WO2019080571A1 - 一种苦楝素的提取方法 - Google Patents

一种苦楝素的提取方法

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Publication number
WO2019080571A1
WO2019080571A1 PCT/CN2018/097854 CN2018097854W WO2019080571A1 WO 2019080571 A1 WO2019080571 A1 WO 2019080571A1 CN 2018097854 W CN2018097854 W CN 2018097854W WO 2019080571 A1 WO2019080571 A1 WO 2019080571A1
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WIPO (PCT)
Prior art keywords
buffer
extracting
filtrate
filter residue
ultrasonic
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PCT/CN2018/097854
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English (en)
French (fr)
Inventor
毛军
陈红霞
陆鹏
袁洪胜
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江苏金太阳纺织科技股份有限公司
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Publication of WO2019080571A1 publication Critical patent/WO2019080571A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J71/00—Steroids in which the cyclopenta(a)hydrophenanthrene skeleton is condensed with a heterocyclic ring
    • C07J71/0005—Oxygen-containing hetero ring
    • C07J71/001—Oxiranes
    • C07J71/0021—Oxiranes at position 14(15)

Definitions

  • the invention relates to the technical field of plant active ingredient extraction, in particular to a method for extracting bitterin.
  • azadirachtin which is mainly extracted from the skin and fruit of tartary buckwheat tree. It has a variety of physiological activities. Its main component is tetracyclic triterpenoids, which have certain medical therapeutic effects and have Good broad-spectrum insecticidal and antibacterial properties, non-destructive to human body, easy to degrade, can be used to control crop diseases and insect pests, is a promising plant-derived pesticide.
  • the extraction methods of tartary buckwheat are mainly conventional extraction method, ultrasonic extraction method, microwave method, rapid extraction method, supercritical and subcritical solvent method, etc., but the above methods all have different degrees of disadvantages, which are mainly manifested in leaching.
  • ethanol is used as a solvent extraction method, and the yield is low;
  • the bitterin product obtained by the rapid extraction method using an organic solvent contains various oily components and impurities, and the high temperature process in the supercritical and subcritical extraction methods will be printed on the ink.
  • the effective chemical activity of the hormone produces a destructive effect.
  • the object of the present invention is to provide a method for extracting tartary buckwheat, which has mild extraction conditions, simple operation and high extraction rate, and is suitable for industrial large-scale production demand.
  • a method for extracting tartary buckwheat comprising the steps of:
  • Step 1 Wash and dry the bitter buckwheat or tartary buckwheat seeds, pulverize to 100-200 mesh, dissolve the obtained tartary buckwheat powder in water, and then add cellulase and pectinase to adjust the pH to 3.5-5.5, 40. -50 ° C reaction 1-3h, filtered to obtain filtrate 1 and filter residue 1;
  • Step 2 take the filter residue 1, add to the pH value of 2.0-3.0 buffer, mix and extract, filter to obtain filter residue 2 and filtrate 2;
  • Step 3 taking the filter residue 2, adding to a pH of 5.0-7.0 buffer, mixing and extracting, filtering to obtain filter residue 3 and filtrate 3;
  • Step 4 take the filter residue 3, add to the pH value of 8.0-8.5 buffer, mix and extract, filter to obtain filter residue 4 and filtrate 4;
  • Step 5 the filtrate 1, the filtrate 2, the filtrate 3 and the filtrate 4 obtained in the steps 1 to 4 are combined, and after extraction, the upper aqueous phase is obtained;
  • Step 6 membrane separation and purification: using a membrane separation technique to perform a membrane separation process on the aqueous phase liquid obtained in the step 5, to obtain a trapped solution,
  • step 7 the cut-off solution obtained in the step 6 is concentrated at a low temperature into a concentrated solution of tarthonin.
  • the mass ratio of bitter buckwheat powder to water in step 1 is 1:50-100.
  • the mass ratio of cellulase, pectinase and tartary buckwheat powder in step 1 is 1:1:10-20.
  • the bitter powder is dissolved by ultrasonic technology
  • the ultrasonic field ultrasonic power is 100W-400W
  • the ultrasonic frequency is 20KHz-80KHz.
