| Study background and objectiveChronic nonspecific ulcerative colitis (UC) is a kind of common disease of digestive system. It's etiopathogenisis still remains unclear. It mainly involves rectum and colon sigmoideum, even other parts of colon and the whole colon. It's clinical manifestation are abdominal pain, diarrhea, hemafecia, rectal tenesmus, bloody purulent stool and inordinate bowel evacuation habit etc. intestinal tract organ involved symptom. Tendency of this disease is chronic, iterative and alternative attack, it's course comparatively long. At present it has been listed as one of modern stubborn diseases by World Health Organization.Usually modern medicine think UC relates to heredity, immunization, infection and consciousness and so on many aspect factors. Effect of immunization factor at the morbility of UC has been confirmed, and become a study hot spot. In modern pharmacology's view TCM of invigorate the spleen and benefiting vital energy such as Astragalus mongholicus, atractylodes macrocephala and licorice root have favourable immunological regulation effect. Especially chemical effective parts Astragalus mongholicus saponin and licoflavone have obvious immunological regulation effect. According to pharmacology experiment they have favourable pharmacologic action on anti-peptic ulcer and protection for gastrointestinal tract tunica mucosa. We can these reports that they are been used as immunomodulator and to treat peptic ulcer in clinic. In our early study we found that Astragalus mongholicus and licoflavone have the effect of promoting proliferation of small intestine epithelium cell (EEC-6), extraction of Baizhu has effect of inducing differentiation to maturity of it. Astragalus mongholicus has effect of regulating secretion of cytokine of IEC-6. So we think these three effective parts have possible curative effect on UC, and according to our initial animal experiment this has been confirmed. One of four great scholars in Jinyuan dynasty Liu Wansu said: "Baizhuhuangqi soup, after administrating previous formula. Although dysentery has been depressed, attemper it with this formula." The three TCM are atractylodes macrocephala, Astragalus mongholicus and licorice root in this soup. It is thus evident that it is long-standing to use these three traditional Chinese drug to treat dysentery.Now there are many TCM and Western patent medicine for treating UC. But they have a common defect of not precise target, large drag consumption and unreliable curative effect. At present there aren't compound preparation of TCM effective parts internal and overseas. Therefore, in order to study pharmacology of astragali, atractylodes macrocephala and licorice root active component infurther step, search for reasonable extraction technology of effective parts, develop safe and reliable,exact curative effect new drug for treating UC fit to clinic, we plan to use advanced colon-target andadhesion releasing technology to make up complex prescription colon-target capsule preparation ofabove-mentioned three effective parts. This will have important significance for developing new drugstudy on treating UC with TCM.Study method1. pharmaceutical research1.1 study on extraction and abstraction technology of Astragalus mongholicus saponinAdopt ethanol backstreaming to extract Astragalus mongholicus saponin, compare extraction percentage of different concentration ethanol. Adopt resin adsorptive process to depurate it, inspect resin dynamic adsorption curve, sample size, concentration and dosage of eluting ethanol and concentration of eluting lye and so on technology parameter. Determine the best extraction and abstraction technology. And inspect analytical method of Astragalus mongholicus saponin.1.2 study on extraction and abstraction technology of glycyrrhizae total flavonoidsInspect various kinds of quantitative analysis of glycyrrhizae total flavonoids, include globulariacitrin- aluminum nitrate colouration system, naringin- NaOH colouration system and naringin direct scanning method. Compare different extraction method of licoflavone to determine the best extraction technology. Adopt macroporous resin adsorptive process to depurate total licoflavone, and inspect resin dynamic adsorption curve, sample size, concentration and dosage of eluting ethanol and so on technology parameter to determine the best adsorbing depuration technology.1.3 study on extraction and abstraction technology of atractylodes macrocephala glycoseInspect the two quantitative analysis of glycose which are UV spectrophotometer and high efficiency liquid chromatography, extract monosaccharide component in AM with water extraction. Depurate AM Ex with resin bleaching method. Inspect different resin's bleaching capability and bleaching technology parameter and then determine the best bleaching technology.1.4 semi-works production study of extraction and abstraction of every TCM effective partsAccording to the technology parameter determined in lab, we carry out semi-works production to establish the base for industry production.1.5 manufacture of colon-target Baizhuhuangqi capsule to be used specially for small animalAccording to the screened best effective parts compatibility proportion by pharmacodynamics experiment, screen adjuvant and it's dosage, fill in the colon-target capsule with admixture to make up Baizhuhuangqi capsule colon-target praeparatum, and verify it's colon-target characteristic. 