WO2014131213A1 - Process for preparing epigallocatechin gallate and application thereof - Google Patents

Process for preparing epigallocatechin gallate and application thereof Download PDF

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Publication number
WO2014131213A1
WO2014131213A1 PCT/CN2013/072763 CN2013072763W WO2014131213A1 WO 2014131213 A1 WO2014131213 A1 WO 2014131213A1 CN 2013072763 W CN2013072763 W CN 2013072763W WO 2014131213 A1 WO2014131213 A1 WO 2014131213A1
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Prior art keywords
green tea
epigallocatechin gallate
extract
anemia
tea extract
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PCT/CN2013/072763
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French (fr)
Chinese (zh)
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黄璐
杨爽
张思佳
牟峰
汪建
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武汉华大药业有限公司
深圳华大基因健康科技有限公司
武汉华大基因科技有限公司
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Publication of WO2014131213A1 publication Critical patent/WO2014131213A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
    • C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
    • C07D311/04—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
    • C07D311/58—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring other than with oxygen or sulphur atoms in position 2 or 4
    • C07D311/60—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring other than with oxygen or sulphur atoms in position 2 or 4 with aryl radicals attached in position 2
    • C07D311/62—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring other than with oxygen or sulphur atoms in position 2 or 4 with aryl radicals attached in position 2 with oxygen atoms directly attached in position 3, e.g. anthocyanidins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/06—Antianaemics

Definitions

  • the invention belongs to the technical field of medicine, and particularly relates to a preparation method of epigallocatechin gallate and use thereof. Background technique
  • tea polyphenol also known as tea tannin
  • catechin which is a general term for catechins, flavonoids, phenolic acids and anthocyanidic compounds in tea.
  • the dry weight of tea is 18-36%.
  • catechins According to the chemical constituents of tea polyphenols, the main component of tea polyphenols is catechins, which account for 70% ⁇ 80% of the total amount of tea polyphenols, mainly containing catechins (C) and epicatechins.
  • a variety of active substances such as EC (EC), epicatechin gallate (ECG), epigallocatechin (EGC), and epigallocatechin gallate (EGCG).
  • Anemia usually refers to the lowest value of hemoglobin, red blood cell and/or hematocrit in peripheral blood, which is lower than that of normal animals of the same sex and age. The hemoglobin value is the most valuable value.
  • Common clinical anemia is iron deficiency anemia and megaloblastic anemia. Iron deficiency anemia can be treated with iron such as ferrous sulfate, ferric ammonium citrate, iron dextran, and megaloblastic anemia can be treated with folic acid and vitamin B 12 .
  • Aplastic anemia is caused by inhibition of bone marrow hematopoietic function, and it is difficult to treat. The main treatment for it is supportive therapy, blood-promoting and bone marrow transplantation.
  • Supportive therapy is mainly to infusion of red blood cells and platelets to maintain blood cell count, and repeated blood transfusion is easy to cause transfusion hemochromatosis, and only use hematopoietic growth factors such as G-CSF, EPO and other hematopoietic growth factors to promote hematopoietic treatment, clinically no significant effect.
  • Drugs for the treatment of aplastic anemia are expensive, costly to treat, and unsatisfactory.
  • Thalassemia also known as globin-producing anemia, is due to an imbalance between the amount of globin peptide synthesis, so correcting the imbalance of globin peptide chains is the key to the treatment of thalassemia.
  • Medications such as globin gene regulators and auxiliary antioxidants, bismuth iron and other high-throughput blood transfusions can alleviate the disease and improve symptoms.
  • the treatment is easy to master, but patients must take symptomatic drugs for life or rely on blood transfusion, so find a safe Effective anemia treatments are very meaningful.
  • the present invention is directed to solving at least some of the above technical problems or at least providing a useful commercial choice. Accordingly, it is an object of the present invention to provide a process for the preparation of epigallocatechin gallate and its novel use.
  • Green tea has the functions of clearing away heat and detoxifying, astringent dehumidification, etc., and it has been proved by modern medical research that green tea also has the functions of anti-virus, antibacterial, anti-oxidation, anti-proliferation and anti-anemia.
  • tea polyphenols, catechins and epicatechins in the treatment of anemia.
  • This patent provides a new traditional Chinese medicine monomer component for the treatment of anemia, epigallocatechin gallate.
  • the composition has been extensively studied in recent years, and it is the monomer component with the highest content in green tea, and has high development value.
  • epigallocatechin gallate has a good effect in treating hemolytic anemia, iron deficiency anemia, aplastic anemia, thalassemia, and excellent
  • the green tea polyphenols and catechins and epicatechins were reported in the report.
  • epigallocatechin gallate is used in the treatment of type B thalassemia, Heinz body hemolytic anemia, iron deficiency anemia, and the curative effect is significantly better than green tea polyphenols and catechins.
  • epicatechin can be prepared into an excellent anti-anemia drug.
  • the invention provides a method of preparing epigallocatechin gallate.
  • the method comprises: subjecting green tea to alcohol extraction to obtain a green tea extract; purifying the green tea extract to obtain a green tea extract; and purifying the green tea extract to obtain An epigallocatechin gallate crude extract; and the crude epigallocatechin gallate extract is recrystallized to obtain epigallocatechin gallate.
  • epigallocatechin gallate in the form of a monomer compound can be efficiently produced at a relatively low cost.
  • the purity of the epigallocatechin gallate prepared according to the method of the present invention was determined to be as high as 93% or more by the HPLC method.
  • the above method may also have the following additional technical features:
  • the green tea is subjected to alcohol extraction to obtain the green tea extract further comprising: heating the green tea medicinal material with 6-15 times by weight of 30%-80% ethanol by heating and refluxing for 1-4 times, each 0.5 to 3 hours later, the separately obtained extracts were combined to obtain the green tea extract.
  • the efficiency of preparing epigallocatechin gallate can be further improved.
  • the green tea medicinal material is heated and refluxed with 8-12 times by weight of 50%-70% ethanol for 1-3 times, each time for 1-3 hours, and the extract is combined to obtain a green tea extract. .
  • the efficiency of preparing epigallocatechin gallate can be further improved.
  • the green tea medicinal material is heated and refluxed twice with 10 times the amount of 60% ethanol for 1.5 times each time. In hours, combine the extracts. Thereby, the efficiency of preparing epigallocatechin gallate can be further improved.
  • the purifying the green tea extract further comprises: performing the first decompression treatment on the green tea extract to remove ethanol; and dissolving the obtained residue in water and extracting with ethyl acetate And separating an ethyl acetate phase containing epigallocatechin gallate; and subjecting the ethyl acetate phase containing epigallocatechin gallate to a second decompression treatment to obtain the green tea extract. Thereby, the efficiency of preparing epigallocatechin gallate can be further improved.
  • the first reduced pressure treatment is carried out at a temperature of 35 to 70 ° C, preferably 50 to 60 ° C, more preferably 50 ° C.
  • the obtained residue is dissolved in water and extracted with ethyl acetate 1 to 4 times, preferably 2 to 3 times, more preferably 2 times.
  • ethyl acetate 1 to 4 times preferably 2 to 3 times, more preferably 2 times.
  • the residue obtained is dissolved in water and extracted with an equal volume of ethyl acetate.
  • the efficiency of preparing epigallocatechin gallate can be further improved.
  • the second reduced pressure treatment is carried out at a temperature of 40 to 90 degrees Celsius, preferably 40 to 60 degrees Celsius, more preferably 50 degrees Celsius.
  • a temperature of 40 to 90 degrees Celsius preferably 40 to 60 degrees Celsius, more preferably 50 degrees Celsius.
  • the green tea extract is purified to obtain a crude extract of epigallocatechin gallate further comprising: dissolving the green tea extract in water, passing through a macroporous resin column, collecting The ethanol eluted fraction was dried under reduced pressure to obtain the crude gallage of gallic catechin gallate.
  • the efficiency of preparing epigallocatechin gallate can be further improved.
  • the oversized resin column is subjected to a ratio of 0.5 to 3 g of green tea / g resin, preferably 1.0 to 2.5 g of medicinal material / g of resin, more preferably 2 g of medicinal material / g of resin.
  • a ratio of 0.5 to 3 g of green tea / g resin preferably 1.0 to 2.5 g of medicinal material / g of resin, more preferably 2 g of medicinal material / g of resin.
  • the macroporous resin column is first eluted with 2-6 column volumes of water, preferably 3-5 column volumes of water, more preferably 3 column volumes of water, and then used. 4-8 column volumes, preferably 5-7 column volumes, more preferably 6 column volumes of 5%-15% ethanol, preferably 8%-12% ethanol, more preferably 10% ethanol to the macroporous resin column Elution is performed. Thereby, the efficiency of preparing epigallocatechin gallate can be further improved.
  • the recrystallizing the epigallocatechin gallate crude extract further comprises: formulating the epigallocatechin gallate crude extract into a saturated aqueous solution; An organic solvent is added to the saturated aqueous solution to carry out recrystallization. Thereby, the purity of the preparation of epigallocatechin gallate can be further improved.
  • the organic solvent is a mixture of methanol and isopropanol, wherein methanol and The volume ratio of propanol is 2:1.
  • the recrystallization is carried out at a temperature of 0 to 15 degrees Celsius, preferably 5 to 10 degrees Celsius, more preferably 8 degrees Celsius. Thereby, the purity of the preparation of epigallocatechin gallate can be further improved.
  • the weight ratio of the epigallocatechin gallate to the organic solvent is l g: 2-5 ml.
  • the weight ratio of epigallocatechin gallate to organic solvent is 1 g: 3 ml.
  • the invention provides the use of epigallocatechin gallate in the preparation of an anti-anaemia product, preferably the epigallocatechin gallate is prepared according to the method described above of.
  • the type of the anti-anemia product is not particularly limited.
  • the anti-anemia product may be a drug, a health care product or a food product for preventing or treating anemia.
  • the drug may be at least one of a tablet, a capsule, a granule, or an injection.
  • the anemia which can be treated or prevented by the anti-anemia product of the present invention may be at least one selected from the group consisting of iron deficiency anemia, hemolytic anemia, aplastic anemia, and thalassemia.
  • the anemia which can be treated or prevented is at least one selected from the group consisting of iron deficiency anemia, beta thalassemia, and Heinz small body hemolytic anemia.
  • epigallocatechin gallate in the treatment of anemia is superior to other active ingredients of green tea, green tea polyphenols, epicatechin, catechins, epicatechin gallic acid Ester, epigallocatechin catechin.
  • the pharmaceutically active ingredient epigallocatechin gallate according to an embodiment of the present invention is preferably used for the treatment of anemia.
  • EGCG significantly increased red blood cell count (RBC) in hemolytic anemia mice and significantly increased hemoglobin (Hb) in hemolytic anemia mice.
  • RBC red blood cell count
  • Hb hemoglobin
  • EGCG can be used to treat hemolytic anemia.
