【摘要】：白復(fù)凝是以渭北野生羊角蔓中抗白癜風(fēng)有效部位為功效成分制備的一種復(fù)凝膠劑。渭北野生羊角蔓為蘿呮科蘿呮屬蘿呮的一個(gè)變種,渭北民間有人用其白汁治療白癜風(fēng),《廣濟(jì)方》有同種植物治療白癜風(fēng)的記載。羊角蔓資源十分豐富,多年來一直是自生自滅,為了開發(fā)利用這一自然資源尋求治療頑疾白癜風(fēng)新藥,本文對(duì)其進(jìn)行了有效部位的篩選、成分檢定、制備工藝、劑型制備及初步評(píng)價(jià)四方面的研究。本研究首先采用化學(xué)方法對(duì)羊角蔓所含化學(xué)成分進(jìn)行系統(tǒng)預(yù)測,依據(jù)檢測結(jié)果,用植物化學(xué)的研究方法得出單體成分并進(jìn)行結(jié)構(gòu)檢定;其次綜合研究結(jié)果,按極性分為4大類,用4種極性不同的溶劑進(jìn)行分類提取,提取物采用活性導(dǎo)向分離法,篩選出有效部位;再次采用4因子3水平響應(yīng)面法對(duì)有效部位的浸出工藝進(jìn)行優(yōu)化研究;最后采用試驗(yàn)對(duì)比、綜合評(píng)價(jià)的方法進(jìn)行制劑工藝研究,并用生物活性法進(jìn)行藥效學(xué)初步評(píng)價(jià)。主要結(jié)果如下:(1)羊角蔓治療白癜風(fēng)有效部位篩選通過系統(tǒng)預(yù)測,定性鑒別得出羊角蔓中含有皂苷、三萜類、甾體類、甾醇、黃酮類、生物堿和有機(jī)酸等14類化學(xué)成分。通過驗(yàn)證建立的酪氨酸酶活性測定法對(duì)石油醚提取物、乙酸乙酯提取物、正丁醇提取物、水提20%、40%、60%、80%、95%醇沉物8類不同極性組合物進(jìn)行生化測定,得出在試驗(yàn)范圍內(nèi),羊角蔓正丁醇提取物對(duì)酪氨酸酶激活效果最為顯著,其激活率為30.71%,陽性對(duì)照補(bǔ)骨脂素激活率為39.34%。(2)羊角蔓有效部位的植化研究正丁醇提取物,經(jīng)理化檢測,富含皂苷,薄層色譜檢測含2個(gè)皂苷斑點(diǎn);經(jīng)柱色譜分離、結(jié)晶純化等處理,得到4個(gè)化合物單體,經(jīng)理化、色譜、光譜等方法檢定,得出化合物1為胡蘿卜苷,化合物2為β-谷甾醇,化合物3為刺槐苷(山奈酚-3-O-葡萄糖基-鼠李糖基-7-O-鼠李糖苷),化合物4為肌醇。(3)羊角蔓有效部位的浸出研究經(jīng)顯微觀察得出羊角蔓藤莖組織結(jié)構(gòu)主要為表皮薄壁細(xì)胞、皮層薄壁細(xì)胞、次生韌皮部薄壁細(xì)胞、次生木質(zhì)部薄壁細(xì)胞及髓細(xì)胞;葉片組織結(jié)構(gòu)主要為表皮薄壁細(xì)胞和葉肉細(xì)胞。組織化學(xué)定位得出羊角蔓藤莖中皂苷主要分布在其皮層薄壁細(xì)胞、次生韌皮部薄壁細(xì)胞和髓細(xì)胞中;葉片中皂苷主要分布在葉肉細(xì)胞中。浸出工藝研究,在單因素研究的基礎(chǔ)上,以總皂苷的標(biāo)示量為效應(yīng)指標(biāo),通過響應(yīng)面中心設(shè)計(jì)試驗(yàn)進(jìn)行工藝優(yōu)化,得出在試驗(yàn)范圍內(nèi)的最佳提取工藝條件為:乙醇濃度76%,液料比10.32:1(mL/g),回流溫度70.2℃,回流時(shí)間2.52 h;5次試驗(yàn),固形物收率為4.32%,產(chǎn)物中總皂苷的標(biāo)示量為60.39%。經(jīng)驗(yàn)證,該工藝的相對(duì)標(biāo)準(zhǔn)偏差RSD為1.50%。(4)制劑研究及藥效初步評(píng)價(jià)以正丁醇提取物的理化性質(zhì)為依據(jù),以臨床適用性為判定標(biāo)準(zhǔn),經(jīng)文獻(xiàn)調(diào)研和6種外用劑型對(duì)比分析得出w/o/w復(fù)乳凝膠劑對(duì)本產(chǎn)品較為適合。制劑處方和成型工藝參數(shù)經(jīng)L9(34)正交試驗(yàn)優(yōu)化得出,以1.0 g藥物,加1.2 g單硬脂酸甘油酯,0.5 g白凡士林,2.7 g液體石蠟,0.25 g硬脂酸,0.2 g司盤-80,0.1 g吐溫-80,0.1 g CMC-Na,0.1 g PVA124及少量助劑和適量蒸餾水,80℃乳化,制成的產(chǎn)品呈米黃色,穩(wěn)定性良好,功效成分以刺槐苷計(jì),其標(biāo)示量不低于8.0%,釋藥性為0~8 h內(nèi),藥物累積釋放率達(dá)75.6%,體外釋藥方式中一級(jí)模型的相關(guān)系數(shù)較高。藥效初步評(píng)價(jià)試驗(yàn)結(jié)果表明,該產(chǎn)品對(duì)皮膚無刺激;對(duì)酪氨酸酶的激活率為30.62%,陽性對(duì)照復(fù)方補(bǔ)骨脂酊激活率為57.34%。由此得出結(jié)論:渭北野生羊角蔓治療白癜風(fēng)有效部位為苷類物質(zhì),以總皂苷為指標(biāo),標(biāo)示量不低于60%;羊角蔓中含有胡蘿卜苷、β-谷甾醇、刺槐苷、肌醇和2個(gè)皂苷類化合物,其中肌醇為在同種植物中初次發(fā)現(xiàn);乙醇提取正丁醇萃取工藝穩(wěn)定,可操作性強(qiáng);復(fù)凝膠劑對(duì)酪氨酸酶激活作用可靠;本研究的結(jié)果為開發(fā)治療白癜風(fēng)新藥提供了可靠的試驗(yàn)依據(jù)。
[Abstract]:Leucoconjugation is a kind of compound gel which is prepared from the effective part of the wild Ramulus Ramulus in Weibei. Wild Ramulus Ramulus Ramulus in Weibei is a variety of Ramulus Ramulus. Some people in Weibei have used its white juice to treat vitiligo. There are records of treating vitiligo with the same plant. In order to exploit and utilize this natural resource to seek a new medicine for vitiligo, this paper studied its effective parts screening, ingredient identification, preparation process, preparation and preliminary evaluation of four aspects. According to the test results, the monomer components were obtained by phytochemistry and their structures were examined; secondly, according to the polarity, the monomer components were divided into four categories and extracted by four different polar solvents, and the active fractions were screened out by activity-oriented separation method; and