Views on Key Technical Problems in Research and Development of Famous Classical Formulas / 中国实验方剂学杂志

In this paper， the key technical problems in the research and development of famous classical formulas are analyzed. Firstly， the puzzled problem for a long-time， which is conversion relationship from medicinal metrology of Han dynasty （HD） to that of modern （gram，g）， is comprehensively expounded that one Liang （两） of HD=3 g is more appropriate. Secondly， the model and principles of quality consistency evaluation are given for the transformation from the quality of authoritative basic sample prepared by casserole （ABS-C） to the quality consistency in Laboratory process， pilot-scale process and industrial production. The consistency evaluation model is ξABS-X=K1（Q1ABS-X/Q1ABS-C）+K2（Q2ABS-X/Q2ABS-C）+……+Ki（QiABS-X/QiABS-C）=∑Ki（QiABS-X/QiABS-C）（i=1，2，3……n）. In the formula， ABS-X means laboratory reference sample ABS-C （ABS-L）， pilot-scale ABS-C （ABS-mP） or industrial production ABS-C （ABS-P）， ξABS-X means the quality consistency rate or similarity degree of ABS-L， ABS-mP and ABS-P processes with ABS-C， Ki means the weight of each quality evaluation index （i）， QiABS-X is the data of i in ABS-L， ABS-mP， ABS-P samples， and QiABS-C is the data （or mean） of i in ABS-C sample. Thirdly， in order to control the quality of the herbal medicines whose active ingredients were unknown， their chemical constituents should be studied deeply， and if necessary， the bioassay research should be carried out according to the main efficacy or indication of famous classical formulas. Finally， for the special processing of some herbal medicines， it is difficult to formulate the processing method， technology and standard of prepared slices. It is suggested that the scientific connotation and historical evolution of the special processing method should be thoroughly sorted out， and its technological characteristics are summarized， the modern processing technology and production processes are simulated， and then the corresponding processing methods and quality standards are formulated.

Analysis of Chemical Constituents in Euodiae Fructus by UPLC-Q-TOF-MS/MS / 中国实验方剂学杂志

Objective:To analyze the chemical constituents in Euodiae Fructus by ultra-high performance liquid chromatography quadrupole time-of-flight mass spectrometry （UPLC-Q-TOF-MS/MS）. Method:The chromatographic separation was performed on an ACQUITY UPLC BEH C₁₈ column （2.1 mm×100 mm， 1.7 μm） with acetonitrile （A）-0.1% formic acid aqueous solution （B） as mobile phase （0-3 min， 6%A； 3-4 min， 6%-10%A； 4-7 min， 10%-12%A； 7-8 min， 12%-14%A； 8-13 min， 14%-15%A； 13-15 min， 15%-20%A； 15-18 min， 20%-30%A； 18-21 min， 30%-49%A； 21-25 min， 49%-51%A； 25-27 min， 51%-73%A； 27-30 min， 73%-80%A； 30-31 min， 80%-100%A； 31-32 min， 100%A） for gradient elution. The column temperature was 35 ℃， and the flow rate was 0.4 mL·min^-1. Mass spectrometry was performed using an electrospray ionization and data were collected in positive and negative ion modes， and the detection range was <italic>m</italic>/<italic>z</italic> 100-1 200. The chemical constituents in Euodiae Fructus were identified rapidly and comprehensively based on the accurate relative molecular mass and combined with literature data and reference substances. Result:A total of 92 chemical constituents were speculatively identified from the 70% methanol extract of Euodiae Fructus， including 39 alkaloids， 19 flavonoids， 12 limonoids， 20 phenolic acids and 2 organic acids. Among them， 26 compounds were confirmed by the reference substances. Conclusion:The compound types of Euodiae Fructus are multiple and quite different in polarity. The chemical compositions of Euodiae Fructus from different regions and species are similar. The established method is rapid and accurate， with which the chemical compositions of Euodiae Fructus have been identified comprehensively. Therefore， this study provides an experimental reference for further clarifying active and toxic constituents of Euodiae Fructus.