  • the synthesis of hemoglobin peptide chains in RBC is increased, which can be used for the treatment of thalassemia.
  • EGCG significantly increased RBC in iron-deficient anemia mice and significantly increased Hb in iron-deficient anemia mice.
  • Description EGCG can be used to treat iron deficiency anemia.
  • the ethyl acetate layer was subjected to reduced pressure at 50 ° C to obtain a green tea extract; After dissolving in pure water, filtering, preparing a solution of 2 g of medicinal material/g resin on a macroporous resin column, eluting with 3 column volumes of water, and eluting with 6 column volumes of 10% ethanol, collecting ethanol wash The part is dehydrated and dried under reduced pressure to obtain a crude extract of epigallocatechin gallate; a methanol/isopropyl alcohol mixture is added to a saturated aqueous solution of epigallocatechin gallate crude extract
  • the volume ratio of methanol to isopropanol is 2: 1, the weight ratio of EGCG to the mixed solution is lg: 3 ml), and recrystallization is repeated once at 8 ° C to obtain epigallocatechin gallate solid. 1.23g, purity is 95.5%
  • mice 120 Kunming mice, half male and half female, weighing 20 ⁇ 2 g, are in good health. They are provided by the Experimental Animal Center of Wuhan University Medical College. Production license number: SCXK (E) 2008-0004. EGCG (prepared according to Example 1, batch number 20110901);
  • mice were randomly divided into 6 groups, 20 in each group, labeled as: blank control group, EGCG group, tea polyphenol group, epicatechin group, catechin group, model control group, except blank Outside the control group, the other 5 groups of mice were intraperitoneally injected with a physiological saline solution of phenylhydrazine at a dose of 90 mg_kg- 1 , and the mice in each group were intragastrically administered once a day for 1 week, and given the last time. Blood samples were taken 1 h after the drug to measure hemoglobin (Hb) and red blood cell count (RBC).
  • Hb hemoglobin
  • RBC red blood cell count
  • the blank control group and the model control group were given an equal volume of normal saline, the EGCG group was administered at a dose of 30 mg/kg body weight, the tea polyphenol group was administered at a dose of 60 mg/kg body weight, and the epicatechin group was administered at a dose of 18.8.
  • the mg/kg body weight and the catechin group were administered at a dose of 20 mg/kg body weight.
  • Hb and RBC of the model control group were significantly lower (P ⁇ 0.01), indicating that the animal model was successful.
  • EGCG group can significantly increase RBC in hemolytic anemia mice (P ⁇ 0.05), and can significantly increase Hb in hemolytic anemia mice (P ⁇ 0.01), catechin group and epicatechin group in mice with hemolytic anemia.
  • the RBC value was significantly improved, and the catechin group had a significant effect on Hb (P ⁇ 0.05), while the epicatechin group and the tea polyphenol group had no significant effects on RBC and Hb.
  • Example 7 Therapeutic effect of epigallocatechin gallate on iron deficiency anemia
  • mice 140 Kunming mice, male and female, weighing 20 ⁇ 2 g, in good health, provided by Experimental Animal Center of Wuhan University Medical College, production license number: SCXK (E) 2008-0004.
  • EGCG prepared according to Example 1, batch number 20110901);
  • mice were randomly divided into 7 groups, 20 in each group, labeled as: blank control group, EGCG group, tea polyphenol group, epicatechin group, catechin group, ferrous sulfate group (positive Control group), model control group, model control group
  • the mice in each administration group were fed with low-iron feeding, normal drinking water, and blood was taken twice a week for 0.4 ml each time for 2 weeks.
  • the blank control group was fed with normal feeding materials and normal drinking water.
  • the other groups continued to use low-iron feeding, and were administered by intragastric administration once a day for seven consecutive days. At the end of the last administration, blood was collected for Hb and RBC.
  • the blank control group and the model control group were given an equal volume of normal saline, the EGCG group was administered at a dose of 30 mg/kg body weight, the tea polyphenol group was administered at a dose of 60 mg/kg body weight, and the epicatechin group was administered at a dose of 18.8 mg/kg body weight, the catechin group was administered at a dose of 20 mg/kg body weight, and the ferrous sulfate group was administered at a dose of 90 mg/kg body weight.
  • Hb (P ⁇ 0.05) and RBC of the model control group were significantly lower (P ⁇ 0.01), indicating that the animal model was successful.
  • EGCG group can significantly increase RBC in mice with iron deficiency anemia (P ⁇ 0.05), and can significantly increase Hb (P ⁇ 0.01) in iron deficiency anemia mice, ferrous sulfate tablets, catechin group, epicatechin
  • Both the group and the tea polyphenol group significantly improved the RBC (P ⁇ 0.05), and the ferrous sulfate group significantly increased the Hb in the iron-deficient anemia mice (P ⁇ 0.05), while the catechin group, the table
  • the effects of the tea group and tea polyphenols on Hb were not significant.
  • Example 8 Therapeutic effect of EGCG on aplastic anemia
  • mice 120 Kunming mice, half male and half female, weighing 20 ⁇ 2 g, are in good health. They are provided by the Experimental Animal Center of Wuhan University Medical College. Production license number: SCXK (E) 2008-0004. EGCG (prepared according to Example 1, batch number 20110901);
  • mice were randomly divided into 6 groups, 20 in each group, labeled as: blank control group, EGCG group, tea polyphenol group, epicatechin group, catechin group, model control group, except blank Outside the control group, the other 5 groups of model mice were pressed Busulfan was administered at a dose of 15 mg/kg once a week for 10 weeks.
  • the blank control group and the model control group were given an equal volume of normal saline, the EGCG group was administered at a dose of 5.6 mg/kg body weight, the tea polyphenol group was administered at a dose of 10 mg/kg body weight, and the epicatechin group was administered at a dose of 10 mg/kg body weight.
  • the catechin group was administered at a dose of 3.75 mg/kg body weight once daily for 20 days.
  • MNC refers to mononuclear cells (Mononuclear Cell)
  • Preparation method Prepare EGCG solid according to Example 5, add starch, dextrin, mix and sieve, granulate, pass 16 mesh sieve, dry at 50 degrees Celsius, whole grain, mix with appropriate amount of magnesium stearate, and obtain capsules.
  • EGCG was prepared according to Example 2, EGCG was dissolved in water for injection, all dissolved, sodium chloride was added to dissolve, and water for injection was added to 1000 ml, filtered, potted in a 5 mL ampoule, 100 ° C. The bacteria are available for 30 minutes.
  • the description of the terms “one embodiment”, “some embodiments”, “example”, “specific example”, or “some examples” and the like means a specific feature described in connection with the embodiment or example.
  • a structure, material or feature is included in at least one embodiment or example of the invention.
  • the schematic representation of the above terms does not necessarily mean the same embodiment or example.
  • the particular features, structures, materials, or characteristics described may be combined in a suitable manner in any one or more embodiments or examples.

Abstract

A process for preparing epigallocatechin gallate and application thereof. The process comprises: extracting green tea with alcohol so as to obtain green tea extract; purifying the green tea extract so as to obtain green tea extract; purifying the green tea extract so as to obtain crude extract of epigallocatechin gallate; and recrystallizing the crude extract of epigallocatechin gallate so as to obtain epigallocatechin gallate.

Description

表没食子儿茶素没食子酸酯的制备方法及其用途  Preparation method of epigallocatechin gallate and use thereof
优先权信息 Priority information
本申请请求 2013 年 02 月 28 日向中国国家知识产权局提交的、 专利申请号为 201310065322.X的专利申请的优先权和权益， 并且通过参照将其全文并入此处。 技术领域  The present application claims priority to and the benefit of the patent application No. 201310065322.X filed on Jan. 28, 2013, the disclosure of which is hereby incorporated by reference. Technical field
本发明属于医药技术领域， 具体涉及表没食子儿茶素没食子酸酯的制备方法及其用途。 背景技术  The invention belongs to the technical field of medicine, and particularly relates to a preparation method of epigallocatechin gallate and use thereof. Background technique
绿茶的主要有效成分是茶多酚 （tea polyphenol, TP ), 又名茶单宁、 儿茶酸， 是茶叶 中儿茶素类、 黄酮类、 酚酸类和花色素类化合物的总称， 约占茶叶干重的 18-36%， 大量 研究表明茶多酚具有很强的抗氧化能力， 一直作为天然的食品抗氧化添加剂。 通过对 茶多酚的化学成分研究表明， 茶多酚的主要成分是儿茶素类化合物， 约占茶多酚总量 的 70%〜80%， 主要含有儿茶素 （C )、 表儿茶素 （EC)、 表儿茶素没食子酸酯 （ECG)、 表没食子酸儿茶素 （EGC ) 及表没食子儿茶素没食子酸酯 （EGCG) 等多种活性物质。  The main active ingredient of green tea is tea polyphenol (TP), also known as tea tannin, catechin, which is a general term for catechins, flavonoids, phenolic acids and anthocyanidic compounds in tea. The dry weight of tea is 18-36%. A large number of studies have shown that tea polyphenols have strong antioxidant capacity and have been used as natural food antioxidant additives. According to the chemical constituents of tea polyphenols, the main component of tea polyphenols is catechins, which account for 70%~80% of the total amount of tea polyphenols, mainly containing catechins (C) and epicatechins. A variety of active substances such as EC (EC), epicatechin gallate (ECG), epigallocatechin (EGC), and epigallocatechin gallate (EGCG).
