WO2008138243A1 - Procédé de préparation de l'icariine - Google Patents

Procédé de préparation de l'icariine Download PDF

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Publication number
WO2008138243A1
WO2008138243A1 PCT/CN2008/070723 CN2008070723W WO2008138243A1 WO 2008138243 A1 WO2008138243 A1 WO 2008138243A1 CN 2008070723 W CN2008070723 W CN 2008070723W WO 2008138243 A1 WO2008138243 A1 WO 2008138243A1
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WO
WIPO (PCT)
Prior art keywords
icariin
ethanol
epimedium
preparing
water
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PCT/CN2008/070723
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English (en)
Chinese (zh)
Inventor
Kun Meng
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Beijing Shenogen Biomedical Co., Ltd
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Publication date
Application filed by Beijing Shenogen Biomedical Co., Ltd filed Critical Beijing Shenogen Biomedical Co., Ltd
Publication of WO2008138243A1 publication Critical patent/WO2008138243A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/44—Preparation of O-glycosides, e.g. glucosides
    • C12P19/60—Preparation of O-glycosides, e.g. glucosides having an oxygen of the saccharide radical directly bound to a non-saccharide heterocyclic ring or a condensed ring system containing a non-saccharide heterocyclic ring, e.g. coumermycin, novobiocin
    • C12P19/605—Preparation of O-glycosides, e.g. glucosides having an oxygen of the saccharide radical directly bound to a non-saccharide heterocyclic ring or a condensed ring system containing a non-saccharide heterocyclic ring, e.g. coumermycin, novobiocin to a 1-benzopyran-2-on (or the chalcones and hydrogenated chalcones thereof, e.g. coumermycin, novobiocin, novenamin)