  • the pH value of the buffer in step 2 is 2.0-3.0, and the buffer is selected from the group consisting of disodium hydrogen phosphate-phosphate buffer, acetic acid-sodium acetate buffer or acetic acid-ammonium acetate buffer; and the pH value is 5.0-7.0 buffer in step 3.
  • the pH value is 8.0-8.5 buffer selected from the group consisting of Ammonia-ammonium chloride buffer, disodium hydrogen phosphate-sodium dihydrogen phosphate buffer or disodium hydrogen phosphate-potassium dihydrogen phosphate buffer.
  • the ultrasonic technique is used in the step 2-4 to assist the extraction
  • the ultrasonic field ultrasonic power is 100W-400W
  • the ultrasonic frequency is 20KHz-80KHz.
  • the extraction solvent in step 5 is selected from the group consisting of methanol, ethanol, acetone, ethyl acetate, chloroform or dichloromethane.
  • the membrane separation in step 6 is a separation membrane having a molecular weight of ⁇ 1000 Da, the membrane separation operating temperature is 20 ° C - 40 ° C, and the operating pressure is 0.3 MPa - 1.5 MPa;
  • the low temperature concentration in step 7 is low temperature evaporation concentration, and the evaporation temperature is between 50 ° C and 60 ° C.
  • the invention adopts "semi-bionic"-membrane separation to extract bitterin.
  • “Semi-bionic” is a mimicking the transit process of oral drugs in the gastrointestinal tract and the environment in the gastrointestinal tract of animals, wherein the gastric juice pH is 2.0-3.5, the intestinal fluid pH is 5.0-7.0, and the large intestinal fluid pH is 7.5-8.5.
  • the acidic water and the alkaline water of the selected pH are successively and sequentially extracted, and the purpose is to extract an "active mixture" containing a high index component.
  • the invention firstly utilizes the semi-bionic technology to extract the medicinal materials by using an acid solution, a neutral solution or an alkali solution with different pH values, and simulates the absorption environment of the gastrointestinal tract in the animal, so that the extractable components of the animal body can be completely extracted and separated from the traditional purification.
  • the extraction condition is mild, the operation is simple, the extraction rate is high, and it is suitable for industrial large-scale production demand; the membrane separation technology is also used for secondary purification of the isolated tartary buckwheat solution, preferably the bitter macromolecule with molecular weight ⁇ 1000Da is intercepted.
  • the final product with a molecular weight of ⁇ 1000 Da has a good pharmacological effect and is more favorable for efficient absorption of the animal body.
  • the present invention also adds a biological enzyme in the extraction process, and utilizes the degradation of cellulase and pectinase to destroy the cellulose and pectin contained in the powder of tartary buckwheat or tartary buckwheat seed, destroying its structure and making it more loose. It accelerates the dissolution rate of bitterin, which is more conducive to the extraction of tartary buckwheat, greatly improving the extraction efficiency and extraction rate.
  • Ultrasonic technology is used in the extraction process, and the mechanical effect, cavitation effect and internal heating of the ultrasonic wave can effectively break the cell wall, increase the solubility of bitterin in the solvent, increase the diffusion speed and the concentration gradient during the leaching process. Accelerate the dissolution of active ingredients for high efficiency extraction.
  • the invention creatively uses the semi-biomimetic technology to separate and extract the tartary buckwheat raw material, and obtains the chuanxiong solution which can absorb the passive object with high effective component; the latter combines the membrane separation and interception technology to obtain the product of molecular weight ⁇ 1000Da tartaryin in semi-bionic technology. Synergistically with the membrane separation technology, the final content of the bitterin product obtained is higher.
  • the filter residue 1 was added to a disodium hydrogen phosphate-phosphate buffer solution having a pH of 2.2, and uniformly mixed at a normal temperature, and the filtrate 2 and the filter residue 2 were filtered after being sufficiently extracted in an ultrasonic field having an ultrasonic power of 200 W and an ultrasonic frequency of 20 KHz for 2 hours.
  • the filter residue 2 is added to a phthalic acid-sodium hydroxide buffer solution having a pH of 5.5, and uniformly mixed at a normal temperature, and the filtrate is filtered after being sufficiently extracted in an ultrasonic field having an ultrasonic power of 200 W and an ultrasonic frequency of 20 kHz for 2 hours.