贫血， 通常指外周血液中血红蛋白、 红细胞和 （或） 红细胞压积低于同性别、 同 年龄正常动物的最低值， 其中以血红蛋白值最具参考价值。 临床常见贫血为缺铁性贫血 和巨幼红细胞性贫血。 缺铁性贫血可用铁剂如硫酸亚铁、 枸橼酸铁铵、 右旋糖酐铁， 巨幼 细胞性贫血可用叶酸和维生素 B12治疗。再生障碍性贫血是骨髓造血功能抑制所致， 治疗比 较困难， 对其治疗目前主要是支持疗法、 促造血和骨髓移植。 支持疗法主要是输注红细胞 和血小板以维持血细胞计数， 而反复输血容易引发输血性血色病， 而仅使用 G-CSF、 EPO 等造血生长因子对再障患者行促造血治疗， 临床无显著效果。 治疗再生障碍性贫血的药物 价格昂贵、 治疗费用高、 治疗效果不理想。 地中海贫血又称珠蛋白生成障碍性贫血， 是由 于珠蛋白肽链合成量之间不平衡所致， 所以纠正珠蛋白肽链失衡成为地中海贫血治疗研究 的关键。 最有效的治疗方法是采用各类造血干细胞移植治疗， 但是相应治疗费用和医疗风 险也最高。 珠蛋白基因调节剂和辅助性抗氧化剂、 祛铁剂等药物治疗及高通量输血可以缓 解病情、 改善症状， 治疗方法容易掌握， 但是患者必须终生服用对症药物或依赖输血， 因 此寻找一种安全有效的贫血症治疗药物非常有意义。 Anemia, usually refers to the lowest value of hemoglobin, red blood cell and/or hematocrit in peripheral blood, which is lower than that of normal animals of the same sex and age. The hemoglobin value is the most valuable value. Common clinical anemia is iron deficiency anemia and megaloblastic anemia. Iron deficiency anemia can be treated with iron such as ferrous sulfate, ferric ammonium citrate, iron dextran, and megaloblastic anemia can be treated with folic acid and vitamin B 12 . Aplastic anemia is caused by inhibition of bone marrow hematopoietic function, and it is difficult to treat. The main treatment for it is supportive therapy, blood-promoting and bone marrow transplantation. Supportive therapy is mainly to infusion of red blood cells and platelets to maintain blood cell count, and repeated blood transfusion is easy to cause transfusion hemochromatosis, and only use hematopoietic growth factors such as G-CSF, EPO and other hematopoietic growth factors to promote hematopoietic treatment, clinically no significant effect. Drugs for the treatment of aplastic anemia are expensive, costly to treat, and unsatisfactory. Thalassemia, also known as globin-producing anemia, is due to an imbalance between the amount of globin peptide synthesis, so correcting the imbalance of globin peptide chains is the key to the treatment of thalassemia. The most effective treatment is the use of various types of hematopoietic stem cell transplantation, but the corresponding treatment costs and medical risks are also the highest. Medications such as globin gene regulators and auxiliary antioxidants, bismuth iron and other high-throughput blood transfusions can alleviate the disease and improve symptoms. The treatment is easy to master, but patients must take symptomatic drugs for life or rely on blood transfusion, so find a safe Effective anemia treatments are very meaningful.
然而， 目前表没食子儿茶素没食子酸酯的制备方法及其用途仍有待进一步研究。 发明内容 However, the preparation method and use of epigallocatechin gallate are still to be further studied. Summary of the invention
本发明旨在至少在一定程度上解决上述技术问题之一或至少提供一种有用的商业选择。 为此， 本发明的一个目的在于提供一种表没食子儿茶素没食子酸酯的制备方法及其新用 途。  The present invention is directed to solving at least some of the above technical problems or at least providing a useful commercial choice. Accordingly, it is an object of the present invention to provide a process for the preparation of epigallocatechin gallate and its novel use.
本发明是基于发明人的下列发现而完成的：  The present invention has been completed based on the following findings of the inventors:
近年来， 运用中医药对贫血症的治疗的研究日趋深入， 中药具有疗效好毒副作用小的 优点。 绿茶具有清热解毒， 收敛除湿等功效， 而且经现代医学研究证明， 绿茶还具有抗病 毒、 抗菌、 抗氧化、 抗增生、 抗贫血的作用。 目前国内有关于茶多酚及儿茶素和表儿茶素 在治疗贫血方面的专利报道， 本专利提供了一种治疗贫血症的新的中药单体成分表没食子 儿茶素没食子酸酯， 该成分近年来研究及其广泛， 且是绿茶中含量最高的单体成分， 具有 很高的开发价值。  In recent years, the use of traditional Chinese medicine for the treatment of anemia has become more and more intensive, and Chinese medicine has the advantages of good efficacy and small side effects. Green tea has the functions of clearing away heat and detoxifying, astringent dehumidification, etc., and it has been proved by modern medical research that green tea also has the functions of anti-virus, antibacterial, anti-oxidation, anti-proliferation and anti-anemia. At present, there are patent reports on tea polyphenols, catechins and epicatechins in the treatment of anemia. This patent provides a new traditional Chinese medicine monomer component for the treatment of anemia, epigallocatechin gallate. The composition has been extensively studied in recent years, and it is the monomer component with the highest content in green tea, and has high development value.
本发明的发明人经过艰苦的劳动， 结合发明人最新的研究研究显示表没食子儿茶 素没食子酸酯在治疗溶血性贫血、 缺铁性贫血、 再生障碍性贫血、 地中海贫血方面疗 效较好， 优于报道所见的绿茶茶多酚及儿茶素和表儿茶素。 特别是表没食子儿茶素没 食子酸酯应用于乙型地中海贫血、 海恩兹小体性（Heinz body)溶血性贫血、 缺铁性贫血 的治疗，疗效明显优于绿茶茶多酚及儿茶素和表儿茶素，可制备成优良的抗贫血的药物。  The inventor of the present invention, after arduous labor, combined with the latest research by the inventor, showed that epigallocatechin gallate has a good effect in treating hemolytic anemia, iron deficiency anemia, aplastic anemia, thalassemia, and excellent The green tea polyphenols and catechins and epicatechins were reported in the report. In particular, epigallocatechin gallate is used in the treatment of type B thalassemia, Heinz body hemolytic anemia, iron deficiency anemia, and the curative effect is significantly better than green tea polyphenols and catechins. And epicatechin, can be prepared into an excellent anti-anemia drug.
在本发明的一个方面， 本发明提出了一种制备表没食子儿茶素没食子酸酯的方法。 根据本发明的实施例， 该方法包括： 将绿茶进行加醇提取， 以便获得绿茶提取液； 将所述 绿茶提取液进行纯化， 以便获得绿茶提取物； 将所述绿茶提取物进行纯化， 以便获得表没 食子儿茶素没食子酸酯粗提物； 以及将所述表没食子儿茶素没食子酸酯粗提物进行重结 晶， 以便获得表没食子儿茶素没食子酸酯。 由此， 利用该方法， 能够以较低成本有效地制 备单体化合物形式的表没食子儿茶素没食子酸酯。 通过 HPLC方法进行检测， 确定根据 本发明的方法所制备的表没食子儿茶素没食子酸酯的纯度可以高达 93%以上。  In one aspect of the invention, the invention provides a method of preparing epigallocatechin gallate. According to an embodiment of the present invention, the method comprises: subjecting green tea to alcohol extraction to obtain a green tea extract; purifying the green tea extract to obtain a green tea extract; and purifying the green tea extract to obtain An epigallocatechin gallate crude extract; and the crude epigallocatechin gallate extract is recrystallized to obtain epigallocatechin gallate. Thus, with this method, epigallocatechin gallate in the form of a monomer compound can be efficiently produced at a relatively low cost. The purity of the epigallocatechin gallate prepared according to the method of the present invention was determined to be as high as 93% or more by the HPLC method.
根据本发明的实施例， 上述方法还可以具有下列附加技术特征：  According to an embodiment of the invention, the above method may also have the following additional technical features:
根据本发明的一个实施例， 将绿茶进行加醇提取， 以便获得绿茶提取液进一步包括： 将绿茶药材用 6-15倍重量的 30%-80%的乙醇进行加热回流提取 1-4次， 每次 0.5-3小时， 并将分别获得的提取液进行合并， 以便获得所述绿茶提取液。 由此， 可以进一步提高制备 表没食子儿茶素没食子酸酯的效率。  According to an embodiment of the present invention, the green tea is subjected to alcohol extraction to obtain the green tea extract further comprising: heating the green tea medicinal material with 6-15 times by weight of 30%-80% ethanol by heating and refluxing for 1-4 times, each 0.5 to 3 hours later, the separately obtained extracts were combined to obtain the green tea extract. Thereby, the efficiency of preparing epigallocatechin gallate can be further improved.
根据本发明的一个实施例， 将绿茶药材用 8-12倍重量的 50%-70%乙醇进行加热回流提 取 1-3次， 每次 1-3小时， 并合并提取液， 以便获得绿茶提取液。 由此， 可以进一步提高制 备表没食子儿茶素没食子酸酯的效率。  According to an embodiment of the present invention, the green tea medicinal material is heated and refluxed with 8-12 times by weight of 50%-70% ethanol for 1-3 times, each time for 1-3 hours, and the extract is combined to obtain a green tea extract. . Thereby, the efficiency of preparing epigallocatechin gallate can be further improved.
根据本发明的一个实施例，绿茶药材用 10倍量的 60%乙醇加热回流提取 2次，每次 1.5 小时， 合并提取液。 由此， 可以进一步提高制备表没食子儿茶素没食子酸酯的效率。 根据本发明的一个实施例， 将所述绿茶提取物进行纯化进一步包括： 将所述绿茶提取 液进行第一减压处理， 以便除去乙醇； 将所得到的残留物加水溶解后用乙酸乙酯萃取， 并 分离含有表没食子儿茶素没食子酸酯的乙酸乙酯相； 将所述含有表没食子儿茶素没食子 酸酯的乙酸乙酯相进行第二减压处理， 以便获得所述绿茶提取物。 由此， 可以进一步提高 制备表没食子儿茶素没食子酸酯的效率。 According to one embodiment of the present invention, the green tea medicinal material is heated and refluxed twice with 10 times the amount of 60% ethanol for 1.5 times each time. In hours, combine the extracts. Thereby, the efficiency of preparing epigallocatechin gallate can be further improved. According to an embodiment of the present invention, the purifying the green tea extract further comprises: performing the first decompression treatment on the green tea extract to remove ethanol; and dissolving the obtained residue in water and extracting with ethyl acetate And separating an ethyl acetate phase containing epigallocatechin gallate; and subjecting the ethyl acetate phase containing epigallocatechin gallate to a second decompression treatment to obtain the green tea extract. Thereby, the efficiency of preparing epigallocatechin gallate can be further improved.
根据本发明的一个实施例，所述第一减压处理是在 35〜70摄氏度的温度下， 优选 50〜60 摄氏度温度下， 更优选 50摄氏度温度下进行的。 由此， 可以进一步提高制备表没食子儿茶 素没食子酸酯的效率。  According to an embodiment of the present invention, the first reduced pressure treatment is carried out at a temperature of 35 to 70 ° C, preferably 50 to 60 ° C, more preferably 50 ° C. Thereby, the efficiency of preparing epigallocatechin gallate can be further improved.
根据本发明的一个实施例， 将所得到的残留物加水溶解后用乙酸乙酯萃取 1〜4次， 优 选 2〜3次， 更优选 2次。 由此， 可以进一步提高制备表没食子儿茶素没食子酸酯的效率。  According to an embodiment of the present invention, the obtained residue is dissolved in water and extracted with ethyl acetate 1 to 4 times, preferably 2 to 3 times, more preferably 2 times. Thereby, the efficiency of preparing epigallocatechin gallate can be further improved.
根据本发明的一个实施例， 将所得到的残留物加水溶解后用等体积的乙酸乙酯萃取。 由此， 可以进一步提高制备表没食子儿茶素没食子酸酯的效率。  According to one embodiment of the invention, the residue obtained is dissolved in water and extracted with an equal volume of ethyl acetate. Thereby, the efficiency of preparing epigallocatechin gallate can be further improved.
