Compatibility And Pharmacokinetics Of Citrus Aurantium And Paeonol In Treating IBS Rats With Liver Depression And Spleen Deficiency

ObjectiveIrritable bowel syndrome is a functional gastrointestinal disorder disease. Zhishi-baishao herb-pair was used on the treatment of liver stagnation and spleen deficiency. By explore the changes in the chemical composition of zhishi-baishao herb-pair, the dose-effect relationship, the change of the pharmacological effects and metabolic mechanisms and compatibility discipline in vivo, it will not only further to validate compatibility-relationship between zhishi and baishao and also play a role in finding the best compatibility proportion. Furthermore, by finding out the relationship of dose-efficiency and time-efficiency, thus will to bring into full play the effect of drugs, and it is also provide the basis for the clinical treatment of IBS with stagnation of liver and spleen deficiency, development of new herb-pairs and drugs.Methods1. The study on material basis of zhishi-baishao herb-pairAccording to the complexity of zhishi-baishao herb-pair, chemical compositions of chromatographic mobile phase, proportion detected wavelength, chromatographic column temperature were optimized and applied to the whole party HPLC unified analysis method, and made instrument precision, stability and method repeatability investigtion samples. To establish a reliable method for controlling the quality of zhishi and baishao basing on cluster analysis and principal component analysis, HPLC was applied to establish the chromatographic fingerprint. The HPLC with UV switch contents determination method of Flavonoids Components (Naringin, Hesperidin, Neohesperidin) and Paconiflorin was established. Using multiple index comprehensive evaluation method and HPLC, the content of Naringin, Hesperidin, Neohesperidin and Paconiflorin were prepared by different decoction methods and different compatibility ratio, determined optimum decoction method and compatibility ratio. To improve purification process of active ingredients(Naringin, Hesperidin, Neohesperidin and Paconiflorin), single-factor、test orthogonaltest and central composite design-response surface methodology were adopted to optimize preparation conditions.2. Researching on the major pharmacological effect of zhishi-baishao herb-pair on the different compatibility ratioThe couplet medicines zhishi-baishao in Zhi-Shao-San were taken as study object, the compatibility ratio of zhishi-baishao was duplication floating up and down basing on the ratio of the couplet medicines reported in the literatue. Design into three ratios of1:1,1:2and2:1three compatibility groups. With the side effects, gastrointestinal effect、analgesia effect and behavioral effect as related indicators, studing on the major pharmacological effect of couplet medicines zhishi-baishao on the different compatibility ratio.Researching on pharmacodynamic test of gastrointestinal effectIrrigation stomach to IBS rats with different compatibility ratio, the small intestinal propulsion of rates, the amount of defecation in24h and the abdominal withdrawal reflex were observed as pharmacologic index.Researching on pharmacodynamic test of analgesia effectObserve and study the analgesia effect of different compatibility ratio of Zhishi-baishao herb-pairs, by using hot-plate analgesia test and writhing analgesia test with normal mice.Researching on pharmacodynamic test of behavioral effectIrrigation stomach to IBS rats with different compatibility ratio, Behaviors within an open field of IBS rats were observed as pharmacologic index.3. The correlation between the chemical components and pharmacologic actions of the compatibility of Zhishi-BaiShao herb-pairIBS model was established by intracolonic instillation of acetic acid and stimulated with forceps clip on tails and fetter. The small intestinal propulsion of rates, the amount of defecation in24h and the abdominal withdrawal reflex were observed as pharmacologic index. Design different compatibility and different decocting methods of zhishi-baishao herb-pair on HPLC determination, and the correlation of11chromatographic peaks with pharmacologic and index was analyzed by Grey Relational Analysis and Partial Least Square Regression.4. The study on Zhishi-baishao herb-pair on the kinetics of in vitro and in vivo drug index components in rats(1) Development of a HPLC-MS/MS method for simultaneous determination of four analytes of Zhi-Shao-San decoction in rat plasma and its application to a comparative pharmacokinetic study in normal and irritable bowel syndrome ratsEstablish and application flavonoids and paeoniflorin ingredients like rats plasma samples processing method and HPLC-MS/MS conditions, detection big pile of gas fluids Fried liquid of flavonoids and paeoniflorin into blood, through the analysis of rat medicated knowable, application of detection plasma pharmacokinetic software components DAS2.1calculation pharmacokinetics parameters (AUC、Tmax、T1/2、Cma、CL、MRT0-t). A comparative study was designed and conducted to compare the pharmacokinetic difference of paeoniflorin, naringin, hesperidin, and neohesperidin after oral administration of Zhi-Shao-San decoction to normal rats and IBS rats induced by acetic acid and restraint stress.