then the effective fractions were extracted by 4-factor-3 horizontal response surface methodology. The main results are as follows: (1) Screening the effective parts of Vitiligo Vitiligo treatment by systematic prediction, qualitative identification of Vitiligo Vitiligo contains saponins, triterpenoids, steroids, steroids, steroids, steroids. Tyrosinase activity assay was established to determine petroleum ether extract, ethyl acetate extract, n-butanol extract, water extract 20%, 40%, 60%, 80%, 95% alcohol sediment and eight kinds of different polar compositions. The activation rate of tyrosinase was 30.71%. The activation rate of Psoralen in positive control was 39.34%. (2) Phytochemical study on the effective fraction of Aristolochia tenuifolia L. Physicochemical, chromatographic and spectroscopic analysis showed that compound 1 was carotene, compound 2 was beta-sitosterol, compound 3 was Acacia glycoside (kaempferol-3-O-glucosyl-rhamnosyl-7-O-rhamnosyl glycoside), compound 4 was inositol. (3) Microscopic observation of the effective parts of Ramulus angustifolia stem tissue structure was mainly surface. Histochemical localization showed that saponins were mainly distributed in cortical parenchyma cells, secondary phloem parenchyma cells, secondary xylem parenchyma cells and pulp cells. Saponins were mainly distributed in mesophyll cells. On the basis of single factor study, the extraction process was optimized by response surface methodology. The optimum extraction conditions were as follows: ethanol concentration 76%, liquid-to-material ratio 10.32:1 (mL/g), reflux temperature 70.2 C and reflux temperature 70.2 C. The relative standard deviation (RSD) was 1.50%. (4) The preparation study and pharmacodynamics preliminary evaluation were based on the physicochemical properties of n-butanol extract, and the clinical applicability was taken as the criterion. Literature investigation and comparison of six external dosage forms were carried out. The formulation and molding parameters were optimized by L9 (34) orthogonal test. 1.0 g drug, 1.2 g glycerin monostearate, 0.5 g white vaseline, 2.7 g liquid paraffin, 0.25 g stearic acid, 0.2 g spark-80, 0.1 g Tween-80, 0.1 g CMC-Na, 0.1 g PVA124 and a small amount of additives and suitable additives were obtained. The results of preliminary pharmacodynamic evaluation showed that the product had no irritation to the skin, and the cumulative release rate was 75.6% within 0-8 hours. The activation rate of tyrosinase was 30.62%. The activation rate of positive control compound psoralen tincture was 57.34%. It was concluded that the effective parts of wild Ramulus amurensis in Weibei were glycosides, and the total saponins were used as indicators, the labeling quantity was not less than 60%. Ramulus amurensis contained carotene, beta-sitosterol, Acacia glycosides, inositol and two saponins. Mesoinositol was found in the same plant for the first time; ethanol extraction of n-butanol extraction process is stable and operable; compound gel can activate tyrosinase reliably; the results of this study provide a reliable experimental basis for the development of new drugs for vitiligo.
【學(xué)位授予單位】：陜西科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：TQ460.6

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