根据本发明的一个实施例，所述第二减压处理是在 40〜90摄氏度的温度下， 优选 40〜60 摄氏度的温度下， 更优选在 50摄氏度的温度下进行的。 由此， 可以进一步提高制备表没食 子儿茶素没食子酸酯的效率。  According to an embodiment of the invention, the second reduced pressure treatment is carried out at a temperature of 40 to 90 degrees Celsius, preferably 40 to 60 degrees Celsius, more preferably 50 degrees Celsius. Thereby, the efficiency of preparing epigallocatechin gallate can be further improved.
根据本发明的一个实施例， 将所述绿茶提取物进行纯化， 以便获得表没食子儿茶素没 食子酸酯粗提物进一步包括： 将所述绿茶提取物用水溶解后， 过大孔树脂柱， 收集乙醇洗 脱部分， 减压干燥， 以便获得所述没食子儿茶素没食子酸酯粗提物。 由此， 可以进一步提 高制备表没食子儿茶素没食子酸酯的效率。  According to an embodiment of the present invention, the green tea extract is purified to obtain a crude extract of epigallocatechin gallate further comprising: dissolving the green tea extract in water, passing through a macroporous resin column, collecting The ethanol eluted fraction was dried under reduced pressure to obtain the crude gallage of gallic catechin gallate. Thereby, the efficiency of preparing epigallocatechin gallate can be further improved.
根据本发明的一个实施例， 所述过大孔树脂柱是按照 0.5-3g绿茶 /g树脂， 优选 1.0-2.5g 药材 /g树脂， 更优选 2g药材 /g树脂的比例加样而进行的。 由此， 可以进一步提高制备表没 食子儿茶素没食子酸酯的效率。  According to an embodiment of the present invention, the oversized resin column is subjected to a ratio of 0.5 to 3 g of green tea / g resin, preferably 1.0 to 2.5 g of medicinal material / g of resin, more preferably 2 g of medicinal material / g of resin. Thereby, the efficiency of preparing epigallocatechin gallate can be further improved.
根据本发明的一个实施例， 首先用 2-6个柱体积的水， 优选 3-5个柱体积的水， 更优选 3个柱体积的水对所述大孔树脂柱进行洗脱， 再用 4-8个柱体积， 优选 5-7个柱体积， 更优 选 6个柱体积的 5%-15%乙醇， 优选 8%-12%乙醇， 更优选 10%的乙醇对所述大孔树脂柱进 行洗脱。 由此， 可以进一步提高制备表没食子儿茶素没食子酸酯的效率。  According to one embodiment of the invention, the macroporous resin column is first eluted with 2-6 column volumes of water, preferably 3-5 column volumes of water, more preferably 3 column volumes of water, and then used. 4-8 column volumes, preferably 5-7 column volumes, more preferably 6 column volumes of 5%-15% ethanol, preferably 8%-12% ethanol, more preferably 10% ethanol to the macroporous resin column Elution is performed. Thereby, the efficiency of preparing epigallocatechin gallate can be further improved.
根据本发明的一个实施例，将所述表没食子儿茶素没食子酸酯粗提物进行重结晶进一 步包括： 将所述表没食子儿茶素没食子酸酯粗提物配制成饱和水溶液； 以及向所述饱和水 溶液中加入有机溶剂， 以便进行重结晶。 由此， 可以进一步提高制备表没食子儿茶素没食 子酸酯的纯度。  According to an embodiment of the present invention, the recrystallizing the epigallocatechin gallate crude extract further comprises: formulating the epigallocatechin gallate crude extract into a saturated aqueous solution; An organic solvent is added to the saturated aqueous solution to carry out recrystallization. Thereby, the purity of the preparation of epigallocatechin gallate can be further improved.
根据本发明的一个实施例， 所述有机溶剂为甲醇与异丙醇的混合物， 其中， 甲醇与异 丙醇的体积比为 2:1。 由此， 可以进一步提高制备表没食子儿茶素没食子酸酯的纯度。 根据本发明的一个实施例， 所述重结晶是在 0〜15摄氏度， 优选 5〜10摄氏度， 更优选 8 摄氏度的温度下进行的。 由此， 可以进一步提高制备表没食子儿茶素没食子酸酯的纯度。 According to an embodiment of the present invention, the organic solvent is a mixture of methanol and isopropanol, wherein methanol and The volume ratio of propanol is 2:1. Thereby, the purity of the preparation of epigallocatechin gallate can be further improved. According to an embodiment of the invention, the recrystallization is carried out at a temperature of 0 to 15 degrees Celsius, preferably 5 to 10 degrees Celsius, more preferably 8 degrees Celsius. Thereby, the purity of the preparation of epigallocatechin gallate can be further improved.
根据本发明的一个实施例，所述表没食子儿茶素没食子酸酯与有机溶剂的重量体积比 为 l g:2-5ml。 由此， 可以进一步提高制备表没食子儿茶素没食子酸酯的纯度。  According to an embodiment of the present invention, the weight ratio of the epigallocatechin gallate to the organic solvent is l g: 2-5 ml. Thereby, the purity of the preparation of epigallocatechin gallate can be further improved.
根据本发明的一个实施例，优选表没食子儿茶素没食子酸酯与有机溶剂的重量体积比 为 1 g:3ml。  According to an embodiment of the present invention, it is preferred that the weight ratio of epigallocatechin gallate to organic solvent is 1 g: 3 ml.
在本发明的第二方面，本发明提出了表没食子儿茶素没食子酸酯在制备抗贫血产品 中的用途， 优选地， 所述表没食子儿茶素没食子酸酯是根据前面所述的方法制备的。  In a second aspect of the invention, the invention provides the use of epigallocatechin gallate in the preparation of an anti-anaemia product, preferably the epigallocatechin gallate is prepared according to the method described above of.
根据本发明的实施例， 所述抗贫血产品的类型并不受特别限制。 根据本发明的具 体示例， 优选地抗贫血产品可以为用于预防或者治疗贫血的药物、 保健品或食品。 具 体的， 所述药物可以呈片剂、 胶囊剂、 颗粒剂、 或注射剂的至少一种。  According to an embodiment of the present invention, the type of the anti-anemia product is not particularly limited. According to a specific example of the present invention, preferably the anti-anemia product may be a drug, a health care product or a food product for preventing or treating anemia. Specifically, the drug may be at least one of a tablet, a capsule, a granule, or an injection.
根据本发明的实施例， 可以采用本发明的抗贫血产品可以治疗或者预防的贫血可 以为选自缺铁性贫血、 溶血性贫血、 再生障碍性贫血和地中海贫血的至少一种。 优选 地， 可以治疗或者预防的贫血为选自缺铁性贫血、 乙型地中海贫血、 海恩兹小体性溶 血性贫血的至少一种。  According to an embodiment of the present invention, the anemia which can be treated or prevented by the anti-anemia product of the present invention may be at least one selected from the group consisting of iron deficiency anemia, hemolytic anemia, aplastic anemia, and thalassemia. Preferably, the anemia which can be treated or prevented is at least one selected from the group consisting of iron deficiency anemia, beta thalassemia, and Heinz small body hemolytic anemia.
发明人发现利用本发明药物活性成分表没食子儿茶素没食子酸酯治疗贫血症时， 效果优于绿茶的其它活性成分绿茶茶多酚、表儿茶素、儿茶素、表儿茶素没食子酸酯、 表没食子酸儿茶素。 因而， 优选将根据本发明实施例的药物活性成分表没食子儿茶素 没食子酸酯用于贫血症的治疗。  The inventors have found that the use of the active ingredient of the present invention, epigallocatechin gallate, in the treatment of anemia is superior to other active ingredients of green tea, green tea polyphenols, epicatechin, catechins, epicatechin gallic acid Ester, epigallocatechin catechin. Thus, the pharmaceutically active ingredient epigallocatechin gallate according to an embodiment of the present invention is preferably used for the treatment of anemia.
在表没食子儿茶素没食子酸酯对溶血性贫血小鼠的治疗作用实验中， EGCG 可显 著增加溶血性贫血小鼠红细胞计数 （RBC ) , 并可显著增加溶血性贫血小鼠血红蛋白 ( Hb )。 说明 EGCG可用于治疗溶血性贫血。 另外， 经 EGCG作用于贫血小鼠后， 单 位 RBC中血红蛋白肽链的合成有所增加， 可用于地中海贫血的治疗。  In the therapeutic effect of epigallocatechin gallate on mice with hemolytic anemia, EGCG significantly increased red blood cell count (RBC) in hemolytic anemia mice and significantly increased hemoglobin (Hb) in hemolytic anemia mice. Description EGCG can be used to treat hemolytic anemia. In addition, after EGCG is applied to anemia mice, the synthesis of hemoglobin peptide chains in RBC is increased, which can be used for the treatment of thalassemia.
在表没食子儿茶素没食子酸酯对患有缺铁性贫血小鼠的治疗作用实验中， EGCG 可显著增加缺铁性贫血小鼠 RBC， 并可显著增加缺铁性贫血小鼠 Hb。 说明 EGCG可 用于治疗缺铁性贫血。  In the therapeutic effect of epigallocatechin gallate on mice with iron deficiency anemia, EGCG significantly increased RBC in iron-deficient anemia mice and significantly increased Hb in iron-deficient anemia mice. Description EGCG can be used to treat iron deficiency anemia.
在表没食子儿茶素没食子酸酯对患有再生障碍性贫血小鼠的治疗作用实验中， 给 与 EGCG 后， 白细胞计数 （WBC )、 Hb、 RBC、 血小板计数 （PLT ) 均有明显升高。 用骨髓造血肝细胞培养方法观察 EGCG、 儿茶素、 表儿茶素、 茶多酚对造血损伤小鼠 股骨多向性造血细胞 （CFU-mix)、 红系祖细胞 （CFU-E )、 粒单系祖细胞 （CFU-GM) 的影响， 结果表明， EGCG可显著性提高 CFU-mix值、 CFU-E值、 以及 CFU-GM值。 说明 EGCG可用于治疗再生障碍性贫血。 In the therapeutic effect of epigallocatechin gallate on mice with aplastic anemia, white blood cell count (WBC), Hb, RBC, and platelet count (PLT) were significantly increased after administration of EGCG. Using bone marrow hematopoietic hepatocyte culture method to observe EGCG, catechin, epicatechin and tea polyphenols in hematopoietic mice, femur multi-directional hematopoietic cells (CFU-mix), erythroid progenitor cells (CFU-E), granules The effects of single progenitor cells (CFU-GM) showed that EGCG significantly increased CFU-mix values, CFU-E values, and CFU-GM values. This indicates that EGCG can be used to treat aplastic anemia.
需要说明的是， 本发明的药物组合物及其用途是本申请的发明人经过艰苦的创造 性劳动和优化工作才完成的。  It should be noted that the pharmaceutical composition of the present invention and its use are completed by the inventor of the present application through arduous creative labor and optimization work.
