Study On The Chemical Change Of Compound Compatibility Of TCM And Quality Evaluation Of The Compound Traditional Chinese Medicine

Traditional Chinese medicine (TCM) is an important component of excellent Chinese traditional cultures. It has played an indispensable role in the prevention and treatment of diseases in China. Most TCM are administered orally as decoction of compound traditional Chinese medicine in clinics. Compatibility is just the essence of complex prescription of TCM. The components of complex prescription of TCM are not a simple addition of components from each herb. The therapeutic effect is also not a simple merging of each herb. Complex prescription will achieve the effects of detoxication and synergism owing to the interaction of the components.Nowadays, some design modes and contents have been used in studying on the compound compatibility law of traditional Chinese medicine, such as the chemical and efficacy substances of traditional Chinese prescription, the pharmacology of compound Chinese herb, the pharmacokinetics of the compound traditional Chinese medicine, and so on. A lot of authors are interested in pharmacological and clinical area. However, few emphasis is laid on the study of chemical substances, compatibility mechanism and quality evaluation of multi-component in compound traditional Chinese. In particularly, the quality control method which only an individual component is needed to limit its content of compound traditional Chinese medicine is very popular. So it was absolutely insufficient to evaluate the quality thoroughly. Separation and determination of multi-component in compound traditional Chinese medicine are of great importance for quality evaluation and control. The contents and proportion of components can not only reflect the manufacture process level of preparation, but also evaluate and ensure the drug effect.In this paper, the scientific connotation of compound compatibility of TCM was revealed by studying the change of the contents of index components in vitro and in vivo in Long-Dan-Xie-Gan decoction, Gui-Zhi decoction, respectively. And two HPLC methods for determination multi-component in Long-Dan-Xie-Gan pill and Gui-Zhi granules were developed to explain the necessity and feasibility on the multi-component quality evaluation of compound traditional Chinese medicine.From the study, the conclusion can be reached that: (1) the content of aristolochic acid A in Caulis Aristolochiae Manshuriensis was decreased significantly after compatibility in Long-Dan-Xie-Gan decoction (n=6,P<0.05). The content of aristolochic acid A in Caulis Aristolochiae Manshuriensis could be inhibited significantly after combining with Radix Angelicae Sinensis. (2)There were no significant changes about the contents of cinnamic acid and peoniflorin before and after compatibility in Gui-Zhi decoction (n=6,P>0.05).However, the content of total polysaccharide increased when all herbs in Gui-Zhi decoction were put together to decoct (n=6,P<0.01). The content of glycyrrhizic acid in Radix Glycyrrhizae preparata was decreased markedly after combining with Raidix Paeoniae Alba (n=6,P<0.01). (3)The metabolism procedure of cinnamic acid in decoction of individual Ramulus Cinnamomi was essentially in agreement with that of Gui-Zhi decoction. But significant difference appeared in the metabolism process of glycyrrhizic acid in decoction of individual Radix Glycyrrhizae preparata and Gui-Zhi decoction. The parameters Cmax and AUC of glycyrrhizic acid of Gui-Zhi decoction were significant larger than that of decoction of Radix Glycyrrhizae preparata (n=6,P<0.01). The parameters T1/2Ke and Tmax of glycyrrhizic acid of Gui-Zhi decoction were noticeably decreased after compatibility (n=6,P<0.01). (4) Two quick, accurate and reliable HPLC methods were established for determination the contents of multi-component in Long-Dan-Xie-Gan pill and Gui-Zhi granules which can be used for quality control of preparation.PART 1 Study on the Chemical Change of Compound Compatibility of TCM(1) Influence of Compatibility on the Content of Aristolochic Acid A in Long-Dan-Xie-Gan Decoction①The Differences of the Contents of Aristolochic Acid A in Caulis Aristolochiae Manshuriensis before and after CompatibilityObjective: The differences of the contents of aristolochic acid A were analyzed in Caulis Aristolochiae Manshuriensis and Long-Dan-Xie-Gan decoction which was composed of Caulis Aristolochiae Manshuriensis and other traditional Chinese medicines according to TCM theory. The rationalization about the compatibility of Long-Dan-Xie-Gan decoction was explored in some respect.Methods: An HPLC method was applied to determine the contents of aristolochic acid A in