Investigation on in Vitro Antioxidant Activity and Chemical Composition of Different Polar Parts of Extract of Rosa cymosa Roots / 中国实验方剂学杂志

Objective:To investigate the antioxidant activity and chemical composition of 75% ethanol extract of <italic>Rosa cymosa</italic> roots and its different polar parts. Method:The 75% ethanol extract of <italic>R. cymosa</italic> roots was divided into dichloromethane， ethyl acetate， <italic>n</italic>-butanol and water parts by organic solvent extraction. <italic>In vitro</italic> antioxidant activity of each fraction was determined by 1，1-diphenyl-2-picrylhydrazyl （DPPH） and 2，2'-azino-bis（3-ethylbenzothiazoline-6-sulfonic acid） （ABTS） free radical scavenging assays， as well as ferric reducing antioxidant power （FRAP） test. The contents of total triterpenes， total phenols， total tannins and condensed tannins in each fraction were determined by spectrophotometry. SPSS 24.0 software was used to conduct Pearson correlation analysis between the antioxidant activity of each fraction and the content of the main components， and then the main active fraction and the main active components were determined. The chemical constituents of the active fraction was analyzed by ultra performance liquid chromatography-quadrupole-time-of-flight mass spectrometry （UPLC-Q-TOF-MS/MS）， and the structures of the main chromatographic peaks were predicted. Result:Each fraction of <italic>R. cymosa</italic> roots had certain antioxidant activity， and there was a significant dose-effect relationship within a certain concentration range， but the antioxidant activity of different polar parts was different. In DPPH and ABTS free radical scavenging tests， the antioxidant activity of each fraction and vitamin C （VC， the positive drug） was ranked as ethyl acetate fraction>VC><italic>n</italic>-butanol fraction>ethanol extract>water fraction>dichloromethane fraction. In FRAP test， the activity of ethyl acetate fraction was weaker than that of VC， and the other order was unchanged. The contents of total triterpenes， total phenols， total tannins and condensed tannins in ethyl acetate fraction were 3.81%， 50.33%， 3.32%， and 39.79%， in <italic>n</italic>-butanol fraction were 0.88%， 41.42%， 2.25% and 23.55%， in ethanol extract were 2.90%， 41.95%， 3.43% and 20.14%， in water fraction were 0， 26.80%， 16.90% and 7.57%， and in dichloromethane fraction were 21.23%， 12.90%， 1.59%， and 6.17%， respectively. Correlation analysis results showed that the contents of total phenols and condensed tannins were positively correlated with the antioxidant activity， the contents of total triterpenes were negatively correlated with the antioxidant activity， and the correlation between total tannins and antioxidant activity was not obvious. A total of 26 compounds were identified from the ethyl acetate fraction by UPLC-Q-TOF-MS/MS， including 11 condensed tannins， 4 hydrolysable tannins， 6 triterpenes， 3 flavonoids， 1 benzoic acid derivative and 1 chlorogenic acid analogue. Conclusion:Ethyl acetate fraction is the main antioxidant active site of <italic>R. cymosa</italic> roots， and phenols mainly composed of condensed tannins are the main active components. The results can provide experimental basis for the development of natural antioxidants.

Anti-acetylcholinesterase Active Ingredients from Aconitum tanguticum by UPLC-Q-TOF-MS/MS / 中国实验方剂学杂志

Objective:To identify the anti-acetylcholinesterase active ingredients in <italic>Aconitum tanguticum</italic>， so as to lay the foundation for finding new anti-Alzheimer's disease （AD） drugs. Method:The anti-acetylcholinesterase active fractions of <italic>A. tanguticum</italic> were screened by the modified Ellman's method， and the chemical composition of the active fraction was analyzed by ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry （UPLC-Q-TOF-MS/MS）. The chromatographic separation was performed on an ACQUITY UPLC BEH C₁₈ column （2.1 mm×50 mm， 1.7 μm） with acetonitrile （A）-0.4% ammonia aqueous solution （B） as mobile phase for gradient elution， and the column temperature was set at 30 ℃ with the flow rate of 0.4 mL·min^-1. Phase A of the dichloromethane fraction changed with time as follows：0-3 min， 5%A； 3-7 min， 5%-20%A； 7-11.5 min， 20%-33%A； 11.5-15.5 min， 33%-50%A； 15.5-20.5 min， 50%-80%A； 20.5-23 min， 80%-85%A； 23-25 min， 85%-95%A. Phase A of the <italic>n</italic>-butanol fraction changed with time as follows：0-2 min， 5%A； 2-8 min， 5%-20%A； 8-11 min， 20%-33%A； 11-15 min， 33%-95%A. Mass spectrometry was performed on electrospray ionization， data were collected in positive ion mode， and the detection range was <italic>m</italic>/<italic>z</italic> 100-1 500. Result:Both the dichloromethane and <italic>n</italic>-butanol fractions had a certain inhibitory effect on acetylcholinesterase， their half inhibitory concentration （IC₅₀） values were （64±4.4） mg·L^-1 and （85.7±3.8） mg·L^-1， respectively. By UPLC-Q-TOF-MS/MS analysis， a total of 21 alkaloids were identified from the dichloromethane fraction， and 11 alkaloids were identified from <italic>n</italic>-butanol fraction. Guan-fu base Ⅰ， found in both fractions， was first discovered in <italic>A. tanguticum</italic>. Conclusion:Diterpene alkaloids are the main anti-acetylcholinesterase substances of <italic>A. tanguticum</italic>， which is worth further exploration.