Definitions

  • the invention relates to the field of traditional Chinese medicine, in particular to a method for extracting the active ingredient icariin from Chinese traditional medicine Epimedium.
  • Epimedium is a commonly used Chinese medicinal material. It is a dry part of the plants of the genus Epimedium genus of the Berberridaceae family.
  • the Pharmacopoeia of the People's Republic of China (2000 edition) contains the following five kinds of Epimedium medicinal herbs.
  • Epimedium brevicornum Maxim Epimedium sagittatum, Epimedium pubescens
  • Epimedium wushanenes Epimedium koreanum Nakai.
  • Epimedium has the effects of invigorating the kidney and strengthening the yang, strengthening the muscles and strengthening the bones, and removing the phlegm and dehumidification.
  • icariin The molecular structure of icariin is:
  • the chemical name is: 3, 5, 7-trihydroxy-4' methoxy-8-isopentenyl flavonoid-L-pyran rhamnose ⁇ -D-glucopyranoside.
  • icariin contains two glycosyl groups, namely the glucosyl group at the 3-position and the rhamnosyl group at the 7-position, its activity is poor, and the degasting product of icariin is lascivious.
  • the activity of alizarin is very high.
  • Most of the domestic acid hydrolysis methods are used.
  • hydrochloric acid hydrolysis easily forms an addition with the double bond on the 8-substituent, resulting in a product with a low yield and no use value.
  • sulfuric acid hydrolysis can only remove the 7-position glycosyl group.
  • the invention provides a preparation method of icariin, which has complete deglycation reaction and high yield, simple operation of the whole process, convenient treatment after reaction and simple purification.
  • a method for preparing icariin which is characterized in that icariin is obtained by enzymatic hydrolysis using ⁇ -glucosidase.
  • the weight ratio of the ⁇ -glucosidase to icariin is 1 : 1-10, and the enzymatic reaction conditions are carried out in an alcohol-water solution at 40-60 ° C for 20-30 hours.
  • the weight ratio of ⁇ -glucosidase to icariin is 1:5, and the enzymatic reaction conditions are carried out in an alcohol-water solution at 50 ° C for 24 hours, and the alcohol is ethanol.
  • the post-treatment of the reaction mixture after the enzymatic hydrolysis is carried out by a centrifugal treatment method in which the reaction mixture is centrifuged to discard the supernatant, dissolved in acetone, centrifuged, filtered, discarded, and the filtrate is steamed. dry.
  • the preparation method of the icariin further comprises purification of icariin, which is recrystallized by acetone-water.
  • the preparation method of icariin further comprises the steps of preparing icariin, wherein the preparation step of icariin comprises extracting total flavonoids of Epimedium and purifying icariin, and the total flavonoids of Epimedium Extraction steps include
  • the concentrated solution content was 0.2 g of Epimedium Herbs / ml.
  • the D101 macroporous resin is used in an amount of 1-2 times that of the epimedium raw material, the upper column flow rate of the concentrated liquid is 5 column volumes/h, and the eluent is used in an amount of 3-4 column volumes.
  • the purification of the icariin comprises a separation and purification method using a silica gel column, which includes using 10:0, 9:1, 8:1, 7:1, 6:1 chloroform-methanol gradient elution; Concentration of fractions containing icariin by TLC.
  • the purification of the icariin further comprises recrystallizing the concentrate obtained above with methanol, and the filtrate is washed successively with acetone and methanol.
  • ⁇ -glucosidase ⁇ -Glycosidase can hydrolyze the sugar substituents in the above two positions in one step, and the enzymatic hydrolysis yield is high (up to about 55%), and the subsequent treatment is simple, the whole enzymatic hydrolysis process is simple, and industrial production is easy.
  • the ratio of the amount of ⁇ -glucosidase added to the weight of the reactant is 1:1-10, and the enzymatic reaction conditions are carried out in an alcohol-water solution at 40-60 ° C for 20-30 hours, since the icariin can be dissolved.
  • an aqueous alcohol solution is used as the reaction liquid, and the alcohol is selected from commonly used ethanol, which has low cost and low pollution. Since the hydrolysis reaction is carried out in ethanol, the enzyme is relatively easy to be inactivated, so the reaction time should not be too long, so the selection time is 20-30 hours, at which time the glycolytic reaction is substantially completed.
  • the weight ratio of ⁇ -Glycosidase to icariin is 1:5, and the enzymatic reaction conditions are carried out in an alcohol-water solution at 50 ° C for 24 hours.
  • the reaction mixture is centrifuged to discard the supernatant, then dissolved in acetone, centrifuged, filtered, and the filter residue is discarded.
  • the filtrate is evaporated to dryness to obtain icariin, which is very simple and convenient, and is easy to industrialize.
  • Icariin can also be further purified by recrystallization to obtain a product of high purity, and the recrystallization solvent is an acetone-water mixed solvent.
  • the purified ampicillin obtained in the present invention has a yield of 55%.
  • the invention can be used for enzymatic hydrolysis of icariin, and can also be self-extracted from Epimedium medicinal materials.
  • the advantage of self-extraction is that it can save cost, and can also monitor impurity content, thereby adopting various kinds of targeted Way to remove some impurities.
  • the preparation of icariin comprises the extraction of total flavonoids of Epimedium and the purification of icariin, and the extraction step of the total flavonoids of Epimedium includes boiling the extract of Epimedium with water, and then concentrating the extract to 0.2 g.
  • Astragalus medicinal herbs/ml, adsorbed with D101 macroporous resin column (D101 macroporous resin column should be cleaned and repacked with some organic solvents adsorbed on it before use.
  • D101 macroporous resin is used for epimedium raw materials. 1-2 times, the upper column flow rate of the concentrate is 5 column volumes / h, the amount of eluent is 3-4 column volumes, so that the separation effect is better.), eluted with an ethanol-water gradient.
  • the eluate is the total flavonoid product of Epimedium, collected and concentrated.
  • the yield is about 2% (that is, 100 g of Epimedium can obtain about 2 g of total flavonoids of Epimedium).
  • the D101 macroporous resin column was rinsed back with 90% ethanol and water.
  • the total flavonoids of Herba Epimedii are detected by HPLC and contain about 25% of icariin. Further purification is required for enzymatic hydrolysis.
  • the purification of icariin was carried out by silica gel column separation and purification using a 10:0, 9:1, 8:1, 7:1, 6:1 gradient of chloroform-methanol;
  • the obtained icariin was further purified, recrystallized from methanol, and the filtrate was washed with acetone and methanol.
  • the purity of the product icariin thus obtained is about 95%, which fully satisfies the requirements of the next enzymatic reaction.
  • the invention adopts ⁇ -glucosidase to carry out enzymatic hydrolysis reaction of icariin, and has complete deglycosylation and high yield, and adopts self-designed extraction of icariin from Epimedium medicinal material, and the whole process operation It is simple, easy to handle after reaction, and the purity of the product is in full compliance with the medicinal standards.
  • Example 1 is commercially available products, and the purity is 98% or more.
  • Example 3 is a self-made product, and ⁇ -glucosidase ( ⁇ -Glycosidase) is an Amano enzyme preparation company. Product.)
  • Example 1 is a self-made product, and ⁇ -glucosidase ( ⁇ -Glycosidase) is an Amano enzyme preparation company. Product.)
  • Post-treatment of the reaction solution Centrifuge the reaction solution and discard the supernatant. The precipitate was dissolved in 2,500 ml of acetone, and the solution was centrifuged. The supernatant was taken out, filtered, and the insoluble matter was discarded, and the supernatant was evaporated to dryness.
  • Post-treatment of the reaction solution Centrifuge the reaction solution and discard the supernatant. The precipitate was dissolved in 2,500 ml of acetone, and the solution was centrifuged. The supernatant was taken out, filtered, and the insoluble matter was discarded, and the supernatant was evaporated to 25 ml per gram (relative to icariin).
  • the total yield is 55.5 %.
  • the yield of total flavonoids is about 2% (that is, 2 g of total flavonoids can be obtained from 100 g of medicinal materials), and the content of icariin is about 25% by HPLC. See Figure 3. (For the determination method, see the first edition of the 2005 Chinese Pharmacopoeia, page 229)
  • the yield of icariin in this step is about 10%. (ie 100g total flavonoids can get 10g icariin) 3.3 Preparation of icariin
  • the concentrated supernatant was taken, and an equal amount of water was added to recrystallize, placed, crystallized, filtered, and dried to obtain 6 g of icariin.
  • the yield of this step is 55%.
  • the self-made icariin has a purity of only 95% or more, and the enzymatic hydrolysate is also completely complete, and the obtained icariin can fully meet the medicinal standard.