(2)Metabolism research of active ingredients of zhishi-baishao herb-pair in intestinal floraIntestinal flora of rats were cultured at37℃in vitro. After being extracted by acetic ether, the main metabolites of flavonoids components and paeoniflorin were separated and quantified by HPLC. It was investigated the degradation characteristics and possible covariates of flavonoids components and paeoniflorin when being incubated with rat faeces.(3) Metabolism research of active ingredients of zhishi-baishao herb-pair in urine and faeces ratsApplied HPLC unified analysis method, the contents of flavonoids components and paeoniflorin in urine and faeces of rats were detected.Results1.The study on material basis of Zhishi-baishao herbA HPLC method was developed for determination of four indicative components (paconiflorin, naringin, hesperidin and neohesperidin) in decoction of Zhishi-baishao herb. Method:Syncronis C18(4.6mm×250mm,5μm) was adopted and the mobile phase was0.3%phosphoric acid solution (A)-acetonitrile (B) with gradient elution at a flow rate of1.0mL·min-1and the detection wavelength was230nm (0-30min) for paconiflorin,283nm(30-60min) for naringin, hesperidin, neohesperidin. The method was dependable, simple, practical, and may be use to the quality controll of zhishi-baishao herb.The optimized purification process for general flavones was following:sample concentration was3.5mg/mL, ethanol concentration of eluent was88%and eluent volume was72mL. The purity of general flavones in Fructus Aurantii Immaturus was79%. The optimized purification process of paconiflorin weas the following:adsorption time was1h, ethanol concentration of eluent was80%and eluent volume was80mL. The recovery of paconiflorin was91.78%. The optimum process, simple in methodology, yet yields reliable results.The mutual model of zhishi-baishao herb-pair was established. There were11peaks was the comom peaks of HPLC fingerprints in10batch of samples and4peaks of them were identified.The best ratio of zhishi-baishao herb-pair is1:1. It was shown by direct analysis that10times of water, boiled decocting in40minutes twice after soaked in30minutes was the optimum method.2. Researching on the major pharmacological effect of couplet medicines zhishi-baishao on the different compatibility ratio Researching on pharmacodynamic test of gastrointestinal effectBy doing pharmacodynamic studies in rat found that different compatibility ratio of Zhishi-baishao herb-pairs was significantly decreased defecation number, increased the growth rate of body weight, restrain the rate of the small intestine to promote. The scores of abdominal withdrawal reflex under the dilatation pressures of20,40,60and80mm Hg were all significantly higher in the model group than in the normal group. The significant difference of treatment group compared with the model group showed that the Zhishi-baishao herb-pairs can reduce diarrhea irritable bowel syndrome model rats visceral nerve hypersensitivity.Researching on pharmacodynamic test of analgesia effectZhishi-baishao herb-pairs produced significantly analgesia in hot-plate test and writhing test. Zhishi-baishao herb-pairs prolonged the latency of pain response of mice in hot-plate test. All ratios of Zhishi-baishao herb-pairs showed anti-nociceptive effects, but with significant differences among Zhishi-baishao herb-pairs1:2.Researching on pharmacodynamic test of behavioral effectIn the open field test, the total score of model rats decreased significantly, compared with the normal rats. Those of Baishao group and Zhishi-Baishao1:2increased significantly, compared with model group.3. The correlation between the chemical components and pharmacologic actions of the compatibility of zhishi-baishao herb-pairChromatographic peak1,2,3,5,7,8,9,10and11had a close correlation with the pharmacologic index. Based on Grey Relational Analysis determining contribution degree to the peaks, Naringin, Hesperidin, Neohesperidin and partial unknown compounds had high close correlation with pharmacologic index(correlation degree>0.80correlation degreecorrelation degreecorrelation degree). The compatibility of zhishi-baishao herb-pair on improvement of IBS rat gastrointestinal tact, related to their containing a wide range of chemical substances.4. The study on Zhishi-BaiShao herb-pair on the kinetics of in vitro and in vivo drug index components