Long-Dan-Xie-Gan decoction co-boiling extracts and Caulis Aristolochiae Manshuriensis individual boiling extracts. The differences of the contents of aristolochic acid A were compared.Results: The calibration curve of aristolochic acid A was shown to be linear over the range from 0.76 to 51.0μg·mL-1. The recovery of the method in individual boiling extracts and co-boiling extracts were 98.6% and 96.5%, respectively, the content of aristolochic acid A was lower in co-boiling extracts than that in individual boiling extracts (n=6, P<0.05).Conclusion: The results suggested that the content of aristolochic acid A in Caulis Aristolochiae Manshuriensis can be decreased significantly after compatibility in Long- Dan-Xie-Gan decoction. ②Change of the Content of Aristolochic Acid A in the Decomposed Recipes of Long- Dan-Xie-Gan decoction Objective: To reveal the cause of change about the contents of aristolochic acid A before and after compatibility.Methods: The formula of Long-Dan-Xie-Gan decoction was decomposed in term of TCM theory. An HPLC method was adopted to determine the contents of aristolochic acid A in different experimental groups. The data was analyzed statistically by ANOVA.Results: The content of aristolochic acid A in Caulis Aristolochiae Manshuriensis is decreased after its combining with Radix Angelicae Sinensis and (or) Radix Rehmanniae. Conclusion: A significant decrease on the content of aristolochic acid A in Caulis Aristolochiae Manshuriensis came into being due to compatibility of TCM according to TCM theory.③The Content of Aristolochic Acid A in Caulis Aristolochiae Manshuriensis after its Combining with Radix Angelicae Sinensis and (or) Radix Rehmanniae Objective: To explore the reason why the content of aristolochic acid A in Caulis Aristolochiae Manshuriensis was decreased after its combining with Radix Angelicae Sinensis and(or) Radix Rehmanniae.Methods: The HPLC method was used for determination the contents of aristolochic acid A in different experimental groups. The recovery ratio of aristolochic acid A was calculated by assaying the contents of aristolochic acid A in the dregs and decoction.Results: The recovery ratio of each group was no less than 96.7%. The content of aristolochic acid A of Caulis Aristolochiae Manshuriensis was lowered 41.3% after its combining with Radix Angelicae Sinensis (n=5, P<0.01). Conclusion: The results indicated that aristolochic acid A was not transformed into new substances or decomposed during the boiling. The results demonstrated that the formula of Long-Dan-Xie-Gan decoction is composed scientificly, and the content of aristolochic acid A in Caulis Aristolochiae Manshuriensis can be inhibited significantly after combining with Radix Angelicae Sinensis.(2) Influence of Compatibility on the Content of Cinnamic Acid, Peoniflorin, Glycyrrhizic Acid and total Polysaccharides in Gui-Zhi decoction①Influence of Compatibility on the Content of Cinnamic Acid, Peoniflorin and Glycyrrhizic Acid in Gui-Zhi decoction Objective: To compare the variance of the contents of cinnamic acid, peoniflorin and glycyrrhizic acid before and after compatibility in Gui-Zhi decoction.Methods: The contents of the three index components in different decoction were determined by HPLC.Results: Good linear relationship was shown between peak area and concentration of the three components. The mean recoveries of the three components were all within 98.0%~101.0%. There were no significant changes about the contents of cinnamic acid and peoniflorin before and after compatibility (n=6,P>0.05). But the content of glycyrrhizic acid in Radix Glycyrrhizae preparata was reduced about 50% after its combining with other herbs in Gui-Zhi decoction (n=6,P<0.01).Conclusion: Changes such as chemical, physical and microenvironment in decoction may be lead to the result that the content of some index components in TCM is changed for their compatibility use.②Change of the Content of Glycyrrhizic Acid in the Decomposed Recipes of Gui-Zhi decoctionObjective: To explore the reason why the content of glycyrrhizic acid in Radix Glycyrrhizae preparata was decreased after compatibility in Gui-Zhi decoction.Methods: The Chinese medical formula of Gui-Zhi decoction was discomposed by the orthogonal design method. The contents of glycyrrhizic acid in various groups were determined by HPLC. In the same time, pH in each decoction was recorded to explain the difference.Results: The calibration curve of glycyrrhizic acid was in good linearity over the range of 8.75~280μg·mL-1 (r= 0.999 9), and the average recovery was 99.4%. Influence of factor B (Raidix Paeoniae Alba was used or not) on the content of glycyrrhizic acid was significant (n=8,P<0.05). The pH in different decoction was also of varieties.Conclusion: The content of glycyrrhizic acid in Radix Glycyrrhizae preparata was decreased after combining with Raidix Paeoniae Alba. The results suggested that reduction of pH in decoction was probably related to the loss of content of glycyrrhizic acid.