本文關(guān)鍵詞：銀杏葉中銀杏內(nèi)酯的分離純化及標準樣品研制　出處：《山東農(nóng)業(yè)大學》2017年碩士論文　論文類型：學位論文

  更多相關(guān)文章： 銀杏內(nèi)酯 響應(yīng)面 亞臨界水 高速逆流色譜 標準樣品

【摘要】：銀杏(Ginkgo biloba L.)是一種裸子植物落葉喬木,我國自古以來就有銀杏葉入藥和食療的記載,20世紀70年代初在國外特別是歐洲利用銀杏葉提取物(extract of Ginkgo biloba,EGB)來治療心腦血管疾病包括腦血管動脈粥樣硬化和中樞外周血流紊亂的研究出現(xiàn),其藥用價值受到了更為廣泛的關(guān)注。銀杏葉提取物是經(jīng)現(xiàn)代提取工藝從銀杏葉中提取的活性物質(zhì),其主要活性化學成分主要包括兩大類:黃酮類及萜類。銀杏內(nèi)酯A、B、C等是二萜類內(nèi)酯,白果內(nèi)酯屬于半萜類內(nèi)酯,是血小板活化因子(platelet activating factor,PAF)拮抗劑。臨床上使用銀杏內(nèi)酯治療心血管疾病、急性胰腺炎和血栓等。本研究主要進行了銀杏內(nèi)酯的分析方法、提取工藝的優(yōu)化,并進行高純度銀杏內(nèi)酯制備分離,得到的銀杏內(nèi)酯樣品進行定值分析,證明結(jié)果符合標準樣品的要求,可以使用。主要研究成果如下:1.銀杏內(nèi)酯分析方法的優(yōu)化:采用Agilent 1260串聯(lián)385-ELSD檢測器及YMC-Pack ODS-A(5μm,4.6×250mm)的反相色譜柱進行分析,依據(jù)分離度最大(實際測得分離度R7.2)、檢測時間適宜、流動相安全等要求,優(yōu)選異丙醇:水(10:90,v/v)作為流動相,流速為1ml/min,氣體流速為1.60L/min(N2),漂移管穩(wěn)定溫度為90℃,可以更加安全準確地定量測定銀杏內(nèi)酯。2.優(yōu)化銀杏內(nèi)酯提取方法:在單因素試驗的基礎(chǔ)上,采用四因素三水平的響應(yīng)曲面分析法,建立乙醇提取銀杏葉中銀杏內(nèi)酯得率的二次多項數(shù)學模型,并以銀杏內(nèi)酯提取率為響應(yīng)值作響應(yīng)曲面和等高線,考察提取次數(shù)、提取時間(h)、乙醇的濃度(%)和固液比(g/ml)對銀杏內(nèi)酯提取率的影響。結(jié)果表明,銀杏內(nèi)酯提取的優(yōu)化工藝條件為:提取次數(shù)為3次,提取時間2h,乙醇濃度44%,固液比0.04g/ml,在此工藝條件下,銀杏內(nèi)酯的提取率為0.46%。同時對比亞臨界水提取效果,為了提高銀杏內(nèi)酯的提取率及提取效率,進行單因素實驗分析四種因素:提取溫度,提取時間,提取次數(shù)和料液比對銀杏葉中銀杏內(nèi)酯提取率的影響并確定因素水平。運用響應(yīng)面實驗方法優(yōu)化亞臨界水提取銀杏葉中銀杏內(nèi)酯的工藝,并對各影響因素的重要性進行排序。研究表明當提取溫度180℃,料液比(g/ml)為1∶25,提取時間30min,提取次數(shù)為3時,銀杏內(nèi)酯的提取率為0.47%。亞臨界水萃取具有綠色環(huán)保、無有機溶劑殘留、提取率高等特點。3.銀杏內(nèi)酯的前處理。經(jīng)過提取得到銀杏內(nèi)酯粗提物,用石油醚進行脫脂至石油醚相接近無色,用乙酸乙酯萃取。乙酸乙酯提取液加入弱堿NaHCO3溶液洗滌3次脫除黃酮類物質(zhì)。硅膠脫除極性較大的小分子雜質(zhì)比活性炭,硅藻土,酸性氧化鋁吸附效果好。利用重結(jié)晶法對銀杏內(nèi)酯B進行純化,在實驗中用乙醇和水對銀杏內(nèi)酯進行重復(fù)結(jié)晶可以得到銀杏內(nèi)酯B的純度為95%。4.高速逆流色譜法純化銀杏內(nèi)酯,選擇溶劑系統(tǒng)為正己烷-乙酸乙酯-甲醇-水(4/5/3/5,v/v/v/v),正向旋轉(zhuǎn),轉(zhuǎn)速為800r/min、流速為5ml/min、操作溫度25℃得到白果內(nèi)酯、銀杏內(nèi)酯A、B、C制備純度為99%以上。5.對銀杏內(nèi)酯進行結(jié)構(gòu)鑒定。通過薄層色譜、質(zhì)譜儀、核磁共振圖譜等對銀杏內(nèi)酯結(jié)構(gòu)進行鑒定。6.對樣品進行定值分析。本品按照GB/T15000.3-2008標準要求,采用多個實驗室協(xié)作試驗定值,參加實驗室的數(shù)目為8個。按照標準樣品定值程序,用高效液相色譜儀測定,采用峰面積歸一化法進行純度定值,經(jīng)過統(tǒng)計計算得到樣品的標準值及不確定度。
[Abstract]:Ginkgo biloba (Ginkgo biloba L.) is a species of gymnosperms, deciduous trees, since ancient times China's ginkgo leaf is used as medicine and diet records, at the beginning of 1970s in foreign countries especially in Europe using the extract of Ginkgo biloba (extract of Ginkgo biloba, EGB) to the treatment of cardiovascular and cerebrovascular diseases including cerebral vascular atherosclerosis and central peripheral blood flow disorder there, its medicinal value attracted more attentions. Ginkgo biloba extract (Ginkgo biloba extract) is an active substance extracted from the leaves of Ginkgo biloba by modern extraction technology. Its main active chemical components include two major categories: flavonoids and terpenoids. Ginkgolide A, B, C and so on are two terpene lactone, and the ginkgo lactone belongs to the terpene lactone, and it is a platelet activating factor (platelet activating factor, PAF) antagonist. Ginkgolide is used clinically for the treatment of cardiovascular disease, acute pancreatitis and thrombus. This study mainly carried out ginkgolide analysis methods, optimization of extraction technology, and made high-purity Ginkgolides separation and ginkgolide samples obtained by fixed value analysis. The results showed that the results accorded with the requirements of standard samples, and they could be used. The main research results are as follows: 1. analysis methods for optimization of Ginkgolides: using Agilent 1260 385-ELSD detector and YMC-Pack ODS-A series (5 m, 4.6 x 250mm) reversed-phase HPLC column was analyzed, based on the separation of the largest (measured resolution R7.2), mobile phase, detection time for security requirements, selection of isopropyl alcohol water: (10:90, v/v) as mobile phase, the flow rate was 1ml/min, the gas flow rate is 1.60L/min (N2), stable drift tube temperature is 90 DEG C, can be more safe and accurate quantitative determination of ginkgolides. 