本发明的附加方面和优点将在下面的描述中部分给出， 部分将从下面的描述中变 得明显， 或通过本发明的实践了解到。 具体实施方式  The additional aspects and advantages of the invention will be set forth in part in the description which follows. detailed description
下面详细描述本发明的实施例， 所述实施例的示例在附图中示出， 其中自始至终相同 或类似的标号表示相同或类似的元件或具有相同或类似功能的元件。 下面通过参考附图描 述的实施例是示例性的， 仅用于解释本发明， 而不能理解为对本发明的限制。 除非特殊说 明， 在实施例中所进行的操作均为按照 《中华人民共和国药典》 2010年版以及本领域中公 知的方法进行的。  The embodiments of the present invention are described in detail below, and the examples of the embodiments are illustrated in the drawings, wherein the same or similar reference numerals are used to refer to the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the drawings are intended to be illustrative of the invention and are not to be construed as limiting. Unless otherwise stated, the operations performed in the examples were carried out in accordance with the 2010 edition of the Pharmacopoeia of the People's Republic of China and methods well known in the art.
实施例 1: 表没食子儿茶素没食子酸酯的制备  Example 1: Preparation of epigallocatechin gallate
绿茶药材 50g，加入 6倍量的 80%乙醇加热回流提取 4次，每次 0.5小时，合并提取液。 提取液在 35摄氏度下减压回收除去乙醇， 加水溶解后用等体积乙酸乙酯萃取 1次， 萃取后 乙酸乙酯层经过 40摄氏度进行减压处理以便获得绿茶提取物，将绿茶提取物用纯水溶解后， 过滤， 配成 0.5g药材 /g树脂的溶液上大孔树脂柱， 用 6个柱体积的水洗脱， 再用 4个柱体 积的 15%的乙醇洗脱， 收集乙醇洗脱部分， 减压干燥， 得表没食子儿茶素没食子酸酯粗提 物； 在上述表没食子儿茶素没食子酸酯粗提物饱和水溶液中加入甲醇 /异丙醇混合液 （甲 醇与异丙醇的体积比为 2: 1， EGCG的重量与加入的混合液体积比为 lg: 2ml), 在 5摄氏 度下搅拌重结晶， 得固体 1.04g， 纯度为 93.5% (HPLC法）。  50 g of green tea medicinal material was added to a 6-fold amount of 80% ethanol and heated under reflux for 4 times for 0.5 hour each time, and the extracts were combined. The extract was recovered under reduced pressure at 35 ° C to remove ethanol, dissolved in water and extracted once with an equal volume of ethyl acetate. After extraction, the ethyl acetate layer was subjected to reduced pressure at 40 ° C to obtain a green tea extract, and the green tea extract was pure. After the water was dissolved, it was filtered, and a 0.5 g medicinal material/g resin solution was applied to a macroporous resin column, eluted with 6 column volumes of water, and then eluted with 4 column volumes of 15% ethanol to collect ethanol. Partially, dried under reduced pressure, to obtain a crude extract of epigallocatechin gallate; a methanol/isopropyl alcohol mixture (methanol and isopropanol) is added to a saturated aqueous solution of the crude gallage of gallic catechin gallate The volume ratio was 2:1, and the volume ratio of EGCG to the mixed liquid was lg: 2 ml), and recrystallization was carried out at 5 ° C to obtain a solid of 1.04 g, and the purity was 93.5% (HPLC method).
实施例 2: 表没食子儿茶素没食子酸酯的制备  Example 2: Preparation of epigallocatechin gallate
绿茶药材 50g，加入 15倍量的 30%乙醇加热回流提取 1次，每次 3小时，合并提取液。 提取液在 70摄氏度下减压回收除去乙醇， 加水溶解后用等体积乙酸乙酯萃取 4次， 萃取后 乙酸乙酯层经过 90摄氏度进行减压处理以便获得绿茶提取物，将绿茶提取物用纯水溶解后， 过滤， 配成 3g药材 /g树脂的溶液上大孔树脂柱， 用 2个柱体积的水洗脱， 再用 8个柱体积 的 5%的乙醇洗脱，收集乙醇洗脱部分，减压干燥，得表没食子儿茶素没食子酸酯粗提物； 在上述表没食子儿茶素没食子酸酯粗提物饱和水溶液中加入甲醇 /异丙醇混合液 （甲醇与 异丙醇的体积比为 2: 1， EGCG的重量与加入的混合液体积比为 lg: 5ml), 在 8摄氏度下 重结晶纯化， 得到固体 1.15g， 纯度为 95.1% (HPLC法）。  50 g of green tea medicinal material was added to a 15-fold amount of 30% ethanol and refluxed for 1 hour for 3 hours, and the extracts were combined. The extract was recovered under reduced pressure at 70 ° C to remove ethanol, dissolved in water and extracted with an equal volume of ethyl acetate 4 times. After extraction, the ethyl acetate layer was subjected to reduced pressure at 90 ° C to obtain green tea extract, and the green tea extract was pure. After the water was dissolved, it was filtered, and a solution of 3 g of the drug/g resin was placed on a macroporous resin column, eluted with 2 column volumes of water, and then eluted with 8 column volumes of 5% ethanol to collect the ethanol eluting portion. , drying under reduced pressure, to obtain a crude extract of epigallocatechin gallate; adding a methanol/isopropanol mixture (methanol and isopropanol volume) to a saturated aqueous solution of the crude gallage catechin gallate The ratio of 2:1, the weight ratio of EGCG to the mixed liquid to be added was lg: 5 ml), and recrystallization was carried out at 8 ° C to obtain a solid 1.15 g and a purity of 95.1% (HPLC method).
实施例 3: 表没食子儿茶素没食子酸酯的制备  Example 3: Preparation of epigallocatechin gallate
绿茶药材 50g， 加入 8倍量的 70%乙醇加热回流提取 3次， 每次 1小时， 合并提取液。 提取液在 60摄氏度下减压回收除去乙醇， 加水溶解后用等体积乙酸乙酯萃取 3次， 萃取后 乙酸乙酯层经过 60摄氏度进行减压处理以便获得绿茶提取物，将绿茶提取物用纯水溶解后， 过滤， 配成 2g药材 /g树脂的溶液上大孔树脂柱， 用 6个柱体积的水洗脱， 再用 8个柱体积 的 10%的乙醇洗脱，收集乙醇洗脱部分，减压干燥，得表没食子儿茶素没食子酸酯粗提物； 在上述表没食子儿茶素没食子酸酯粗提物饱和水溶液中加入甲醇 /异丙醇混合液 （甲醇与 异丙醇的体积比为 2: 1， EGCG的重量与加入的混合液体积比为 lg: 4ml), 在 0摄氏度下 重结晶纯化， 得到固体 1.19g， 纯度为 93.9% (HPLC法）。 50 g of green tea medicinal material was added to an 8-fold amount of 70% ethanol and refluxed for 3 times for 1 hour, and the extracts were combined. The extract was recovered under reduced pressure at 60 ° C to remove ethanol, dissolved in water and extracted with an equal volume of ethyl acetate three times. After extraction, the ethyl acetate layer was subjected to reduced pressure at 60 ° C to obtain green tea extract, and the green tea extract was pure. After the water was dissolved, it was filtered, and a solution of 2 g of medicinal material/g resin was placed on a macroporous resin column, eluted with 6 column volumes of water, and then eluted with 8 column volumes of 10% ethanol to collect the ethanol eluting fraction. , drying under reduced pressure, to obtain a crude extract of epigallocatechin gallate; adding a methanol/isopropanol mixture (methanol and isopropanol volume) to a saturated aqueous solution of the crude gallage catechin gallate The ratio was 2: 1, and the volume ratio of EGCG to the mixed liquid was lg: 4 ml), and recrystallization was carried out at 0 ° C to obtain 1.19 g of a solid having a purity of 93.9% (HPLC method).
实施例 4: 表没食子儿茶素没食子酸酯的制备  Example 4: Preparation of epigallocatechin gallate
绿茶药材 50g， 加入 10倍量的 50%乙醇加热回流提取 2次， 每次 1.5小时， 合并提取 液。 提取液在 50摄氏度下减压回收除去乙醇， 加水溶解后用等体积乙酸乙酯萃取 2次， 萃 取后乙酸乙酯层经过 60摄氏度进行减压处理以便获得绿茶提取物， 将绿茶提取物用纯水溶 解后， 过滤， 配成 1.5g药材 /g树脂的溶液上大孔树脂柱， 用 4个柱体积的水洗脱， 再用 5 个柱体积的 10%的乙醇洗脱， 收集乙醇洗脱部分， 减压干燥， 得表没食子儿茶素没食子酸 酯粗提物； 在上述表没食子儿茶素没食子酸酯粗提物饱和水溶液中加入甲醇 /异丙醇混合 液 （甲醇与异丙醇的体积比为 2: 1， EGCG的重量与加入的混合液体积比为 lg: 3ml), 在 15摄氏度下重结晶纯化， 得到固体 1.09g， 纯度为 94.3% (HPLC法）。  50 g of green tea medicinal material, and added with 10 times the amount of 50% ethanol and refluxed for 2 times for 1.5 hours each time, and the extracts were combined. The extract was recovered under reduced pressure at 50 ° C to remove ethanol, dissolved in water and extracted twice with an equal volume of ethyl acetate. After extraction, the ethyl acetate layer was subjected to reduced pressure at 60 ° C to obtain green tea extract, and the green tea extract was pure. After the water is dissolved, it is filtered, and a 1.5 g medicinal material/g resin solution is applied to a macroporous resin column, eluted with 4 column volumes of water, and then eluted with 5 column volumes of 10% ethanol to collect ethanol elution. Partially, dried under reduced pressure, to obtain a crude extract of epigallocatechin gallate; a methanol/isopropyl alcohol mixture (methanol and isopropanol) is added to a saturated aqueous solution of the crude gallage of gallic catechin gallate The volume ratio was 2:1, and the volume ratio of EGCG to the mixed solution was lg: 3 ml), and recrystallization was carried out at 15 ° C to obtain a solid of 1.09 g and a purity of 94.3% (HPLC method).
