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Based on the heat-clearing and detoxifying effects of Gentianae Radix et Rhizoma, the network pharmacology is mainly used to predict the potential targets of Gentianae Radix et Rhizoma for anti-inflammatory activity and to perform the experimental verification. A method for detecting the biological potency of Gentianae Radix et Rhizoma based on verifiable targets has been established to provide a reference for improving the quality evaluation and control standards of Gentianae Radix et Rhizoma. High performance liquid chromatography can be used to construct chemical fingerprints of different batches of Gentianae Radix et Rhizoma. Constructing a component-target-disease network of Gentianae Radix et Rhizoma for its anti-inflammatory activity was applied to screen potential anti-inflammatory components and related targets of Gentianae Radix et Rhizoma, and to verify the target of Gentianae Radix et Rhizoma by using biological evaluation methods. Detecting the biological potency of different batches of Gentianae Radix et Rhizoma extracts was used to inhibit COX-2 enzyme activity based the verifiable target cyclooxygenase-2(COX-2). The results showed that different batches of Gentianae Radix et Rhizoma accorded with the pharmacopoeia testing regulations, and the chemical fingerprints have a high similarity(similarity>0.93), suggesting that there is no significant difference in the characteristics of the chemical components. Based on network pharmacology predictions, 18 candidate targets were found to have potential direct interactions with the ingredients in Gentianae Radix et Rhizoma. Among them, the most important target is COX-2. Based on the experimental verification of recombinant human COX-2 protease activity inhibition, Gentianae Radix et Rhizoma can inhibit the COX-2 enzyme activity in a dose-dependent manner. It can function with a low concentration(0.75 mg·mL~(-1)), which preliminarily confirmed the accuracy of network pharmacology prediction. The biological potency detection method of Gentianae Radix et Rhizoma based on COX-2 inhibitory activity was optimized and established. The qualitative response parallel line method was used to calculate the biological potency of anti-inflammatory activity, which ranged from 23.04 to 46.60 U·mg~(-1). For network pharmacology prediction, it can screen and clarify the possible targets of traditional Chinese medicine rapidly, which can guide the establishment of a biological evaluation method for the quality of medicinal materials with related activities. Compared with chemical fingerprints, the biological potency testing can better detect quality fluctuations of traditional Chinese medicine.
Subject(s)
Humans , Anti-Inflammatory Agents/pharmacology , Biological Assay , Drugs, Chinese Herbal/pharmacology , Medicine, Chinese Traditional , Quality Control , RhizomeABSTRACT
Xiaojin pills, the first choice for clinical treatment of breast hyperplasia, were selected to explore the suitability of a bioactivity assay with chemical fingerprinting for the development of an overall quality evaluation assay. The liposoluble and water-soluble fraction fingerprints of Xiaojin pills were established. The ability to inhibit platelet aggregation and the rate of inhibition of cyclooxygenase-2 (COX-2) for 16 batches of Xiaojin pills from several manufacturers was analyzed; the chemical fingerprints of these samples were correlated with the bioactivity and chemical analysis. The animal protocol was approved by the Committee on the Ethics of Animal Experiments of Affiliated Hospital of Chengdu University of Traditional Chinese Medicine Approval, ID: 2018BL-002. Results showed that the antiplatelet aggregation activity of 16 batches was 0.712-1.278 U∙mg-1, with a relative standard deviation (RSD) of 15.4%. COX-2 inhibition was 52.07%-68.95% and the RSD was 8.91%. The results showed that there was little difference in the biological effects of these samples. However, the chemical fingerprint consistency of these 16 batches of Xiaojin pills was poor, and the similarity of nearly half of the samples was less than 0.9. The total peak area of Xiaojin pills was 32.74%-165.37% across samples, showing very poor chemical consistency. In order to explore the reasons for the poor chemical consistency despite good consistency in the biological assays, the fingerprint chromatogram was analyzed by multivariate statistical analysis. The main chromatographic peaks were identified. The results showed that the similarity of Xiaojin pills was mainly determined by the prominent chromatographic peaks 17, 18, 20, 23 and 27 in the liposoluble fingerprints, which were identified from Liquidambaris resina and Angelica sinensis Radix. However, Liquidambaris resina and Angelicae sinensis Radix had almost no anti-platelet aggregation activity or COX-2 inhibitory effect at the normal prescription ratio. As a result, the ability to utilize chemical fingerprints to evaluate the quality consistency of Xiaojin pills is limited. The selection of biological evaluation methods that reflect clinical efficacy could make up for the shortcomings of chemical evaluation methods for quality assessment, and provide new ideas and methods for the overall quality evaluation of complex Chinese patent medicines.
