本文選題：淫羊藿 + 低糖苷組分�。� 參考：《南京中醫(yī)藥大學(xué)》2016年碩士論文

【摘要】：淫羊藿是傳統(tǒng)的補(bǔ)益類中藥,有強(qiáng)筋骨,祛風(fēng)濕,補(bǔ)腎陽(yáng)之功效。本文較全面的查閱了淫羊藿藥理作用的相關(guān)報(bào)道,發(fā)現(xiàn)淫羊藿在治療骨質(zhì)疏松上得到了廣泛的應(yīng)用,其發(fā)揮藥效的主要成分為黃酮類化合物。結(jié)合本實(shí)驗(yàn)室對(duì)淫羊藿十多年來(lái)的研究基礎(chǔ),制備了溶解性和滲透性均較好的淫羊藿低糖苷組分。那么淫羊藿多糖苷向低糖苷轉(zhuǎn)化時(shí)存在怎樣的規(guī)律；所得低糖苷組分與淫羊藿總提取物相比,在藥效上是否具有優(yōu)效性呢?本課題帶著這些疑問(wèn)展開(kāi)了探討,具體研究?jī)?nèi)容如下：第二章我們選用四種具有代表性的淫羊藿品種,分別用紫外分光光度法和HPLC法分析了不同品種淫羊藿黃酮成分含量及各成分組成結(jié)構(gòu)差別。使用纖維素酶酶解淫羊藿多糖苷黃酮,以脫去一個(gè)或兩個(gè)糖苷而轉(zhuǎn)化為低糖苷：進(jìn)一步采用AB-8大孔樹(shù)脂分離純化,最終獲得淫羊藿低糖苷組分。采用UPLC/Q-TOF-MS分析、表征該組分,結(jié)合本實(shí)驗(yàn)室前期研究及文獻(xiàn)報(bào)道,辨析出組分包含8種化合物,分別為淫羊藿苷A、大花淫羊藿苷F、寶藿苷II、箭藿苷A、箭藿苷B、鼠李糖基淫羊藿次苷、7-O-rhamnosyl icariside Ⅱ和寶藿苷Ⅰ。在組分酶解的基礎(chǔ)上,進(jìn)一步單獨(dú)對(duì)朝藿定A、朝藿定B、朝藿定C及淫羊藿苷酶解,朝藿定A7位脫去一分子的葡萄糖生成箭藿苷A；朝藿定B先是7位脫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位脫去一分子木糖生成寶藿苷Ⅰ；朝藿定C7位脫去一分子的葡萄糖生成鼠李糖基淫羊藿次苷；淫羊藿苷7位脫去一分子的葡萄糖生成了寶藿苷Ⅰ第三章通過(guò)上述生物轉(zhuǎn)化工藝獲得了低糖苷組分,本部分進(jìn)一步評(píng)價(jià)該組分活性。采用MTT法優(yōu)選了淫羊藿低糖苷組分對(duì)大鼠成骨樣細(xì)胞UMR-106細(xì)胞作用的最佳時(shí)間,并分析比較了不同品種淫羊藿低糖苷組分對(duì)UMR-106細(xì)胞的作用強(qiáng)度,結(jié)果表明,朝鮮淫羊藿低糖苷組分在濃度為30 μg/mL,作用時(shí)間為48 h時(shí),對(duì)細(xì)胞的增殖作用最為明顯。細(xì)胞內(nèi)、外堿性磷酸酶(ALP)活性的測(cè)定結(jié)果顯示,淫羊藿低糖苷組分(10、20 μg·mL-1)能夠顯著增強(qiáng)細(xì)胞內(nèi)、外ALP活性,說(shuō)明淫羊藿低糖苷組分能夠促進(jìn)成骨細(xì)胞的增殖分化。第四章獲得的低糖苷組分具有較好的活性,本部分進(jìn)一步探討、比較了淫羊藿低糖苷組分(HEFF)相對(duì)于淫羊藿提取物(HEE)的優(yōu)效性。首先以切除卵巢的骨質(zhì)疏松大鼠為模型,分別測(cè)定各組大鼠血清中堿性磷酸酶(ALP)和抗酒石酸酸性磷酸酶(StrACP)活性,子宮及股骨濕重,脛骨生物力學(xué)性質(zhì),股骨骨小梁參數(shù),骨保護(hù)素(OPG)、破骨細(xì)胞分化因子(RANKL)蛋白表達(dá)。實(shí)驗(yàn)結(jié)果表明,淫羊藿淫羊藿提取物和低糖苷組分均能較好的改善骨質(zhì)疏松所致的指標(biāo)變化,但是同等生藥劑量的HEFF與HEE相比,除中劑量組血清StrACP活性和股骨內(nèi)OPG蛋白表達(dá)有顯著差異外,其他指標(biāo)并無(wú)顯著性差異,這與我們的預(yù)期不相吻合。