The Mechanism Research On Processed ESW Enhance The ESW Of "Warming The Kidney And Spleen, Relieving Diarrhea With Astringents"

Decoction pieces processing, Chinese herbal formula are the important characteristics of clinical medication in Traditional Chinese Medicine (TCM). In recent years, the studies on the mechanism of Chinese Herbal Medicine preparation and processing have increased gradually. However, these studies mainly focused on the single drug usage, seldom brought into research of Chinese herbal formula, leading to mutual detachment of the Chinese Herbal Medicine processing study and clinical formula use in TCM. It can not reveal the significance of Chinese herbal medicine processing comprehensively. Under the financial aid of the National’s 11th five-year science and technology supporting plan, our research group have been summarized the warming kidney and spleen, strengthening anti-diarrhea effect of "Er Shen Wan" (abbreviate ESW) which, in the formula, fructus psoraleae (FP) (the sovereign drug of formula "ESW) was stir-baked with salt. Based on the previous study. We try to investigate the synergy mechanism and scientific connotation of TCM processing from the perspective of Chinese herbal formula.OBJECTIVESUnder the financial aid of the National Natural Science Fund Project "Research on Efficacy Increasing Mechanism of Processing for Chinese Medicine in the Formula of ESW" (81274085), we study on the material basis and mechanism of the formula ESW as the astringent on relieving diarrhea. In the previous study, we used spleen kidney Yang deficiency (SKYD) mouse model as the research object, we confirmed that the formula composed salt-processed FP and wheat bran simmer myristica fragrans (MF) has the best anti-diarrhea effect to the model mouse. The most important result is that the effective part from ESW solvent extract for anti-diarrhea is the petroleum ether extract (PE). In this study, we established the rat model conforming to the SKYD syndrome, meanwhile validated and evaluated the effective extract parts of anti-diarrhea effect. Based on the TCM dialectical treatment principle, from "the cause-the SKYD" (essence), "disease-diarrhea" (symptom) two aspects, we researched the mechanism of ESW which composed of the processed FP and MF on "warming the kidney and spleen" and " relieving diarrhea with astringents". It provides a scientific theory for guiding clinical rational drug use.METHODSThe before and after the processing fomula comparative study mode (Efficacy-effective group’s fingerprint-metabolic and excrete process-mechanism of effective constituent) was utilized to discuss the synergistic mechanism of PF and MF processing in formula by multi-angle and multi-level.1. The method of literature research was used to organize the ESW root and intention, the etiology and pathogenesis of attending the Wu Gengxie, and the modern research progress of SKYD animal model. It provided the oretical basis for the mechanism research.2. On the basis of literature, the method of combining the pathology and etiology causes were used, through a number of symptoms index, to evaluate the animal model, to establish the SKYD rats model in accordance with TCM syndrome.3. The SKYD rat was used as the research object. HE staining was used for the main organ pathology detection and analysis, flow cytometry was used to investigate T cell subsets (CD3/CD4/CD8) level of SKYD rats before and after drug administration, in order to explore the enhanced effect of processing in ESW formula on "warm the kidney and spleen".4. ELISA was used to measure the gastrointestinal hormones; 16SrRNA Real-time fluorescent quantitative PCR was used to detect intestinal flora and the number of species, in order to observe the recovery on intestinal flora of SKYD rat when treated with ESW; IHC, RT-PCR and Western blot were used respectively, from the level of organization, gene and protein level, to discuss the influence of ESW on the colon AQP3 expression of SKYD rats, to explore the mechanism of ESW on relieving diarrhea with astringents.5. Through biological function experiment system, the effect of ESW on rabbit gastrointestinal smooth muscle contractions, and the feedback of 4 kinds gastrointestinal smooth muscle stimulant were investigated to clarify the target of ESW on inhibition of gastric contraction.6. We used formula pharmacokinetic method to qualitatively and quantitatively analyze the chemical composition of ESW in the