Research On The Quality Control Of Chinese Medicine Cassia And Uncaria

Objective:Chinese medicine cassia,is the dry and mature seed of the legume Cassia obtusifolia L.or Cassia tora L.It has the functions of improving vision,liver-protection and laxative.Modern pharmacological studies have found that Cassia has the effects of lowering blood pressure,lowering blood fat,anti-aging,anti-oxidation,immunity and anti-tumor.It is widely planted all over the country and overseas.However,due to differences in cultivation environment,processing and transportation,there are some differences in the quality of the medicinal materials.The Chinese Pharmacopoeia 2015 edition stipulated the sputum contained in it.The content of the constituents such as aurantio-obtusin and chrysophanol was determined as the standard for quality control of cassia seed.However,due to its wide range of pharmacological effects,the main medicinal ingredients are many,and the determination of the content of the two components alone as an indicator of quality control cannot fully reflect its quality.Therefore,it is necessary to continue to supplement and improve the standards for its quality control.In this experiment,the UPLC fingerprint of Cassia and the determination of five anthraquinones were determined and combined with LC-MS technology to fast and comprehensive analyze the chemical components contained in Cassia to supplement the existing Cassia quality standards.The zebrafish hyperlipidemia model,based on the intensity of zebrafish tail lipid staining as a quantitative indicator,preliminarily evaluated the differences in pharmacological activities of Cassia medicinal materials between different batches,and supplemented and improved the quality control system of traditional Chinese medicine.Chinese medicinal uncaria,is Uncaria rhynchophylla(Miq.)Miq.ex Havil、Uncaria macrophylla Wall、Uncaria hirsute Havil、Uncaria sinensis(Oliv)Havil、or Uncaria sessilifiuctus Roxb.Dry with hooked stems.At present,the varieties of Uncaria medicinal materials on the market are more complex,and the chemical constituents and pharmacological effects of Uncaria medicinal materials among different varieties are also different.The 2015 edition of the Chinese Pharmacopoeia has not yet included the standard for the determination of Uncaria,and only qualitative identification has seriously affected the quality control and application of Uncaria.In this experiment,two species of Uncaria rhynchophylla(Miq.)Miq.ex Havil.and Uncaria macrophylla Wall were collected,and their UPLC fingerprints were established under the same chromatographic conditions.The significant pharmacological activity content of rhynchophylline and isorhynchophylline in Uhcaria macrophylla Wall was determined.Provide reference for the differentiation and quality control of two varieties of Uncaria.Methods:(1)28 batches of Cassia medicinal materials from different origins were collected.UPLC method combined with cluster analysis,principal component analysis and similarity evaluation were used to establish the fingerprint of Chinese Cassia.(2)UPLC method was used to simultaneously determine the content of five kinds of terpenoids of emodin,chrysophanol,physcion,ether,aurantio-obtusin and chryso-ob-tusin in 28 batches of cassia seed.Provide reference for the improvement of existing quality standards(3)LC-MS technology was used to analyze the chemical constituents contained in Cassia medicinal materials,which provided a reference for the quality control of Cassia medicinal materials and the basic research of pharmacodynamic substances(4)Using the zebrafish hyperlipidemia model,the zebrafish tail blood lipid staining intensity was used as the quantitative analysis index of the experiment,and the pharmacological activity difference of different batches of Cassia medicinal materials was preliminarily evaluated,which provided a reference for the supplement of quality control.(5)12 batches of Uncaria rhynchophylla(Miq.)Miq.ex Havil.medicinal materials and 10 batches of Uncaria macrophylla Wall were collected from different producing areas.The fingerprints of Uncaria rhynchophylla(Miq.)Miq.ex Havil.and Uncaria macrophylla Wall were established by ultra performance liquid chromatography(UPLC).Provide reference for the differentiation and quality control of the variety of Uncaria.(6)The content of rhynchophylline and isorhynchophylline in 10 batches of Uncaria macrophylla Wall was determined by UPLC method to supplement the quality control of Uncaria.(7)Based on the results of the above experiments,preliminary drafts of quality standards for Cassia and Uncaria medicinal materials were prepared.Results:(1)UPLC fingerprints of 28 batches of Cassia medicinal materials from different producing areas were established by using UPLC method.A total of 40 common peaks were matched.By comparison with standard products,10 components were identified,and 28 batches of medicinal materials were identified.The similarity is higher,and the similarity range is between 0.827 and 0.990.In general,the quality of Chinese cassia seed is relatively stable.Through cluster analysis and principal component analysis,it is concluded that although the cassia seed is widely planted throughout the country,the quality produced by Hainan and Shandong is good,which may be related to the local climatic conditions.