Qualitative and quantitative analysis of nucleosides and nucleobases in Phylloporia ribis by UPLC-Q-TOF-MS and HPLC / 中国中药杂志

The dried fruit body of Phylloporia ribis(Hymenochaetaceae), which prefers to live on the stumps of Lonicera japonica(Caprifoliaceae), has a variety of activities, whereas its pharmacodynamic material basis is not completely clear and there are few reports on its quality control and evaluation. In this study, an UPLC-Q-TOF-MS method was used to analyze the nucleosides and nucleobases in P. ribis and a HPLC method was established for simultaneous determination of 10 nucleosides and nucleobases. MS and MS/MS data were acquired in positive ion mode. Based on the data comparison of the sample and the reference substance, the literature data and the compound databases of ChemSpider and PubChem, 18 nucleosides and nucleobases were identified qualitatively from the water extract of P. ribis for the first time. After optimization, the HPLC was performed using a Welch Ultimate AQ C_(18) column(4.6 mm×250 mm, 5 μm) by gradient elution with acetonitrile and water as mobile phase, the flow rate of 1.0 mL·min~(-1), the detection wavelength of 260 nm, and the column temperature of 30 ℃. Through the investigation of the extraction method, solvent and time, it was determined that the test solution should be obtained by cold water extraction for 18 h. At the present HPLC conditions, 10 components of uracil, cytidine, hypoxanthine, uridine, thymine, inosine, guanosine, 2'-deoxyinosine, 2'-deoxyguanosine and thymidine could be well separated(R > 1.5) and showed good linearity(r > 0.999 9) in the concentration ranges of 0.247-24.7, 0.283-28.3, 0.273-27.3, 0.256-25.6, 0.257-25.7, 0.318-31.8, 0.245-24.5, 0.267-26.7, 0.250-25.0 and 0.267-26.7 mg·L~(-1), respectively. The average reco-veries of 10 components were 95.78%-104.5%, and the RSDs were 2.2%-5.2%(n=6). The contents of 10 nucleosides and nucleobases in different samples of P. ribis varied greatly, which were 0.021-0.122, 0.004-0.029, 0.014-0.226, 0.009-0.442, 0.003-0.014, 0.002-0.146, 0.007-0.098, 0-0.054, 0.005-0.069, 0.004-0.081 and 0.072-1.28 mg·g~(-1) for uracil, cytidine, hypoxanthine, uridine, thymine, inosine, guanosine, 2'-deoxyinosine, 2'-deoxyguanosine, thymidine and total 10 components, respectively. These results demonstrated that the components had significant differences in the internal quality, and good quality control was needed to ensure the medical efficacy. This study provides a scientific basis for the discovery of pharmacodynamic ingredients, quality control and evaluation of P. ribis.

Research progress of tannins in traditional Chinese medicines in recent ten years / 中国中药杂志

In this paper, the newly isolated tannins were sorted after a review of the literature concerning tannins in recent 10 years, and their research progress was summarized in terms of extraction, isolation, pharmacological activity and metabolism. Hydrolysable tannins and condensed tannins are the main structural types. Modern research shows that tannins have many pharmacological effects, such as bacteriostasis, antioxidation, antitumor, antivirus and blood glucose reduction, and have broad development prospects. They are usually extracted by water, ethanol and acetone and isolated and purified by macroporous resin and gel column chromatography. The packings commonly adopted for the column chromatography mainly included Sephadex LH-20, Diaion HP-20, MCI-gel CHP-20 and Toyopearl HW-40. Modern analytical techniques such as nuclear magnetic resonance spectroscopy(NMR), fast atom bombardment mass spectrometry(FAB-MS) and circular dichroism(CD) are generally used for the structural identification of tannins. Howe-ver, their isolation, purification and structural identification are still challenging. It is necessary to use a variety of high-throughput screening methods to explore their pharmacological activities and to explore the material basis responsible for their functions through experiments in vivo.

