| The compatibility application of Chinese herbal is the main form of Chinese medicine treatment. In the process of clinical, the mixing of two or more drugs is called compatibility; while, when several drugs are mixed by certain regulation and proportional component, and there are processing and using methods for relatively determined diseases is called the formula. The formula is the development of drug compatibility, and also is an advanced form of the application of drug compatibility. However, the complex chemical compositions of the traditional Chinese medicine and the change of their quality and quantity with the process of the compatibility bring some difficulties for the study of the mechanism of traditional Chinese medicine compatibility or formula. Thus, this prevents the development and application of Chinese medicine. In this paper, the chemical constituents from the compatibility of Panax ginseng and Veratrum nigrum which comes from "eighteen incompatible medicaments of traditional Chinese medicine" and the Gancaofuzi decoction are analyzed by modern analytical methods and technology. And the intestinal transport and pharmacology are also studied. The main contents include several aspects as follows:1. The isolation and identification of chemical constituents from the compatibility of Panax ginseng and Veratrum nigrum.The method for separation of Panax ginseng and Veratrum nigrum separately at the positive ion mode by HPLC/UPLC-ESI-MS was established. 12 kinds of ginsenosides and 8 kinds of alkaloids were detected and identified by the precise molecular weight and the pieces obtained from secondary ion mass spectrometry according to the regular fractures in the related literatures. After compatibility with large proportion of Veratrum nigrum, the peak area of several ginsenosides reduced so that the dissolution of these ginsenosides was cut back. Thereby the pharmacological effects of ginsenosides were decreased, which people involved that Veratrum nigrum has "reduce efficient" role to Panax ginseng. On the other side, we found that when a large quantity of Panax ginseng was added to Veratrum nigrum, the dissolution of alkaloids increased, thereby the toxicityof Veratrum alkaloid increased. That is to say, Panax ginseng has "increasing toxic" role to Veratrum nigrum. These results accorded to incompatibility principle from "eighteen incompatible medicaments of traditional Chinese medicine". And the results laid the material foundation for the study of transport mechanism in vitro of the main chemical ingredients from this "anti-drug couple".2. The transport study of Panax ginseng and Veratrum nigrum before and after compatibilityThe Caco-2 monolayer model was established, which is simulated to the human intestinal epithelial cells, to study the transport of main chemical ingredients from Panax ginseng and Veratrum nigrum combined with HPLC/UPLC-ESI-MS. Firstly, the effects of time and concentration on the ginsenosides and Veratrum alkaloids from both absorption and efflux were studied. The results showed that the two-way transport of ginsenosides and alkaloids are time-dependent and the absorption is concentration-dependent. The efflux of ginsenoside Rb1, Rc, Rb2, Rd and Veratrum alkaloid did not have a concentration-dependent manner, which may be the role of some kind of transporter protein substrates. By the study of recovery from absorption transport, we found that ginsenosides and other six kinds of Veratrum alkaloids showed high recovery at about 100% in addition to jervine and veratramine, suggesting that it may be related to cell specific binding or enzyme system in the cell metabolism. After compatibility with different proportions of Veratrum nigrum, the bioavailability of ginsenosides decreased, indicating that Veratrum nigrum prevent the pharmacological effects of ginsenosides. And when a certain proportion of Panax ginseng was added to Veratrum nigrum, the bioavailability of Veratrum alkaloid increased, indicating that the toxic effects of Veratrum alkaloid can be promoted by Panax ginseng. The results proved the incompatibility principle again from the perspective of transport.3. Study on the saponins in Gancaofuzi decoctionThe quantity changes of saponins from single herb extract, herb-pair extract and Gancaofuzi decoction was detected by the modern analytical methods and techniques. Firstly, the total contents of saponins were detected by UV spectrophotometry. We found that the total saponins contents increased when R.glycyrrhizae was compared with A. macrocephalae Rhizoma and Cinnamomum cassia Presl. While, Radix