三元双相氯化铝水解制备薯蓣皂素方法Method for preparing diosgenin by hydrolysis of ternary two-phase aluminum chloride
技术领域Technical field
本发明涉及植物提取技术领域,具体涉及利用薯蓣资源提取皂素,更具体地,涉及一种三元双相AlCl
3水解制备薯蓣皂素的方法。
The present invention relates to the technical field of plant extracts, particularly relates resource extraction diosgenin saponin, more particularly, it relates to a process for preparing Ternary AlCl 3 biphasic hydrolysis diosgenin.
背景技术Background technique
甾体激素类药物是仅次于抗生素的全球第二大药品种类,而薯蓣皂素的结构和甾体激素类药物相似,是合成甾体激素类药物的最理想前体。以薯蓣皂素为原料合成的肾上腺皮质激素、性激素和蛋白同化激素三大类甾体激素药物多达上百种。因此,对于薯蓣资源提取皂素的研究和生产制备具有良好的市场前景和发展价值。Steroid hormone drugs are the world's second largest drug category after antibiotics. Diosgenin has a structure similar to steroid hormone drugs and is the most ideal precursor for the synthesis of steroid hormone drugs. There are as many as hundreds of three types of steroid hormone drugs synthesized from diosgenin as raw materials of adrenal cortex hormones, sex hormones and anabolic hormones. Therefore, the research and production preparation of saponins extracted from yam resources has a good market prospect and development value.
薯蓣皂苷元和不同种类、数目的糖通过糖苷键结合形成不同的薯蓣皂苷,因此,薯蓣皂苷的水解是提取制备薯蓣皂素的关键。现行的制备薯蓣皂素的技术主要基于化学水解和生物水解两种方法,并由此衍生出一些辅助提取制备技术或者多种方法结合的技术。Diosgenin and different types and numbers of sugars are combined by glycosidic bonds to form different diosgenin. Therefore, the hydrolysis of diosgenin is the key to the extraction and preparation of diosgenin. The current preparation technology of diosgenin is mainly based on two methods, chemical hydrolysis and biological hydrolysis, and some auxiliary extraction preparation techniques or techniques combining multiple methods have been derived from this.
化学水解法主要是指利用如硫酸、盐酸等传统无机强酸水解糖苷键释放出薯蓣皂素的提取制备方法。传统的酸水解提取薯蓣皂素的工艺由Rothrock等提出,即直接用无机酸水解薯蓣根茎,用水洗涤至中性后,烘干并用石油醚等有机溶剂进行水浴循环萃取,最后结晶得到皂素成品。张裕卿等考察了用硫酸水解提取盾叶薯蓣皂素的效果,结果表明:在2mol/L的浓度下水解4h,索式提取8h后可得皂素收率为1.28%。直接酸水解法工艺简单,操作简便,适用于大规模工业化生产。但无机酸水解工艺皂素得率一般较低,为1.6%左右,用无机强酸水解时,不仅会造成设备腐蚀和洗涤时耗水量大,排出的酸性废水难以进行回收处理,并且废渣中有一部分皂苷未能与大量存在的淀粉和纤维素类物质在酸解后分离。这直接造成了环境的污染和资源的浪费,同时还影响薯蓣皂素的得率。而生物水解法提取皂素是利用一些霉菌微生物产生的生物酶直接转化皂苷生成皂素,优点是对环境友好无污染。一些研究者分别用米曲霉、哈茨木霉、泡盛曲霉对盾叶薯蓣进行接种培养来发酵直接利用皂苷转化提取皂素,研究结果证实薯蓣皂素的产量从17.06mg/g~74.26mg/g不等,差异显著,生物转化利用皂苷水解的过程及机 理复杂,皂素收率偏低,且生成的副产物较多。尽管因利用生物法和酶解相结合将皂苷生物转化为薯蓣皂素对环境无污染而受到众多研究者关注,然而,与传统的直接酸水解法相比,由于酶制剂成本的不经济性和生物转化的低效性导致其在薯蓣皂素工业中的应用有限。由于传统制备技术皂素面临着收率低、酸耗高、排污量大、淀粉废渣和酸性废液无法回收或回收成本高等资源消耗过巨等问题,因此,亟需找寻一种皂素收率高、酸耗低、排污量小的薯蓣皂素的制备方法。The chemical hydrolysis method mainly refers to the extraction and preparation method of using traditional strong inorganic acids such as sulfuric acid and hydrochloric acid to hydrolyze glycosidic bonds to release diosgenin. The traditional process of extracting diosgenin by acid hydrolysis was proposed by Rothrock et al., that is, hydrolyzing the rhizome of dioscorea directly with inorganic acid, washing it with water to neutrality, drying and using petroleum ether and other organic solvents for water bath circulation extraction, and finally crystallizing to obtain the finished saponin product . Zhang Yuqing et al. investigated the effect of extracting diosgenin by hydrolysis with sulfuric acid, and the results showed that the yield of saponin was 1.28% after hydrolysis at a concentration of 2mol/L for 4h, 8h after Soxhlet extraction. The direct acid hydrolysis method has simple process and simple operation, and is suitable for large-scale industrial production. However, the yield of saponin in the inorganic acid hydrolysis process is generally low, about 1.6%. When using a strong inorganic acid to hydrolyze, it will not only cause equipment corrosion and large water consumption during washing, the discharged acidic wastewater is difficult to recycle, and there is a part of the waste residue. The saponins could not be separated from the starch and cellulosic substances present in a large amount after acid hydrolysis. This directly causes environmental pollution and waste of resources, and also affects the yield of diosgenin. The biohydrolysis method for extracting saponin is the use of biological enzymes produced by some mold microorganisms to directly convert saponin to produce saponin, which has the advantage of being environmentally friendly and pollution-free. Some researchers used Aspergillus oryzae, Trichoderma harzianum, and Aspergillus awamori to inoculate Dioscorea zingiberensis to ferment and directly use saponin conversion to extract saponin. The research results confirmed that the yield of diosgenin varies from 17.06mg/g to 74.26mg/g. The difference is significant, the process and mechanism of biotransformation using saponin hydrolysis are complicated, the yield of saponin is low, and more by-products are generated. Although the combination of biological methods and enzymatic hydrolysis to convert saponins into diosgenin has no pollution to the environment, it has attracted the attention of many researchers. However, compared with the traditional direct acid hydrolysis method, the cost of enzyme preparations is uneconomical and biological. The inefficiency of transformation leads to its limited application in the diosgenin industry. As the traditional preparation technology of saponin is faced with the problems of low yield, high acid consumption, large sewage discharge, starch waste and acid waste liquid cannot be recycled, or high recycling cost, and other problems such as excessive resource consumption, it is urgent to find a saponin yield The preparation method of diosgenin is high, low in acid consumption and small in sewage discharge.
