WO2009109109A1 - 茶多酚在制备预防或治疗肿瘤药物中的应用 - Google Patents

茶多酚在制备预防或治疗肿瘤药物中的应用 Download PDF

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WO2009109109A1
WO2009109109A1 PCT/CN2009/000233 CN2009000233W WO2009109109A1 WO 2009109109 A1 WO2009109109 A1 WO 2009109109A1 CN 2009000233 W CN2009000233 W CN 2009000233W WO 2009109109 A1 WO2009109109 A1 WO 2009109109A1
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tea
use according
tea polyphenol
epigallocatechin
cells
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PCT/CN2009/000233
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English (en)
French (fr)
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张莉
刘蕴华
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Zhang Li
Liu Yunhua
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Priority to CN2009801045844A priority Critical patent/CN101965185A/zh
Priority to JP2010549002A priority patent/JP2011513340A/ja
Priority to EP09716802.5A priority patent/EP2263668B1/en
Priority to US12/921,353 priority patent/US20110021620A1/en
Publication of WO2009109109A1 publication Critical patent/WO2009109109A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • A61K31/353—3,4-Dihydrobenzopyrans, e.g. chroman, catechin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia

Definitions

  • the present invention relates to the use of a tea polyphenol or a pharmaceutically acceptable salt thereof or a pharmaceutical composition containing any of them for the preparation of a medicament for preventing or treating a tumor.
  • methods for treating tumors mainly include cytotoxicity, induction of apoptosis and differentiation, and biological targeted therapy.
  • toxic side effects such as inhibition of normal cell proliferation and growth, peripheral neurotoxicity, and impaired vital organ function are important factors limiting the application of these drugs, and their pharmacoeconomics is an important factor to be considered.
  • Biotargeted therapy uses "tailor-made" drugs for tumor pathogenesis, including imatinib mesylate (tyrosineb inhibitor, tyrosine kinase inhibitor, for chronic myeloid leukemia), rituximab, etc. Drug prices are an important barrier to universal access, and drug resistance caused by target mutations is a fatal weakness.
  • the drugs currently used for cancer treatment often adopt a combination scheme, but the median lethal dose (LD50) of some drugs is close to the onset concentration, which is likely to cause obvious bone marrow suppression, vital organ damage and other toxic effects.
  • the reaction is poorly tolerated, and the toxic and side effects between the various drugs have synergistic effects, so the curative effect can only be improved to a certain extent.
  • a considerable number of patients still have drug resistance, which is characterized as refractory and relapse.
  • the limited efficacy of some products with high biochemical content is not directly proportional to the high price.
  • other factors such as high preservation requirements and inconvenient use are the root causes of human beings to explore new ingredients and methods of cancer treatment. .
  • Tea leaves in China are rich in vitamin C, chlorophyll, carotene, tea polyphenols and other nutrients.
  • Tea polyphenols also contain catechins.
  • the catechins include: Tea galactate (EGCG), epigallocatechin (EGC), epicatechin gallate (ECG) and epicatechin (EC), of which EGCG is the most abundant and biologically active, The antioxidant effect of EGC is stronger than EGCG. Another strong epidemic
  • the present invention is mainly based on the idea that although studies have shown that catechins such as epigallocatechin gallate (EGCG) have an antitumor effect, tea phenols from plant extracts are purified to obtain various catechin pure products. The cost is very high, and if the tea polyphenol which is not purified and rich in catechins such as EGCG can be directly used as an antitumor drug, the production cost can be greatly reduced.
  • EGCG epigallocatechin gallate
  • the present invention provides the use of a tea polyphenol or a pharmaceutically acceptable salt thereof or a pharmaceutical composition containing any of them for the preparation of a medicament for preventing or treating a tumor.
  • the tea polyphenol is a tea extract comprising catechin mainly composed of epigallocatechin gallate (EGCG), epicatechin gallate (ECG) and epigallocatechin (EGC). Class of substances.
  • the pharmaceutical agent may be in the form of a tablet, a capsule, a buccal tablet, an orally disintegrating tablet, an oral instant tablet, a dispersible tablet, a chewable agent, a granule, a dry suspension, an injection,
  • a pharmaceutically acceptable arbitrary dosage form such as a solution, a sustained release agent, a controlled release agent or an immediate release sustained release preparation.
  • the pharmaceutical composition further comprises a pharmaceutically acceptable adjuvant or carrier.
  • the tea polyphenol has been removed from the tannic acid contained in the tea leaves, and the content of the undesirable component caffeine in the prepared medicament is less than 0.2% by weight.
  • EGCG EGCG
  • ECG ECG
  • EGC ECG
  • the capsule is a green tea glue of Pharmanex Corporation of the United States. Pouch Tegreen97®.
  • the agent of the present invention can be used alone for the treatment of tumors without being combined with other drugs.
  • the invention directly prepares the antitumor agent by using the tea extract tea polyphenol, and does not need to separate the pure product of the active ingredients such as EGCG, EGC, ECG and the like from the tea polyphenol by complicated and expensive purification process, thereby greatly reducing the production cost of the medicament.
  • the tea polyphenols involved in the present invention have significant inhibitory effects on various human tumor cells, especially hematopoietic malignant cells, and even have a good inhibitory effect on primary cells of some blood tumors. It not only adds a new drug with high efficacy and low toxicity to the treatment of malignant tumors, but also provides the possibility for patients with secondary vasculitis after long-term intravenous infusion of cytotoxic drugs to continue treatment with other routes.
  • the efficacy test of the mouse tumor model found that the medicament of the present invention has an obvious therapeutic effect on mouse tumors, can reduce the tumor size, and prolong the survival of tumor model mice.
  • the tea polyphenol is basically used without any other support for symptomatic treatment, and can be used alone to achieve the above pharmaceutical effects.