  • filter residue 3 is added to a pH 8.0 disodium hydrogen phosphate-potassium dihydrogen phosphate buffer solution, uniformly mixed at room temperature, and fully extracted in an ultrasonic field with an ultrasonic power of 200 W and an ultrasonic frequency of 20 KHz for 2 hours. Filtered filtrate 4 and filter residue 4.
  • the filtrates 1-4 are combined, extracted with ethanol, allowed to stand, layered, and the upper aqueous phase is taken, and the above operation is repeated three times; the aqueous phase solution obtained in the above step is added to the membrane separation device for separation and purification, preferably for extraction.
  • the tartary buckwheat seeds are washed, dried, pulverized to 100-200 mesh, mixed with water, and the mass ratio of tartary buckwheat powder to water is 1:80; after thorough mixing, cellulase and pectinase, cellulase and pectin are added.
  • the mass ratio of the enzyme to the tartary buckwheat powder is 1:1:15, and the pH of the mixed system is adjusted to 3.5-5.5, the temperature is 40-50 ° C, and the reaction is fully reacted for 2 h in an ultrasonic field with an ultrasonic power of 200 W and an ultrasonic frequency of 20 KHz. Filtrate 1 and filter residue 1 were obtained by filtration.
  • the filter residue 1 is added to a buffer solution of disodium hydrogen phosphate-potassium dihydrogen phosphate at a pH of 6.0, and uniformly mixed at a normal temperature, and the filtrate 3 is filtered after being sufficiently extracted in an ultrasonic field having an ultrasonic power of 200 W and an ultrasonic frequency of 20 KHz for 2 hours.
  • Filter residue 3 the filter residue 3 is added to the ammonia water-ammonium chloride buffer solution with a pH of 8.2, and the mixture is uniformly mixed at normal temperature, and the filtrate 4 is filtered after being sufficiently extracted in an ultrasonic field with an ultrasonic power of 200 W and an ultrasonic frequency of 20 KHz for 2 hours. Filter residue 4.
  • the filtrates 1-4 are combined, extracted with ethanol, allowed to stand, layered, and the upper aqueous phase is taken, and the above operation is repeated 3 times; the aqueous phase solution obtained in the above step is added to the membrane separation device for separation and purification again, preferably Obtain an ultrafiltration membrane with a molecular weight of ⁇ 1000 Da, adjust the membrane separation operating temperature to 30 ° C, and operate at a pressure of 0.3 MPa to 0.5 MPa; transfer the trapped solution to an evaporation concentration device, stir well, allow to stand, filter, and concentrate at low temperature to form scutellarin Concentrated solution.
  • the filter residue 1 is added to an acetic acid-ammonium acetate buffer solution having a pH of 3.0, and uniformly mixed at a normal temperature, and the filtrate 2 and the filter residue 2 are filtered after being sufficiently extracted in an ultrasonic field having an ultrasonic power of 200 W and an ultrasonic frequency of 20 KHz for 2 hours;
  • the filter residue 2 is added to a buffer solution of disodium hydrogen phosphate-potassium dihydrogen phosphate at a pH of 6.5, and uniformly mixed at a normal temperature, and the filtrate 3 is filtered after being sufficiently extracted in an ultrasonic field having an ultrasonic power of 200 W and an ultrasonic frequency of 20 KHz for 2 hours.
  • Filter residue 3 The filter residue 3 is added to a buffer solution of disodium hydrogen phosphate-sodium dihydrogen phosphate at a pH of 8.5, uniformly mixed at room temperature, and fully extracted in an ultrasonic field with an ultrasonic power of 200 W and an ultrasonic frequency of 20 KHz for 2 hours. Filtrate 4 and filter residue 4.
  • the filtrates 1-4 are combined, extracted with ethanol, allowed to stand, layered, and the upper aqueous phase is taken, and the above operation is repeated 3 times; the aqueous phase solution obtained in the above step is added to the membrane separation device for separation and purification again, preferably Obtain the ultrafiltration membrane with molecular weight ⁇ 1000Da, adjust the membrane separation operation temperature to 40 °C, and operate the pressure to 0.3MPa-0.5MPa; transfer the trapped solution to the evaporation concentration equipment, stir well, let stand, filter, and concentrate at low temperature into bitter Concentrated solution.