发明内容Summary of the invention
本发明的目的在于克服现有技术中存在的上述缺陷和不足,提供一种三元双相AlCl
3水解制备薯蓣皂素的方法。
Object of the present invention to overcome the prior art drawbacks and deficiencies exist, there is provided a process for preparing Ternary AlCl 3 biphasic hydrolysis diosgenin.
本发明的上述目的是通过以下技术方案给予实现的:The above objectives of the present invention are achieved through the following technical solutions:
一种三元双相AlCl
3水解制备薯蓣皂素的方法,将干燥后的薯蓣粉末投入到反应釜中,然后加入由三氯化铝水溶液、乙醇和石油醚组成的三元双相溶液,混匀,于130~180℃反应0.5~3.0h,将反应液过滤,滤去残渣,分离出石油醚层进行旋转蒸发,重结晶后得到薯蓣皂素。
A method for preparing diosgenin by hydrolysis of ternary biphasic AlCl 3 , put the dried dioscorea powder into a reaction kettle, and then add a ternary biphasic solution composed of aluminum trichloride aqueous solution, ethanol and petroleum ether, and mix After homogenization, react at 130-180°C for 0.5-3.0h, filter the reaction solution, filter off the residue, separate the petroleum ether layer, perform rotary evaporation, and obtain diosgenin after recrystallization.
本发明方法采用非传统酸的易斯酸三氯化铝在乙醇/水/石油醚的三元双相体系中酸解制备菊叶薯蓣皂素,路易斯酸三氯化铝是水溶性的无机盐类,水溶液呈酸性,安全无毒易操作,可用来替代无机强酸硫酸水解制备皂素。由于薯蓣皂素的极性与甾体皂苷的极性有很大差异,与甾体皂苷相比,薯蓣皂素由于不存在糖链所以极性较弱,这使得难以同时提取皂素和各种薯蓣皂苷,通过往反应体系中加入石油醚,利用皂苷和皂素溶解性的差异在皂苷水解的同时同步萃取皂素到石油醚有机相中,使得酸解皂苷和提取皂素同步进行;同时,氯化铝的弱酸性和部分皂苷被淀粉包裹或者连接在细胞壁上难以被释放出来,菊叶薯蓣中甾体皂苷并未完全被利用来转化皂素,本发明通过选择乙醇和水的混合溶剂有利于总皂苷的萃取,使皂苷可以更大程度通过酸水解制备得到皂素。本发明乙醇提取剂辅助的三元双相AlCl
3水解制备皂素的方法显著的提高了皂素得率,缩短了提取周期,同时消耗的酸用量大大减少,在不使用浓H
2SO
4的情况下,对环境的污染也将变得最小。
The method of the present invention adopts non-traditional acid aluminum trichloride easy acid hydrolysis in a ternary biphasic system of ethanol/water/petroleum ether to prepare diosgenin. Lewis acid aluminum trichloride is a water-soluble inorganic salt Class, the aqueous solution is acidic, safe, non-toxic and easy to operate, and can be used to replace the strong inorganic acid and sulfuric acid hydrolysis to prepare saponin. Since the polarity of diosgenin is very different from the polarity of steroidal saponins, compared with steroidal saponins, diosgenin has weaker polarity due to the absence of sugar chains, which makes it difficult to extract both saponin and various saponins at the same time. Diosgenin, by adding petroleum ether to the reaction system, the difference in solubility of saponins and saponin is used to simultaneously extract saponin into the petroleum ether organic phase while saponin is hydrolyzed, so that the acid hydrolysis of saponin and the extraction of saponin are performed simultaneously; at the same time, The weak acidity of aluminum chloride and part of the saponins are encapsulated by starch or attached to the cell wall and are difficult to be released. The steroidal saponins in dioscorea chrysanthemum have not been fully utilized to convert saponin. The present invention selects a mixed solvent of ethanol and water. Conducive to the extraction of total saponin, so that saponin can be prepared by acid hydrolysis to a greater extent to obtain saponin. The method for preparing saponin by the ternary two-phase AlCl 3 hydrolysis assisted by the ethanol extractant of the present invention significantly improves the yield of saponin, shortens the extraction period, and at the same time greatly reduces the consumption of acid, and does not use concentrated H 2 SO 4 In this case, the pollution to the environment will also be minimized.
优选地,所述反应温度为140~170℃。合适而有利的温度可以加速反应进程,与此同时还可以减少提取时间并提高提取效率。然而,极端过高的温度又有可能促使菊叶薯蓣中高含量的淀粉部分糊化,从而导致一些淀粉包裹的皂苷不能 水解转化为皂素,引起皂素得率损失。更重要的是,适当的温度会降低三元双相溶剂的粘度,同时增加三元双相溶剂的扩散系数,从而加速水层中皂苷的水解并提高转移到石油醚中的薯蓣皂素的提取效率。Preferably, the reaction temperature is 140-170°C. Appropriate and favorable temperature can accelerate the reaction process, while at the same time it can also reduce the extraction time and improve the extraction efficiency. However, the extremely high temperature may promote the partial gelatinization of the high content of starch in Dioscorea zingiberensis, causing some starch-coated saponin not to be hydrolyzed and converted into saponin, causing the loss of saponin yield. More importantly, an appropriate temperature will reduce the viscosity of the ternary two-phase solvent and increase the diffusion coefficient of the ternary two-phase solvent, thereby accelerating the hydrolysis of saponin in the water layer and improving the extraction of diosgenin transferred to petroleum ether. effectiveness.
更优选地,所述反应温度140~160℃(优选150℃)。More preferably, the reaction temperature is 140-160°C (preferably 150°C).
优选地,所述反应时间为1~2.5h(优选2.0h)。Preferably, the reaction time is 1 to 2.5 hours (preferably 2.0 hours).