  • the tea polyphenols of the present invention have almost no toxic side reactions of the above-mentioned conventional drugs, and the tea polyphenols are widely distributed in plants, and the raw material supply is abundant, and the economic benefits are obvious.
  • the agent may be a capsule, which is very convenient to use, and avoids the damage and stimulating effect on the tissues and blood vessels of the conventionally used injection type cytotoxic drugs. '
  • the tea polyphenol is extracted from plants, due to the standardization of the production process, the content of the main components in the extract is kept stable, and the difference in the content of the components in the different batch products is overcome, thereby correspondingly
  • the study has good repeatability and comparability and is a promising product for the treatment of tumors. Since the treatment of the tumor agent made of the tea polyphenol in combination with other drugs does not synergistically increase the toxic side effects, especially the normal hematopoiesis of the bone marrow, and provides a promising combination of other drugs to improve the therapeutic effect. Drug.
  • Fig. 1 is a graph showing the inhibition of proliferation of various cell lines by different concentrations of tea polyphenol solution for one day;
  • Fig. 2 is a graph showing the inhibition of proliferation of various cell lines by the action of 129.6 mol/L concentration of drug solution for 1 to 3 days;
  • Figure 3 is a photomicrograph of the cell morphology observed under light microscope, showing the effect of different concentrations of tea polyphenol solution on the morphology of RPMI8226 cell line;
  • Figure 4a to Figure 4c are scatter plots showing the effect of different concentrations of drug solution on RPMI8226 cells for 1 day. The influence of the apoptotic rate of the strain;
  • Figure 5a to Figure 5c are scatter plots showing the effect of different concentrations of drug solution on the apoptotic rate of U937 cell line for 2.5 days;
  • Fig. 6a to Fig. 6d are peak shape diagrams showing the effects of different concentrations of drug solution on the cell cycle of RPMI8226 cell line for 2 days;
  • Figure 7 is a scatterplot showing the effect of different concentrations of drug solution on the mitochondrial membrane potential of RPMI8226 cell line at different times;
  • Figure 8 is a photomicrograph of cell morphology under light microscope, showing the effect of different concentrations of drug solution for 1.5 days on the morphology of primary cells of ALL-L2;
  • Figure 9 is a scatterplot showing the effect of 1.5 days of different concentrations of drug solution on the apoptosis rate of primary cells of ALL-L2;
  • Figure 10 is a photograph of cell morphology in a mouse tumor tissue section under a microscope. detailed description
  • the present invention provides a novel use of plant extracting natural tea polyphenols as an antitumor agent.
  • the tea polyphenol of the present invention is a tea extract, which is rich in catechins, including epigallocatechin gallate (EGCG) and epicatechin gallate (ECG). And epigallocatechin (EGC).
  • the antitumor agent made of tea polyphenol may be any pharmaceutically acceptable dosage form, such as tablets, capsules, buccal tablets, orally disintegrating tablets, orally dissolving tablets, dispersible tablets, chewables, granules, dry blending. Agent, injection, solution, sustained release, controlled release or immediate release sustained release preparation.
  • the change in the content of catechins in the tea polyphenol does not significantly affect its anti-tumor effect.
  • the catechins in the antitumor solid agent made of tea polyphenol are controlled by the production process.
  • the content of the substance is 60 to 70% by weight.
  • the catechins are mainly composed of three monomers: EGCG, ECG and EGC, all of which have certain anti-tumor effects. Therefore, the ratio of EGCG, ECG and EGC in the tea polyphenols used in the present invention is not particularly different. limits.
  • EGCG is the main part of the three, for example, more than 50% of the total weight of the three
  • the EGC is the least, for example, about 10% or less of the total weight.
  • EGCG, ECG and EGC respectively account for the total weight of the three therapeutic remedies in the tea polyphenols used in the invention or prepared by the production process control. 50 to 60%, 20 to 25% and 8 to 10%.
  • the adverse reaction components such as citric acid and caffeine in the tea leaf can be removed during the production process of the tea polyphenol, and the final treatment is preferably ensured.
  • the caffeine content of the tumor agent is less than 0.5 mg / 250 mg of the agent. Almost all people can tolerate such caffeine levels.
  • the tea polyphenol used in the present invention can be prepared into various pharmaceutical preparations such as a powder, a tablet, a capsule or a solvent by a conventional pharmaceutical method.
  • the drug When the drug is used, it can be taken orally, or it can be dissolved in water and used.
  • This tea polyphenol mixture can inhibit the proliferation of a variety of tumor cells in vitro, and its mechanism may induce apoptosis through caspase-3 and other pathways, and its inhibition compared with EGCG pure products and certain cytotoxic drugs. The role of proliferation is even worse. This mixture has a good inhibitory effect on even the primary cells of some tumors. Oral administration of this tea polyphenol preparation can achieve an effective tumor suppressing concentration, and preferably inhibit the growth of human multiple myeloma RPMI8226 cells, lymphoma Jurkat cells, Sudhl-4 cells and the like in nude mice.
  • the Chinese names of the tumor cell lines used are as follows:
  • ⁇ 4 human acute promyelocytic leukemia cell line
  • NB4-R1 resistant to all-trans retinoic acid human acute promyelocytic leukemia cell line 1 ;
  • NB4-R2 resistant to all-trans retinoic acid human acute promyelocytic leukemia cell line 2;
  • U937 human acute monocytic leukemia cell line
  • ⁇ 562 human chronic myeloid leukemia cell line
  • RPMI8226 Human multiple myeloma cell line.
  • mice nude mice: Entrusted the Experimental Animal Science Department of Shanghai Jiao Tong University School of Medicine to purchase the proceeds from Haislake Experimental Animals Co., Ltd. (SCXK (Shanghai) 2003-0003).