  • This embodiment differs from Example 1 in that no membrane separation and purification is performed.
  • the filter residue 1 was added to a disodium hydrogen phosphate-phosphate buffer solution having a pH of 2.2, and uniformly mixed at a normal temperature, and the filtrate 2 and the filter residue 2 were filtered after being sufficiently extracted in an ultrasonic field having an ultrasonic power of 200 W and an ultrasonic frequency of 20 KHz for 2 hours.
  • the filter residue 2 is added to a phthalic acid-sodium hydroxide buffer solution having a pH of 5.5, and uniformly mixed at a normal temperature, and the filtrate is filtered after being sufficiently extracted in an ultrasonic field having an ultrasonic power of 200 W and an ultrasonic frequency of 20 kHz for 2 hours.
  • filter residue 3 is added to a pH 8.0 disodium hydrogen phosphate-potassium dihydrogen phosphate buffer solution, uniformly mixed at room temperature, and fully extracted in an ultrasonic field with an ultrasonic power of 200 W and an ultrasonic frequency of 20 KHz for 2 hours. Filtered filtrate 4 and filter residue 4.
  • the filtrates 1-4 are combined, extracted with ethanol, allowed to stand, layered, and the upper aqueous phase is taken, and the above operation is repeated 3 times; the aqueous phase solution obtained in the above step is transferred to an evaporation concentration device, stirred, and allowed to stand, filtered. And concentrated at low temperature into a concentrated solution of tarthonin.
  • Embodiment 1 The difference between this embodiment and Embodiment 1 is that the extraction is not performed using the semi-bionic technique.
  • the filtrate 1 is extracted with ethanol, allowed to stand, and then separated, and the upper aqueous phase is taken, and the above operation is repeated 3 times; the aqueous phase solution obtained in the above step is added to the membrane separation device for separation and purification, preferably, the molecular weight is ⁇ 1000 Da.
  • the ultrafiltration membrane is adjusted to have a separation temperature of 20 ° C and an operating pressure of 0.3 MPa to 0.5 MPa.
  • the retentate solution is transferred to an evaporation concentration apparatus, stirred, allowed to stand, filtered, and concentrated to a concentrated solution of tartary buckwheat.
  • the present invention uses a method of plotting a standard curve of a bitterin solution at a specific concentration, and measures the concentration of the final extracted product by measuring the absorbance value of the tartary buckwheat solution at an unknown concentration.
  • the specific data is shown in the following table.
  • the concentration of bitterin in the tartary buckwheat solution extracted by the semi-bionic method is higher because the mirabiine is present in the stomach of the animal.
  • the bitterin solution obtained by the conditional extraction process is more likely to be stably present and has more active ingredient content.
  • the above results indicate that the concentration of tartary buckwheat can be further enhanced by the membrane separation technique, and the excess impurities can be removed by the filtration retention of the macromolecular membrane, so that the concentration of the active component of the bitterin which can act with the organism is higher.