实施例 5: 表没食子儿茶素没食子酸酯的制备  Example 5: Preparation of epigallocatechin gallate
绿茶药材 50g， 加入 10倍量的 60%乙醇加热回流提取 2次， 每次 1.5小时， 合并提取 液。 提取液在 50摄氏度下减压回收除去乙醇， 加水溶解后用等体积的乙酸乙酯萃取 2次， 萃取后乙酸乙酯层经过 50摄氏度进行减压处理， 以便获得绿茶提取物； 将绿茶提取物用纯 水溶解后， 过滤， 配成 2g药材 /g树脂的溶液上大孔树脂柱， 用 3个柱体积的水洗脱， 再用 6个柱体积的 10%的乙醇洗脱， 收集乙醇洗脱部分， 减压干燥， 得表没食子儿茶素没食子 酸酯粗提物； 在表没食子儿茶素没食子酸酯粗提物饱和水溶液中加入甲醇 /异丙醇混合液 50 g of green tea medicinal material, added with 10 times the amount of 60% ethanol and refluxed for 2 times for 1.5 hours each time, and the extracts were combined. The extract was recovered under reduced pressure at 50 ° C to remove ethanol, dissolved in water, and extracted twice with an equal volume of ethyl acetate. After extraction, the ethyl acetate layer was subjected to reduced pressure at 50 ° C to obtain a green tea extract; After dissolving in pure water, filtering, preparing a solution of 2 g of medicinal material/g resin on a macroporous resin column, eluting with 3 column volumes of water, and eluting with 6 column volumes of 10% ethanol, collecting ethanol wash The part is dehydrated and dried under reduced pressure to obtain a crude extract of epigallocatechin gallate; a methanol/isopropyl alcohol mixture is added to a saturated aqueous solution of epigallocatechin gallate crude extract
(甲醇与异丙醇的体积比为 2: 1， EGCG的重量与加入的混合液体积比为 lg: 3ml), 在 8 摄氏度下搅拌重结晶 1 次， 得到表没食子儿茶素没食子酸酯固体 1.23g， 纯度为 95.5%(The volume ratio of methanol to isopropanol is 2: 1, the weight ratio of EGCG to the mixed solution is lg: 3 ml), and recrystallization is repeated once at 8 ° C to obtain epigallocatechin gallate solid. 1.23g, purity is 95.5%
(HPLC法）。 (HPLC method).
实施例 6: 表没食子儿茶素没食子酸酯对溶血性贫血小鼠的治疗作用  Example 6: Therapeutic effect of epigallocatechin gallate on mice with hemolytic anemia
一、 材料  First, the material
实验药物及试剂： 昆明小鼠 120只， 雌雄各半， 体重 20±2 g， 健康状况良好， 由武汉 大学医学院实验动物中心提供， 生产许可证号： SCXK (鄂） 2008-0004。 EGCG (按照实施 例 1制备， 批号 20110901 );  Experimental drugs and reagents: 120 Kunming mice, half male and half female, weighing 20±2 g, are in good health. They are provided by the Experimental Animal Center of Wuhan University Medical College. Production license number: SCXK (E) 2008-0004. EGCG (prepared according to Example 1, batch number 20110901);
二、 动物造模及给药方法 将实验动物小鼠随机分为 6组， 每组各 20只， 分别标记为： 空白对照组、 EGCG组、 茶多酚组、 表儿茶素组、 儿茶素组、 模型对照组， 除空白对照组外， 其余 5 组小鼠腹腔注 射苯肼的生理盐水溶液， 注射剂量为 90mg_kg- 1造模， 当天各组小鼠灌胃给药， 每日 1次， 连续 1周， 并于末次给药后 1 h采血测定血红蛋白 （Hb) 及红细胞计数 （RBC)。 其中空白 对照组及模型对照组都给予等体积的生理盐水， EGCG组给药剂量为 30mg/kg体重， 茶多 酚组给药剂量为 60mg/kg体重，表儿茶素组给药剂量为 18.8mg/kg体重，儿茶素组给药剂量 为 20mg/kg体重。 Second, animal modeling and drug delivery methods The experimental animal mice were randomly divided into 6 groups, 20 in each group, labeled as: blank control group, EGCG group, tea polyphenol group, epicatechin group, catechin group, model control group, except blank Outside the control group, the other 5 groups of mice were intraperitoneally injected with a physiological saline solution of phenylhydrazine at a dose of 90 mg_kg- 1 , and the mice in each group were intragastrically administered once a day for 1 week, and given the last time. Blood samples were taken 1 h after the drug to measure hemoglobin (Hb) and red blood cell count (RBC). The blank control group and the model control group were given an equal volume of normal saline, the EGCG group was administered at a dose of 30 mg/kg body weight, the tea polyphenol group was administered at a dose of 60 mg/kg body weight, and the epicatechin group was administered at a dose of 18.8. The mg/kg body weight and the catechin group were administered at a dose of 20 mg/kg body weight.
三、 结果  Third, the results
上述几种药物对溶血性贫血小鼠 RBC及 Hb的影响见表 1。  The effects of the above several drugs on RBC and Hb in hemolytic anemia mice are shown in Table 1.
表 1 几种药物对溶血性贫血小鼠 RBC及 Hb的影响 （ ±s)  Table 1 Effects of several drugs on RBC and Hb in hemolytic anemia mice (±s)
Figure imgf000008_0001
Figure imgf000008_0001
注： 与空白对照组比较， *P<0.05， **P<0.01 ; 与模型对照组比较： AP<0.05， A AP <0.01。 Note: Compared with the blank control group, *P<0.05, **P<0.01; compared with the model control group: A P<0.05, AA P <0.01.
结果显示： 与空白对照组比较， 模型对照组 Hb、 RBC显著降低 （P<0.01)， 说明动物造 模成功。 EGCG组可显著增加溶血性贫血小鼠 RBC(P<0.05)， 并可显著增加溶血性贫血小鼠 Hb (P<0.01), 儿茶素组及表儿茶素组对溶血性贫血小鼠模型的 RBC值有显著的提升作用， 儿茶素组对 Hb的影响显著（P<0.05 )， 而表儿茶素组和茶多酚组则对 RBC和 Hb的影响都 不具有显著性特征。  The results showed that compared with the blank control group, the Hb and RBC of the model control group were significantly lower (P<0.01), indicating that the animal model was successful. EGCG group can significantly increase RBC in hemolytic anemia mice (P<0.05), and can significantly increase Hb in hemolytic anemia mice (P<0.01), catechin group and epicatechin group in mice with hemolytic anemia. The RBC value was significantly improved, and the catechin group had a significant effect on Hb (P<0.05), while the epicatechin group and the tea polyphenol group had no significant effects on RBC and Hb.
实施例 7: 表没食子儿茶素没食子酸酯对缺铁性贫血的治疗作用  Example 7: Therapeutic effect of epigallocatechin gallate on iron deficiency anemia
一、 材料  First, the material
实验药物及试剂： 昆明小鼠 140只， 雌雄各半， 体重 20±2 g， 健康状况良好， 由武汉 大学医学院实验动物中心提供， 生产许可证号： SCXK (鄂） 2008-0004。 EGCG (按照实施 例 1制备， 批号 20110901 );  Experimental drugs and reagents: 140 Kunming mice, male and female, weighing 20±2 g, in good health, provided by Experimental Animal Center of Wuhan University Medical College, production license number: SCXK (E) 2008-0004. EGCG (prepared according to Example 1, batch number 20110901);
二、 动物造模及给药方法  Second, animal modeling and drug delivery methods
将实验动物小鼠随机分为 7组， 每组各 20只， 分别标记为： 空白对照组、 EGCG组、 茶多酚组、 表儿茶素组、 儿茶素组、 硫酸亚铁组 （阳性对照组）、 模型对照组， 模型对照组 及各给药组小鼠采用低铁伺料喂养， 正常饮水， 同时每周眼眶取血 2次， 每次 0.4ml， 连续 2周， 空白对照组采用普通伺料喂养， 正常饮水。 造模成功后除空白对照组外其余各组继续 采用低铁伺料喂养，且灌胃给药，每日 1次，连续七天，末次给药后 lh采血测定 Hb及 RBC。 其中空白对照组及模型对照组都给与等体积的生理盐水， EGCG组给药剂量为 30mg/kg体 重， 茶多酚组给药剂量为 60mg/kg体重， 表儿茶素组给药剂量为 18.8mg/kg体重， 儿茶素组 给药剂量为 20mg/kg体重， 硫酸亚铁组给药剂量为 90mg/kg体重。 The experimental animal mice were randomly divided into 7 groups, 20 in each group, labeled as: blank control group, EGCG group, tea polyphenol group, epicatechin group, catechin group, ferrous sulfate group (positive Control group), model control group, model control group The mice in each administration group were fed with low-iron feeding, normal drinking water, and blood was taken twice a week for 0.4 ml each time for 2 weeks. The blank control group was fed with normal feeding materials and normal drinking water. After successful modeling, the other groups continued to use low-iron feeding, and were administered by intragastric administration once a day for seven consecutive days. At the end of the last administration, blood was collected for Hb and RBC. The blank control group and the model control group were given an equal volume of normal saline, the EGCG group was administered at a dose of 30 mg/kg body weight, the tea polyphenol group was administered at a dose of 60 mg/kg body weight, and the epicatechin group was administered at a dose of 18.8 mg/kg body weight, the catechin group was administered at a dose of 20 mg/kg body weight, and the ferrous sulfate group was administered at a dose of 90 mg/kg body weight.
三、 结果  Third, the results
上述几种药物对缺铁性贫血小鼠 RBC及 Hb的影响见表 2。  The effects of these drugs on RBC and Hb in iron-deficient anemia mice are shown in Table 2.
表 2 几种药物对缺铁性贫血小鼠 RBC及 Hb的影响 （ ±s)  Table 2 Effects of several drugs on RBC and Hb in iron-deficient anemia mice (±s)
Figure imgf000009_0001
Figure imgf000009_0001
注： 与空白对照组比较， *P<0.05， **P<0.01 ; 与模型对照组比较： AP<0.05， A AP <0.01。 Note: Compared with the blank control group, *P<0.05, **P<0.01; compared with the model control group: A P<0.05, AA P <0.01.
结果显示： 与空白对照组比较， 模型对照组 Hb (P<0.05 )、 RBC显著降低 （P<0.01)， 说明动物造模成功。 EGCG组可显著增加缺铁性贫血小鼠 RBC(P<0.05)， 并可极显著增加缺 铁性贫血小鼠 Hb (P<0.01)， 硫酸亚铁片组、 儿茶素组、 表儿茶素组和茶多酚组均对 RBC有 显著的提升作用 （P<0.05 )， 硫酸亚铁片组可显著增加缺铁性贫血小鼠 Hb (P<0.05)， 而儿茶 素组、 表儿茶素组和茶多酚对 Hb的影响都不具有显著性特征。  The results showed that compared with the blank control group, the Hb (P<0.05) and RBC of the model control group were significantly lower (P<0.01), indicating that the animal model was successful. EGCG group can significantly increase RBC in mice with iron deficiency anemia (P<0.05), and can significantly increase Hb (P<0.01) in iron deficiency anemia mice, ferrous sulfate tablets, catechin group, epicatechin Both the group and the tea polyphenol group significantly improved the RBC (P<0.05), and the ferrous sulfate group significantly increased the Hb in the iron-deficient anemia mice (P<0.05), while the catechin group, the table The effects of the tea group and tea polyphenols on Hb were not significant.