优选地,所述三氯化铝水溶液的浓度为1.25g/L~15g/L。Preferably, the concentration of the aluminum trichloride aqueous solution is 1.25 g/L to 15 g/L.
优选地,所述三氯化铝水溶液的浓度为2.5g/L~15g/L。Preferably, the concentration of the aluminum trichloride aqueous solution is 2.5 g/L to 15 g/L.
当AlCl
3浓度过低时,低浓度的AlCl
3溶液明显的不足以完全水解甾体皂苷,仅能断裂甾体皂苷部分糖苷键,使皂苷水解为一些中间产物,如薯蓣皂苷元-三糖苷、薯蓣皂苷元-二糖苷和延龄草苷等;而过高浓度AlCl
3溶液有可能引起了副反应的发生,使得皂素脱水环化进而影响了皂素的得率。因此,所述三氯化铝水溶液的浓度优选为3.75~6.25g/L(更优选为5.0g/L)。
When the AlCl 3 concentration is too low, the low-concentration AlCl 3 solution is obviously not enough to completely hydrolyze the steroidal saponins, and can only break the steroidal saponins part of the glycosidic bond, so that the saponins are hydrolyzed into some intermediate products, such as diosgenin-triglycoside, Diosgenin-diglycoside and trillium glucoside, etc.; while too high concentration of AlCl 3 solution may cause side reactions, which will cause the dehydration and cyclization of saponin and affect the yield of saponin. Therefore, the concentration of the aluminum trichloride aqueous solution is preferably 3.75 to 6.25 g/L (more preferably 5.0 g/L).
优选地,乙醇/水/石油醚溶液体积比为6~15:16:8~32;例如6:16:32、10:16:25、15:16:20。Preferably, the volume ratio of the ethanol/water/petroleum ether solution is 6-15:16:8-32; for example, 6:16:32, 10:16:25, 15:16:20.
更优选地,乙醇/水/石油醚溶液体积比为15:16:20。More preferably, the volume ratio of ethanol/water/petroleum ether solution is 15:16:20.
优选地,所述薯蓣粉末在反应体系中的添加量为18~20g/L。(优选19.6g)Preferably, the addition amount of the yam powder in the reaction system is 18-20 g/L. (Preferably 19.6g)
优选地,所述薯蓣为菊叶薯蓣;由于菊叶薯蓣基于自身皂素含量高的特点,是更理想的皂素生产原料。Preferably, the dioscorea dinosaur is dioscorea dinosaur; due to its high saponin content, dioscorea dinosaur is a more ideal raw material for saponin production.
作为一种优选的实施方式,所述三元双相AlCl
3水解制备薯蓣皂素的方法为将干燥后的5.0g薯蓣粉末加入密闭反应器中,然后加入由80mL 5.0g/L三氯化铝水溶液、75mL乙醇、100mL石油醚,混匀,于160℃反应2.0h,将反应液过滤,滤去残渣,分离出石油醚层进行旋转蒸发,重结晶后得到薯蓣皂素,皂素得率可达到3.21%。
As a preferred embodiment, the method for preparing diosgenin by hydrolysis of ternary two-phase AlCl 3 is to add 5.0 g of dioscorea powder after drying into a closed reactor, and then add 80 mL of 5.0 g/L aluminum trichloride. Aqueous solution, 75mL ethanol, 100mL petroleum ether, mix well, react at 160℃ for 2.0h, filter the reaction solution, filter off the residue, separate the petroleum ether layer for rotary evaporation, recrystallize to obtain diosgenin, the saponin yield can be Reached 3.21%.
与现有技术相比,本发明具有以下有益效果:Compared with the prior art, the present invention has the following beneficial effects:
本发明方法采用非传统酸的易斯酸三氯化铝在乙醇/水/石油醚的三元双相体系中酸解制备菊叶薯蓣皂素,使用本发明三元双相AlCl
3水解的新方法得到的皂素最高得率达到了3.21%,皂素得率增幅比传统硫酸法高22.05%,比没有使用乙醇的双相AlCl
3水解法高67.19%,甚至是双相硫酸水解法的2.7倍多。本发明乙醇提取剂辅助的三元双相AlCl
3水解制备皂素的方法显著的提高了皂素得率,缩短了提取周期,同时消耗的酸用量大大减少。更重要的,在不使用浓H
2SO
4 的情况下,对环境的污染也将变得最小,具有较大的应用前景。
The method of the present invention adopts the non-traditional acidic acid aluminum trichloride in the ternary biphasic system of ethanol/water/petroleum ether to prepare diosgenin, and uses the new ternary biphasic AlCl 3 hydrolysis of the present invention. The highest yield of saponin obtained by the method reached 3.21%, and the increase rate of saponin was 22.05% higher than that of the traditional sulfuric acid method, 67.19% higher than that of the two-phase AlCl 3 hydrolysis method without ethanol, and even 2.7 of the two-phase sulfuric acid hydrolysis method. Times more. The method for preparing saponin by ternary two-phase AlCl 3 hydrolysis assisted by the ethanol extractant of the present invention significantly improves the saponin yield, shortens the extraction period, and greatly reduces the consumption of acid. More importantly, without the use of concentrated H 2 SO 4 , the pollution to the environment will also become minimal, which has a larger application prospect.
具体实施方式Detailed ways
以下结合具体实施例来进一步说明本发明,但实施例并不对本发明做任何形式的限定。除非特别说明,本发明采用的试剂、方法和设备为本技术领域常规试剂、方法和设备。The present invention will be further described below in conjunction with specific embodiments, but the embodiments do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the technical field.
除非特别说明,以下实施例所用试剂和材料均为市购。Unless otherwise specified, the reagents and materials used in the following examples are all commercially available.
实验材料:三年生菊叶薯蓣块茎,取自广东省翁源县菊叶薯蓣种植基地。然后将三年生菊叶薯蓣根茎切成薄片,用冷冻干燥机冷冻干燥24h去除多余水分,之后用研磨机磨成粉,装入密封袋,置于恒温除水密封器保存备用。Experimental material: Three-year-old chrysanthemum leaf dioscorea tuber, taken from the chrysanthemum leaf dioscore plant base in Wengyuan County, Guangdong Province. Then cut the three-year-old chrysanthemum leaf yam rhizome into thin slices, freeze-dried for 24 hours with a freeze dryer to remove excess water, then grind it into powder with a grinder, put it into a sealed bag, and store it in a thermostatic dewatering sealer for later use.