Content determination and transferring rules of seven components in Angelicae Sinensis Radix and its processed products / 中国中药杂志

A HPLC method was established for simultaneous determination of two organic acids(chlorogenic acid and ferulic acid) and five phthalides(senkyunolide I, senkyunolide H, senkyunolide A, ligustilide, and butylidenephthalide) in Angelicae Sinensis Radix and its processed products to clarify the underlying material transferring rules. The analysis was performed on a Welch Ultimate C_8 column(4.6 mm×250 mm, 5 μm) with acetonitrile(A)-0.085% phosphoric acid water(B) as the mobile phase in a gradient elution mode at the flow rate of 1.1 mL·min~(-1), the column temperature of 25 ℃, the detection wavelength of 280 nm, and the injection volume of 10 μL. Under these conditions, the content of the above-mentioned seven components was analyzed in 15 batches of Angelicae Sinensis Radix and its processed products, and the transfer rate of each compound was calculated. As a result, in the processed products, the average content of chlorogenic acid was slightly decreased and that of ferulic acid was equivalent to the medicinal materials. The content of senkyunolide I, senkyunolide H, senkyunolide A, and butylidenephthalide showed an increasing trend in the processed products as compared with the medicinal materials. The mass fraction of ligustilide in the medicinal materials was above 0.7%(0.94% on average), meeting the requirement of 0.6% in the Hong Kong Chinese Materia Medica Standards, but was 0.47% on average in the processed products, which was decreased by 50% approximately. Further investigation showed that the content of ligustilide in freshly made processed products of Angelicae Sinensis Radix did not change significantly compared with that in the medicinal materials, indicating that the loss of ligustilide in the processed products mainly occurred in the storage. Therefore, Angelicae Sinensis Radix is suitable for storing in the form of medicinal materials and the freshly made processed products should be used except for special cases. Additionally, it is recommended to control the content of volatile oils or ligustilide in medicinal materials and processed products of Angelicae Sinensis Radix to ensure its effectiveness in clinical medication.

Research progress on Polygalae Radix / 中国中药杂志

Polygalae Radix has long been used in China for calming the mind, promoting intelligence, communicating the heart and kidney, eliminating phlegm, and reducing swelling. At present, it is used to treat amnesia, insomnia, and malaise. Modern research has revealed that Polygalae Radix mainly contains triterpenoid saponins, xanthone, oligosaccharide esters, etc., with the activities of improving memory, resisting dementia, protecting the brain, relieving cough, and removing phlegm, as well as sedation and hypnosis. The present study reviews the research progress on chemical composition, pharmacological action, quality control, and metabolism of Polygalae Radix in the past 30 years, to provide a theoretical basis for further research and development.

HPLC fingerprint of Tibetan medicinal plant ″Bangga″ and chemical components analysis of its two original plants by LC-MS/MS / 中国中药杂志

To obtain the chemical profile of Tibetan medicinal plant ″Bangga″, the present study established the HPLC fingerprint of ″Bangga″ and inferred common chemical constituents of its two original plants, Aconitum tanguticum and A. naviculare by LC-MS. The HPLC analysis was performed on a Kromasil 100 C_8 column(4.6 mm×250 mm, 5 μm) with acetonitrile(A)-0.1% formic acid in water(B) as mobile phase in a gradient elution mode. Besides, the flow rate was set at 1 mL·min~(-1) and the column temperature was 35 ℃. The detection wavelength was set at 255 nm and the injection volume was 10 μL. Seventeen batches of ″Bangga″ samples were analyzed and the HPLC fingerprint was established under the above conditions. Similarity evaluation was performed using Similarity Evaluation System for Chromatographic Fingerprint of Traditional Chinese Medicine(2012). As a result, 16 common peaks were selec-ted and the similarity values of 17 batches of ″Bangga″ were in the range of 0.702-0.966. Furthermore, one batch of A. tanguticum and one batch of A. naviculare were analyzed by LC-MS/MS and 74 common compounds were inferred, including 10 phenolic acids, 26 flavonoids, and 38 alkaloids. The established method, with good separation and strong specificity, is simple and feasible, and can be used for the quality control of ″Bangga″ and identification of its two original plants. A. tanguticum and A. naviculare are similar in chemical composition and component content, but are quite different in the content of flavonoids.

Study on determination and quantity transfer of multi index components in Wenjing Decoction / 中国中药杂志

Based on the textual research on literature, the key information of Wenjing Decoction were tested and identified, and 15 batches of lyophilized powder samples of Wenjing Decoction were prepared. The specific components, including paeoniflorin, glycyrrhizin, ginsenosides(Rg_1, Re and Rb_1), glycyrrhizic acid, and paeonol, were used as indexes to establish the HPLC method for quantitative evaluation, and the content ranges and transfer rates of these components were determined. The results showed that the contents of paeoniflorin, glycyrrhizin, ginsenosides Rg_1 + Re, ginsenoside Rb_1, glycyrrhizic acid, and paeonol in the 15 batches of samples were 0.62%-0.86%, 0.25%-0.76%, 0.14%-0.30%, 0.07%-0.21%, 0.63%-1.16%, and 0.09%-0.25%, respectively, and their transfer rates from the decoction pieces to the reference materials were 14.99%-19.42%, 28.11%-40.93%, 25.92%-61.88%, 25.03%-64.06%, 23.43%-35.53%, and 5.34%-10.44%, respectively. The consistency of the transfer rates between batches indicated that the preparation process was stable. It is suggested that the contents of paeoniflorin, glycyrrhizin, ginsenosides Rg_1 + Re, ginsenoside Rb_1, glycyrrhizic acid, and paeonol in Wenjing Decoction should not be less than 0.52%, 0.35%, 0.15%, 0.10%, 0.63%, and 0.12%, respectively. In this study, we determined the contents and analyzed the quantity transfer process of the index components in Wenjing Decoction, which can provide a basis for the follow-up development of Wenjing Decoction and the quality control of related preparations.