2 pharmacology study 2.1 pharmacodynamic action observation of Baizhuhuangqi soup and it's effective parts formula anddetermination of the best compatibilityObserve curative effect of Baizhuhuangqi soup and it's effective parts formula on UC model in mice. Adopt TNBS clysmata to make UC model in mice, design different compatibility proportion formula according to mixture uniformity design. Observe undermentioned indexes, damage of gross morphous and histology mucosa, activity of myeloperoxidase(MPO), general evaluate it's curative effect, compare difference of curative effect of each formula, determine regression equation by regression analysis and determine the best curative effect compatibility ratio.2.2 curative effect observation of small colon-target capsule in rats TNBS-UC modelSixty rats, grouped 6 groups at random, 10 rats a group, normal group, model group, SASP group (0.25g/kg), three small capsule groups (1(K 2(k 40mg/kg). absolute diet 24h, anesthetized by aether, except the normal group, other rats are given an enema by mixed liquor which contains T N B S lOOmg/kg and 50% ethanol to make experimental UC model, the normal groups mitte tales doses 50% ethanol. After making model for 24h, SASP group was administrated by 0.25g/kg SASP suspl, the rest three groups were lightly anesthetized by diethyl ether, and insert polyethylene catheter whose inner diameter is 3mm by mouth, drench small capsules via catheter with water, the normal and model groups were administrated hollow small capsules with the same method. Administer for 14 successive days. Sacrifice rats to detect all indexes. These indexes are DAI, damage index of mucous membrane of colon, pathological section, MPO, SOD and 11^6 level in serum.2.3 acute toxicity testing of Baizhuhuangqi CapsuleAccoring to pharmacology experiment method observe acute toxicity test of Baizhuhuangqi Capsule in mice, determine the formula's safe. result 1. result of each effective parts' extracting and abstracting technologyThe final technology for extracting and abstracting Astragalus mongholicus saponin is that to extract Astragalus mongholicus coarse powder twice with 60% ethanol, the dosage are 10 times and 8 times. After filtrating the extract enriching recovery ethanol, dissolve the concentration with 10 tmes water, filter, throw away sedimentum, supernatant pass through AB-8 resin Collumn, respectively elute Collumn with water, 0.1% NaOH solution, 20% ethanol, 80% ethanol in turn. Collect the 80% ethanol elution parts which contain abundant saponin, recover ethanol, the concentration was vacuum dried at 60 °C and we can get Astragalus mongholicus saponin extractive. Compared with literature reports, we add two steps of low concentration and lye elution, so significantly increase purity coefficient of Astragalus mongholicus saponin in the extractive.The final technology for extracting and abstracting total flavonoids of licorice root is that to to extract licorice root coarse powder twice with 95% ethanol, the first dosage is 8 times and the second is 6 times, combine extract and filter, decompress and recover ethanol. dissolve the concentration with 5times water, filter it and throw away sedimentum, make supernatant pass through AB-8 resin Collumn, then elute Collumn with water, 80% ethanol in turn. Their dosage is 4 times column volume of resin. Collect the 80% ethanol elution parts which contain abundant flavonoids, recover ethanol, the concentration was vacuum dried at 60°C and we can get total flavonoids extractive.The final technology for extracting and abstracting atractylodes macrocephala monosaccharide is that to filter enrichment after water extracted, add 95% ethanol to precipitate for 24h, filter and get supernatant, retrieve ethanol and filter, filter liquor be passed through D900 lewatit Ml to decolor, decolorized solution be retrieved ethanol and condense, add dehydrated alcohol in and to heat to seethe, then put it in the place where temperature is 0°C to educe atractylodes macrocephala monosaccharides.2. semi-works production study of extraction and abstraction of every TCM effective partsThe semi-works production result shows, total amount of Astragalus mongholicus administered is 250kg, outcome of mastic is 2.5kg, yield is 1%, content of Astragalus mongholicus saponin is 55.64%. total amount of licorice root is 150kg, outcome of mastic is 3.6kg, yield is 2.4, purity coefficient of total flavonoids is 63.29%.3. manufacture of colon-target capsule to be used specially for small anim&l-Baizhuhuangqi capsuleAccording to the result of screening colon adherent adjuvant, carbomer 934 P's adhesive power is maximum. So we use carbomer 934 P as the adhesive adjuvant. According to the requirement of commonly used amount of carbomer and adhesive power, we think more suitable ratio of medicine and carbomer is 10:4. out-of-body disintegration and releasing degree experiment shows, small colon-target capsule is very easily disaggregate and release on the condition of pH7.8 or above. Verification experiment of colon-target in rats' body shows that the small capsule disintegrate in small intestine end or colon of rats.4. pharmacodynamic action of Baizhuhuangqi soup and it's effective parts formula and the best compatibility proportion.We treat the UC mice model with Baizhuhuangqi soup, and found that it really can promote reparation of damaged colon mucosa, lighten the inflammation degree, especially each effective parts formula after refinement has more conspicuous curative effect. According to uniformity design proposal, ran regression analysis of effective parts compatibUity ratio and MPO activity and get the regression equation:y=0.787856872-0.009377347656X2-0.0015522986525X3+0.00006687741172X22-0.000035 00827982XiX2+0.000006068904215XiX3o by analysis F =1378.86191, P =0.0204<0.05, it illustrate the quantity and effect equations are confident. According to calculation the best abstract compatibility proportion is: B13: AS: GF^O. 455: 0. 272:0.273, collate the practical prescription result, we determined the best proportion is AM: Astragalus mongholicus: licorice root=4:3:3.5. curative effect OB of colon-target capsule in treating rats TNBS-UC model... |
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