(2)The UPLC method was used to determine the content of five anthraquinones,emodin,chrysophanol,physcion,aurantio-obtusin and chryso-ob-tusin in the cassia seed.The results showed that aurantio-obtusin and chrysophanol are in accordance with the Pharmacopoeia.The content of chryso-ob-tusin is 0.1809～1.1311 mg·g-1,the content of emodin is 0.1952～0.8940·mg-1,and the content of physcion is 0.1917～0.8752 mg·g-1.Through methodological investigation,it shows that the precision of the instrument used in this experiment is good,the stability and repeatability of the sample and the recovery rate of the sample can meet the standards stipulated by the pharmacopoeia,and provide reference for the further improvement of the quality standard of Chinese Pharmacopoeia.(3)LC-MS technology was used to estimate the chemical components contained in Cassia.In the positive ion and negative ion modes,the experimental data were analyzed and Speculated 39 chemical components.The anthraquinone component contained in cassia seed is present in the form of free quinone and conjugated glucoside,and the glucoside component is diversified due to its position of attachment to the carbohydrate and the type of glycosyl group.At the same time,naphthopyrone compounds exist in a large number in cassia seeds,and their pharmacological activities need further development and utilization.This method can quickly infer Cassia chemical components.The chemical components contained in the medicinal materials can also provide a reference for the quality control of Cassia.(4)Used the zebrafish hyperlipidemia model to verify the prevention and treatment of Cassia medicinal materials for hyperlipidemia,and analyzed the activity of different batches of medicinal materials.The results showed that Cassia medicinal materials were within the experimental concentration range.The pharmacological activity increased with the increase of the concentration of the drug.Different batches of total extracts of Cassia seed can inhibit the fat absorption of zebrafish juveniles to varying degrees.Among them,the similarity of the S27 in the fingerprint is weaker than that of S25 and S26 in the effect of pharmacodynamics,which indicates that the herb with higher similarity is more obvious in the effect of pharmacodynamics.(5)UPLC fingerprints of 12 batches of Uncaria rhynchophylla(Miq.)Miq.ex Havil medicinal materials and 10 batches of Uncaria macrophylla Wall medicinal materials were established under the same chromatographic conditions in this experiment.Analysis by similarity evaluation software,12 batches of Uncaria rhynchophylla(Miq.)Miq.ex Havil medicinal materials on the UPLC fingerprints matched a total of 22 common peaks.In the 12 batches of Uncaria rhynchophylla(Miq.)Miq.ex Havil,the similarity of the 11 batches was greater than 0.9,and only one batch was lower than 0.9,which was 0.737.The 10 batches of Uncaria macrophylla Wall matched 12 common peaks.The similarity was above 0.9.By comparing the control fingerprints of the two varieties of herbs,the chemical composition,rhynchophylline and isorhynchophylline content were significantly different.The content of rhynchophylline and isorhynchophylline in Uncaria macrophylla Wall was higher.Therefore,the method established in this experiment can be used to distinguish between the two different varieties of Uncaria rhynchophylla(Miq.)Miq.ex Havil and Uncaria macrophylla Wall,and also can be used for quality control of two varieties.(6)The content of rhynchophylline and isorhynchophylline in the medicinal materials of Uncaria macrophylla Wall was determined by UPLC method.The content of rhynchophylline was in the range of 0.2214~0.6469 mg·g-1,while the isorhynchophylline was in the ranged of 0.0424～0.1309 mg·g-1.the sum of the two ranges from 0.2657 to 0.7559 mg ·-1.Through methodological investigation,it shows that the precision,stability,repeatability and sample recovery rate of this experiment can reach the relevant standards stipulated by the Chinese Pharmacopoeia.Therefore,the method can be combined with the relevant experiments of the previous fingerprint.Provide reference for the quality control of Uncaria macrophylla Wall.(7)Based on the above experimental results,increase the fingerprint and the content of the three components on the basis of the existing Cassia obtusifolia L.quality standards;and increase the fingerprint and the content of the two components on the Uncaria macrophylla Wall.To increase the fingerprint of Uncaria rhynchophylla(Miq.)Miq.ex Havil.Preliminary drafting of the quality standard for Chinese herbal medicine Cassia and Uncaria.Conclusion:UPLC was used to establish the fingerprint of Chinese Cassia seed and the content of five anthraquinone chemical components was determined.The chemical components contained in Cassia were analyzed by LC-MS technology,which was beneficial to the further development of Cassia.The experiment verified the hyperlipidemia model of zebrafish,and preliminarily evaluated the pharmacological activity of Cassia medicinal materials between different batches,which provided new ideas and methods for the improvement of the quality standard of Chinese Cassia.This paper established the UPLC fingerprint of Uncaria rhynchophylla(Miq.)Miq.ex Havil and Uncaria macrophylla Wall,and determined the content of rhynchophylline and isorhynchophylline in the Uncaria macrophylla Wall.Provide reference for the differentiation of medicinal materials and the improvement of quality control.