③Comparison of the Contents of total Polysaccharide in Single Medicine Decoction and Mixed Medicine Decoction of Gui-Zhi decoctionObjective: The contents of total polysaccharide in the addition of decoction of individual herb were compared with the co-boiling extracts of Gui-Zhi decoction.Methods: Total polysaccharide was assayed at wavelength of 490 nm by phenol-sulfuric acid method. The content of total polysaccharide in different decoction was analyzed by ANOVA.Results: The content of glucose was determined with linear range of 20~120μg·mL-1.The mean converting factor of polysaccharide was 1.884. In the addition of each herb decoction and the co-boiling extracts of Gui-Zhi decoction, the content of polysaccharide was 16.3 mg·mL-1, 22.1 mg·mL-1, respectively.Conclusion: The content of total polysaccharide increased when all herbs in Gui-Zhi decoction were put together to decoct.(3) Effect of Compatibility on Pharmacokinetics of Cinnamic Acid and Glycyrrhizic Acid in Gui-Zhi decoction Objective: To compare the data of pharmacokinetics parameters of cinnamic acid and glycyrrhizic acid after an intragastric administration of extractive of Gui-Zhi, Gan-Cao and Gui-Zhi decoction in rats.Methods: The rats were given water extractive of Ramulus Cinnamomi, Radix Glycyrrhizae preparata and Gui-Zhi decoction, respectively. The plasma concentration of cinnamic acid and glycyrrhizic acid were analyzed by HPLC. The main pharmacokinetics parameters were obtained by 3P87Results: Both cinnamic acid and glycyrrhizic acid pharmacokinetics in different decoction in rats were shown to fit two-compartent model. The metabolism procedure of cinnamic acid in decoction of Ramulus Cinnamomi was essentially coincidence with that of Gui-Zhi decoction. But significant difference appeared in the metabolism process of glycyrrhizic acid in decoction of Radix Glycyrrhizae preparata and Gui-Zhi decoction. The parameters Cmax and AUC of glycyrrhizic acid in Gui-Zhi decoction were significantly larger than that in decoction of Radix Glycyrrhizae preparata (n=5,P<0.01). The values of T1/2Ke and Tmax of glycyrrhizic acid in Gui-Zhi decoction were noticeably decreased after compatibility (n=5,P<0.01).Conclusion: The absorption of glycyrrhizic acid in Radix Glycyrrhizae preparata was promoted after compatibility in Gui-Zhi decoction. It is rational to apply the compound formulae.PART 2 Study on the Quality Evaluation of the Compound Traditional Chinese Medicine(1) Determination of the Contents of Gentiopicrin, Geniposide and Baicalin in Long-Dan-Xie-Gan Pills Simultaneously by HPLCObjective: A simple HPLC method was established for determination the contents of gentiopicrin, geniposide and baicalin in Long-Dan-Xie-Gan pills.Methods: The separation was achieved on a Hypersil C18 (250×4.6 mm, 5.0μm) column at room temperature. The eluent consisted of acetonitrile (A)-1% glacial acetic acid at a flow rate of 1.0 mL·min-1. A gradient method was set up. The UV detection wavelength was set at 254 nm.Results: The calbration curves of gentiopicrin, geniposide and baicalin were shown to be linear over the ranges of 4.60~147μg·mL-1, 9.05~290μg·mL-1 and 8.40~268μg·mL-1, respectively. Mean recoveries were not less than 98.1%. Conclusion: The analytical results demonstrate that this method is accurate and reliable with good reproducibility. It can be used to indicate the quality of this preparation directly.(2) Assay for the Contents of Peoniflorin, Cinnamic Acid and Glycyrrhizic Acid in Gui-Zhi Granules by HPLC Objective: To establish a simple HPLC method for simultaneously determination the contents of peoniflorin, cinnamic acid and glycyrrhizic acid in Gui-Zhi granules.Methods: The separation was carried out on a Phenomenex C18 column (250 mm×4.6 mm, 5μm) at room temperature. The mobile phase consisted of acetonitrile and 1% glacial acetic acid (pH 3.7) using a gradient program. The analysis was performed at a flow rate of 1.0 mL·min-1 with detection at 315 nm. The injection volume was 20μL.Results: The calibration curves for determination of peoniflorin, cinnamic acid and glycyrrhizic acid were linear over the range of 1.898~121.5μg·mL-1, 1.969~126μg·mL-1 and 2.5~160μg·mL-1, respectively. The average recoveries of peoniflorin, cinnamic acid and glycyrrhizic acid were 99.7%, 99.3% and 98.8%, respectively.Conclusion: A selective and sensitive HPLC method is presented for the rapid quality control analysis of Gui-Zhi Granules.