淫羊藿低糖苷组分转化规律、生物药剂学性质及其软胶囊的初步研究

发布时间：2018-06-17 17:54

本文选题：淫羊藿 + 低糖苷组分　；参考：《南京中医药大学》2016年硕士论文

【摘要】：淫羊藿是传统的补益类中药,有强筋骨,祛风湿,补肾阳之功效。本文较全面的查阅了淫羊藿药理作用的相关报道,发现淫羊藿在治疗骨质疏松上得到了广泛的应用,其发挥药效的主要成分为黄酮类化合物。结合本实验室对淫羊藿十多年来的研究基础,制备了溶解性和渗透性均较好的淫羊藿低糖苷组分。那么淫羊藿多糖苷向低糖苷转化时存在怎样的规律；所得低糖苷组分与淫羊藿总提取物相比,在药效上是否具有优效性呢?本课题带着这些疑问展开了探讨,具体研究内容如下：第二章我们选用四种具有代表性的淫羊藿品种,分别用紫外分光光度法和HPLC法分析了不同品种淫羊藿黄酮成分含量及各成分组成结构差别。使用纤维素酶酶解淫羊藿多糖苷黄酮,以脱去一个或两个糖苷而转化为低糖苷：进一步采用AB-8大孔树脂分离纯化,最终获得淫羊藿低糖苷组分。采用UPLC/Q-TOF-MS分析、表征该组分,结合本实验室前期研究及文献报道,辨析出组分包含8种化合物,分别为淫羊藿苷A、大花淫羊藿苷F、宝藿苷II、箭藿苷A、箭藿苷B、鼠李糖基淫羊藿次苷、7-O-rhamnosyl icariside Ⅱ和宝藿苷Ⅰ。在组分酶解的基础上,进一步单独对朝藿定A、朝藿定B、朝藿定C及淫羊藿苷酶解,朝藿定A7位脱去一分子的葡萄糖生成箭藿苷A；朝藿定B先是7位脱去一分子的葡萄糖生成箭藿苷B,然后又有一部分箭藿苷B3位脱去一分子木糖生成宝藿苷Ⅰ；朝藿定C7位脱去一分子的葡萄糖生成鼠李糖基淫羊藿次苷；淫羊藿苷7位脱去一分子的葡萄糖生成了宝藿苷Ⅰ第三章通过上述生物转化工艺获得了低糖苷组分,本部分进一步评价该组分活性。采用MTT法优选了淫羊藿低糖苷组分对大鼠成骨样细胞UMR-106细胞作用的最佳时间,并分析比较了不同品种淫羊藿低糖苷组分对UMR-106细胞的作用强度,结果表明,朝鲜淫羊藿低糖苷组分在浓度为30 μg/mL,作用时间为48 h时,对细胞的增殖作用最为明显。细胞内、外碱性磷酸酶(ALP)活性的测定结果显示,淫羊藿低糖苷组分(10、20 μg·mL-1)能够显著增强细胞内、外ALP活性,说明淫羊藿低糖苷组分能够促进成骨细胞的增殖分化。第四章获得的低糖苷组分具有较好的活性,本部分进一步探讨、比较了淫羊藿低糖苷组分(HEFF)相对于淫羊藿提取物(HEE)的优效性。首先以切除卵巢的骨质疏松大鼠为模型,分别测定各组大鼠血清中碱性磷酸酶(ALP)和抗酒石酸酸性磷酸酶(StrACP)活性,子宫及股骨湿重,胫骨生物力学性质,股骨骨小梁参数,骨保护素(OPG)、破骨细胞分化因子(RANKL)蛋白表达。实验结果表明,淫羊藿淫羊藿提取物和低糖苷组分均能较好的改善骨质疏松所致的指标变化,但是同等生药剂量的HEFF与HEE相比,除中剂量组血清StrACP活性和股骨内OPG蛋白表达有显著差异外,其他指标并无显著性差异,这与我们的预期不相吻合。我们推测可能有以下两个原因：在此给药剂量下,大鼠肠道菌群数量能够较好的使多糖苷转化为低糖苷而发挥药效；淫羊藿低糖苷分溶解性较差,限制了其生物利用度。为了探究是否由于第一种原因所造成的,作者又选用无完整肠道的骨质疏松斑马鱼为模型来进行淫羊藿低糖苷组分优效性的评价。将受精后3d(3dfp)的斑马鱼幼鱼随机分为空白培养基组、0.5%DMSO溶媒对照组、泼尼松龙组、依替膦酸二钠组、不同浓度的淫羊藿低糖苷组分(生药量分别为6.56,13.125,26.25,52.5,105 mg.L-1)组和淫羊藿提取物组(生药量分别为6.56,13.125,26.25,52.5,105 mg.L-1)。每天换液至9 dfp,麻醉致死后采用茜素红对幼鱼骨骼进行染色,然后对骨染色面积及累积光密度进行定量分析。实验结果显示HEFF和HEE均可增加骨质疏松斑马鱼头部骨染色面积和累积光密度,具有一定的浓度依赖性,HEFF组与HEE组相比,在生药浓度为13.125,26.25,52.5,105 mg·L-1时,头部骨累积光密度具有显著性差异。第五章淫羊藿低糖苷组分的优效性与其生物药剂学性质密切相关。本部分对淫羊藿低糖苷组分的生物药剂学性质进行了研究,并对其进行分类。我们首先测定了组分中八种化合物在水及不同pH的缓冲盐中的平衡溶解度及油水分配系数；进一步对其相似性进行分析,采用质量权重系数法对组分整体性质进行整合表征,计算出组分整体的平衡溶解度及油水分配系数。结果表明组分平衡溶解度较差,且对pH较为敏感,油水分配系数LogP介于1.91～3.04之间,提示药物在肠道内具有较好的吸收性。根据中药BCS分类系统,淫羊藿低糖苷组分具有低溶解性,高渗透性,属于II类中药组分。第六章为了解决淫羊藿低糖苷组分在水中溶解性低的问题,遵循中药实际应用中经济、方便的原则,我们将低糖苷组分设计为以大豆油为基质,蜂蜡和大豆磷脂分别为助悬剂和润湿剂的混悬剂。分别单因素考察处方中各成分的用量,然后用正交法确定了最佳比例为组分：基质：蜂蜡：大豆磷脂=1.0：1.5：0.1：0.04,该比例下所得的内容物具有较好的稳定性和流动性。综合上述实验可知,笔者成功的制备出了淫羊藿低糖苷组分,并对淫羊藿多糖苷向低糖苷的酶解转化规律进行探讨,然后分别以骨质疏松大鼠和骨质疏松斑马鱼为模型,评价了淫羊藿低糖苷组分相对于淫羊藿提取液的优效性,最后结合淫羊藿低糖苷组分的生物药剂学性质,制备出了可以灌装入软胶囊中的混悬剂以提高其生物利用度。
[Abstract]:Epimedium is the traditional tonifying Chinese medicine, which has the effect of strengthening the bone, removing wind and dampness and nourishing the kidney yang. This article has a comprehensive review of the related reports of the pharmacological action of epimedium, and found that Epimedium has been widely used in the treatment of osteoporosis. The main ingredient of Epimedium is the yellow ketone compound. It combines with Epimedium for more than 10 years in our laboratory. On the basis of the study, the low glycosides of Epimedium are prepared with good solubility and permeability. Then, what is the rule of the transformation of Epimedium Polysaccharide glycosides to low glycosides; is the advantage of the low glycoside components compared with the total extract of Epimedium? The contents are as follows: in the second chapter, we choose four representative Herba Epimedium species, using UV spectrophotometry and HPLC method to analyze the composition and composition difference of different