薯蓣皂素标准品(≥99.9%,HPLC级),购自美国Fluka公司;甲醇,色谱纯,购自美国Sigma–Aldrich公司;石油醚(60-90℃),分析纯,购自天津富宇精细化工有限公司;无水乙醇、葡萄糖、三氯化铝,分析纯,购自上海国药集团化学试剂有限公司;氢氧化钠,分析纯,购自广州化学试剂厂;酒石酸钾钠、亚硫酸钠、重蒸酚、3,5-二硝基水杨酸,分析纯,均购自阿拉丁试剂(上海)有限公司。Diosgenin standards (≥99.9%, HPLC grade), purchased from Fluka, USA; methanol, chromatographically pure, purchased from Sigma-Aldrich, USA; petroleum ether (60-90℃), analytically pure, purchased from Tianjin Fuyu Fine Chemical Co., Ltd.; absolute ethanol, glucose, aluminum trichloride, analytically pure, purchased from Shanghai Sinopharm Chemical Reagent Co., Ltd.; sodium hydroxide, analytically pure, purchased from Guangzhou Chemical Reagent Factory; potassium sodium tartrate, sodium sulfite, heavy Distilled phenol and 3,5-dinitrosalicylic acid, analytically pure, were purchased from Aladdin Reagent (Shanghai) Co., Ltd.
实施例1Example 1
精确称取5.0g菊叶薯蓣粉末投入300mL Parr反应釜中,然后加入由80mL 4.0g/L AlCl
3水溶液、75mL的乙醇和100mL石油醚组成的三元双相溶液,搅拌均匀后,设置反应釜转速为150rpm,于150℃的提取温度下反应2.0h。结束后,将石油醚有机相中的薯蓣皂素提取物旋蒸、浓缩、结晶,用色谱级甲醇溶解制样备用。然后进行薯蓣皂素分析定量。收集底部的废水溶液,便于进行随后的COD分析。
Accurately weigh 5.0g of Dioscorea chrysanthemum powder into a 300mL Parr reactor, then add a ternary two-phase solution consisting of 80mL of 4.0g/L AlCl 3 aqueous solution, 75mL of ethanol and 100mL of petroleum ether. After stirring, set the reactor The rotation speed is 150 rpm, and the reaction is carried out for 2.0 h at an extraction temperature of 150°C. After the end, the diosgenin extract in the petroleum ether organic phase was rotary evaporated, concentrated, crystallized, and dissolved in chromatographic grade methanol to prepare a sample for later use. Then quantify the diosgenin analysis. Collect the waste water solution at the bottom for subsequent COD analysis.
薯蓣皂素的分析方法为:将上述甲醇定容溶解后的薯蓣皂素提取物,稀释一定倍数后,用0.22μm的滤膜过滤后收集到1mL的样品瓶,制样备用。以Fluka公司的皂素标准品(纯度≥99.9%)为对照,进行高效液相色谱检测。具体过程为:薯蓣皂素标准品用甲醇溶解后,用高效液相色谱对浓度为4.0×10
-3mg/mL、2.0×10
-2mg/mL、1.0×10
-1mg/mL和0.5mg/mL的薯蓣皂素标准品进行测定,以皂素标准品的色谱峰面积为依据,绘制标准曲线,以峰面积作为样品定量依据,皂素得率的计算通过以下公式进行计算:
其中 W:菊叶薯蓣原料重量,g;C:皂素浓度,mg/mL;N:稀释倍数。高效液相色谱法测定参数:UV检测器;C18色谱柱(4.6mm×250mm,5μm);柱温箱温度:40℃;流动相:甲醇/水;流速为1.0mL/min;上样体积为10μL。每次测定重复三次。
The analysis method of diosgenin is: dilute the diosgenin extract dissolved in methanol at a constant volume, dilute it by a certain multiple, filter it with a 0.22μm filter membrane, and collect it into a 1mL sample bottle for sample preparation. With Fluka's saponin standard product (purity ≥99.9%) as a control, high performance liquid chromatography was performed. The specific process is as follows: After the standard substance of diosgenin is dissolved in methanol, the concentrations of 4.0×10 -3 mg/mL, 2.0×10 -2 mg/mL, 1.0×10 -1 mg/mL and 0.5 are determined by high performance liquid chromatography. The mg/mL diosgenin standard was determined. Based on the chromatographic peak area of the saponin standard, a standard curve was drawn, and the peak area was used as the quantitative basis for the sample. The saponin yield was calculated by the following formula: Where W: Dioscorea daisy raw material weight, g; C: Saponin concentration, mg/mL; N: Dilution multiple. HPLC measurement parameters: UV detector; C18 chromatographic column (4.6mm×250mm, 5μm); column oven temperature: 40℃; mobile phase: methanol/water; flow rate: 1.0mL/min; sample volume: 10μL. Each measurement was repeated three times.
结果显示,实施例1所述方法制备得到的皂素得率可达到3.08%。The results show that the yield of saponin prepared by the method described in Example 1 can reach 3.08%.