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  • Organic Chemistry (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Microbiology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Biotechnology (AREA)
  • Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicines Containing Plant Substances (AREA)

Abstract

Cette invention se rapporte à un procédé de préparation de l'icariine qui comprend les étapes consistant à traiter l'icariine avec la β-glucosidase dans une solution constituée d'éthanol et d'eau, puis à obtenir l'icariine par centrifugation et recristallisation. L'invention a également trait à un procédé de préparation de l'icariine qui comprend les étapes consistant à absorber l'extrait aqueux de l'épimède concentré en utilisant une résine macroporeuse D101, à éluer la résine avec différentes concentrations d'éthanol, puis à séparer la fraction d'élution d'éthanol à 50 % par chromatographie sur gel de silice et à obtenir l'icariine par recristallisation.
PCT/CN2008/070723 2007-05-09 2008-04-16 Procédé de préparation de l'icariine WO2008138243A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN2007100990251A CN101302548B (zh) 2007-05-09 2007-05-09 淫羊藿素的制备方法
CN2007100990251 2007-05-09

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111560409A (zh) * 2020-05-08 2020-08-21 陕西嘉禾生物科技股份有限公司 一种淫羊藿甙的制备方法
CN112961891A (zh) * 2021-03-25 2021-06-15 西安巨子生物基因技术股份有限公司 利用双相酶促反应制备淫羊藿苷的方法