实施例 8: EGCG对再生障碍型贫血的治疗作用  Example 8: Therapeutic effect of EGCG on aplastic anemia
一、 材料  First, the material
实验药物及试剂： 昆明小鼠 120只， 雌雄各半， 体重 20±2 g， 健康状况良好， 由武汉 大学医学院实验动物中心提供， 生产许可证号： SCXK (鄂） 2008-0004。 EGCG (按照实施 例 1制备， 批号 20110901 );  Experimental drugs and reagents: 120 Kunming mice, half male and half female, weighing 20±2 g, are in good health. They are provided by the Experimental Animal Center of Wuhan University Medical College. Production license number: SCXK (E) 2008-0004. EGCG (prepared according to Example 1, batch number 20110901);
二、 动物造模及给药方法  Second, animal modeling and drug delivery methods
将实验动物小鼠随机分为 6组， 每组各 20只， 分别标记为： 空白对照组、 EGCG组、 茶多酚组、 表儿茶素组、 儿茶素组、 模型对照组， 除空白对照组外， 其余 5 组造模小鼠按 15mg/kg的剂量给予白消安， 每周一次， 腹腔注射， 共 10周。 The experimental animal mice were randomly divided into 6 groups, 20 in each group, labeled as: blank control group, EGCG group, tea polyphenol group, epicatechin group, catechin group, model control group, except blank Outside the control group, the other 5 groups of model mice were pressed Busulfan was administered at a dose of 15 mg/kg once a week for 10 weeks.
其中空白对照组及模型对照组都给与等体积的生理盐水， EGCG组给药剂量为 5.6mg/kg 体重， 茶多酚组给药剂量为 10mg/kg体重， 表儿茶素组给药剂量为 3.5mg/kg体重， 儿茶素 组给药剂量为 3.75mg/kg体重， 每日一次， 连续灌胃 20d。  The blank control group and the model control group were given an equal volume of normal saline, the EGCG group was administered at a dose of 5.6 mg/kg body weight, the tea polyphenol group was administered at a dose of 10 mg/kg body weight, and the epicatechin group was administered at a dose of 10 mg/kg body weight. For the 3.5 mg/kg body weight, the catechin group was administered at a dose of 3.75 mg/kg body weight once daily for 20 days.
三、 结果  Third, the results
在给药前、 给药后每周及末次给药后次日每只小鼠尾静脉取血， 观察小鼠 WBC、 Hb、 RBC、 PLT的计数， 结果见表 3。 并用骨髓造血肝细胞培养方法， 观察 EGCG、 儿茶素、 表 儿茶素、 茶多酚对造血损伤小鼠股骨多向性造血细胞 （CFU-mix)、 红系祖细胞 （CFU-E)、 粒单系祖细胞（CFU-GM) 的影响， 停药次日测定小鼠股骨内 CFU-mix、 CFU-GM、 CFU-E 数量， 结果见表 4。  Blood was taken from the tail vein of each mouse before administration, weekly after administration, and the day after the last administration, and the counts of WBC, Hb, RBC, and PLT of the mice were observed. The results are shown in Table 3. Using bone marrow hematopoietic hepatocyte culture method, EGCG, catechin, epicatechin and tea polyphenols were observed for hematopoietic mice with multi-directional hematopoietic cells (CFU-mix) and erythroid progenitor cells (CFU-E). The effects of granulocyte-monoblastic progenitor cells (CFU-GM) were measured. The number of CFU-mix, CFU-GM and CFU-E in the femur of the mice was determined on the next day. The results are shown in Table 4.
表 3 几种药物对再生障碍性贫血小鼠外周血的影响 （ ±s)  Table 3 Effects of several drugs on peripheral blood in mice with aplastic anemia (±s)
Figure imgf000010_0001
Figure imgf000010_0001
注： 与空白对照组比较： *P<0.05; 与模型对照组比较： AP<0.05 Note: Compared with the blank control group: *P<0.05; compared with the model control group: A P<0.05
表 4 几种药物对再生障碍性贫血小鼠 CFU-mix、 CFU-GM CFU-E的影响 （ ±s)  Table 4 Effects of several drugs on CFU-mix and CFU-GM CFU-E in aplastic anemia mice (±s)
Figure imgf000010_0002
Figure imgf000010_0002
注： （1 ) 与空白对照组比较： *P<0.05; 与模型对照组比较： AP<0.05 Note: (1) Compared with the blank control group: *P<0.05; compared with the model control group: A P<0.05
(2) MNC指单个核细胞 （Mononuclear Cell)  (2) MNC refers to mononuclear cells (Mononuclear Cell)
结果表明， 再生障碍性贫血模型对照组的 WBC、 Hb、 RBC、 PLT较空白对照组都有明 显减少（P<0.05 )， 模型对照组的 CFU-mix、 CFU-GM、 CFU-E较空白对照组都有明显减少 (P<0.05 )， 说明动物建模成功。 给予 EGCG后， WBC、 Hb、 RBC、 PLT与模型对照组相 比均有明显升高 （P<0.05 )， CFU-mix, CFU-GM、 CFU-E较空白对照组都有明显减少 （P <0.05 )。 而儿茶素组对 RBC的指标影响不明显， 与模型对照组相比无明显差异， 而表儿茶 素对 Hb、 RBC、 PLT等指标无明显影响作用，茶多酚组对 Hb、 PLT等指标无明显提升作用。 EGCG对再生障碍性贫血贫血的治疗作用明显。 The results showed that the WBC, Hb, RBC and PLT of the control group of aplastic anemia model were clearer than the blank control group. Significantly decreased (P<0.05), CFU-mix, CFU-GM, CFU-E in the model control group were significantly reduced compared with the blank control group (P<0.05), indicating that the animal model was successful. After EGCG administration, WBC, Hb, RBC and PLT were significantly increased compared with the model control group (P<0.05), and CFU-mix, CFU-GM and CFU-E were significantly lower than the blank control group (P < 0.05). However, the catechin group had no significant effect on the RBC index, and there was no significant difference compared with the model control group. The epicatechin had no significant effect on Hb, RBC, PLT and other indicators. The tea polyphenol group had Hb, PLT, etc. There is no significant improvement in the indicators. EGCG has a significant therapeutic effect on aplastic anemia.
实施例 9: EGCG胶囊的制备  Example 9: Preparation of EGCG capsules
处方：  Prescription:
EGCG 50g  EGCG 50g
淀粉 450g  Starch 450g
糊精 200g  Dextrin 200g
硬脂酸镁 5g  Magnesium stearate 5g
制备方法： 按照实施例 5制备 EGCG固体， 加入淀粉、 糊精， 混匀过筛， 制粒， 过 16 目筛， 50摄氏度干燥， 整粒， 适量硬脂酸镁混匀， 装胶囊即得。  Preparation method: Prepare EGCG solid according to Example 5, add starch, dextrin, mix and sieve, granulate, pass 16 mesh sieve, dry at 50 degrees Celsius, whole grain, mix with appropriate amount of magnesium stearate, and obtain capsules.
实施例 10: EGCG注射剂的制备  Example 10: Preparation of EGCG injection
处方：  Prescription:
EGCG 50g  EGCG 50g
氯化钠 8.5g  Sodium chloride 8.5g
注射用水 加至 1000ml  Water for injection added to 1000ml
制备方法：按照实施例 2制备得到 EGCG,将 EGCG溶解于注射用水中，使全部溶解， 加氯化钠再溶解，加注射用水至 1000ml,滤过，灌封于 5mL安瓿瓶中， 100摄氏度灭菌 30min 即得。 在本说明书的描述中， 参考术语"一个实施例"、 "一些实施例"、 "示例"、 "具体示例"、 或"一些示例"等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含 于本发明的至少一个实施例或示例中。 在本说明书中， 对上述术语的示意性表述不一定指 的是相同的实施例或示例。 而且， 描述的具体特征、 结构、 材料或者特点可以在任何的一 个或多个实施例或示例中以合适的方式结合。  Preparation method: EGCG was prepared according to Example 2, EGCG was dissolved in water for injection, all dissolved, sodium chloride was added to dissolve, and water for injection was added to 1000 ml, filtered, potted in a 5 mL ampoule, 100 ° C. The bacteria are available for 30 minutes. In the description of the present specification, the description of the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" and the like means a specific feature described in connection with the embodiment or example. A structure, material or feature is included in at least one embodiment or example of the invention. In the present specification, the schematic representation of the above terms does not necessarily mean the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in a suitable manner in any one or more embodiments or examples.
尽管已经示出和描述了本发明的实施例， 本领域的普通技术人员可以理解 ： 在不脱离 本发明的原理和宗旨的情况下可以对这些实施例进行多种变化、 修改、 替换和变型， 本发 明的范围由权利要求及其等同物限定。  While the embodiments of the present invention have been shown and described, the embodiments of the invention may The scope of the invention is defined by the claims and their equivalents.

Claims

权利要求书 claims
1、 一种制备表没食子儿茶素没食子酸酯的方法， 其特征在于， 包括： 1. A method for preparing epigallocatechin gallate, which is characterized by comprising:
将绿茶进行加醇提取， 以便获得绿茶提取液； Extract green tea with alcohol to obtain green tea extract;
将所述绿茶提取液进行纯化， 以便获得绿茶提取物； Purifying the green tea extract to obtain green tea extract;
将所述绿茶提取物进行纯化， 以便获得表没食子儿茶素没食子酸酯粗提物； 以及 将所述表没食子儿茶素没食子酸酯粗提物进行重结晶， 以便获得表没食子儿茶素没 食子酸酯。 Purifying the green tea extract to obtain a crude epigallocatechin gallate extract; and recrystallizing the crude epigallocatechin gallate extract to obtain epigallocatechin gallate acid ester.
2、 根据权利要求 1所述的方法， 其特征在于， 将绿茶进行加醇提取， 以便获得绿茶提 取液进一步包括： 2. The method according to claim 1, characterized in that, extracting the green tea with alcohol to obtain the green tea extract further includes:
将绿茶药材用 6-15倍重量的 30%-80%的乙醇进行加热回流提取 1-4次， 每次 0.5-3小 时， 并将分别获得的提取液进行合并， 以便获得所述绿茶提取液。 The green tea medicinal materials are heated and refluxed with 6-15 times the weight of 30%-80% ethanol for 1-4 times, each time for 0.5-3 hours, and the separately obtained extracts are combined to obtain the green tea extract. .
3、 根据权利要求 1所述的方法， 其特征在于， 将绿茶进行加醇提取， 以便获得绿茶提 取液进一步包括： 3. The method according to claim 1, characterized in that, extracting the green tea with alcohol to obtain the green tea extract further includes:
将绿茶药材用 8-12倍重量的 50%-70%乙醇进行加热回流提取 1-3次， 每次 1-3小时， 并合并提取液， 以便获得绿茶提取液。 Extract the green tea medicinal materials with 8-12 times the weight of 50%-70% ethanol under heating and reflux 1-3 times for 1-3 hours each time, and combine the extracts to obtain a green tea extract.