实施例2Example 2
本实施例考察温度对三元双相体系皂素得率的影响。精确称取5.0g菊叶薯蓣粉末投入300mL Parr反应釜中,然后加入由80mL 5.0g/L AlCl
3水溶液、75mL的乙醇和100mL石油醚组成的三元双相溶液,搅拌均匀后,设置反应釜转速为150rpm,,待反应釜温度上升至预定系列温度(130℃、140℃、150℃、160℃、170℃、180℃)后开始计时,反应时间为2.0h。结束后,将石油醚有机相中的薯蓣皂素提取物旋蒸、浓缩、结晶,用色谱级甲醇溶解制样备用。按照实施例1分析的方法对菊叶薯蓣皂素得率进行计算,考察温度对三元双相体系中AlCl
3提取皂素得率的影响。结果如表1所示:
This example examines the influence of temperature on the yield of saponin in the ternary two-phase system. Accurately weigh out 5.0g of Dioscorea chrysanthemum powder into a 300mL Parr reactor, then add a ternary biphasic solution consisting of 80mL of 5.0g/L AlCl 3 aqueous solution, 75mL of ethanol and 100mL of petroleum ether. After stirring, set the reactor. The rotation speed is 150 rpm. After the temperature of the reactor rises to a predetermined series of temperatures (130°C, 140°C, 150°C, 160°C, 170°C, 180°C), the timing starts, and the reaction time is 2.0h. After the end, the diosgenin extract in the petroleum ether organic phase was rotary evaporated, concentrated, crystallized, and dissolved in chromatographic grade methanol to prepare a sample for later use. According to the analysis method in Example 1, the yield of diosgenin of chrysanthemum leaf was calculated, and the influence of temperature on the yield of saponin extracted by AlCl 3 in the ternary two-phase system was investigated. The results are shown in Table 1:
表1Table 1
从表1可以看出,在130~150℃的温度范围内,薯蓣皂素得率随升高的温度显著提高,最大皂素得率为3.21%,而温度超过160℃时,皂素得率又缓慢降低。这是因为合适而有利的温度可以加速反应进程,与此同时还可以减少提取时间并提高提取效率。然而,极端过高的温度又有可能促使菊叶薯蓣中高含量的淀粉部分糊化,从而导致一些淀粉包裹的皂苷不能水解转化为皂素,引起皂素得率损失。更重要的是,适当的温度会降低三元双相溶剂的粘度,同时增加三元双相溶剂的扩散系数,从而加速水层中皂苷的水解并提高转移到石油醚中的薯蓣皂素的提取效率。因此,温度为150℃是最优提取温度并获得最大的薯蓣皂素得率,为3.21%。It can be seen from Table 1 that in the temperature range of 130~150℃, the yield of diosgenin increases significantly with increasing temperature, the maximum yield of saponin is 3.21%, and when the temperature exceeds 160℃, the yield of saponin is Slowly lowered again. This is because a suitable and favorable temperature can accelerate the reaction process, while at the same time it can also reduce the extraction time and improve the extraction efficiency. However, the extremely high temperature may promote the partial gelatinization of the high content of starch in the dioscorea chrysanthemum, causing some starch-coated saponins not to be hydrolyzed and converted into saponin, causing the loss of saponin yield. More importantly, an appropriate temperature will reduce the viscosity of the ternary two-phase solvent and increase the diffusion coefficient of the ternary two-phase solvent, thereby accelerating the hydrolysis of saponin in the water layer and improving the extraction of diosgenin transferred to petroleum ether. effectiveness. Therefore, a temperature of 150°C is the optimal extraction temperature and the maximum yield of diosgenin is 3.21%.
实施例3Example 3
本实施例考察提取时间对三元双相体系皂素得率的影响。精确称取5.0g菊叶薯蓣粉末投入300mL Parr反应釜中,然后加入由80mL 5.0g/L AlCl
3水溶液、 75mL的乙醇和100mL石油醚组成的三元双相溶液,搅拌均匀后,设置反应釜转速为150rpm,于150℃的提取温度下分别反应(0.5h、1.0h、1.5h、2.0h、2.5h、3.0h)。结束后,将石油醚有机相中的薯蓣皂素提取物旋蒸、浓缩、结晶,用色谱级甲醇溶解制样备用。按照实施例1分析的方法对菊叶薯蓣皂素得率进行计算,考察反应时间对三元双相体系中AlCl
3提取皂素得率的影响。结果如表2所示:
This example examines the effect of extraction time on the yield of saponin in the ternary two-phase system. Accurately weigh 5.0g Dioscorea chrysanthemum powder into a 300mL Parr reactor, then add a ternary biphasic solution consisting of 80mL 5.0g/L AlCl 3 aqueous solution, 75mL ethanol and 100mL petroleum ether. After stirring evenly, set the reactor The rotation speed is 150 rpm, and the reaction is performed at an extraction temperature of 150° C. (0.5h, 1.0h, 1.5h, 2.0h, 2.5h, 3.0h). After the end, the diosgenin extract in the petroleum ether organic phase was rotary evaporated, concentrated, crystallized, and dissolved in chromatographic grade methanol to prepare a sample for later use. According to the analysis method in Example 1, the yield of diosgenin from chrysanthemum leaf was calculated, and the effect of reaction time on the yield of saponin extracted by AlCl 3 in the ternary two-phase system was investigated. The results are shown in Table 2:
表2Table 2
从表2可以看出,当提取时间为0.5h时,可以获得2.69%的皂素得率。当提取时间延长后,薯蓣皂素的得率缓慢增加,在提取时间2.0h时,皂素含量达到最大值3.21%,其余时间段皂素得率相差不大。随着提取时间的进一步延长,薯蓣皂素含量略有下降,表明皂素的甾体碳骨架长时间在高温溶液中很可能被逐渐破坏,但损失量不是很大。因此,反应时间选择2.0h为最佳反应时间。It can be seen from Table 2 that when the extraction time is 0.5 h, a saponin yield of 2.69% can be obtained. When the extraction time is prolonged, the yield of diosgenin slowly increases. When the extraction time is 2.0h, the saponin content reaches the maximum value of 3.21%, and the yield of saponin in the rest of the time period is not much different. With the further extension of the extraction time, the content of diosgenin decreased slightly, indicating that the steroidal carbon skeleton of saponin was likely to be gradually destroyed in a high-temperature solution for a long time, but the loss was not very large. Therefore, the best reaction time is 2.0h.