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Objective To study the overall similarity of composition-activity of different kinds of Paridis Rhizoma, and establish a new method for selecting alternative resources. Methods The HPLC method was used to detect the content of polyphyllin I, II, VI, and VII, and pharmacological model of analgesic and hemostatic were used to detect the efficacy. The data were standardized using standard deviation method. Using polyphyllin I, II, VI, and VII at minimum 0.6%, aspirin and Yunnan Baiyao Group analgesic and hemostatic efficacy data as standard controls, we also established component and activity indexes. SPSS 20.0 Software was used to analyze the principal component analysis (PCA) and hierarchical cluster analysis (HCA) of these seven medicinal plants. Results The total content of the main saponins in seven medicinal plants of Paris genus ranged from 0.125% to 1.649%, and the maximum content (1.649%) was found in the P. forrestii while the minimum content (0.125%) was found in P. daliensis. The study also disclosed that P. polyphylla var. yunnanensis has the most active analgesic activity, and the time of bleeding (BT) in mice and the time of coagulation (CT) in mice were the shortest in P. polyphylla. The shortest mice activated partial thrombin time (APTT) was P. forrestii and the longest was P. thibetica. The shortest prothrombin time (PT) in mice was P. vietnamensis. The longest BT in mice was P. daliensis. The longest CT and PT in mice was P. polyphylla. PCA and HCA results showed that P. polyphylla var. yunnanensis, P. vietnamensis and P. thibetica were in the same class, while P. pubescens, P. daliensis, P. polyphylla, and P. forrestii were in another class. It is indicated that the overall similarity of component-activity of P. vietnamensis, P. thibetica, and P. polyphylla var. yunnanensis was higher, thus they can be considered as alternative sources. Conclusion The quality relations of different kinds of Paris genus were evaluated by the overall similarity of the component and activity index, which provides ideas and methods for the search of the replacement resources of the endangered plants.
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The aim of the present study was to explore a sensitive, stable and reliable method for evaluating the phagocytosis, in which RAW264.7 macrophages engulfed GFP-Escherichia coli was tested by high-content screening technology. The study was conducted to optimize the method in evaluation of traditional Chinese medicine in the promotion of macrophage function. By testing macrophages at different ratio of bacteria to cells (multiplicity of infection, MOI), and at different incubation time, we optimized a high content screening method and the experimental parameters to determine the impact of bacteria in macrophages (fluorescence intensity index = be swallowed bacteria/macrophages). The method was used to determine whether Dendrobium moniliforme (DM) have effects on macrophage phagocytosis. The results show that the index has a positive relationship with MOI values, and the highest index was observed at incubation time of 1.5 h. The optimized conditions was 1×104 cells/well with a MOI of 50:1 (bacteria:cells) with incubation of 1.5 h. Under this condition, the relative standard deviation (RSD) was less than 10% in the precision test. Using the method to detect DM regulating macrophage phagocytosis experiment results showed that in 0.31-2.50 g·L-1 concentration range, DM has a dose-response effect in promoting phagocytosis. We successfully established the method for evaluation of macrophage phagocytosis, and proved the activity of DM in promotion of macrophage phagocytosis.
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The biological potency assay and chemical fingerprint chromatogram were applied to quality evaluation of rhubarb. Using the biological potency as indicators, we evaluated the differences in quality of multiple batches of rhubarbs and related products. Using the platelet aggregation analyzer, we determined platelet aggregation rate in the different rhubarbs preparations, and calculated the biological potency based on the simplified probit principle. UPLC was adopted to establish the fingerprint spectra for rhubarbs. The spectral efficiency correlation analysis between chromatograms and biological potencies were conducted using the double variables of SPSS 22.0 software. We used three chemical composition to verify the potency. The biological potency results suggest that Rheum palmatum has a more potent activity than Rheum tanguticum, and wine-treated rhubarb had a higher potentcy than charred. We identified 10 elements in the Fingerprint Spectrum. The relevant elements including rhein-8-O-β-D-glucoside, emodin-8-O-β-D-glucoside and rhein have the strongest activity in the inhibition of platelet aggregation. In conclusion, this study provides a analytical method for rhubarb biological potency based on determination of the maximum antagonism rate model. The rhein may be the effective substance. It may serve as a reference in the quality control of wine processed rhubarb products.