Research progress of pesticide residues in Chrysanthemum / 中国中药杂志

Chrysanthemum is widely used as a type of edible flower and also considered as the important materials of many beverages in China. Due to the occurrence of diseases and pests, and the lack of regulations for species, frequency, dose of pesticides in Chrysanthemum, pesticides have become one of the main pollutants in Chrysanthemum. The pesticide residues in Chrysanthemum were detected frequently and worth noting. This paper focused on the types of pesticides, pesticide residue detection techniques, and risk assessment methods for Chrysanthemums on the basis of relevant literatures. The pesticide residues of traditional Chinese medicine are mainly organochlorines, organophosphorus and pyrethroids, and the detection techniques include gas chromatography(GC), liquid chromatography(LC) or both combined with mass spectrometry(MS). With the increasing use of traditional Chinese medicine, Chrysanthemum is widely circulated in the market. Therefore, it is important to understand the current situation of pesticide residues in different varieties of Chrysanthemum, so as to provide theoretical reference for the control of quality and safety of Chrysanthemum and the formulation of the maximum residue limit.

Optimization of determination of astragaloside Ⅳ in Astragali Radix by continuous single-factor method / 中国中药杂志

This paper aims to solve the problems of complicated-unstable test solution preparation process and insufficient extraction of the active ingredient astragaloside Ⅳ in the legal method for the determination of astragaloside Ⅳ in Astragali Radix. The continuous single-factor analysis of seven main factors affecting the content of astragaloside Ⅳ was carried out by HPLC-ELSD, and then the pre-paration method of test solution was optimized. This optimized method exhibited excellent performance in precision, repeatability and stability. The average recovery rate of astragaloside Ⅳ was 99.65% with RSD 2.2%. Astragaloside Ⅳ showed a good linearity between the logarithm of peak area and the logarithm of injection quantity in the range of 0.46-9.1 μg(r=0.999 6). The contents of astragaloside Ⅳ in 29 batches of Astragali Radix were determined by the new and the legal methods. The results showed that the average content of astragaloside Ⅳ in these Astragali Radix samples determined by the former method was 1.458 times than that of the latter one, indicating the new method was simple, reliable and more adequate to extract target compound. According to the results, it is suggested to improve the content standard of astragaloside Ⅳ in Astragali Radix in the new edition of Chinese Pharmacopeia.

Scientificity of traditional commodity grade of Chinese medicinal materials--taking Lonicerae Japonicae Flos as example / 中国中药杂志

Taking Lonicerae Japonicae Flos as an example, the method of "expert consensus of different regions" was used to screen the representative samples and evaluate their commodity grades. The correlation analysis, hierarchical cluster analysis and partial least squares discriminant analysis(PLS-DA) of "commodity grade-appearance characteristic-component content" were carried out to reveal the scientificity of traditional commodity grade of Chinese medicinal material. By referring to the existing literature and the grade investigation from the sample collection regions, 78 "initial grade" samples were screened out from 118 collected samples. Authoritative experts from four regions(n=4) including Linyi(Shangdong province), Bozhou(Anhui province), Anguo(Hebei province) and Beijing were organized to evaluate their commodity grades, separately. Based on the grade consistency rate(R_i≥70%), 69 "local grade" samples were screened out from the "initial grade" samples. Based on the average grade consistency rate ■ "authoritative grade" samples were screened out from the "local grade" samples, including15 first-grade samples, 9 second-grade samples, 11 third-grade samples and 17 fourth-grade samples. For these "authoritative grade" samples, the main appea-rance characteristics were quantified and the contents of 13 components were determined by ultra performance liquid chromatography(UPLC). Furthermore, the total contents of 6 phenolic acids, 4 flavonoids and 3 iridoids were calculated, respectively. The results of correlation analysis showed that 4 appearance characteristics indices were correlated with the commodity grades: color, rate of yellow bars(including blooming flowers), rate of black heads(including black bars), and rate of stems and leaves(including bud debris). Five component content indices were correlated with the commodity grades: chlorogenic acid, isochlorogenic acid C, sweroside, loganin and the total contents of six phenolic acids. Furthermore, chlorogenic acid, loganin and the total contents of six phenolic acids showed significantly negative correlation with the main appearance characteristics, indicating that the appearance characteristics of Lonicerae Japonicae Flos can reflect its internal quality, and these 3 indices can be used as quality markers(Q-markers). The results of hierarchical cluster analysis showed that the samples of four grades were classified into four categories, and the samples with the same grades and the same categories accounted for 80.8% of the total samples, while the samples with the different grades were obviously classified into different categories. The results of PLS-DA analysis showed that the samples of different grades showed obvious intragroup aggregation and intergroup dispersion. The above results indicated that it was feasible to evaluate the traditional commodity grade of Lonicerae Japonicae Flos by the method of "expert consensus of different regions". For the evaluation of traditional commodity grade of Chinese medicinal material, the samples should be representative, expert conclusions should have enough consensuses, and grade determination should be authoritative. As the crystallization of clinical experience, traditional commodity grade can scientifically reflect the internal quality of Chinese medicinal material.