实施例4Example 4
本实施例考察AlCl
3溶液浓度对三元双相体系皂素得率的影响。精确称取5.0g菊叶薯蓣粉末投入300mL Parr反应釜中,然后加入由80mL不同浓度的AlCl
3水溶液(1.25g/L、2.5g/L、3.75g/L、5.0g/L、6.25g/L、10g/L、15g/L)、75mL的乙醇和100mL石油醚组成的三元双相溶液,搅拌均匀后,设置反应釜转速为150rpm,于150℃的提取温度下分别反应2.0h。结束后,将石油醚有机相中的薯蓣皂素提取物旋蒸、浓缩、结晶,用色谱级甲醇溶解制样备用。按照实施例1分析的方法对菊叶薯蓣皂素得率进行计算,考察不同浓度的AlCl
3溶液对三元双相体系中AlCl
3提取皂素得率的影响。结果如图表3所示:
In this example , the influence of the concentration of the AlCl 3 solution on the yield of saponin in the ternary two-phase system was investigated. Accurately weigh out 5.0g dioscorea chrysanthemum powder into a 300mL Parr reactor, and then add 80mL AlCl 3 aqueous solutions of different concentrations (1.25g/L, 2.5g/L, 3.75g/L, 5.0g/L, 6.25g/ L, 10g/L, 15g/L), 75mL ethanol and 100mL petroleum ether ternary biphasic solution, after stirring evenly, set the reactor speed to 150rpm, and react at the extraction temperature of 150°C for 2.0h respectively. After the end, the diosgenin extract in the petroleum ether organic phase was rotary evaporated, concentrated, crystallized, and dissolved in chromatographic grade methanol to prepare a sample for later use. Calculates Dioscorea saponin yield analysis method according to Example 1 embodiment, the effects of different concentrations of a solution of AlCl 3 AlCl 3 in saponin extraction yield in the ternary phase system. The results are shown in Figure 3:
表3table 3
从表3可以看出,随着AlCl
3溶液浓度从1.25g/L增加到3.75g/L,皂素得率大幅度上升,表明AlCl
3溶液浓度在皂苷水解中起重要作用,直接影响皂素的 产量。当AlCl
3溶液浓度达到5.0g/L时,皂素的得率最高。可是当添加更多的三氯化铝作酸水解催化剂时,皂素得率明显下降,表明高浓度的AlCl
3溶液可能通过过量水解电离的氢离子破坏产生的皂素,导致副反应的发生,即皂素很容易脱水形成△3,5-脱氧替告皂苷元和打开F-螺缩醛环后被修饰成呋喃烷衍生物。因此,浓度为5.0g/L的AlCl
3溶液足以用来提取获得最高皂素得率。
It can be seen from Table 3 that as the concentration of the AlCl 3 solution increases from 1.25 g/L to 3.75 g/L, the saponin yield increases significantly, indicating that the concentration of the AlCl 3 solution plays an important role in the hydrolysis of saponin and directly affects the saponin The output. When the concentration of AlCl 3 solution reaches 5.0 g/L, the yield of saponin is the highest. However, when more aluminum trichloride is added as an acid hydrolysis catalyst, the yield of saponin decreases significantly, indicating that high-concentration AlCl 3 solution may destroy the saponin produced by excessive hydrolysis and ionization of hydrogen ions, leading to side reactions. That is, saponin can be easily dehydrated to form △3,5-deoxysapogenin and modified into furan derivatives after opening the F-spiroacetal ring. Therefore, an AlCl 3 solution with a concentration of 5.0 g/L is sufficient for extraction to obtain the highest yield of saponin.
实施例5Example 5
本实施例考察乙醇/水比例对三元双相体系皂素得率的影响。精确称取5.0g菊叶薯蓣粉末投入300mL Parr反应釜中,然后加入由80mL 5.0g/L AlCl
3水溶液、不同体积的乙醇和100mL石油醚组成的三元双相溶液,搅拌均匀后,设置反应釜转速为150rpm,于150℃的提取温度下分别反应2.0h。结束后,将石油醚有机相中的薯蓣皂素提取物旋蒸、浓缩、结晶,用色谱级甲醇溶解制样备用。按照实施例1中的方法对菊叶薯蓣皂素得率进行计算,考察不同体积比的水/石油醚溶液对三元双相体系中AlCl
3提取皂素得率的影响。结果如表4所示:
This example investigates the influence of the ethanol/water ratio on the yield of saponin in the ternary two-phase system. Accurately weigh 5.0g Dioscorea chrysanthemum powder into a 300mL Parr reactor, then add a ternary biphasic solution consisting of 80mL 5.0g/L AlCl 3 aqueous solution, different volumes of ethanol and 100mL petroleum ether. After stirring evenly, set the reaction The speed of the kettle was 150 rpm, and the reaction was carried out at an extraction temperature of 150° C. for 2.0 hours. After the end, the diosgenin extract in the petroleum ether organic phase was rotary evaporated, concentrated, crystallized, and dissolved in chromatographic grade methanol to prepare a sample for later use. According to the method in Example 1, the yield of diosgenin of chrysanthemum leaf was calculated, and the influence of water/petroleum ether solution of different volume ratios on the yield of saponin extracted by AlCl 3 in the ternary two-phase system was investigated. The results are shown in Table 4:
表4Table 4
在很大程度上来说,更多的甾体皂苷被提取溶剂乙醇从薯蓣原料中提取并溶解在酸性水相中,就意味着皂苷水解生产的皂素含量更高。从表4可以看出,随着乙醇体积比例的增加,皂素的得率也在逐渐增加,皂素得率最大值为3.21%时,乙醇/水比例为15/16(v/v);当乙醇的用量继续增加时,薯蓣皂素的得率略有下降,表示此时甾体皂苷溶解已达饱和。因此,选择比例为15/16(v/v)的乙醇水溶剂作为从菊叶薯蓣中提取皂素的最佳条件。To a large extent, more steroidal saponins are extracted from the dioscorea raw materials by the extraction solvent ethanol and dissolved in the acidic aqueous phase, which means that the content of saponin produced by hydrolysis of saponins is higher. It can be seen from Table 4 that as the volume ratio of ethanol increases, the yield of saponin is gradually increasing. When the maximum yield of saponin is 3.21%, the ratio of ethanol/water is 15/16 (v/v); When the amount of ethanol continued to increase, the yield of diosgenin decreased slightly, indicating that the dissolution of steroidal saponins reached saturation at this time. Therefore, the ethanol water solvent with a ratio of 15/16 (v/v) was selected as the best condition for extracting saponin from Dioscorea chrysanthemum.