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Medicines Containing Plant Substances (AREA)

Abstract

本发明提供了一种苦楝素的提取方法，先将苦楝皮或苦楝种子洗净烘干后粉碎，采用纤维素酶和果胶酶水溶液提取；滤渣依次采用pH值为2.0-3.0、pH值为5.0-7.0和pH值为8.0-8.5的缓冲液萃取，滤液经有机溶剂萃取后再进行膜分离，截留溶液经低温浓缩制成苦楝素。本发明创造性地采用半仿生技术对苦楝素原料进行分离萃取，得到有效成分较高的能够被动物体吸收的川楝素溶液；后结合膜分离截取技术得到分子量≤1000Da苦楝素产物，在半仿生技术和膜分离技术协同作用下，使得最终得到的苦楝素产物有效成分含量更高。

Description

一种苦楝素的提取方法 技术领域
本发明涉及植物有效成分提取技术领域，具体涉及一种苦楝素的提取方法。
背景技术
苦楝素又被称为印楝素，主要是从苦楝树的皮和果实中提取而来，具有多种生理活性，其主要成分为四环三萜类物质，具有一定的医药治疗作用，并有着良好的广谱杀虫抗菌性能，对人体无损，易于降解，可以用来防治农作物病虫害，是一种极具潜力的植物源农药。
目前苦楝素的提取的方法主要为常规浸提法、超声提取法、微波法、快速萃取法、超临界和亚临界溶剂法等，但是上述方法都存在不同程度的弊端，具体主要表现在浸提法中以乙醇为溶剂萃取法产率低；利用有机溶剂的快速萃取法得到的苦楝素产品含有多种油性成分残留，杂质较多；超临界和亚临界提取法中的高温工艺会对印楝素的有效化学活性产生破坏作用。
发明内容
本发明的目的是提供一种苦楝素的提取方法，提取条件温和，操作简单、提取率高，适合工业化大规模的生产需求。
本发明采取的具体方案如下：
一种苦楝素的提取方法，其特征在于：包括以下步骤：
步骤1，将苦楝皮或苦楝种子洗净、烘干，粉碎至100-200目，将得到的苦楝粉末溶于水后再加入纤维素酶和果胶酶，调节pH值为3.5-5.5，40-50℃反应1-3h，过滤得到滤液1和滤渣1；
步骤2，取滤渣1，加至pH值为2.0-3.0缓冲液中，混匀萃取，过滤得到滤渣2和滤液2；
步骤3，取滤渣2，加至pH值为5.0-7.0缓冲液中，混匀萃取，过滤得到滤渣3和滤液3；
步骤4，取滤渣3，加至pH值为8.0-8.5缓冲液中，混匀萃取，过滤得到滤渣4和滤液4；
步骤5，将步骤1至4得到的滤液1、滤液2、滤液3和滤液4合并，萃取后，得到上层水相；
步骤6，膜分离提纯：利用膜分离技术对步骤5中得到的水相液体进行膜分离工艺，得到截留后的的溶液，
步骤7，将步骤6得到的截留溶液低温浓缩成苦楝素浓溶液。
优选地，步骤1中苦楝粉末与水的质量比为1：50-100。
优选地，步骤1中纤维素酶、果胶酶和苦楝粉末的质量比为1:1:10-20。
优选地，步骤1中用超声波技术辅助苦楝粉末溶解，超声场的超声功率为100W-400W，超声频率为20KHz-80KHz。
优选地，步骤2中pH值为2.0-3.0缓冲液选自磷酸氢二钠-磷酸缓冲液、醋酸-醋酸钠缓冲液或醋酸-醋酸铵缓冲液；步骤3中pH值为5.0-7.0缓冲液选自邻苯二甲酸-氢氧化钠缓冲液、磷酸氢二钠-磷酸二氢钾缓冲液或磷酸氢二钠-磷酸二氢钠缓冲液；步骤4中pH值为8.0-8.5缓冲液选自氨水-氯化铵缓冲液、磷酸氢二钠-磷酸二氢钠缓冲液或磷酸氢二钠-磷酸二氢钾缓冲液。
优选地，步骤2-4中使用超声波技术辅助萃取，超声场的超声功率为100W-400W，超声频率为20KHz-80KHz。
优选地，步骤5中的萃取溶剂选自甲醇、乙醇、丙酮、乙酸乙酯、三氯甲烷或二氯甲烷。
优选地，步骤6中膜分离是选用分子量≤1000Da的分离膜，膜分离的操作温度为20℃-40℃、操作压力为0.3MPa-1.5MPa；