  • a therapeutic agent for tumors is prepared from green tea extract tea polyphenols, which is a green tea capsule T e g ree n 97® produced by Pharmanex, USA.
  • the capsule contains tea polyphenol powder, and the total weight of each capsule is 250 mg, of which 162 mg of catechins (including EGCG 95 mg, ECG 37 mg, EGC 15 mg, molecular weights of 458.4, 442.4, 306.3, respectively) and caffeine less than 0.5 mg.
  • catechins including EGCG 95 mg, ECG 37 mg, EGC 15 mg, molecular weights of 458.4, 442.4, 306.3, respectively
  • caffeine less than 0.5 mg.
  • the doses mentioned in the following test are based on the EGCG content of the green tea capsule Tegreen 97®.
  • the doses of 32.4 mol/L, 64.8 mol/L, 129.6 mol/L and 259.2 mol/L are equivalent to one capsule content. 1 : 6400, 1: 3200, 1:1600, 1:800 diluted supernatant concentration.
  • the following anti-tumor tests were carried out using the tea polyphenol solution in the green tea capsule Tegreen 97®:
  • AnnexinV-FITC and PI double standard quantitative detection of apoptotic rate
  • Mitochondrial Membrane Potential Analysis Reveals a possible mechanism for inducing apoptosis.
  • AnnexinV-FITC and PI double standard quantitative detection of apoptotic rate
  • Figure 1 is a graph showing the inhibition of proliferation of various cell lines in a dose of 32.4 mol/L, 64.8 mol/L, 129.6 mol/L, 259.6 ⁇ 1/ ⁇ Tegreen 97® capsule tea polyphenols for 1 day; The curve indicates the inhibition of proliferation of various cell lines by a dose of 129.6 ⁇ mol/L for 1 to 3 days.
  • RPMI8226 As shown in Fig. 3, after treatment of multiple myeloma cell line RPMI8226 with different concentrations of tea polyphenol solution for 1 day, the control group ( ⁇ /L) had uniform nuclear staining, loose chromatin and normal cell ratio; 32.4 mol/L, 64.8 After 1 day of mol/L, 129.6 mol/L, 259.2 mol/L tea polyphenols, RPMI8226 cells showed membrane shrinkage, chromatin condensation, nuclear condensation and apoptotic bodies.
  • Apoptosis rate was quantified by flow cytometry (Annexin V-FITC and PI double labeling).
  • FITC fluorescein isothiocyanate-labeled Annexin-V binds to membrane-phosphorylserine (PS), which is externalized during apoptosis, and apoptotic cells show Annexin-V positive;
  • PS membrane-phosphorylserine
  • PI that is, propidium iodide binds to nucleic acid, and dead cells show PI positive;
  • Figure 4a to Figure 4c show the fluorescence scatter plot displayed by the flow cytometer.
  • the lower left quadrant is Annexin-V FITC and PI double-labeled negative cells (normal cells), and the lower right quadrant is Annexin-V FITC single marker.
  • Positive cells early apoptotic cells
  • the upper right quadrant is Annexin-V FITC and PI double-labeled positive cells (late apoptotic cells)
  • the upper left quadrant is PI single-label positive cells (dead cells).
  • Apoptosis rate was quantified by flow cytometry (Annexin V-FITC and ⁇ double standard method).
  • Figure 5a to Figure 5c show the fluorescence scatter plot of the flow cytometer.
  • the lower left quadrant is Annexin-V FITC and PI double-labeled negative cells (normal cells), and the lower right quadrant is Annexin-V FITC single-label positive.
  • the cells early apoptotic cells
  • the upper right quadrant are Annexin-V FITC and PI double-labeled positive cells (late apoptotic cells)
  • the upper left quadrant is PI single-label positive cells (dead cells).
  • Figure 6a to Figure 6d are peak shape diagrams displayed by flow cytometry, in which FL3 and count represent sub-two The percentage of ploidy cells, an indicator of apoptosis.
  • the proportion of subdiploid cells increased with the increase of the concentration of the drug solution (control group, 32.4 ⁇ 0 1/ ⁇ , 64.8 mol/L, 129.6 mol/L tea polyphenols for 2 days)
  • the proportion of subdiploid cells was 0.5%, 8.3%, 15.7%, and 94.1%, respectively.
  • Mitochondrial membrane potential analysis reveals possible mechanisms of induction of apoptosis
  • Flow cytometry (Rhodamine R 23 fluorescent labeling method) was used to quantitatively detect intracellular mitochondrial membrane potential and explore possible mechanisms of cell proliferation inhibition.
  • the ⁇ ® in Figure 7 gives the proliferation and apoptosis of human multiple myeloma cells in different doses and different time periods. Compared with the control group ( ⁇ /L), 64.8 mol/L, 129.6 mol/L, 259.2 ⁇ 1 ⁇ tea polyphenols acted on human multiple myeloma cells for 3 hours, and the mitochondrial membrane potential began to decrease, and it was time-dependent.
  • the control group As shown in Fig. 8, after 1.5 days of primary cells of acute lymphoblastic leukemia treated with different concentrations of tea polyphenols, the control group ( ⁇ /L) had uniform nuclear staining, loose chromatin and normal cytoplasm ratio; 129.6 mol/L, After 1.5 days of 194.4 mol/L and 259.2 mol/L tea polyphenols, the primary cells showed membrane shrinkage, chromatin condensation, nuclear condensation and eosinophils (apoptotic bodies).
  • tea polyphenols can induce primary cell apoptosis in patients with acute lymphoblastic leukemia, and this effect is obvious with increasing concentration.
  • AnnexinV-FITC and PI double standard quantitative detection of apoptotic rate
  • the rate of apoptosis was quantified by flow cytology (Annexin V-FITC and PI double-label method).