实施例6Example 6
本实施例考察水/石油醚体积比对三元双相体系皂素得率的影响。精确称取5.0g菊叶薯蓣粉末投入300mL Parr反应釜中,然后加入由80mL 5.0g/L AlCl
3水溶液、75mL乙醇和不同比例的石油醚组成的三元双相溶液,搅拌均匀后,设置反应釜转速为150rpm,于150℃的提取温度下分别反应2.0h。结束后,将石油醚有机相中的薯蓣皂素提取物旋蒸、浓缩、结晶,用色谱级甲醇溶解制样备用。 按照实施例1中的方法对菊叶薯蓣皂素得率进行计算,考察不同体积比的水/石油醚溶液对三元双相体系中AlCl
3提取皂素得率的影响。结果如表5所示:
This example examines the influence of the volume ratio of water/petroleum ether on the yield of saponin in the ternary two-phase system. Accurately weigh 5.0g of Dioscorea chrysanthemum powder into a 300mL Parr reactor, and then add a ternary biphasic solution consisting of 80mL of 5.0g/L AlCl 3 aqueous solution, 75mL of ethanol and different proportions of petroleum ether. After stirring, set the reaction. The speed of the kettle was 150 rpm, and the reaction was carried out at an extraction temperature of 150° C. for 2.0 hours. After the end, the diosgenin extract in the petroleum ether organic phase was rotary evaporated, concentrated, crystallized, and dissolved in chromatographic grade methanol to prepare a sample for later use. According to the method in Example 1, the yield of diosgenin of chrysanthemum leaf was calculated, and the influence of water/petroleum ether solution of different volume ratios on the yield of saponin extracted by AlCl 3 in the ternary two-phase system was investigated. The results are shown in Table 5:
表5table 5
从表5可以看出,当水/石油醚为80mL/100mL时的体积比获得了最高的皂素产量,而随着消耗更多的石油醚则带来了皂素得率降低的结果;此外,如果石油醚的用量小于60mL,则皂素得率刚刚达到约2.69%。这表明较少的石油醚溶剂不足以提取和完全溶解水解生成的皂素,而消耗更多的石油醚溶剂是不切实际的且不经济的。因此,综合考虑石油醚在各个方面的影响,水/石油醚的最佳体积比例被确定为80mL/100mL。It can be seen from Table 5 that when the volume ratio of water/petroleum ether is 80mL/100mL, the highest saponin yield is obtained, and as more petroleum ether is consumed, the saponin yield decreases; in addition; If the amount of petroleum ether is less than 60 mL, the saponin yield has just reached about 2.69%. This indicates that less petroleum ether solvent is not enough to extract and completely dissolve the saponin produced by hydrolysis, and it is impractical and uneconomical to consume more petroleum ether solvent. Therefore, considering the influence of petroleum ether in various aspects, the optimal volume ratio of water/petroleum ether is determined to be 80mL/100mL.
实施例7Example 7
本实施例考察搅拌速率(转速)对三元双相体系皂素得率的影响。精确称取5.0g菊叶薯蓣粉末投入300mL Parr反应釜中,然后加入由80mL 5.0g/L AlCl
3水溶液、75mL的乙醇和100mL石油醚组成的三元双相溶液,搅拌均匀后,分别设置反应釜转速为(50rpm、100rpm、150rpm、200rpm、250rpm),于150℃的提取温度下分别反应2.0h。结束后,将石油醚有机相中的薯蓣皂素提取物旋蒸、浓缩、结晶,用色谱级甲醇溶解制样备用。按照实施例1中的方法对菊叶薯蓣皂素得率进行计算,考察不同体积比的水/石油醚溶液对三元双相体系中AlCl
3提取皂素得率的影响。结果如表6所示:
In this example, the effect of the stirring rate (rotation speed) on the yield of saponin in the ternary two-phase system was investigated. Accurately weigh 5.0g Dioscorea chrysanthemum powder into a 300mL Parr reactor, and then add a ternary biphasic solution consisting of 80mL 5.0g/L AlCl 3 aqueous solution, 75mL ethanol and 100mL petroleum ether. After stirring evenly, set the reaction separately The speed of the kettle is (50 rpm, 100 rpm, 150 rpm, 200 rpm, 250 rpm), and the reaction is carried out at an extraction temperature of 150° C. for 2.0 h. After the end, the diosgenin extract in the petroleum ether organic phase was rotary evaporated, concentrated, crystallized, and dissolved in chromatographic grade methanol to prepare a sample for later use. According to the method in Example 1, the yield of diosgenin of chrysanthemum leaf was calculated, and the influence of water/petroleum ether solution of different volume ratios on the yield of saponin extracted by AlCl 3 in the ternary two-phase system was investigated. The results are shown in Table 6:
表6Table 6
从表6可以看出,与其他影响因素相比,搅拌速率对皂素得率仅产生很小的影响。在50~250rpm这些搅拌速度中,皂素得率在100rpm时达到最高,仅比其他更高的搅拌速率略高。较高搅拌速率得到较少的皂素产量的原因可归因于酸解底物与酸催化剂之间的接触和反应时间不足。可以看出,作为影响参数的搅拌速率对菊叶薯蓣皂素提取的影响程度是平缓和温和的,皂素得率最低的为3.01%, 最高为3.21%。It can be seen from Table 6 that compared with other influencing factors, the stirring rate has only a small effect on the saponin yield. Among these stirring speeds of 50 to 250 rpm, the saponin yield reaches the highest at 100 rpm, which is only slightly higher than other higher stirring speeds. The reason why the higher agitation rate results in less saponin production can be attributed to the insufficient contact time between the acidolysis substrate and the acid catalyst and the insufficient reaction time. It can be seen that the stirring rate, which is an influencing parameter, has a gentle and mild influence on the extraction of diosgenin from Chrysanthemum leaves, with the lowest saponin yield being 3.01% and the highest being 3.21%.
通过上述实施例优化后的最佳提取技术参数为干燥的菊叶薯蓣粉末在150℃的温度下使用80mL浓度为5.0g/L的AlCl
3溶液和体积比为15/16/20(v/v/v)乙醇/水/石油醚处理下(即80mL AlCl
3水溶液、75mL的乙醇和100mL石油醚),该方法提取的菊叶薯蓣皂素得率达到了3.21%。
The optimal extraction technical parameters optimized through the above-mentioned examples are that the dried Dioscorea zingiberensis powder uses 80 mL of an AlCl 3 solution with a concentration of 5.0 g/L and a volume ratio of 15/16/20 (v/v) at a temperature of 150°C. /v) Under the treatment of ethanol/water/petroleum ether (ie, 80 mL of AlCl 3 aqueous solution, 75 mL of ethanol and 100 mL of petroleum ether), the yield of diosgenin extracted by this method reached 3.21%.
对比例1Comparative example 1
方法一:双相酸水解法:用0.5mol/L的浓硫酸代替三氯化铝,在最优的反应条件下即140℃下水解1.5h,重复三氯化铝提取菊叶薯蓣皂素的步骤。Method 1: Biphasic acid hydrolysis: replace aluminum trichloride with 0.5mol/L concentrated sulfuric acid, hydrolyze for 1.5h at 140℃ under the optimal reaction conditions, and repeat the extraction of diosgenin from diosgenin with aluminum trichloride step.