2. optimization of Ginkgolides extraction method: on the basis of single factor experiment, the response surface of four factors and three levels of analysis, two multinomial mathematical model of ethanol extraction of Ginkgolides in Ginkgo leaf yield, and the extraction rate of Ginkgolides as response surface and contour as the response value, the effects of extraction times and extraction time (H), ethanol concentration (%) and solid-liquid ratio (g/ml) effect on the extraction rate of ginkgolides. The results showed that the optimum extraction conditions of Ginkgolides were: extraction times 3 times, extraction time 2h, ethanol concentration 44%, solid-liquid ratio 0.04g/ml, under this technological condition, the extraction rate of Ginkgolides was 0.46%. At the same time, the effect of subcritical water extraction was compared. In order to improve the extraction rate and extraction efficiency of Ginkgolides, a single factor experiment was conducted to analyze four factors: extraction temperature, extraction time, extraction times and ratio of material to liquid, and determine the level of Ginkgolides. The process of extracting ginkgolide from Ginkgo biloba leaves by subcritical water was optimized by the response surface method, and the importance of the factors was sorted. The results showed that the extraction rate of ginkgolide was 0.47% when the extraction temperature was 180, the ratio of material to liquid (g/ml) was 1: 25, the extraction time was 30min, and the extraction times were 3. Subcritical water extraction has the characteristics of green environmental protection, no residual organic solvent and high extraction rate. 3. pretreatment of ginkgolide. The crude extract of ginkgolide was extracted, and oil ether was used for degreasing to petroleum ether, which was colorless and extracted with ethyl acetate. Ethyl acetate extract was washed with weak alkali NaHCO3 solution for 3 times to remove flavonoids. The adsorption of silica gel with high polarity is better than that of activated carbon, diatomite and acid alumina. Ginkgolide B was purified by recrystallization. In the experiment, ethanol and water were used to recrystallize Ginkgolides, and the purity of ginkgolide B was 95%. Purification of ginkgolide 4. high-speed countercurrent chromatography, solvent system selection for hexane - ethyl acetate - methanol - water (4/5/3/5, v/v/v/v), the positive rotation speed is 800r/min, the flow rate was 5ml/min, the operating temperature of 25 DEG C by bilobalide, ginkgolide A, B, C preparation purity was more than 99%. 5. the structure of ginkgolide was identified. The structure of ginkgolide was identified by TLC, MS, NMR and so on. 6. to analyze the value of the sample. In accordance with the GB/T15000.3-2008 standard, this product adopts a number of laboratory cooperation tests, and the number of participants in the laboratory is 8. According to the standard sample calibration procedure, the HPLC method was used to determine the purity, and the peak area normalization method was used to determine the purity. After that, the standard value and uncertainty of the samples were obtained.
【學位授予單位】：山東農(nóng)業(yè)大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：TQ464

【參考文獻】

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1 張國松;李東R，

本文編號：1347284