  • the lower left quadrant is Annexin-V FITC and PI double-labeled negative cells (normal cells), and the lower right quadrant is Annexin-V FITC single-label positive cells (early apoptotic cells).
  • the upper right quadrant is Annexin-V FITC and PI double-labeled positive cells Dead cells), the upper left quadrant is PI single-label positive cells (dead cells).
  • the tumor continued to increase after the 16th day and died on the 18th day.
  • the tumor did not increase but decreased after the administration on the 16th day, and the death time of the mouse was delayed at least compared with the control group. It took 3 to 4 days. It indicates that tea polyphenols have obvious tumor treatment effects.
  • mice fed with different doses of tea polyphenols were taken out, paraffin sections were taken, HE staining, and cell morphology changes were observed under a microscope.
  • the tumor cells of Omg/kg * d tea polyphenols were uniformly stained, the chromatin was loose, and the cytoplasm ratio was normal. 12 mg/kg « d, 24 mg/kg * d tea polyphenol feeding After that, the tumor cells showed membrane shrinkage, chromatin condensation, nuclear condensation and apoptotic bodies.

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Description

茶多酚在制备预防或治疗肿瘤药物中的应用 技术领域
本发明涉及茶多酚或其在药学上可以接受的盐或含有它们中任何一种 的药物组合物在制备预防或治疗肿瘤药物中的应用。 技术背景
目前药物治疗肿瘤的方法， 主要包括细胞毒、 诱导凋亡及分化和生物 靶向治疗等。
一些来自天然植物和矿物的成分， 在前两种方法中起着举足轻重的作用。 比如长春新碱、 高三尖杉酯碱、 羟基喜树碱、 紫杉醇以及亚砷酸等。 但不同程 度抑制正常细胞的增殖和生长、 周围神经毒性、 重要脏器功能受损等毒副反应 是限制这些药物应用的重要因素， 且其药物经济学是不得不考虑的一个重要因 素。
生物靶向治疗应用针对肿瘤发病机制而"度身定做"的药物， 包括甲磺 酸伊马替尼 （Imatinib, 酪氨酸激酶抑制剂， 针对慢性粒细胞性白血病） 、 利妥昔单抗等， 药物价格是限制普及应用的重要障碍， 而靶点突变导致的 耐药则是其致命的弱点。
由于以上这些因素， 目前用于肿瘤治疗的药物往往采用联合方案， 但 某些药物的半数致死剂量 （LD50 ) 与起效浓度比较接近， 容易引起明显的 骨髓抑制、 重要脏器功能损伤等毒副反应， 耐受性较差， 而且多种药物之 间的毒副反应具有协同作用， 所以只能在一定程度上提高疗效， 仍有相当 一部分患者出现耐药， 表现为难治、 复发。 而一部分生化科技含量较高的 产品， 其疗效的有限与价格的高昂不成正比， 加之保存要求高、 使用不方 便等其他因素， 都是促使人类不断探索肿瘤治疗的新成分、 新方法的根本 原因。
我国常见的茶叶植物茶叶中富含维生素 C、 叶绿素、 胡罗卜素、 茶多 酚等营养素， 茶多酚中又含有儿茶素（catechins )类物质， 儿茶素类物质包 括： 表没食子儿茶素没食子酸酯 （EGCG) 、 表没食子儿茶素 （EGC) 、 表 儿茶素没食子酸酯（ECG)和表儿茶素（EC ) ， 其中以 EGCG的含量最多、 生物活性最广泛， 而 EGC的抗氧化作用比 EGCG更强。 另有很强的流行病
确认本 学证据也显示增高体内茶多酚浓度能提供强力的保护作用， 消除氧自由基 对机体的损伤， 具有肿瘤预防作用。 喝茶能够抗毒灭菌、 防癌、 长寿， 其 有益健康的作用早已有了共识， 但具体有益到何种程度、 在哪些方面有益 尚未得到进一步的研究证实。