优选地，步骤7中低温浓缩是低温蒸发浓缩，蒸发温度在50℃-60℃。
本发明采用“半仿生”-膜分离结合提取苦楝素。“半仿生”是模仿口服药物在胃肠道的转运过程和在动物胃肠道中的存在环境，其中胃液pH为2.0-3.5、小肠液pH为5.0-7.0、大肠液pH为7.5-8.5。采用选定pH的酸性水和碱性水依次连续提取，其目的是提取含指标成分高的“活性混合物”。
本发明首先利用半仿生技术，采用不同pH值的酸溶液、中性溶液、碱溶液提取药材，模拟动物内的胃肠道吸收环境，使得动物体可吸收利用成分提取完全，与传统的提纯分离方法相比，提取条件温和，操作简单、提取率高，适合工业化大规模的生产需求；还利用膜分离技术对已经分离的苦楝素溶液进行二次提纯，优选截取分子量≤1000Da的苦楝素大分子，原因是分子量≤1000Da的最终产物具有较好的药理作用，同时更加有利于动物体的高效吸收。
此外，本发明在提取过程中还加入了生物酶，利用纤维素酶和果胶酶的降解作用破坏苦楝皮或苦楝种子粉末中含有的纤维素和果胶质，破坏其结构，使其更加疏松，加速苦楝素的溶出速率，更加有利于苦楝素的萃取，大大提升萃取效率和萃取率。
在萃取过程中采用超声波技术，利用超声波的机械效应、空化效应和内加热作用，可有效地使细胞壁破裂，增加苦楝素在溶剂中的溶解度，提高其扩散速度和浸出过程中的浓度梯度，加速有效成分的溶出，实现高效率提取。
本发明创造性地采用半仿生技术对苦楝素原料进行分离萃取，得到有效成分较高的能够被动物体吸收的川楝素溶液；后结合膜分离截取技术得到分子量≤1000Da苦楝素产物，在半仿生技术和膜分离技术协同作用下，使得最终得到的苦楝素产物有效成分含量更高。
具体实施方式
下面结合具体实施例对本发明的技术方案作进一步说明。
实施例1
将苦楝皮洗净、烘干，粉碎至100-200目，加水混合，苦楝粉末与水的质量比为1:50；充分混合后，加入纤维素酶和果胶酶，纤维素酶和果胶酶和苦楝粉末的质量比为1:1:20，调节混合体系调节pH值为3.5-5.5，温度为40-50℃，在超声功率为200W、超声频率为20KHz的超声场中充分反应2h后，过滤得到滤液1和滤渣1。
将滤渣1加入到pH值为2.2的磷酸氢二钠-磷酸缓冲溶液中，常温下混合均匀，在超声功率为200W、超声频率为20KHz的超声场中充分萃取2h后过滤的滤液2和滤渣2；将滤渣2加入到pH值为5.5的邻苯二甲酸-氢氧化钠缓冲溶液中，常温下混合均匀，在超声功率为200W、超声频率为20KHz的超声场中充分萃取2h后过滤的滤液3和滤渣3；将滤渣3加入到pH值为8.0的磷酸氢二钠-磷酸二氢钾缓冲溶液中，常温下混合均匀，在超声功率为200W、超声频率为20KHz的超声场中充分萃取2h后过滤的滤液4和滤渣4。
将滤液1-4合并，用乙醇萃取，静置，分层后，取上层水相，重复上述操作3次；将上述步骤得到的水相溶液加入到膜分离设备中再次进行分离提纯，优选截取分子量≤1000Da的超滤膜，调节膜分离操作温度为20℃、操作压力为0.3MPa-0.5MPa；将截留溶液转移到蒸发浓缩设备，搅匀，静置，过滤，并低温浓缩成苦楝素浓溶液。
实施例2
将苦楝种子洗净、烘干，粉碎至100-200目，加水混合，苦楝粉末与水的质量比为1:80；充分混合后，加入纤维素酶和果胶酶，纤维素酶和果胶酶和苦楝粉末的质量比为1:1:15，调节混合体系调节pH值为3.5-5.5，温度为40-50℃，在超声功率为200W、超声频率为20KHz的超声场中充分反应2h后，过滤得到滤液1和滤渣1。