varieties of epimedium, and use cellulase to hydrolyse Epimedium Polysaccharide flavonoid to remove one or two glycosides and turn into low glycosides. Further using AB-8 macroporous resin to separate and purify the low glycoside component of Epimedium. UPLC/Q-TOF-MS analysis was used to characterize the component. Combining with previous studies and literature reports, 8 compounds were identified, including icariin A, Epimedium F, Epimedium II, arrows heroside A, arrows the B, and rhamnolipid prostitution sheep. Anoin, 7-O-rhamnosyl icariside II and proeoside I. On the basis of the hydrolysate of the component, the A, the Epimedium B, the herba Epimedium C and the icariin were hydrolysated, and the glucose of the herba Epimedium A7 bit was removed from the one molecule of the glucose to produce arrows, and the 7 bits of glucose that removed the one sub of the glucoside B, and then a part of it. The B3 position of arrows was removed from a molecule of xylose and produced by a molecular xylose. The C7 bit of the herba Epimedium was removed from a molecule of glucose to produce the rhameon Epimedium Hyde, and the 7 bits of the glucose of icariin produced the third chapter of the Epimedium I obtained the low glycoside component through the above biotransformation process. This part further evaluated the component of the component. The optimum time for the effect of Epimedium low glucoside on rat osteoblast UMR-106 cells was optimized by MTT method, and the effect of different varieties of Epimedium on UMR-106 cells was analyzed and compared. The results showed that the low glycoside component of Epimedium of Korean Epimedium was 30 mu g/mL and the action time was 48 h. The intracellular and external alkaline phosphatase (ALP) activity of Epimedium (10,20 mu g. ML-1) significantly enhanced the intracellular and external ALP activity, indicating that Epimedium low glycoside components could promote the proliferation and differentiation of osteoblasts. The low glycoside components obtained in the fourth chapter have good activity in this part. The advantages of Epimedium low glycoside component (HEFF) relative to Epimedium extract (HEE) were compared. First, the rat model of ovariectomized osteoporosis rats was used to determine the serum alkaline phosphatase (ALP) and tartrate acid phosphatase (StrACP) activity, the wet weight of the uterus and femur, and the biomechanical properties of the tibia, respectively. The parameters of trabecular bone, osteoprotegerin (OPG) and osteoclast differentiation factor (RANKL) protein expression. Experimental results showed that Epimedium Herba Epimedium extract and low glycoside components can improve the index changes of osteoporosis, but the same dose of HEFF and HEE, in the middle dose group, the serum StrACP activity and the OPG egg in the femur. There are no significant differences in other indicators, which do not coincide with our expectations. We speculate that there may be two reasons for the following: under the dosage, the number of intestinal flora in rats can better make the polysaccharide glycosides converted to low glycosides, and the solubility of Epimedium low glucoside is poor and restricts its birth. In order to explore the result of the first cause, the author chose the osteoporosis zebrafish with no complete intestinal tract