另外， 诸多茶叶植物中所含儿茶素的成分、 含量各不相同， 加上生产 工艺的非标准化， 目前能够摄食的儿茶素类产品名目繁多， 其含量及质量 都不确定， 大致属于保健品的范畴。 国外对 EGCG抑制肿瘤增生的研究主 要为体外试验， 研究的细胞株包括前列腺癌、 胃癌、 结肠癌、 皮肤癌、 乳 腺癌、 肺癌、 平滑肌肉瘤等实体肿瘤细胞， 但其抑制肿瘤增殖的活性并不 很强， 加之 EGCG、 EGC的纯制品相当昂贵， 尚无进一步的体内研究报道。 发明内容
有鉴于现有技术的上述缺陷， 本发明的目的在于提供一种成本低廉、 使用方便且几乎无毒副反应的治疗肿瘤药物。
本发明主要基于这样的构思： 虽然研究表明表没食子儿茶素没食子酸 酯（EGCG)等儿茶素具有抗肿瘤效果， 但从植物提取物茶多酚经纯化得到 各种儿茶素纯制品的代价十分昂贵， 如果能将未经纯化而富含 EGCG等儿 茶素类物质的茶多酚直接用作抗肿瘤药物， 则可大大降低生产成本。
因此， 本发明提供了茶多酚或其在药学上可以接受的盐或含有它们中 任何一种的药物组合物在制备预防或治疗肿瘤药物中的应用。
所述茶多酚为茶叶提取物， 其包括主要由表没食子儿茶素没食子酸酯 ( EGCG) 、 表儿茶素没食子酸酯 （ECG) 和表没食子儿茶素 （EGC ) 组成 的儿茶素类物质。
在本发明的实施方式中， 所述药物的药剂形式可以为片剂、 胶囊、 口 含片、 口腔崩析片、 口腔速溶片、 分散片、 咀嚼剂、 颗粒剂、 干混悬剂、 注射剂、 溶液剂、 缓释剂、 控释剂或速释缓释制剂等药学上可接受的任意 剂型。
在本发明的实施方式中， 所述药物组合物还包含药学上可接受的辅料 或载体。
在本发明优选实施方式中， 所述茶多酚中去除了茶叶所含的鞣酸， 且 制成的药剂中不良成分咖啡因的含量小于 0.2重量％。
在本发明的实施方式中， 所述茶多酚中， EGCG、 ECG和 EGC分别占 所述儿茶素类物质重量 50〜60%、 20〜25 %和 8〜10%。
在本发明具体实施例中， 所述胶囊剂为美国 Pharmanex公司的绿茶胶 囊 Tegreen97®。
本发明所述药剂可以无需与其它药物联合而单独用于治疗肿瘤。
本发明用茶叶提取物茶多酚直接制备抗肿瘤药剂， 而无需经繁复昂贵 的纯化过程从茶多酚中分离 EGCG、 EGC、 ECG等有效成分的纯制品， 从 而可以大大降低药剂的生产成本。
而且， 经细胞水平的药理试验发现本发明涉及的茶多酚对多种人类肿 瘤细胞有明显的抑制作用， 尤其是造血系统恶性肿瘤细胞， 甚至对部分血 液肿瘤的原代细胞也有良好的抑制作用， 不仅为恶性肿瘤的治疗增加了一 种疗效高、 毒性反应低的新药物， 也为长期静脉输注细胞毒药物后继发血 管炎的患者改用其他途径继续治疗提供了可能性。 经小鼠肿瘤模型的疗效 试验发现， 本发明涉及的药剂对小鼠肿瘤具有明显的治疗作用， 可减小肿 瘤大小， 并延长肿瘤模型小鼠的存活期。 并且， 所述茶多酚应用时基本不 需其他的支持对症治疗， 即可单独使用达到上述药学效果。
还有， 本发明涉及的茶多酚几乎没有上述现有药物具有的毒副反应， 而且该茶多酚的来源植物分布广泛， 原料供应丰富， 经济学效益显而易见。
还有， 所述药剂可以是胶囊剂， 使用的时候非常方便， 避免了以往常 用的注射剂型细胞毒药物对组织、 血管的损伤及刺激作用。'
另外， 所述茶多酚尽管是从植物中提取的， 但由于其生产工艺的标准 化， 使该提取物中主要成分的含量保持稳定， 克服了不同批号产品中成分 含量的差异， 因而使相应的研究具有良好的重复性和可比性， 是用于治疗 肿瘤有前景的产品。 由于所述由该茶多酚制成的治疗肿瘤药剂与其他药物 联合使用并不协同增加毒副反应， 尤其对骨髓的正常造血没有抑制作用， 也为联合其他药物共同提高治疗效果提供了有前景的药物。
以下将结合附图对本发明的技术效果作进一步说明， 以充分地了解本 发明。 附图说明
图 1是曲线图， 表示不同浓度茶多酚药液作用 1天对多种细胞株的增 殖抑制作用；
图 2是曲线图，表示 129.6 mol/L浓度药液作用 1~3天对多种细胞株的 增殖抑制作用；
图 3为光镜下观察细胞形态照片， 表示不同浓度茶多酚药液作用 1天 对 RPMI8226细胞株形态的影响；
图 4a〜图 4c为散点图，表示不同浓度药液作用 1天对 RPMI8226细胞 株凋亡率的影响；
图 5a〜图 5c为散点图， 表示不同浓度药液作用 2.5天对 U937细胞株 凋亡率的影响；
图 6a〜图 6d为峰形图，表示不同浓度药液作用 2天对 RPMI8226细胞 株细胞周期的影响；
图 7为散点图表示不周浓度药液作用不同时间对 RPMI8226细胞株线 粒体膜电位的影响；
图 8为光镜下细胞形态照片， 表示不同浓度药液作用 1.5天对 ALL-L2 原代细胞形态的影响；
图 9为散点图，表示不同浓度药液作用 1.5天对 ALL-L2原代细胞凋亡 率的影响； 及
图 10为显微镜下小鼠肿瘤组织切片中细胞形态照片。 具体实施方式
本发明提供植物提取天然茶多酚作为抗肿瘤药剂的新用途。 本发明涉 及的茶多酚为茶叶提取物， 其富含儿茶素类物质， 该儿茶素类物质包括表 没食子儿茶素没食子酸酯 （EGCG) 、 表儿茶素没食子酸酯 （ECG)和表没 食子儿茶素 （EGC ) 。
茶多酚制成的抗肿瘤药剂可以是任意药学可接受的剂型， 如片剂、 胶 囊、 口含片、 口腔崩析片、 口腔速溶片、 分散片、 咀嚼剂、 颗粒剂、 干混. 悬剂、 注射剂、 溶液剂、 缓释剂、 控释剂或速释缓释制剂。
虽然， 该茶多酚中儿茶素的含量变化并不明显影响它的抗肿瘤效果。 但是， 一般地， 为了确保各批产品的稳定性和使用效果的可比性， 通过生 产工艺控制， 在本发明的实施方式中， 使由茶多酚制成的抗肿瘤固体药剂 中儿茶素类物质的含量为 60〜70重量％。