方法二:传统直接酸水解法:将20.0g菊叶薯蓣粉末在200mL 2mol/L浓硫酸水溶液中水解2.0h。过滤混合物后,用200mL热水洗涤固体残余物并在80℃下干燥。然后使用固体残余物通过索氏提取器用石油醚反复回流提取薯蓣皂素4小时。Method 2: Traditional direct acid hydrolysis: 20.0g Dioscorea chrysanthemum powder was hydrolyzed in 200mL 2mol/L concentrated sulfuric acid aqueous solution for 2.0h. After filtering the mixture, the solid residue was washed with 200 mL of hot water and dried at 80°C. Then, the solid residue was used to extract diosgenin through a Soxhlet extractor with petroleum ether repeatedly at reflux for 4 hours.
方法三:双相AlCl
3:将干燥后的5.0g菊叶薯蓣粉末加入密闭反应器中,依次加入4.0g/L三氯化铝溶液80mL,混匀,再加入100mL石油醚,于160℃反应2h,将反应液过滤,滤去残渣,分离出石油醚层进行旋转蒸发,重结晶后得到薯蓣皂素。
Method 3: Biphasic AlCl 3 : Add 5.0g of dried chrysanthemum dioscorea powder into a closed reactor, add 80mL of 4.0g/L aluminum trichloride solution in sequence, mix well, then add 100mL petroleum ether, and react at 160℃ After 2h, the reaction solution was filtered, the residue was filtered off, the petroleum ether layer was separated, and rotary evaporated, and diosgenin was obtained after recrystallization.
1、按照本发明优化后的最佳提取技术参数提取薯蓣皂素,并与上述方法一~方法三制备得到的菊叶薯蓣皂素得率进行比较,并考察了得率增幅,固体残留量,酸消耗量和其他相关变量,其结果如表7所示:1. Extract diosgenin according to the optimal extraction technical parameters optimized in the present invention, and compare the yield of diosgenin prepared by the above-mentioned methods 1 to 3, and investigate the yield increase and solid residues. Acid consumption and other related variables, the results are shown in Table 7:
表7Table 7
表7结果表明,本发明使用三元双相AlCl
3溶液的方法得到的皂素得率增幅比传统硫酸法高22.05%,甚至是双相硫酸水解法的2.7倍多。与传统硫酸法中要经过6小时的提取时间消耗相比,使用三元双相AlCl
3溶液水解的薯蓣皂素提取过程仅需2小时,仅比双相硫酸水解法多0.5h。类似地,在不使用硫酸进行皂 素提取的情况下,三元双相AlCl
3溶液水解皂素得率比没有使用乙醇的双相AlCl
3水解法进行对比要高67.19%。
The results in Table 7 show that the method of using the ternary two-phase AlCl 3 solution of the present invention has a 22.05% increase in the yield of saponin obtained from the traditional sulfuric acid method, and even more than 2.7 times that of the two-phase sulfuric acid hydrolysis method. Compared with the extraction time consumption of 6 hours in the traditional sulfuric acid method, the extraction process of diosgenin using ternary two-phase AlCl 3 solution hydrolysis only takes 2 hours, which is only 0.5h longer than the two-phase sulfuric acid hydrolysis method. Similarly, without the use of sulfuric acid for saponin extraction, the hydrolysis yield of saponin in the ternary two-phase AlCl 3 solution was 67.19% higher than that of the two-phase AlCl 3 hydrolysis method without ethanol.
与传统硫酸法中要经过6小时的提取时间消耗相比,使用三元双相AlCl
3水解制备薯蓣皂素的过程仅需2小时。本发明的三元双相AlCl
3水解法使用的酸用量为0.5g的AlCl
3,明显低于传统硫酸法的39.21g的浓H
2SO
4用量,这也使得其废液pH值要高于传统硫酸法,使其更易处理以便于后续的发酵实验。使用乙醇提取剂辅助的三元双相AlCl
3水解制备皂素的方法显著的提高了皂素得率,缩短了提取周期,同时消耗的酸用量大大减少,更重要的,在不使用浓H
2SO
4的情况下,对环境的污染也将大大降低。也表明了乙醇提取剂辅助的三元双相AlCl
3水解提皂素的方法显著的改善了皂素得率并缩短了酸水解时间,同时没有浓H
2SO
4的使用和消耗意味着环境污染也将变得最小。
Compared with the extraction time consumption of 6 hours in the traditional sulfuric acid method, the process of preparing diosgenin using ternary two-phase AlCl 3 hydrolysis only takes 2 hours. The ternary two-phase AlCl 3 hydrolysis method of the present invention uses an acid dosage of 0.5 g of AlCl 3 , which is significantly lower than the traditional sulfuric acid method of 39.21 g of concentrated H 2 SO 4 , which also makes the pH value of the waste liquid higher than The traditional sulfuric acid method makes it easier to handle for subsequent fermentation experiments. The method of preparing saponin by using ethanol extractant-assisted ternary two-phase AlCl 3 hydrolysis significantly improves the yield of saponin, shortens the extraction period, and at the same time greatly reduces the amount of acid consumed. More importantly, it does not use concentrated H 2 In the case of SO 4 , the pollution to the environment will also be greatly reduced. It also shows that the method of ternary two-phase AlCl 3 hydrolysis to extract saponin assisted by ethanol extractant significantly improves the yield of saponin and shortens the acid hydrolysis time. At the same time, no use and consumption of concentrated H 2 SO 4 means environmental pollution. It will also become the smallest.
因此,本发明乙醇提取剂辅助的三元双相AlCl
3水解制备皂素的方法显著的提高了皂素得率,缩短了提取周期,同时消耗的酸用量大大减少。更重要的,在不使用浓H
2SO
4的情况下,对环境的污染也将变得最小,具有较大的应用前景。
Therefore, the ternary two-phase AlCl 3 hydrolysis method for preparing saponin assisted by the ethanol extractant of the present invention significantly improves the saponin yield, shortens the extraction period, and greatly reduces the consumption of acid. More importantly, without the use of concentrated H 2 SO 4 , the pollution to the environment will also become minimal, which has a larger application prospect.