as a model to evaluate the efficiency of the low glycosides of Epimedium. The young fish of 3D (3dfp) after fertilization were randomly divided into the blank culture group, the 0.5%DMSO solvent control group, the prednisolone group, and eepinosine. The group of Epimedium with different concentrations (6.56,13.125,26.25,52.5105 mg.L-1) and epimedium extract group (6.56,13.125,26.25,52.5105 mg.L-1 respectively) and Herba Epimedium extract group (6.56,13.125,26.25,52.5105 mg.L-1 respectively). A daily liquid was changed to 9 DFP. After the anesthesia, alizarin red was used to stain the bones of the young fish, and then the area and accumulation of the bone were stained and accumulated. The experimental results showed that HEFF and HEE could increase the bone staining area and cumulative light density in the head of the osteoporotic zebra fish, and have a certain concentration dependence. Compared with the HEE group, the HEFF group was significantly different from the HEE group in the concentration of 13.125,26.25,52.5105 mg. L-1. The fifth chapter of Epimedium The advantages of low glucoside components are closely related to their biopharmaceutical properties. This part studies the Biological Pharmaceutics properties of Epimedium low glycoside components and classifies them. We first measured the equilibrium solubility and oil and water distribution coefficient of eight compounds in the water and different pH buffer salts. The results showed that the equilibrium solubility of the components and the oil and water distribution coefficient were calculated. The results showed that the equilibrium solubility of the components was poor, and it was more sensitive to pH, and the oil and water distribution coefficient LogP was between 1.91 and 3.04, suggesting that the drug had a better absorption in the intestinal tract. According to the BCS classification system of traditional Chinese medicine, the low solubility and high permeability of Epimedium is of low solubility and high permeability. The sixth chapter is to solve the problem of low solubility of Epimedium low glucoside components in water, and follow the principle of economic and convenience in the practical application of Chinese medicine, we design low glycosides into soybean oil as the matrix. The wax and soybean phosphatide were suspended suspension and wetting agent respectively. The dosage of each ingredient in the prescription was investigated by single factor, and the optimum proportion was determined by orthogonal method: Base: beeswax: soybean phospholipid =1.0:1.5:0.1:0.04, which had good stability and fluidity under this proportion. It is known that the author successfully prepared Epimedium low glycoside components, and discussed the transformation of Epimedium Polysaccharide glycoside into low glycoside, and then used osteoporosis rats and osteoporotic zebrafish as models to evaluate the advantages of Epimedium low glycoside components relative to the extract of Herba Epimedium. Finally, it was combined with low sugar of Epimedium. The biopharmaceutical properties of glucoside components have been prepared, and the suspensions can be filled into soft capsules to improve their bioavailability.
【学位授予单位】：南京中医药大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R284

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