另外， 儿茶素类物质主要由 EGCG、 ECG和 EGC三种单体组成， 三者 均有一定抗肿瘤作用， 因此本发明对所用茶多酚中 EGCG、 ECG和 EGC相 互之间的比例没有特别的限制。 通常， 三者中 EGCG为主要部分， 比如占 三者总重量的 50%以上， EGC最少， 比如占总重量的 10%左右或以下。 但 是， 为了确保各批产品的稳定性和使用效果的可比性， 通过生产工艺控制 使本发明所用茶多酚中或由其制备的治疗肿瘤药剂中， EGCG、ECG和 EGC 分别占三者总重量的 50〜60%、 20〜25 %和 8〜10%。
为了增加由上述茶多酚制成的药剂的适用性， 在茶多酚生产过程中可 去除茶叶中的鞣酸、 咖啡因等不良反应成分， 且最好保证最后制成的治疗 肿瘤药剂中咖啡因含量小于 0.5mg/250mg药剂。 几乎所有人群都可耐受这 样的咖啡因水平。
本发明所用茶多酚可用常规的制药方法制成各种药学制剂， 例如粉剂、 片剂、 胶囊剂或溶剂。 在药剂使用时， 可以直接口服， 也可将其溶于水后 使用。
这种茶多酚混合剂体外能很好地抑制多种肿瘤细胞的增殖， 其机制可 能通过 caspase-3等途径诱导细胞凋亡， 而且与 EGCG纯制品、 某些细胞毒 药物相比， 其抑制增殖的作用有过之而无不及。 该种混合剂甚至对一些肿 瘤的原代细胞具有良好的抑制作用。 口服这种茶多酚制剂能达到有效的抑 瘤浓度， 较好地抑制人多发性骨髓瘤 RPMI8226细胞、 淋巴瘤 Jurkat细胞、 Sudhl-4细胞等在裸鼠体内的生长。 实施例
试验材料来源
1. 细胞株 NB4、 NB4-R NB4-R2、 U937、 562> RPMI8226由上海 交通大学医学院附属瑞金医院、 上海血液研究所赠送；
所用肿瘤细胞株的中文名称如下：
ΝΒ4: 人急性早幼粒性白血病细胞株；
NB4-R1： 耐全反式维甲酸人急性早幼粒性白血病细胞株 1 ;
NB4-R2: 耐全反式维甲酸人急性早幼粒性白血病细胞株 2;
U937: 人急性单核细胞白血病细胞株；
Κ562: 人慢性粒细胞性白血病细胞株；
RPMI8226: 人多发性骨髓瘤细胞株。
2. 试验用小鼠、 裸鼠： 委托上海交通大学医学院实验动物科学部向上 海斯莱克实验动物有限责任公司 （SCXK (沪） 2003-0003 ) 购买所得。
3. 由绿茶提取物茶多酚制成治疗肿瘤药剂， 该药剂为美国 Pharmanex 公司出产的绿茶胶囊 Tegreen 97®。 该胶囊内包含茶多酚粉末， 每颗胶囊总 重 250mg， 其中儿茶素类物质 162mg (含 EGCG 95mg、 ECG 37mg、 EGC 15mg, 分子量分别为 458.4、 442.4、 306.3 ) ， 咖啡因小于 0.5mg。 （更详细 的成分可参见美国的医师桌上手册： Physicians' desk reference®: Ingredients of Tegreen97®. Pharmanex, 2005， 59Edition:2782.)。
下面试验提及的剂量是以绿茶胶囊 Tegreen 97®中的 EGCG含量为标准 的， 剂量 32.4 mol/L、 64.8 mol/L、 129.6 mol/L和 259.2 mol/L分别相当 于 1颗胶囊内容物按 1 :6400、 1 :3200、 1 :1600、 1 :800稀释后的上清液浓度。 采用该绿茶胶囊 Tegreen 97®配置的茶多酚溶液进行了以下抗肿瘤试 验：
(一）对 NB4、 NB4-R NB4-R2、 U937、 K562、 RPMI8226细胞株的影响：
1. 细胞生长抑制试验： ΜΤΤ检测细胞生存率
2. 瑞氏染色： 观察细胞的形态学改变
3. AnnexinV-FITC和 PI双标： 定量检测凋亡率
4. 细胞周期、 细胞核内 DNA浓度分析： 揭示诱导细胞凋亡的可能机 制
5. 线粒体膜电位分析： 揭示诱导细胞凋亡的可能机制。
(二） 对急性淋巴细胞白血病、 急性单核细胞白血病患者原代细胞的影响：
1. 瑞氏染色： 观察细胞的形态学改变
2. AnnexinV-FITC和 PI双标： 定量检测凋亡率
(三） 绿茶胶囊 Tegreen 97®配置的茶多酚溶液对裸鼠肿瘤模型的疗效。 下面详细说明以上各试验及试验结果。
(一） 对 NB4、 NB4-R1、 NB4-R2、 U937、 K562、 RPMI8226细胞株的 影响。
1.细胞生长抑制试验： ΜΤΤ检测细胞生存率
图 1的曲线表示在 32.4 mol/L、 64.8 mol/L、 129.6 mol/L、 259.2μπιο1/ί 剂量 Tegreen 97®胶囊茶多酚作用 1天， 对多种细胞株的增殖抑制作用； 图 2的曲线表示 129.6μ mol/L剂量作用 1~3天对多种细胞株的增殖抑 制作用。
结果表明： 129.6和 259.2 mol/L的剂量下作用 2〜3天，对各细胞株的 增殖均起到明显的抑制作用。
2. 瑞氏染色： 观察细胞凋亡的形态学改变
经瑞氏染色， 观察 （光镜 x lOO倍） 细胞凋亡的形态学改变。
如图 3所示，不同浓度茶多酚溶液作用多发性骨髓瘤细胞株 RPMI8226 1天后， 对照组 （Ομιηοΐ/L)细胞核均匀淡染、 染色质疏松、 胞装比例正常； 32.4 mol/L、 64.8 mol/L、 129.6 mol/L、 259.2 mol/L茶多酚作用 1天后， RPMI8226细胞出现胞膜皱缩、 染色质浓集、 细胞核固缩及凋亡小体。