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CN102070688A (zh) * 2010-12-20 2011-05-25 大兴安岭林格贝有机食品有限责任公司 一种富集纯化淫羊藿中淫羊藿苷的方法
CN102718819B (zh) * 2012-07-10 2013-07-03 刘志强 一种制备淫羊藿苷的工艺
EP2808016B1 (fr) 2013-05-31 2018-10-17 Beijing Shenogen Pharma Group Ltd. Utilisation de l'icaritine pour la préparation d'une composition de traitement du cancer
CN103305564B (zh) * 2013-07-05 2014-10-15 西安瑞天生物科技有限公司 将淫羊藿苷转化为淫羊藿素的方法
CN104127466B (zh) * 2013-08-23 2018-05-22 江苏省中医药研究院 一种淫羊藿总黄酮口服肠溶制剂及其应用
CN104711300B (zh) * 2013-12-13 2018-07-24 北京珅奥基医药科技有限公司 淫羊藿素的制备方法
CN104059116A (zh) * 2014-04-30 2014-09-24 西安岳达植物科技有限公司 一种从淫羊藿中分离淫羊藿甙的方法
CN103936705B (zh) * 2014-05-05 2015-10-21 北京盛诺基医药科技有限公司 阿可拉定化合物的晶型、含有该晶型的药物及用途
CN104230870A (zh) * 2014-09-16 2014-12-24 北京盛诺基医药科技有限公司 一种阿可拉定化合物以及该化合物的用途
CN105476957B (zh) * 2014-12-18 2021-04-20 北京珅奥基医药科技有限公司 一种阿可拉定注射剂及其制备方法和用途
CN106148454B (zh) * 2015-03-24 2021-01-26 北京珅奥基医药科技有限公司 一种宝藿苷ⅰ的制备方法
CN104711301B (zh) * 2015-03-24 2018-07-24 北京盛诺基医药科技有限公司 一种淫羊藿素的制备方法
CN106995829B (zh) * 2017-05-12 2021-03-30 南京林业大学 一种酶法转化淫羊藿总黄酮制备淫羊藿苷元的方法
CN107641621B (zh) * 2017-06-14 2021-07-23 江苏康缘药业股份有限公司 一种糖苷酶组合物及酶法制备淫羊藿苷元的方法
CN108440620A (zh) * 2018-04-26 2018-08-24 赣州禾绿康健生物技术有限公司 一种高含量淫羊藿苷提取物的制备方法
CN110143942B (zh) * 2019-05-30 2021-04-09 苏州禾研生物技术有限公司 一种有机酸水解淫羊藿苷制备淫羊藿素及鼠李糖糖浆的方法
CN110699263B (zh) * 2019-10-29 2021-05-11 浙江工业大学 黑曲霉yh-6及其应用于提高淫羊藿中淫羊藿素的含量
CN112138017A (zh) * 2020-08-27 2020-12-29 上海中医药大学 淫羊藿苷的酶解产物及其主要组分宝藿苷i的医药用途
CN112553264A (zh) * 2020-12-16 2021-03-26 金凤燮 酶转化高效制备淫羊藿苷元的方法

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111560409A (zh) * 2020-05-08 2020-08-21 陕西嘉禾生物科技股份有限公司 一种淫羊藿甙的制备方法
CN111560409B (zh) * 2020-05-08 2023-04-28 陕西嘉禾生物科技股份有限公司 一种淫羊藿甙的制备方法
CN112961891A (zh) * 2021-03-25 2021-06-15 西安巨子生物基因技术股份有限公司 利用双相酶促反应制备淫羊藿苷的方法
CN112961891B (zh) * 2021-03-25 2024-01-26 西安巨子生物基因技术股份有限公司 利用双相酶促反应制备淫羊藿苷的方法

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