Study On The Dissolution Of The Precipitation From Gegenqinlian Decoction And The Absorption Changes Of Berberine And Puerarin In The Intestine

TCM " Ge-Gen-Qin-Lian " (GGQL) decoction came from the classical herbal-combined prescription which was established by great doctor Zhang Zhongjing and composed from Radix puerariae, Rhizoma coptidis, Radix scutellariae, and Radix glycyrrhizae Preparata. This prescription is used for treating abdominal pain, diarrhea resulting from damp heat. We have studied on its compatibility, active compounds, quality control, pharmacological effect under the support by National Science Foundation of China、Guangdong province. The synthetic study confirmed that some side effects of Coptis can be reduced and the synthesis curative effect of medicine can be enhanced after drug compatibility. However, The synergy-attenuation mechanism after compatibility waits for further studying.Many Traditional Chinese medicine decoction have the precipitation production after drug matching, and the precipitation production include the main physiological effect ingredient of herbal. So, the precipitation has some pharmacological action. But there is rarely report about the basic research of the precipitation in vivo environment. More and more research proved that some ingredients in traditional Chinese medicine, such as flavanone class, coumarin class, biological alkalis are the P-glycoprotein substrates. Therefore, traditional chinese medicine between some ingredients will possibly have interaction, Thereby, drug compatibility will affect their absorption and the curative effect from each other. Researching intestinal tract absorption rule and the interaction relations of representative chemical composition from the prescription is a new breakthrough point to elaborate the mechanism of the traditional Chinese medicine compatibility prescription.Objective:The compatibility of prescription in Traditional Chinese medicine is the special feature and superiority of Chinese Materia Medica,, and expounding the rule of prescription has the important guiding sense to clinical dialectical treatment. The Traditional Chinese medicine takes orally primarily, and the intestinal absorption is one of the important factors to contribute the bioavailability and the pharmaco-dynamic. In order to study the prescription principles of GGQL, we study the dissolution of GGQL decoction precipitate in gastrointestinal fluid environment, and also the in situ perfusing method in rats was used to research the intestinal absorption changes of berbine and puerarin by combination. The research will lay the groundwork for the further research of GGQL decoction precipitate. And also provides a new breakthrough point to elaborate the mechanism of the GGQL prescription.1、To summarize and analyse the international literatures of GGQL prescription、Traditional Chinese medicine precipitate and the impact of Chinese Medicine on P-gp. Combine with the result of the research on compatibility, active compounds, quality control, pharmacological effect of GGQL prescription, and make a rational analysis on the synergy-attenuation mechanism of GGQL prescription.2、By using simulated gastrointestinal tract environment to study the dissolution of GGQL decoction precipitate in gastrointestinal fluid. Lay a foundation for the basic research of the precipitation in vivo environment.3、By using in situ perfusing method in rats to study the absorption characteristics of berberine and jatrorrhizine chloride. The indexes including the Pa and Ka. 4、To research the intestines absorption changes of berberine and puerarin when combined with each other. Provide an evidence for the synergy-attenuation mechanism of GGQL prescription.Methods:1、Using computer or handworked searches, by full using of CA, BP, CNKI, CMCI, WEB and so on, to get plentiful and all-sided literatures of GGQL prescription、Traditional Chinese medicine precipitate and the impact of Chinese Medicine on P-gp, in order to analyse it’s research-overview.2、In this study, using RP-HPLC to analysis pueraria isoflavones, skullcapflavones, coptis alkaloids and the glycyrr(h)izin, glycyrrhizic acid content in the GGQL Decoction、precipitated and supernate, using simulated gastrointestinal tract environment to study the dissolution of GGQL decoction precipitate in gastrointestinal fluid.3、The intestinal perfusion experiment was performed on rats in vivo to observe the effects of absorption sites and drug concentration on the intestinal absorption characteristics of berberine and jatrorrhizine, and using