结果表明： 茶多酚能诱导 RPMI8226 细胞株凋亡， 而且该作用随浓度 增高而明显。 3. AnnexinV-FITC和 PI双标： 定量检测凋亡率
多发性骨髓瘤细胞株 RPMI8226
经流式细胞仪 （AnnexinV-FITC和 PI双标的方法） 定量检测细胞凋亡 率。
FITC, 即荧光素异硫氰酸酯标记的 Annexin-V 与凋亡时外化的膜磷 脂酰丝氨酸 （PS ) 结合， 凋亡细胞显示 Annexin-V阳性；
PI, 即碘化丙啶与核酸结合， 死亡细胞显示 PI阳性；
图 4a〜图 4c为该流式细胞仪显示的荧光散点图， 散点图中左下象限 为 Annexin-V FITC 及 PI 双标记阴性细胞 （正常细胞） ， 右下象限为 Annexin-V FITC单标记阳性细胞（早期凋亡细胞），右上象限为 Annexin-V FITC及 PI双标记阳性细胞 （晚期凋亡细胞） ， 左上象限为 PI单标记阳 性细胞 （死亡细胞） 。
结果表明： 不同浓度茶多酚作用多发性骨髓瘤细胞株 RPMI8226 1 天 后， 对照组 ( Ομπιοΐ/L) 中 95%为正常细胞， 64.8μιηοΙ 129.6μιηο1^ 茶 多酚作用 1天后， 早期凋亡和晚期凋亡细胞明显增多。 人急性单核细胞白血病细胞株 U937
经流式细胞仪 （AnnexinV-FITC和 ΡΙ双标的方法） 定量检测细胞凋亡 率。
图 5a〜图 5c该流式细胞仪显示的荧光散点图， 散点图中左下象限为 Annexin-V FITC及 PI双标记阴性细胞（正常细胞），右下象限为 Annexin-V FITC单标记阳性细胞（早期凋亡细胞） ， 右上象限为 Annexin-V FITC及 PI双标记阳性细胞（晚期凋亡细胞），左上象限为 PI单标记阳性细胞（死 亡细胞） 。
结果表明： 不同浓度茶多酚作用人急性单核细胞白血病细胞株 U937 2.5天后，对照组（Ομπιοΐ/L)中 94.7%为正常细胞， 129.6 mol/L、 259.2μπιο1/Ι 茶多酚作用 2.5天后， 早期凋亡和晚期凋亡细胞明显增多。
4. 细胞周期、 细胞核内 DNA浓度分析： 揭示细胞凋亡的可能机制 经流式细胞仪检测， 不同浓度药液作用人多发性骨髓瘤细胞株
RPMI8226 2天后， Gl、 S、 G2期的比例没有显著的区别 （分别为 32%， 64.4%和 3.5%) 。
图 6a〜图 6d是流式细胞仪显示的峰形图， 图中 FL3和 count表示亚二 倍体细胞的百分比， 反映细胞凋亡的一个指标。通过分析核内 DNA含量显 示， 随着药液浓度的增高， 亚二倍体细胞的比例增多（对照组、 32.4μπι01/ί、 64.8 mol/L、 129.6 mol/L茶多酚作用 2天后， 亚二倍体细胞的比例分别为 0.5%, 8.3%， 15.7%, 94.1%) 。
结果表明： 茶多酚是通过诱导细胞凋亡的途径抑制人多发性骨髓瘤细 胞增殖， 并且与浓度相关。
5. 线粒体膜电位分析： 揭示诱导细胞凋亡的可能机制
经流式细胞仪 （罗丹明 R 23荧光标记的方法） 定量检测细胞内线粒 体膜电位， 探索细胞增殖抑制的可能机制。
图 7 中的 ©〜®给出了不同用药剂量及不同作用时间下， 人多发性骨 髓瘤细胞的增殖和凋亡情况。 与对照组 （Ομηιοΐ/L ) 比较， 64.8 mol/L、 129.6 mol/L、 259.2μπιο1^茶多酚作用人多发性骨髓瘤细胞 3小时起， 线粒 体膜电位开始降低， 并具有时间依赖性。
结果表明： 茶多酚抑制人多发性骨髓瘤细胞增殖的机制与诱导细胞凋 亡相关， 而且与线粒体受损可能相关。
(二）对急性淋巴细胞白血病、 急性单核细胞白血病患者原代细胞的影 响。
1. 瑞氏染色： 观察细胞的形态学改变
经瑞氏染色， 观察细胞的形态学改变。
如图 8所示， 不同浓度茶多酚溶液作用急性淋巴细胞白血病患者原代 细胞 1.5天后， 对照组 （Ομπιοΐ/L) 细胞核均匀淡染、 染色质疏松、 胞浆比 例正常； 129.6 mol/L、 194.4 mol/L、 259.2 mol/L茶多酚作用 1.5天后， 原 代细胞出现胞膜皱缩、染色质浓集、细胞核固縮及嗜酸性小体（凋亡小体）。
结果表明： 茶多酚能诱导急性淋巴细胞白血病患者原代细胞凋亡， 而 且该作用随浓度增高而明显。
2. AnnexinV-FITC和 PI双标： 定量检测凋亡率
经流式细胞学 （AnnexinV-FITC和 PI双标的方法） 定量检测细胞凋亡 率。
如图 9所示， 散点图中左下象限为 Annexin- V FITC及 PI双标记阴性 细胞 （正常细胞） ， 右下象限为 Annexin-V FITC单标记阳性细胞 （早期 凋亡细胞） ， 右上象限为 Annexin-V FITC及 PI双标记阳性细胞（晚期凋 亡细胞） ， 左上象限为 PI单标记阳性细胞 （死亡细胞） 。
结果表明： 不同浓度茶多酚作用急性淋巴细胞白血病原代细胞 1.5 天 后， 对照组 ( Ομπιοΐ/L ) 中 86.8%为正常细胞， 129.6 mol/L、 194.4 mol/L、 259.2 mol/L茶多酚作用 1.5天后， 早期凋亡和晚期凋亡细胞明显增多， 正 常增殖的细胞量明显减少， 分别为 67.1%， 21%和 5.76°/。。