我們推測(cè)可能有以下兩個(gè)原因：在此給藥劑量下,大鼠腸道菌群數(shù)量能夠較好的使多糖苷轉(zhuǎn)化為低糖苷而發(fā)揮藥效；淫羊藿低糖苷分溶解性較差,限制了其生物利用度。為了探究是否由于第一種原因所造成的,作者又選用無(wú)完整腸道的骨質(zhì)疏松斑馬魚為模型來(lái)進(jìn)行淫羊藿低糖苷組分優(yōu)效性的評(píng)價(jià)。將受精后3d(3dfp)的斑馬魚幼魚隨機(jī)分為空白培養(yǎng)基組、0.5%DMSO溶媒對(duì)照組、潑尼松龍組、依替膦酸二鈉組、不同濃度的淫羊藿低糖苷組分(生藥量分別為6.56,13.125,26.25,52.5,105 mg.L-1)組和淫羊藿提取物組(生藥量分別為6.56,13.125,26.25,52.5,105 mg.L-1)。每天換液至9 dfp,麻醉致死后采用茜素紅對(duì)幼魚骨骼進(jìn)行染色,然后對(duì)骨染色面積及累積光密度進(jìn)行定量分析。實(shí)驗(yàn)結(jié)果顯示HEFF和HEE均可增加骨質(zhì)疏松斑馬魚頭部骨染色面積和累積光密度,具有一定的濃度依賴性,HEFF組與HEE組相比,在生藥濃度為13.125,26.25,52.5,105 mg·L-1時(shí),頭部骨累積光密度具有顯著性差異。第五章淫羊藿低糖苷組分的優(yōu)效性與其生物藥劑學(xué)性質(zhì)密切相關(guān)。本部分對(duì)淫羊藿低糖苷組分的生物藥劑學(xué)性質(zhì)進(jìn)行了研究,并對(duì)其進(jìn)行分類。我們首先測(cè)定了組分中八種化合物在水及不同pH的緩沖鹽中的平衡溶解度及油水分配系數(shù)；進(jìn)一步對(duì)其相似性進(jìn)行分析,采用質(zhì)量權(quán)重系數(shù)法對(duì)組分整體性質(zhì)進(jìn)行整合表征,計(jì)算出組分整體的平衡溶解度及油水分配系數(shù)。結(jié)果表明組分平衡溶解度較差,且對(duì)pH較為敏感,油水分配系數(shù)LogP介于1.91～3.04之間,提示藥物在腸道內(nèi)具有較好的吸收性。根據(jù)中藥BCS分類系統(tǒng),淫羊藿低糖苷組分具有低溶解性,高滲透性,屬于II類中藥組分。第六章為了解決淫羊藿低糖苷組分在水中溶解性低的問(wèn)題,遵循中藥實(shí)際應(yīng)用中經(jīng)濟(jì)、方便的原則,我們將低糖苷組分設(shè)計(jì)為以大豆油為基質(zhì),蜂蠟和大豆磷脂分別為助懸劑和潤(rùn)濕劑的混懸劑。分別單因素考察處方中各成分的用量,然后用正交法確定了最佳比例為組分：基質(zhì)：蜂蠟：大豆磷脂=1.0：1.5：0.1：0.04,該比例下所得的內(nèi)容物具有較好的穩(wěn)定性和流動(dòng)性。綜合上述實(shí)驗(yàn)可知,筆者成功的制備出了淫羊藿低糖苷組分,并對(duì)淫羊藿多糖苷向低糖苷的酶解轉(zhuǎn)化規(guī)律進(jìn)行探討,然后分別以骨質(zhì)疏松大鼠和骨質(zhì)疏松斑馬魚為模型,評(píng)價(jià)了淫羊藿低糖苷組分相對(duì)于淫羊藿提取液的優(yōu)效性,最后結(jié)合淫羊藿低糖苷組分的生物藥劑學(xué)性質(zhì),制備出了可以灌裝入軟膠囊中的混懸劑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.
【學(xué)位授予單位】：南京中醫(yī)藥大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：R284

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1 趙冰潔;淫羊藿低糖苷組分轉(zhuǎn)化規(guī)律、生物藥劑學(xué)性質(zhì)及其軟膠囊的初步研究[D];南京中醫(yī)藥大學(xué);2016年

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