4、 根据权利要求 1所述的方法， 其特征在于， 将绿茶进行加醇提取， 以便获得绿茶提 取液进一步包括： 4. The method according to claim 1, characterized in that, alcoholic extraction of green tea to obtain the green tea extract further includes:
将绿茶药材用 10倍量的 60%乙醇加热回流提取 2次， 每次 1.5小时， 合并提取液。 Extract the green tea medicinal materials by heating and refluxing 10 times the amount of 60% ethanol twice, for 1.5 hours each time, and combine the extracts.
5、 根据权利要求 2-4所述的方法， 其特征在于， 将所述绿茶提取液进行纯化进一步包 括： 5. The method according to claims 2-4, characterized in that purifying the green tea extract further includes:
将所述绿茶提取液进行第一减压处理， 以便除去乙醇； The green tea extract is subjected to a first pressure reduction treatment to remove ethanol;
将所得到的残留物加水溶解后用乙酸乙酯萃取， 并分离含有表没食子儿茶素没食子酸 酯的乙酸乙酯相； The obtained residue was dissolved in water and extracted with ethyl acetate, and the ethyl acetate phase containing epigallocatechin gallate was separated;
将所述含有表没食子儿茶素没食子酸酯的乙酸乙酯相进行第二减压处理， 以便获得所 述绿茶提取物。 The ethyl acetate phase containing epigallocatechin gallate is subjected to a second reduced pressure treatment to obtain the green tea extract.
6、 根据权利要求 5所述方法， 其特征在于， 所述第一减压处理是在 35〜70摄氏度的温 度下进行的。 6. The method according to claim 5, characterized in that the first decompression treatment is performed at a temperature of 35 to 70 degrees Celsius.
7、 根据权利要求 5所述方法， 其特征在于， 所述第一减压处理是在 50〜60摄氏度的温 度下进行的。 7. The method according to claim 5, characterized in that the first decompression treatment is performed at a temperature of 50 to 60 degrees Celsius.
8、 根据权利要求 5所述方法， 其特征在于， 所述第一减压处理是在 50摄氏度的温度 下进行的。 8. The method according to claim 5, characterized in that the first pressure reduction treatment is performed at a temperature of 50 degrees Celsius.
9、 根据权利要求 5所述方法， 其特征在于， 将所得到的残留物加水溶解后用乙酸乙酯 萃取 1〜4次。 9. The method according to claim 5, characterized in that the obtained residue is dissolved in water and extracted with ethyl acetate 1 to 4 times.
10、 根据权利要求 5 所述方法， 其特征在于， 将所得到的残留物加水溶解后用乙酸乙 酯萃取 2〜3次。 10. The method according to claim 5, characterized in that the obtained residue is dissolved in water and extracted with ethyl acetate 2 to 3 times.
11、 根据权利要求 5 所述方法， 其特征在于， 将所得到的残留物加水溶解后用乙酸乙 酯萃取 2次。 11. The method according to claim 5, characterized in that the obtained residue is dissolved in water and extracted twice with ethyl acetate.
12、 根据权利要求 5 所述方法， 其特征在于， 将所得到的残留物加水溶解后用等体积 的乙酸乙酯萃取。 12. The method according to claim 5, characterized in that the obtained residue is dissolved in water and then extracted with an equal volume of ethyl acetate.
13、 根据权利要求 5所述方法， 其特征在于， 所述第二减压处理是在 40〜90摄氏度的 温度下进行的。 13. The method according to claim 5, characterized in that the second reduced pressure treatment is performed at a temperature of 40 to 90 degrees Celsius.
14、 根据权利要求 5所述方法， 其特征在于， 所述第二减压处理是在 40〜60摄氏度的 温度下进行的。 14. The method according to claim 5, characterized in that the second pressure reduction treatment is performed at a temperature of 40 to 60 degrees Celsius.
15、 根据权利要求 5所述方法， 其特征在于， 所述第二减压处理是在 50摄氏度的温度 下进行的。 15. The method according to claim 5, characterized in that the second pressure reduction treatment is performed at a temperature of 50 degrees Celsius.
16、 根据权利要求 1 所述的方法， 其特征在于， 将所述绿茶提取物进行纯化， 以便获 得表没食子儿茶素没食子酸酯粗提物进一步包括： 16. The method according to claim 1, wherein purifying the green tea extract to obtain a crude epigallocatechin gallate extract further includes:
将所述绿茶提取物用水溶解后， 过大孔树脂柱， 收集乙醇洗脱部分， 减压干燥， 以便 获得所述没食子儿茶素没食子酸酯粗提物。 After the green tea extract is dissolved in water, it is passed through a macroporous resin column, and the ethanol elution fraction is collected and dried under reduced pressure to obtain the crude extract of gallate gallate.
17、 根据权利要求 16所述的方法， 其特征在于， 所述过大孔树脂柱是按照 0.5-3g绿茶 提取物 /g树脂的比例加样而进行的。 17. The method according to claim 16, characterized in that the over-macroporous resin column is loaded at a ratio of 0.5-3g green tea extract/g resin.
18、 根据权利要求 16所述的方法， 其特征在于， 所述过大孔树脂柱是按照 1.0-2.5g绿 茶提取物 /g树脂的比例加样而进行的。 18. The method according to claim 16, characterized in that the over-macroporous resin column is loaded according to a ratio of 1.0-2.5g green tea extract/g resin.
19、 根据权利要求 16所述的方法， 其特征在于， 所述过大孔树脂柱是按照 2g绿茶提 取物 /g树脂的比例加样而进行的。 19. The method according to claim 16, characterized in that the over-macroporous resin column is loaded according to a ratio of 2g green tea extract/g resin.
20、 根据权利要求 16所述的方法， 其特征在于， 首先用 2-6个柱体积的水对所述大孔 树脂柱进行洗脱， 再用 4-8个柱体积的 5%-15%乙醇对所述大孔树脂柱进行洗脱。 20. The method according to claim 16, characterized in that, first, the macroporous resin column is eluted with 2-6 column volumes of water, and then 5%-15% of 4-8 column volumes of water are used. The macroporous resin column was eluted with ethanol.
21、 根据权利要求 16所述的方法， 其特征在于， 首先用 3-5个柱体积的水对所述大孔 树脂柱进行洗脱， 再用 5-7个柱体积的 8%-12%乙醇对所述大孔树脂柱进行洗脱。 21. The method according to claim 16, characterized in that, first, the macroporous resin column is eluted with 3-5 column volumes of water, and then 8%-12% of 5-7 column volumes of water are used. The macroporous resin column was eluted with ethanol.
22、 根据权利要求 16所述的方法， 其特征在于， 首先用 3个柱体积的水对所述大孔树 脂柱进行洗脱， 再用 6个柱体积的 10%的乙醇对所述大孔树脂柱进行洗脱。 22. The method according to claim 16, characterized in that, first, the macroporous resin column is eluted with 3 column volumes of water, and then the macroporous resin column is eluted with 6 column volumes of 10% ethanol. The resin column is eluted.
23、 根据权利要求 1所述的方法， 其特征在于， 将所述表没食子儿茶素没食子酸酯粗 提物进行重结晶进一步包括： 将所述表没食子儿茶素没食子酸酯粗提物配制成饱和水溶液； 以及 23. The method according to claim 1, wherein recrystallizing the crude epigallocatechin gallate extract further includes: Preparing the crude epigallocatechin gallate extract into a saturated aqueous solution; and
向所述饱和水溶液中加入有机溶剂， 以便进行重结晶。 An organic solvent is added to the saturated aqueous solution to perform recrystallization.
24、 根据权利要求 23所述的方法， 其特征在于， 所述有机溶剂为甲醇与异丙醇的混合 物， 其中， 甲醇与异丙醇的体积比为 2: 1。 24. The method according to claim 23, wherein the organic solvent is a mixture of methanol and isopropanol, wherein the volume ratio of methanol to isopropanol is 2:1.
25、 根据权利要求 23所述的方法， 其特征在于， 所述重结晶是在 0〜15摄氏度的温度 下进行的。 25. The method according to claim 23, characterized in that the recrystallization is performed at a temperature of 0 to 15 degrees Celsius.
26、 根据权利要求 23所述的方法， 其特征在于， 所述重结晶是在 5〜10摄氏度的温度 下进行的。 26. The method according to claim 23, characterized in that the recrystallization is carried out at a temperature of 5 to 10 degrees Celsius.
27、 根据权利要求 23所述的方法， 其特征在于， 所述重结晶是在 8摄氏度的温度下进 行的。 27. The method according to claim 23, characterized in that the recrystallization is performed at a temperature of 8 degrees Celsius.
28、 根据权利要求 23所述的方法， 其特征在于， 所述表没食子儿茶素没食子酸酯与 所述有机溶剂的比为 1 g:2-5ml。 28. The method according to claim 23, characterized in that the ratio of the epigallocatechin gallate to the organic solvent is 1 g: 2-5 ml.
29、 根据权利要求 23所述的方法， 其特征在于， 所述表没食子儿茶素没食子酸酯与 所述有机溶剂的比为 l g:3ml。 29. The method according to claim 23, characterized in that the ratio of the epigallocatechin gallate to the organic solvent is 1 g: 3 ml.
30、 表没食子儿茶素没食子酸酯在制备抗贫血产品中的用途， 其特征在于， 所述表 没食子儿茶素没食子酸酯是根据权利要求 1〜29任一项所述的方法制备的。 30. The use of epigallocatechin gallate in the preparation of anti-anemia products, characterized in that the epigallocatechin gallate is prepared according to the method described in any one of claims 1 to 29.
31、 根据权利要求 30所述的用途， 其特征在于， 所述抗贫血产品为用于预防或者 治疗贫血的药物、 保健品或食品。 31. The use according to claim 30, characterized in that the anti-anemia product is a drug, health product or food used to prevent or treat anemia.
32、 根据权利要求 31所述的用途， 其特征在于， 所述药物呈片剂、 胶囊剂、 颗粒 剂、 或注射剂的至少一种。 32. The use according to claim 31, characterized in that the drug is in the form of at least one of tablets, capsules, granules, or injections.
33、 根据权利要求 31所述的用途， 其特征在于， 所述贫血为选自缺铁性贫血、 溶 血性贫血、 再生障碍性贫血和地中海贫血的至少一种。 33. The use according to claim 31, characterized in that the anemia is at least one selected from the group consisting of iron deficiency anemia, hemolytic anemia, aplastic anemia and thalassemia.
34、 根据权利要求 31所述的用途， 其特征在于， 所述贫血为选自缺铁性贫血、 乙 型地中海贫血、 海恩兹小体性溶血性贫血的至少一种。 34. The use according to claim 31, wherein the anemia is at least one selected from the group consisting of iron deficiency anemia, beta thalassemia, and Heinz body hemolytic anemia.
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