in rats(1) Development of a HPLC-MS/MS method for simultaneous determination of four analytes of Zhi-Shao-San decoction in rat plasma and its application to a comparative pharmacokinetic study in normal and irritable bowel syndrome IBS ratsTo our relief, the validated method has been successfully applied to comparing pharmacokinetic profiles of analytes in rats plasma. The pharmacokinetic parameters of naringin, hesperidin, neohesperidin, and paeoniflorin in plasma can be instructive for clinic application. After ig of Zhishi-shaoyao-san dection6g/Kg, AUC0-∞(μg·h/L) were723±190、782±201、4403±2044,11670±2769; MRT0.t (h) were6.1±0.97,7.8±1.0、12.6±2.4.7.2q0.38;T1/2(h)were1.9±0.63、4.6±3.6,13.2±6.4、2.9±0.99; Tmax(h) were2.2±0.82、3.4±2.2、2.5±1.1、3.4±0.73; Cmax(μg/L) were340±228、158±73.632±232、1220±345; CLz(L/Kg/h)were8.8±2.1、8.2±2.3、1.7±0.99、0.51±0.12in blood plasma of normal rats. The chemical components from compatibility of Dachengqi decoction were defecated through stools of rats. AUC0-∞(μg-h/L) were1229±374、1446±493、7757±3101、13508±6142; MRT0-t(h)were8.4±1.1、7.3±4.2、10.6±0.78、8.8±0.67; T1/2(h) were 1.8±0.16、4.5±4.7、9.9±3.7、2.4±1.4;Tmax(h) were1.1±0.38、1.3±0.27、1.5±0.44、8.3±5.7; Cmax(μg/L)were349±256、673±556、1455±454、963±317; CLz(L/Kg/h) were5.4±1.9、4.5±1.3、0.93±0.47、0.58±0.36in blood plasma of IBS model rats with stagnation of liver and spleen deficiency. The results showed that the absorptions of naringin, hesperidin, and neohesperidin in IBS group were all significantly higher than those in normal group and no obviously difference for paeoniflorin between two groups, which is helpful for improving clinical therapeutic efficacy and further pharmacological studies of Zhi-Shao-San.(2) Metabolism of Zhishi-baishao herb-pairs active ingredient in intestinal floraThe main metabolites of Zhishi-baishao herb-pairs were separated and quantified by HPLC. After compatibility of zhishi-baishao herb-pair, however the metabolic rates of hesperidin and neohesperidin were reduced in normal mice, but inhibited in IBS rats. The metabolisms of naringin, hesperidin and neohesperidin were inhibited in all rats.4kinds of active ingredient were fastest metabolized in zhishi-baishao1:1group and had the differences between normal mice and IBS mice.(3) Excretion research of Zhishi-baishao herb-pairs in ratsHesperidin and neohesperidin mainly eliminated from faeces way, however little paconiflorin eliminated from urine way. To ig to Zhishi-baishao herb-pairs, then dectecting24h urine and feces of rats in the analysis, which can display are naringin, hesperidin, neohesperidin, hesperitin, neohesperitin and paconiflorin in faeces, however can not display are naringin and paconiflorin. Overall, quercetin yuan form the amount of eliminated more than nucleoside proportion.Conclusion1. The stable, reliable and reproducible methods have established the entire party HPLC. Select naringin, hesperidin, neohesperidin and paconiflorin as a quality control and process optimization index has certain scientific. This study in traditional Chinese medicine theory and practice will be under the guidance of analytical chemistry, science of Chinese materia medica, statistics and computer technologies combining, further clarify big material base of zhishi-baishao herb-pair discussed the compatibility of the mechanism.2. The results indicate that could inhibit defecation, increase movement of intestine, reduce visceral hypersensitivity and show the antianxiety effect. Prove that Zhishi-baishao herb-pairs on the analgesic effect of acetic acid writhing test and hot-plate test. In conclusion, all of these prove Zhishi-baishao herb-pairs on the effect of spleen deficiency and liver depression remission in IBS rats.3. The compatibility of Zhishi-baishao herb-pair on improvement of IBS rat gastrointestinal tact, related to their containing a wide range of chemical substances. Select naringin, hesperidin, neohesperidin, and paeoniflorin as parts of a quality control, process optimization index and efficacy material foundation has certain scientific.4. The stable, controllable reproducible and accurately HPLC conditions and processing methods of biological samples were established. It was found that dynamic changes of different compatibility caused by excretion and absorption into the blood and intestinal bacteria metabolism process were positive correlation. From the absorption metabolism and excretion confirmed the rationality of Zhishi-baishao herb-pairs.This study in traditional Chinese medicine theory and practice will be under the guidance of TCM, pharmacological, Statistics and computer technologies combining bearing, further clarified Zhishi-BaiShao herb-pair material base and discussed the compatibility of the mechanism. It compatibility and diagnose the methodology ofthe study provides theory basis.