(三） 绿茶胶囊 Tegreen 97®配置的茶多酚溶液对裸鼠肿瘤模型的疗效 制备 4χ 108个细胞 /mL的 RPMI8226细胞株， 向每只小鼠背部皮肤注入 0.5mL， 10天左右长出肿瘤， 接种后第 16天起连续 5天给予小鼠口服胶囊 茶多酚溶液上清， EGCG浓度为 48mg/mL， 口服， 1次 /天。 对照组口服生 理盐水。 第 16天起， 每天测量小鼠肿瘤大小。 结果见下表。
Figure imgf000010_0001
对照组中， 第 16天后肿瘤继续增大， 并于第 18天死亡； 给药组中， 从第 16天给药后肿瘤不但没有增大， 反而不断缩小， 小鼠死亡时间比对照 组至少推迟了 3〜4天。 表明茶多酚有明显的肿瘤治疗效果。
取出不同剂量茶多酚喂食的小鼠肿瘤组织， 做成石蜡切片， HE染色， 显微镜下观察细胞形态变化。
如图 10所示， Omg/kg * d茶多酚喂食小鼠体内的肿瘤组织细胞核均匀 淡染、 染色质疏松、 胞浆比例正常； 12mg/kg « d、 24mg/kg * d茶多酚喂食 后， 肿瘤细胞出现胞膜皱缩、 染色质浓集、 细胞核固缩及凋亡小体。
结果表明： 茶多酚能在小鼠体内引起肿瘤细胞凋亡， 而且该作用随浓 度增高而明显。

Claims

1 . 茶多酚或其在药学上可以接受的盐或含有它们中任何一种的药物组合物 在制备预防或治疗肿瘤药物中的应用。
2. 如权利要求 1所述的应用， 其特征在于， 所述茶多酚包括主要由表没食 子儿茶素没食子酸酯、 表儿茶素没食子酸酯和表没食子儿茶素组成的儿 茶素类物质。
3. 如权利要求 2所述的应用， 其特征在于， 所述茶多酚为茶叶提取物。
4. 如权利要求 3所述的应用， 其特征在于， 在提取时， 所述茶多酚中的主 要活性成分表没食子儿茶素没食子酸酯、 表儿茶素没食子酸酯和表没食 子儿茶素勿需进一步分离纯化， 可直接使用。
5. 如权利要求 4所述的应用， 其特征在于， 所述茶多酚中咖啡因的含量小 于 0.2重量％。
6. 如权利要求 5所述的应用， 其特征在于， 在提取时， 所述茶多酚中去除 了茶叶中所含的鞣酸。
7. 如权利要求 6所述的应用， 其特征在于， 所述茶多酚中， 表没食子儿茶 素没食子酸酯、 表儿茶素没食子酸酯和表没食子儿茶素分别占所述儿茶 素类物质重量的 50-60%， 20-25%和 8-10%。
8. 如权利要求 7所述的应用， 其特征在于， 所述儿茶素类物质的总含量占 所述茶多酚重量的 60-70%。
9. 如权利要求 8所述的应用， 所述药物包括所述茶多酚， 每 lOOOmg所述 药剂中所述儿茶素类物质的含量为 648mg， 其中表没食子儿茶素没食子 酸酯、 表儿茶素没食子酸酯和表没食子儿茶素分别为 380mg、 148mg和 60mgo
10. 如权利要求 1所述的应用， 其特征在于， 所述茶多酚为美国 Pharmanex 公司的绿茶胶囊 Tegreen 97®。
1 1. 如权利要求 10所述的应用， 其特征在于， 所述绿茶胶囊 Tegreen 97® 每颗重 250mg, 包括 162mg所述儿茶素类物质， 其中表没食子儿茶素没 食子酸酯、 表儿茶素没食子酸酯和表没食子儿茶素分别为 95mg、 37mg 和 15mg。
12. 如权利要求 1所述的应用， 其特征在于，所述药物还包含药学上可接受 的辅料或载体。
13. 如权利要求 1所述的应用， 其特征在于， 所述茶多酚可以单独使用。
4. 如权利要求 1所述的应用， 其特征在于， 所述药物的制剂形式为片剂、 胶囊、 口含片、 口腔崩析片、 口腔速溶片、 分散片、 咀嚼剂、 颗粒剂、 干混悬剂、 注射剂、 溶液剂、 缓释剂、 控释剂或速释缓释制剂。
PCT/CN2009/000233 2008-03-06 2009-03-05 茶多酚在制备预防或治疗肿瘤药物中的应用 WO2009109109A1 (zh)

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JP2010549002A JP2011513340A (ja) 2008-03-06 2009-03-05 腫瘍の予防または治療用医薬の製造における茶ポリフェノールの使用
EP09716802.5A EP2263668B1 (en) 2008-03-06 2009-03-05 Use of tea polyphenols in preparing medicaments for prevention or treatment of tumors
US12/921,353 US20110021620A1 (en) 2008-03-06 2009-03-05 Use of tea polyphenols in preparing medicaments for prevention or treatment of tumors

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JP6592010B2 (ja) * 2014-12-22 2019-10-16 シーシーアイホールディングス株式会社 癌細胞増殖抑制組成物および癌細胞増殖の抑制方法
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CN111944771B (zh) * 2019-10-29 2022-06-14 华南农业大学 茶多酚或其组分在制备粪肠球菌噬菌体钝化剂中的应用
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