body. We used the research technical route of "The serum fingerprint-Positioning into the blood components-Pharmacokinetic study of the composition from blood" to research the metabolic process and the composition from blood. NMR was used to identify the structure of discharge product in order to clarify up on the influence of the processing of ESW to the pharmaceutical ingredients metabolic process in the body.7. In vitro method was used to verify the effective activity of the blood components, discharge components, the antidiarrheal active ingredient which related to the literature reports of ESW, in order to clarify the active ingredient of ESW as astringent to relieve diarrhea, and its concentration-response relationship. RESULTS1. After SKYD modeling on the rat, Urine D-xylose excretion rate was reduced, NOS activity and cGMP content was increased, the viscera index was increased (Spleen, Kidneys), and kidney had significant pathological features. We consider this method conforms to the symptoms of SKYD diarrhea model.2. ESW (processed petroleum ether extract) P-PE could significantly reduce the diarrhea index of SKYD model rat (P<0.05) and small intestine propulsion rate, it verifies the material basis for relieving diarrhea with ESW is the PE. ESW PE improved pathological state of renal enlargement, severe kidney lesions (such as glomerular capillary plexus closed renal tubular necrosis, interstitial hyperplasia, etc.) caused by SKYD syndrome. In addition, it can significantly increase CD3+and CD3+CD4+ percentage of the peripheral blood, reduce the percentage of CD3+CD8+ (P<0.05-0.01), most importantly, the effect was enhanced when after processing.3. ESW PE can significantly reduce the SKYD model rat MTL、GAS,and increase the content of B.longum and Lac-aci (P<0.01), lower contents of E.coli and E.faecalis (P<0.01), and improve the SKYD rat AQP3 expression in colon, Additionally, AQP3 expression of colon in P-PE group is significantly higher than the (raw ESW petroleum ether extract) S-PE group (P<0.05). Moreover, itshowed significant inhibitory effect (P<0.01-0.05) on normal rabbit duodenum and ileum contraction movement, characterized by the decreaseof average intestinal muscle tension, amplitude, frequency, and the processing ESW exhibited enhanced effect. However, the ESW PE showed obvious antagonistic effect to the excited intestinal muscle caused by HIS.4. The main components in blood of ESW PE are Psoralen^ Isopsoralen and Bakuchiol, the main discharge composition is Bakuchiol. Compared to S-ESW, Psoralen and Isopsoralen in the P-ESW was reached to the Tmax in advance, Cmax was increased; Bakuchiol total discharge rate decreased significantly (P<0.01).5. There were no obvious significance among the Psoralens Isopsoralen and Bakuchiol on the average tension, the amplitude and frequency of the guinea pig ileum smooth muscle compared with the blank group; antidiarrheal active ingredients reported in literatures Methyleugenol and Methyl Iso-Eugenol can significantly reduce the average tension of guinea pig ileum, the amplitude and frequency (P<0.01), their frequency of IC50 on guinea pig ileum smooth muscle movement were 0.949 umol·L-1 and 3.380 μmol·L-1 respectively. CONCLUSIONSESW composed of processed FP and MF attains "warming the kidney and spleen" by improving the SKYD rat abnormal viscera index and renal pathological state, regulating the balance of T cell subgroup level via increasing the percentage of CD3+, CD3+CD4+, reducing CD3+CD8+. In addition, it attains "relieving diarrhea with astringents" by adjusting the SKYD rat abnormal gastrointestinal hormone levels, adjusting the dysbacteriosis via fostering normal flora growth and reducing pathogenic bacteria group, enhancing the colon water absorption via increasing the expression of colon AQP3, and inhibiting gastric contraction through the resistance of H1 receptor.In the ESW formula, FP acts as a sovereign drug, in order to play a role of "Tonic", and MF acts as a ministerial drug which is given priority to the "Antidiarrheal", they both may jointly participate in regulating the balance of intestinal flora. The two processed drugs in ESW could enhance the effect of "warming the kidney and spleen, relieving diarrhea with astringents" may be related to slow Psoralen, Isopsoralen, Bakuchiol elimination and distribution process, prolong the residence time in the body, reduce the total discharge rate of Bakuchiol, then improve the bioavailability.