chemical composition and fingerprint between the genuine Cistanche and Cistanche sinensis.Based on the consideration of effectiveness and safety,the use of Cistanche sinensis instead of the genuine Cistanche has a greater potential risk.But at present,Cistanche sinensis are frequently used as Cistanche deserticola or Cistanche tubulosa in the market,which should attract the attention of relevant departments and increase the corresponding supervision.At the same time,through the study of the standard decoction of Cistanche decoction pieces,we can provide the standard reference for the study of the material standard of the classic prescription and the quality standard of the traditional Chinese medicine formula granules,which can further ensure the clinical efficacy and safety of Cistanche. |
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