将滤渣1加入到pH值为2.6的醋酸-醋酸钠缓冲溶液中，常温下混合均匀，在超声功率为200W、超声频率为20KHz的超声场中充分萃取2h后过滤的滤液2和滤渣2；将滤渣2加入到pH值为6.0的磷酸氢二钠-磷酸二氢钾缓冲溶液中，常温下混合均匀，在超声功率为200W、超声频率为20KHz的超声场中充分萃取2h后过滤的滤液3和滤渣3；将滤渣3加入到pH值为8.2的氨水-氯化铵缓冲溶液中，常温下混合均匀，在超声功率为200W、超声频率为20KHz的超声场中充分萃取2h后过滤的滤液4和滤渣4。
将滤液1-4合并，用有乙醇萃取，静置，分层后，取上层水相，重复上述操作3次；将上 述步骤得到的水相溶液加入到膜分离设备中再次进行分离提纯，优选截取分子量≤1000Da的超滤膜，调节膜分离操作温度为30℃、操作压力为0.3MPa-0.5MPa；将截留溶液转移到蒸发浓缩设备，搅匀，静置，过滤，并低温浓缩成苦楝素浓溶液。
实施例3
将苦楝皮洗净、烘干，粉碎至100-200目，加水混合，苦楝粉末与水的质量比为1:100；充分混合后，加入纤维素酶和果胶酶，纤维素酶和果胶酶和苦楝粉末的质量比为1:1:10，调节混合体系调节pH值为3.5-5.5，温度为40-50℃，在超声功率为200W、超声频率为20KHz的超声场中充分反应2h后，过滤得到滤液1和滤渣1。
将滤渣1加入到pH值为3.0的醋酸-醋酸铵缓冲溶液中，常温下混合均匀，在超声功率为200W、超声频率为20KHz的超声场中充分萃取2h后过滤的滤液2和滤渣2；将滤渣2加入到pH值为6.5的磷酸氢二钠-磷酸二氢钾缓冲溶液中，常温下混合均匀，在超声功率为200W、超声频率为20KHz的超声场中充分萃取2h后过滤的滤液3和滤渣3；将滤渣3加入到pH值为8.5的磷酸氢二钠-磷酸二氢钠缓冲溶液中，常温下混合均匀，在超声功率为200W、超声频率为20KHz的超声场中充分萃取2h后过滤的滤液4和滤渣4。
将滤液1-4合并，用有乙醇萃取，静置，分层后，取上层水相，重复上述操作3次；将上述步骤得到的水相溶液加入到膜分离设备中再次进行分离提纯，优选截取分子量≤1000Da的超滤膜，调节膜分离操作温度为40℃、操作压力为0.3MPa-0.5MPa；将截留溶液转移到蒸发浓缩设备，搅匀，静置，过滤，并低温浓缩成苦楝素浓溶液。
对照例1
本实施例与实施例1的区别在于不进行膜分离提纯。
将苦楝皮洗净、烘干，粉碎至100-200目，加水混合，苦楝粉末与水的质量比为1:50；充分混合后，加入纤维素酶和果胶酶，纤维素酶和果胶酶和苦楝粉末的质量比为1:1:20，调节混合体系调节pH值为3.5-5.5，温度为40-50℃，在超声功率为200W、超声频率为20KHz的超声场中充分反应2h后，过滤得到滤液1和滤渣1。
将滤渣1加入到pH值为2.2的磷酸氢二钠-磷酸缓冲溶液中，常温下混合均匀，在超声功率为200W、超声频率为20KHz的超声场中充分萃取2h后过滤的滤液2和滤渣2；将滤渣2加入到pH值为5.5的邻苯二甲酸-氢氧化钠缓冲溶液中，常温下混合均匀，在超声功率为200W、超声频率为20KHz的超声场中充分萃取2h后过滤的滤液3和滤渣3；将滤渣3加入到pH值为8.0的磷酸氢二钠-磷酸二氢钾缓冲溶液中，常温下混合均匀，在超声功率为200W、超声频率为20KHz 的超声场中充分萃取2h后过滤的滤液4和滤渣4。
将滤液1-4合并，用乙醇萃取，静置，分层后，取上层水相，重复上述操作3次；将上述步骤得到的水相溶液转移到蒸发浓缩设备，搅匀，静置，过滤，并低温浓缩成苦楝素浓溶液。
对照例2
本实施例与实施例1的区别在于不利用半仿生技术进行提取。
将苦楝皮洗净、烘干，粉碎至100-200目，加水混合，苦楝粉末与水的质量比为1:50；充分混合后，加入纤维素酶和果胶酶，纤维素酶和果胶酶和苦楝粉末的质量比为1:1:20，调节混合体系调节pH值为3.5-5.5，温度为40-50℃，在超声功率为200W、超声频率为20KHz的超声场中充分反应2h后，过滤得到滤液1和滤渣1。