HPLC to analysis the berberine and jatrorrhizine in the circulating fluid.4、Using the intestinal perfusion experiment on rats in vivo to research the berberine and puerarin mutual influence in the intestines absorption. The berberine and jatrorrhizine in intestines circulation fluid was analysised by the highly effective liquid phase chromatography. The concrete chromatograph condition is as follows:(1)the chromatograph condition of berberine:KromasilC18 column(4.6mm×150mm, 5μm), mobile phase:CH3CN-0.033mol/LKH2PO4 solution (37:63); column temperature:35℃; flow rate:1.0mL/min;λ: 265nm; Inj:20μl. (2)the chromatograph condition of puerarin:KromasilC18 column(4.6mm×150mm,5μm), mobile phase:CH3OH-H2O (25:75); column temperature:25℃; flow rate: 1.0mL/min;λ:250nm; Inj:10μl. Results:1、Applying various kinds of index tools, we collected more than 100 literatures. Through summarizes the literature, GGQL decoction precipitate is possibly one of the factors to contribute the synthesis curative effect. At present not yet discovered the report that through the basic research of GGQL decoction precipitate in vivo environment or from the intestinal absorption aspect to illustrate the compatibility of GGQL prescription.2、The dissolution of the main effect component of GGQL decoction precipitation in gastrointestinal environment2.1 The assay results of various components in GGQL decoction、precipitation and supernate.(DPueraria isoflavones:The content of puerarin and daidzin in supernatant was the highest, while the content of daidzein in precipitation was the highest. The weight of GGQL decoction was similar to the precipitation plus supernate’s. The amount of puerarin and daidzin in precipitation were significantly less than that in the supernatant, but the amount of daidzein in precipitation was significantly greater than that in supernatant.②Coptis alkaloids:The content of alkaloidal in precipitation was the highest, The weight of berberine and palmatine in precipitation was similar to that in supernate, however, the amount of jatrorrhizine in precipitation was significantly larger than in supernatant.③Scutellaria flavonoid:The content of flavonoid in precipitation was the highest, and the amount of them in precipitation was also greater than that in supernatant. The Scutellaria flavonoid in GGQL decoction was similar to the precipitation plus supernate’s.④glycyrrhizic acid and glycyrrhizin:The amount of glycyrrhizic acid and glycyrrhizin in supernatant were larger than that in precipitation. The amount of lycyrrhizic acid and glycyrrhizin in GGQL decoction was similar to the precipitation plus supernate’s.2.2 The dissolution of the main components in precipitation.①Pueraria isoflavones:Compared to the dissolution in water, the dissolution of Pueraria flavonoids of precipitation in gastric juice and intestinal juice increased in varying degrees, in which the dissolution of Pueraria flavones in gastric juice had obvious advantages. After the jointed action by the gastrointestinal fluid, the dissolution of Pueraria flavonoids was significantly increased, and the dissolution rate increased with the time extended.②Coptis alkaloids:Compared to the dissolution in water, the dissolution of Coptis alkaloids in gastric juice were significantly increased. After the jointed action by the gastrointestinal fluid, the dissolution of Coptis alkaloids were significantly increased, and the dissolution rate increased with the time extended.③Scutellaria flavonoid:The dissolution of Scutellaria flavonoid in gastric juice was significantly different from that in water and intestinal juice. The dissolution of Scutellaria flavonoid between water and intestinal juice was insignificant. After the jointed action by the gastrointestinal fluid, the dissolution of Scutellaria flavonoid were significantly increased, and the dissolution rate increased with the time extended.④glycyrrhizic acid and glycyrrhizin:The dissolution of glycyrrhizic acid and glycyrrhizin in gastric juice were significantly large than that in water and intestinal juice, and the dissolution of glycyrrhizic acid and glycyrrhizin in intestinal were also larger than that in water. The dissolution of glycyrrhizic acid in water and gastric juice were larger than glycyrrhizin. After the jointed action by the gastrointestinal fluid, the dissolution of glycyrrhizic acid and glycyrrhizin were significantly increased, and the dissolution rate increased with the time extended.3、The intestinal absorption characteristic of berberine and jatrorrhizine.3.1 The absorption characteristic of berberine