AconitiPraeparata could decrease the total contents of saponin from R.glycyrrhizae. Secondly, the content of glycyrrhizin was detected by HPLC method. A. macrocephalae Rhizoma and Cinnamomum cassia Presl could promote the dissolution of glycyrrhizin from R.glycyrrhizae as well as Radix Aconiti Praeparata, but the glycyrrhizin took part in attenuation with alkaloid when compared with Radix Aconiti Praeparata. Finally, several kinds of saponins from R.glycyrrhizae were isolated and identified by HPLC-ESI-MS method. And we found that A. macrocephalae Rhizoma and Cinnamomum cassia Presl could increase their content, but Radix Aconiti Praeparata could reduce their content.4. The purification process and antioxidant study of saponin constituents in Gancaofuzi decoctionD101 macroporous resin was applied to attain the most pure and the highest recovery of saponin constituents in Gancaofuzi decoction and its purity achieved about 49%. The results of antioxidant experiment showed that compared with the single decoction of R.glycyrrhizae, Cinnamomum cassia Presl and Radix Aconiti Praeparata could improve the antioxidant ability of saponin constituents, suggesting that the phenolic acids composition from Cinnamomum cassia Presl and the alkaloids from Radix Aconiti Praeparata play synergistic antioxidant effect. It was also observed that the antioxidant capacity of saponin constituents in Gancaofuzi decoction was better than Gancaofuzi decoction. That is to say, the saponin constituents have good antioxidant capacity, and play important role on the treatment of rheumatic and rheumatoid diseases.5. The transport study of saponin in Gancaofizi decoctionComparing the transport of glycyrrhizin G2 and glycyrrhizic acid in the mixture of two herbs decoction, it is showed that A. macrocephalae Rhizoma, Cinnamomum cassia Presl and Radix Aconiti Praeparata could decrease the bioavailability of glycyrrhizin G2, but increase the bioavailability of glycyrrhizic acid. While comparing the transport of glycyrrhizin G2 and glycyrrhizic acid in the herb-pairs boied together, it is showed that A. macrocephalae Rhizoma, Cinnamomum cassia Presl and Radix Aconiti Praeparata could decrease the bioavailability of glycyrrhizin G2. And A. macrocephalae Rhizoma increase the bioavailability of glycyrrhizic acid, while Cinnamomum cassia Presl and Radix Aconiti Praeparata decrease the bioavailability of glycyrrhizic acid.Different proportions of saponins constituents, flavonoid constituents and alkaloidsconstituents in Gancofuzi decoction were mixed when we studied the transport of saponins constituents in the Caco-2 cell monolayers. The flavonoids constituents could prevent the bioavailability of the four kinds of saponins by increasing their efflux in the transport process. And the alkaloids constituents could promote the bioavailability of the four saponins.At last, we also mixed the main standard in Gancaofuzi decoction to investigate the interactions of the three kinds of constituents. The results showed that glycyrrhizin and glycyrrhizin could decrease the bioavailability of glycyrrhizic acid, but the five kinds of alkaloids could increase the bioavailability of glycyrrhizic acid.6. The pharmacological study of saponin in Gancaofizi decoctionAnimal models were used to study the pharmacological effects of the sigle drug, drug-pairs, Gancaofuzi decoction and saponins in Gancaofuzi decoction. The results showed that the sigle drug, drug-pairs, Gancaofuzi decoction and saponins all have effects on analgesic, anti-inflammatory and immunomodulatory. In the study of the sigle drug and drug-pairs, we investigated the influence of A. macrocephalae Rhizoma, Cinnamomum cassia Presl on R.glycyrrhizae and Radix Aconiti Praeparata, and their mutual influence. While, in the study of the saponins in Gancaofuzi decoction, we found that the saponins were superior to Gancaofuzi decoction on the aspects of suppression peripheral and central pain, suppression foot swelling induced by acute inflammation and rat adjuvant arthritis model, and inhibition TNF-α, IL-1 and IL-2 secreted by mouse macrophage. We also found that when mixed with the flavonoids or alkaloids constituents, the pharmacological effects of the saponins would be changed in varying degrees. This may be related to the bioavailability of the saponins influenced by the flavonoids or alkaloids constituents.The above results laid the material foundation for the research of mechanism of compatibility and formula, and provided a new research idea for the Traditional Chinese Medicine. |
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