Study on assay strategy of total tritepenoid saponins in traditional Chinese medicines using total saponins from Akebiae Caulis as an example / 中国中药杂志

Due to lack of reference substances,the content of triterpenoid saponins in traditional Chinese medicines is usually characterized by colorimetric determination of total saponins. However,the specificity of colorimetric method is poor,and the determination result is not accurate enough. So,in this paper,the content determination method of total triterpenoid saponins was studied by taking Akebiae Caulis saponins as an example. The contents of three main saponin aglycones,including arjunolic acid,hederagenin and oleanolic acid,were determined by HPLC method. Referring to the content determination method of total flavonol glycosides in Ginkgo biloba leaves in the 2015 edition of Chinese Pharmacopoeia,the content of Akebiae Caulis saponins was obtained by multiplying the total content of the three above-mentioned aglycones with conversion coefficient. LC-MS/MS analysis results showed that mutongsaponin C and aponin PJIwere the two main triterpene saponins in Akebiae Caulis,and they shared the same molecular formula. So,the average value of the ratios of the molecular weight between mutongsaponin C and the three aglycones was defined as the conversion coefficient.The three aglycones were separated on an ACE Excel 3 C18-AR column( 4. 6 mm×150 mm,3 μm),and methanol-water( containing0. 04% glacial acetic acid and 0. 02% triethylamine) was used as mobile phase with gradient elution. The detection wavelength was set at 210 nm,and the flow rate was 0. 5 m L·min-1. The results showed that there was a good linearity among the ranges of 1. 053-16. 84,0. 200-3. 200 and 1. 515-24. 24 μg for arjunolic acid,hederagenin and oleanolic acid,respectively. Their average recoveries were97. 90%,97. 50% and 100. 5%,with RSD of 2. 0%,2. 9% and 2. 9%,respectively. The results of methodological investigation met the requirements of content determination. The conversion coefficient was 2. 31. This method is simple and reliable,and can be used for the determination of total triterpenoid saponins in Akebiae Caulis. The assay strategy can be used for the determination of total triterpenoid saponins in other traditional Chinese medicines.

Diuretic effect and material basis of Clematidis Armandii Caulis in rats / 中国中药杂志

To search for the active diuretic fractions of Clematidis Armandii Caulis( CAC) and determine its main active chemical components by using liquid chromatography-mass spectrometry( LC-MS) and diuretic activity evaluation. CAC 75% ethanol extracts and extracts from different polar solvents were orally administered to saline-loaded rats at different doses. 6 h urinary volume,p H and contents of electrolyte Na+,K+and Cl-were measured. The chemical components of the active fractions were separated and identified by ultra performance liquid chromatography-electrospray ionization-quadrupole time of flight-mass spectrometry( UPLC-ESI-Q-TOF-MS/MS) method. As compared with the control group,the urine volume was increased by 44%( P< 0. 01) and 34%( P < 0. 05) in CAC75% ethanol extract 57. 74 and 28. 8 mg·kg-1 groups respectively; the Na+excretion was increased by 52%( P< 0. 01) and 45%( P<0. 05),respectively; while the Cl-excretion was increased by 101%( P<0. 01) and 85%( P<0. 05),respectively. The urine volume,Na+excretion and Cl-excretion were increased by 50%( P< 0. 01),58%( P< 0. 05),and 65%( P< 0. 05) respectively in petroleum ether extract 70. 98 mg·kg-1 group as compared with the control group. While for the n-butanol extract 194. 18 mg·kg-1 group,the urine volume,Na+and Cl-excretion were increased by 42%( P<0. 01),41%( P<0. 05) and 97%( P<0. 01),respectively. The diuretic activity of other fractions was not obvious. There was no statistical difference in K+excretion in all groups. The results of LC-MS analysis showed that six compounds,including two sterols,one chromogen and three fatty acids,were identified from petroleum ether extract.Fourteen compounds,including six triterpenoid saponins,six lignin glycosides,one sterol glycoside and one phenolic glycoside,were identified from the n-butanol extract. All the results suggested that the ethanol extract of CAC had remarkable diuretic activity and its main effective components included sterol,triterpenoid saponin and lignin glycosides.