and jatrorrhizine in different intestines sections.The Pa and Ka of berberine in the duodenum section were higher than any other intestines section, and the difference had statistics significance (P<0.01);Both the Pa and Ka of berberine decreased according to the turn of duodenum, jejunum, ileum and colon.Compared with the duodenum, the Pa and Ka of jatrorrhizin in the jejunum, ileum and colon were all reduced and had the significance difference (P<0.01 or P<0.05).The absorption of jatrorrhizin in the colonare was least. Both the Pa and Ka of jatrorrhizin also decreased according to the turn of duodenum, jejunum, ileum and colon.3.2 The absorption of berberine and jatrorrhizine in intestinal tract at different concentration.The Pa of berberine and jatrorrhizine enhance along with the concentration increase. There were not saturated absorption phenomenon in the concentration scope of 22-88 mgL-1. Compared with the 22μg/mL group, the Pa of the other two group had the significance difference (P<0.01); The Ka of berberine and jatrorrhizine did not have the significance difference in 22-88 mgL-1 concentration scope (P>0.05); The above two targets indicated that the mechanism of absorption of berberine and jatrorrhizine were the passive diffusion.4、The intestinal absorption of berberine and puerarin in different compatibility.4.1 The influence of puerarin on berberine in intestines absorption, drugs were combined with group:Coptis extract 44μg/mL (mark A) group, Coptis extract 44μg/mL+puerarin 22μg/mL (mark B) group, Coptis extract 44μg/mL+puerarin 44μg/mL (mark C) group, Coptis extract 44μg/mL+puerarin 88μg/mL (mark D) group, Coptis extract 44μg/mL+cyclosporin 120μg/mL (mark E) positive control group。The results showed that compares with A group, the Pa of berberine in B, C, D groups did not have the significance difference (P>0.05),but if compared with E group, the PA of berberine in A, B, C, D groups had the significance difference (P< 0.05 or P<0.01). However, the Ka of berberine among A, B, C, D, E groups did not have the significance difference each other (P>0.05).4.2 The influence of berberine on puerarin in intestines absorption, drugs were combined with group:puerarin sample50μg/mL(mark A)group, berberine 15μg/mL+ puerarin sample50μg/mL (mark B) group, berberine30μg/mL+puerarin sample 50μg/mL (mark C) group, berberine 60μg/mL+puerarin sample50μg/mL (mark D) group, puerarin sample 50μg/mL+cyclosporin 120μg/mL (mark E) positive control group. Result:Compares with A group, the Pa and Ka of puerarin in B group does not have statistics significance difference (P>0.05), but the Pa and Ka of puerarin in C, D, E group had the significance difference (P<0.01) with A group comparison; Compared with E group, the Pa of puerarin in B, C, D groups had the significance difference (P<0.05 or P<0.01), and the Ka of puerarin in C, D group did not have statistics significance difference (P>0.05) with E group comparison.Conclusion:1、There are many kinds of active constituents in GGQL decoction precipitate,and the solubility of each kind of ingredient in precipitate is affected by gastrointestinal environment. The Solubility of precipitate has certain increase along with the time extension in gastrointestinal fluid. The research indicates that the pharmacology effect ingredient in GGQL decoction precipitate can be released in the gastro-intestinal tract, and displays the efficacy after absorption. This research not only completes the bedding for the further research of precipitate in pharmacokinetics and pharmacodynamics, but also has prompted that the precipitate production may be a principle and scientification of the compatibility prescription in Chinese Materia Medica.2、Concentration have no obvious effect on the absorption kinetics of berberine and jatrorrhizine. Both berberine and jatrorrhizine can be better absorbed in the duodenum,jejunum and ileum than in the colon. The absorption of berberine and jatrorrhizine in the intestine is a first-order process through passive diffusion mechanism.3、Three different concentrations groups of puerarin solution were little affect on the Pa and Ka of berberine in intestinal absorption (P>0.05); When berberine with low concentration (15μg/mL), it has little influence to the absorption of puerarin, but the degree and the speed of absorption of puerarin in small intestine can be gradually increased with the concentration increasing of berberine. Berberine could improve the absorbption of puerarin in the intestine, but the effect was not as obviously as the effect of cyclosporin on puerarin.