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This study attempts to evaluate the quality of Chinese formula granules by combined use of multi-component simultaneous quantitative analysis and bioassay. The rhubarb dispensing granules were used as the model drug for demonstrative study. The ultra-high performance liquid chromatography (UPLC) method was adopted for simultaneously quantitative determination of the 10 anthraquinone derivatives (such as aloe emodin-8-O-β-D-glucoside) in rhubarb dispensing granules; purgative biopotency of different batches of rhubarb dispensing granules was determined based on compound diphenoxylate tablets-induced mouse constipation model; blood activating biopotency of different batches of rhubarb dispensing granules was determined based on in vitro rat antiplatelet aggregation model; SPSS 22.0 statistical software was used for correlation analysis between 10 anthraquinone derivatives and purgative biopotency, blood activating biopotency. The results of multi-components simultaneous quantitative analysisshowed that there was a great difference in chemical characterizationand certain differences inpurgative biopotency and blood activating biopotency among 10 batches of rhubarb dispensing granules. The correlation analysis showed that the intensity of purgative biopotency was significantly correlated with the content of conjugated anthraquinone glycosides (P<0.01), and the intensity of blood activating biopotency was significantly correlated with the content of free anthraquinone (P<0.01). In summary, the combined use of multi-component simultaneous quantitative analysis and bioassay can achieve objective quantification and more comprehensive reflection on overall quality difference among different batches of rhubarb dispensing granules.
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The contents of schisandrol A, schisandrol B, schisantherin A, schisandrin A , schisandrin B, schisandrin C in Schisandrae Chinensis Fructus (SCF) were determined simultaneously by HPLC. Collect 100-seed weight, color, pulp content, longitude and latitude of SCF of different batches were collected. SIMCA-P and SPSS were applied to make PLS-DA analysis of 24 batches of SCF and correlation analysis of relevant parameters. According to the 13 parameters, SCF from three different places of origin could be distinguished effectively. It was found that the content of chemical component of SCF increased with latitude and longitude first, and then decrease. The results provide some theoretical basis for study of SCF genuineness and traditional method of identifying just from experience.
Subject(s)
China , Chromatography, High Pressure Liquid , Drugs, Chinese Herbal , Chemistry , Fruit , Chemistry , Quality Control , Schisandra , Chemistry , ClassificationABSTRACT
The high quality and stability of Chinese materia medica (CMM) are the key factors to ensure clinical curative effect, and the ultimate goal of quality control (QC) for CMM is to ensure clinical efficacy. It is of significance how to establish a method for QC which could be related to clinical efficacy. According to the current situation and major problems of the QC for CMM, we first propose the efficacy-equivalent (EE) of CMM, and the research approach about EE has been elucidated, hoping to provide the reference for the establishment of the method of QC for CMM.
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<p><b>OBJECTIVE</b>Three long QT syndrome(LQTS) pedigrees were brought together for genetic diagnosis by using short tandem repeat(STR) markers.</p><p><b>METHODS</b>Genomic DNA was extracted from blood samples. STR markers (D7S1824, D7S2439, D7S483, D3S1298, D3S1767, D3S3521) in or spanning the HERG and SCN5A gene were amplified; the haplotype analysis for LQTS was performed.</p><p><b>RESULTS</b>Clinical diagnosis showed that 15 are LQTS patients (3 died) and 11 are probable patients. Linkage analysis showed that LQTS patients are linked with the SCN5A gene in family 1, HERG is linked with the disease in family 2 and 3. Fourteen gene carriers were identified, 2 patients and 7 probable patients were excluded.</p><p><b>CONCLUSION</b>Linkage analysis using STR markers can serve as useful tool for presymptomatic diagnosis.</p>