Establishment of "quantitative analysis of multi-components by single marker" method for phenolic acids in compound Danshen Tablets and its application in mass transfer law / 中国中药杂志

To construct a quality management model for the whole industry chain of compound Danshen Tablets,and quality control system for all key links in the production of compound Danshen Tablets. In this paper,with salvianolic acid B as internal reference substance,three batches of mix standards were prepared,and three sets of relative correlation factors between salvianolic acid B and other phenolic acids were calculated in parallel. Finally,the correlation factors are obtained on average. The quality transfer process was studied by optimizing the concentration of Salvia miltiorrhiza extract. The results showed that RSD among three sets of relative correlation factors ranged between 1. 7%-4. 1%,with no significant difference between the quantitative result of two methods. In addition,the quality transfer study showed that with the rise of the concentration temperature,the content of phenolic acid components changed,which had a significant effect on the salvianolic acid B at more than 80 ℃. It was suggested to rationally control the concentration temperature during the industrial production. The results of this study provide a methodology for the establishment of the quality control system for the whole industry chain of compound Danshen Tablets,and quality control methods for the improvement of the quality of medicinal materials and finished medicine products.

Establishment of QAMS method with dehydroandrographolide as internal reference substance for quality control of Andrographis Herba / 中国中药杂志

To solve the problems of the poor resolution of chromatographic separation,the weak durability of the relative correction factors,and the low accuracy of content determination results in the quantitative analysis of multi-components by single-marker( QAMS) method with andrographolide as the internal reference substance in the existing research of Andrographis Herba,a new QAMS method using dehydroandrographolide as the internal reference substance was established for the first time in this study. This new method can be used to simultaneously determine four diterpene lactones,including andrographolide( A),neoandrographolide( B),14-deoxyandrographolide( C),and dehydroandrographolide( S) through the optimization of chromatographic conditions and systematic investigation of methodology. At the present HPLC chromatographic conditions,four components could be well separated( R > 1. 5),and the methodology validations could satisfy the requirement of quantitative analysis. The relative correction factors( RCFs) of fA/S,fB/S,fC/S were determined as 0. 65,0. 54,0. 78,respectively. The relative standard deviations( RSDs) of their RCFs ranged between 1. 3%-5. 1%,0. 25%-0. 33%,0. 070%-0. 15%,0. 070%-0. 22%,respectively with three brands of HPLC instruments,five brands of C18 column,different flow rates( 0. 9,1. 0,1. 1 m L·min~(-1)),and different column temperatures( 25,30,35 ℃),indicating good durability of the RCFs. The relative retention value( RRV) method was used to locate the chromatographic peak of the components to be determined.The RRVs of rA/S,rB/S,and rC/Swere 0. 44,0. 86,0. 97,respectively. The RSDs of the RRVs ranged between 0. 030%-1. 6% with different HPLC instruments and columns,showing accurate peak location. The present QAMS method and the external standard method( ESM)were both used to determine the contents of four diterpene lactones from Andrographis Herba( 6 batches of medicinal materials and 18 batches of cut crude drugs). The relative errors of the determined content results between two methods were less than 2. 0%. It demonstrated that there was no significant difference in content results between these two methods,indicating good accuracy of the present QAMS method. Therefore,in this study,an accurate and highly durable QAMS method using dehydroandrographolide as the internal reference substance was established for simultaneous determination of four diterpene lactones. This method could be used to effectively control the quality of Andrographis Herba and provide technical basis for the formulation of traditional Chinese medicine industry standard and improvement of the Chinese Pharmacopoeia standard of Andrographis Herba.