将滤液1用乙醇萃取，静置，分层后，取上层水相，重复上述操作3次；将上述步骤得到的水相溶液加入到膜分离设备中再次进行分离提纯，优选截取分子量≤1000Da的超滤膜，调节膜分离操作温度为20℃、操作压力为0.3MPa-0.5MPa；将截留溶液转移到蒸发浓缩设备，搅匀，静置，过滤，并低温浓缩成苦楝素浓溶液。
本发明采用绘制特定浓度下苦楝素溶液的标准曲线的方法，通过测量未知浓度苦楝素溶液的吸光度值来检测最终提取产物的浓度，具体数据如下表所示。
Figure PCTCN2018097854-appb-000001
从上表测试的结果可以看出，在其他条件都相同的情况下，通过半仿生法提取得到的苦楝素溶液中苦楝素的浓度更高，原因是在模拟苦楝素在动物体肠胃中存在的条件提取过程得到的苦楝素溶液更加容易稳定存在，有效成分含量更多。同时上述结果表明，通过膜分离技术可以进一步提升苦楝素的浓度，通过大分子膜的过滤截留作用可以除去多余的杂质，因此能够与生物体作用的苦楝素有效成分的浓度会更高。

Claims (9)

  1. 一种苦楝素的提取方法，其特征在于：包括以下步骤：
    步骤1，将苦楝皮或苦楝种子洗净、烘干，粉碎至100-200目，将得到的苦楝粉末溶于水后再加入纤维素酶和果胶酶，调节pH值为3.5-5.5，40-50℃反应1-3h，过滤得到滤液1和滤渣1；
    步骤2，取滤渣1，加至pH值为2.0-3.0缓冲液中，混匀萃取，过滤得到滤渣2和滤液2；
    步骤3，取滤渣2，加至pH值为5.0-7.0缓冲液中，混匀萃取，过滤得到滤渣3和滤液3；
    步骤4，取滤渣3，加至pH值为8.0-8.5缓冲液中，混匀萃取，过滤得到滤渣4和滤液4；
    步骤5，将步骤1至4得到的滤液1、滤液2、滤液3和滤液4合并，萃取后，得到上层水相；
    步骤6，利用膜分离技术对步骤5中得到的水相液体进行膜分离工艺，得到截留后的的溶液，
    步骤7，将步骤6得到的截留溶液低温浓缩成苦楝素浓溶液。
  2. 根据权利要求1所述的苦楝素的提取方法，其特征在于：步骤1中苦楝粉末与水的质量比为1：50-100。
  3. 根据权利要求1所述的苦楝素的提取方法，其特征在于：步骤1中纤维素酶、果胶酶和苦楝粉末的质量比为1:1:10-20。
  4. 根据权利要求1所述的苦楝素的提取方法，其特征在于：步骤1中用超声波技术辅助苦楝粉末溶解，超声场的超声功率为100W-400W，超声频率为20KHz-80KHz。
  5. 根据权利要求1所述的苦楝素的提取方法，其特征在于：步骤2中pH值为2.0-3.0缓冲液选自磷酸氢二钠-磷酸缓冲液、醋酸-醋酸钠缓冲液或醋酸-醋酸铵缓冲液；步骤3中pH值为5.0-7.0缓冲液选自邻苯二甲酸-氢氧化钠缓冲液、磷酸氢二钠-磷酸二氢钾缓冲液或磷酸氢二钠-磷酸二氢钠缓冲液；步骤4中pH值为8.0-8.5缓冲液选自氨水-氯化铵缓冲液、磷酸氢二钠-磷酸二氢钠缓冲液或磷酸氢二钠-磷酸二氢钾缓冲液。
  6. 根据权利要求1所述的苦楝素的提取方法，其特征在于：步骤2-4中使用超声波技术辅助萃取，超声场的超声功率为100W-400W，超声频率为20KHz-80KHz。
  7. 根据权利要求1所述的苦楝素的提取方法，其特征在于：步骤5中的萃取溶剂选自甲醇、乙醇、丙酮、乙酸乙酯、三氯甲烷或二氯甲烷。
  8. 根据权利要求1所述的苦楝素的提取方法，其特征在于：步骤6中膜分离是选用分子量≤1000Da的分离膜，膜分离的操作温度为20℃-40℃、操作压力为0.3MPa-1.5MPa；
  9. 根据权利要求1所述的苦楝素的提取方法，其特征在于：步骤7中低温浓缩是低温蒸发浓缩，蒸发温度在50℃-60℃。
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