Quality evaluation of Poria based on specific chromatogram and quantitative analysis of multicomponents / 中国中药杂志

HPLC specific chromatograms of Poria were established, and the concentrations of 10 triterpenoids(16α-hydroxydehydrotrametenolic acid, poricoic acid B, dehydrotumulosic acid, poricoic acid A, polyporenic acid C, poricoic acid AM, 3-O-acetyl-16α-hydroxydehydrotrametenolic acid, dehydropachymic acid, pachymic acid, and dehydrotrametenolic acid) were simultaneously determined. Chromatographic analysis was conducted on a Welch Ultimate XB C_(18) column(4.6 mm × 250 mm,5 μm). Acetonitrile solution(contain 3% tetrahydrofuran)(A) and 0.1% formic acid aqueous solution(B) were used as the mobile phase with gradient elution at a flow rate of 1.0 mL·min~(-1). The column temperature was 30 ℃ and the injection volume was 20 μL. The experimental data were analyzed by the SPSS 22.0 and GraphPad Prism 7.0. The established triterpenoids fingerprints were specific, and the 10 components were well separated and showed good linearity(r≥0.999 6) within the concentration ranges tested. The mean recoveries were between 98.53%-103.8%(RSD 1.7%-2.7%). The method was specific and repeatable, and could be used for identification and quality evaluation of Poria. The results showed that the contents of 10 triterpenoids were positively correlated with each other. The contents of 10 triterpenoids of samples collected from producing areas were higher than that collected from markets. The total contents of 10 triterpenoids of samples collected from Hubei and Yunnan province were slightly higher than that from Anhui province, but the contents of samples from Anhui province were varied in smaller ranges.

Qualitative and quantitative analysis on non-triterpenoids in Ligustri Lucidi Fructus / 中国中药杂志

In order to improve the quality control level of Ligustri Lucidi Fructus(LLF) and to explore the changes of chemical components after processing,the HPLC method for fingerprint and simultaneous determination of the major polar components in LLF were established. The octadecylsilane bonded silica gel was used as the stationary phase,with acetonitrile as the mobile phase A and0. 2% formic acid as the mobile phase B in a gradient elution procedure at a flow rate of 1. 0 m L·min-1. The detection wavelength was set at 280 nm and the column temperature was 25 ℃. There were 22 common peaks,20 of which were selected from the fingerprint of LLF and its wine-steamed product,respectively,and 14 chromatographic peaks were identified with reference substances. With the same chromatographic conditions,seven components were quantitatively analyzed and the results of system adaptability and methodology investigation all met the requirements of content determination. Compared with the crude LLF,the content of 5-hydroxymethyl furfural and salidroside significantly increased in wine-steamed LLF,while the contents of iridoid glycosides generally decreased. The method provided a basis for quality control of LLF and its processed products as well as the related preparations.

Content change of main components in processed Ligustrum lucidum fruits of different processing time and their characteristic quality standard based on UPLC technology / 中国中药杂志

To investigate the dynamic change law of the main components in Ligustri Lucidi Fructus during the wine-steaming process and attempt to establish the characteristic quality standard of wine-steamed Ligustri Lucidi Fructus by determining the content of salidroside and specnuezhenide using Ultra Performance Liquid Chromatography (UPLC) technology at different processing time points (12, 15, 18, 21, 24 h). The chromatographic separation was performed on Waters Acquity UPLC®BEH C₁₈ column (2.1 mm×50 mm, 1.7 μm) with acetonitrile and 0.2% formic acid aqueous solution as the mobile phase for gradient elution; and the detection wavelength was set at 280 nm; the flow rate was 0.5 mL·min⁻¹, and the column temperature was set at 40 °C. The results showed that the two components were well separated in the above conditions. The salidroside and specnuezhenide showed a good linear relationship within the range of 10.19-326 ng and 49.53-1 585 ng, respectively. Their average recovery was 103.4% and 101.7% with RSD of 0.81% and 0.79%, respectively. With the extension of processing time, the content of specnuezhenide was decreased, while salidroside was gradually increased. For the 27 batches of Ligustri Lucidi Fructus, the content of salidroside was between 0.042 5% and 0.192 4%, and that of specnuezhenide was between 0.829 7% and 5.218 0%. While for the 25 batches of wine-steamed Ligustri Lucidi Fructus, the content of the first one was between 0.229 2% and 1.045 8%, and the latter one was between 0.743 8% and 3.645 4%. As compared with Ligustri Lucidi Fructus, the ratio of specnuezhenide to salidroside was significantly decreased in the wine-steamed Ligustri Lucidi Fructus. According to the experimental results, the quality standard of Ligustri Lucidi Fructus is tentatively fixed as follows: the content of specnuezhenide shall not be less than 0.80%, and the ratio of specnuezhenide content/salidroside content (Sp/Sa) should not be smaller than 15. As for the wine-steamed ones, the content of salidroside should not be less than 0.20%, and specnuezhenide content should not be less than 0.70%; Sp/Sa should not be greater than 8. The method established in this study is simple and reliable, which could be used for the content detection of salidroside and specnuezhenide in Ligustri Lucidi Fructus samples. The characteristic quality standard established in this study could be used to distinguish the Ligustri Lucidi Fructus and wine-steamed Ligustri Lucidi Fructus.