WO2012061954A1 - Preparative method and use of zingiberensis saponin extracted from dioscorea zingiberensis with antitumor activity - Google Patents

Preparative method and use of zingiberensis saponin extracted from dioscorea zingiberensis with antitumor activity Download PDF

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WO2012061954A1
WO2012061954A1 PCT/CN2010/001801 CN2010001801W WO2012061954A1 WO 2012061954 A1 WO2012061954 A1 WO 2012061954A1 CN 2010001801 W CN2010001801 W CN 2010001801W WO 2012061954 A1 WO2012061954 A1 WO 2012061954A1
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cancer
turmeric
methanol
zingiberensis
water
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PCT/CN2010/001801
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French (fr)
Chinese (zh)
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黄文�
吴晓华
倪晋仁
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北京大学
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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/70Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7048Compounds having saccharide radicals and heterocyclic rings having oxygen as a ring hetero atom, e.g. leucoglucosan, hesperidin, erythromycin, nystatin, digitoxin or digoxin
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18Magnoliophyta (angiosperms)
    • A61K36/88Liliopsida (monocotyledons)
    • A61K36/894Dioscoreaceae (Yam family)
    • A61K36/8945Dioscorea, e.g. yam, Chinese yam or water yam
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00Antineoplastic agents
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00Antineoplastic agents
    • A61P35/04Antineoplastic agents specific for metastasis
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07JSTEROIDS
    • C07J71/00Steroids in which the cyclopenta(a)hydrophenanthrene skeleton is condensed with a heterocyclic ring
    • C07J71/0005Oxygen-containing hetero ring

Definitions

  • the invention uses a medicinal plant, Dioscorea zingiberensis ( ) / ⁇ « ⁇ ⁇ / ⁇ / « ⁇ . . Wright ) That is, turmeric is the raw material, and the hydrophobic saponin component is separated by solvent extraction total soap column chromatography.
  • the anti-tumor activity of turmeric and glucoside has been significantly inhibited against colon cancer, lung cancer, breast cancer, cervical cancer, ovarian cancer and other tumors. Background technique
  • Tumors are the main diseases that endanger human health and life. About 5 million people worldwide die from malignant tumors each year. In recent years, due to the deterioration of the environment, the pressure of modern people's work, the spread of diseases, the incidence of malignant tumors is on the rise, and it is the second leading cause of death after cardiovascular disease. Therefore, the world invests a lot of manpower and resources every year to research and develop new drugs for cancer prevention. In recent years, in addition to studying western medicine, pharmacologists have also conducted many studies on traditional Chinese medicine, and also explored the mechanism of malignant tumor formation. The invention and application of advanced technologies such as combinatorial chemistry and genetic engineering have accelerated the development of malignant tumor drugs.
  • saponins have a wide range of anti-tumor activities, and are an ideal anticancer drug with high efficiency and low toxicity.
  • Steroidal saponins are oligosaccharides of a class of spirosanes compound derivatives. It is mainly distributed in plants such as Dioscorea, Liliaceae, Scrophulariaceae, Polygonaceae, Agaveaceae, and other plants such as Amaryllidaceae, Leguminosae and Rhamnaceae also contain saponins.
  • Commonly used traditional Chinese medicine Zhimu, Aspartame, Maimendong, Qiyeyihuahua, etc. all have a large number of saponins.
  • saponins obtained by hydrolysis and de-sugarization of saponins are well-known as the basis of natural artificial semi-synthetic steroids and contraceptives, and the medicinal value of saponins itself has also developed rapidly in the past decade.
  • certain saponins have activities such as lowering blood fat, lowering blood sugar, anti-cancer, anti-virus, anti-bacterial and anti-infective, treating coronary heart disease and regulating physiological functions.
  • ⁇ illin has a significant anti-fungal effect. ⁇ buius terresris)
  • Total saponins are not only used to treat coronary heart disease, but also prevent pulmonary heart disease, lower blood pressure, enhance sexual function and anti-aging.
  • Yellow yam Dioscorea panthaiapraia et Burkill is a kind of saponin total saponin extracted from raw materials. It is used to treat coronary heart disease, angina pectoris, shortness of breath, palpitations, chest tightness, chest pain and other symptoms. The total effective rate is over 90%. It can be seen that the development of modern separation technology and the development of research methods have made the saponins with large polarity and complex sugar chains also have a breakthrough development, which will promote the development and utilization of the medicinal value of steroidal saponins.
  • Dioscin is an important component of saponins, and dioscin has good biological activity.
  • the former Soviet Union researchers found that the saponin extract in the Caucasian yam has a cholesterol-lowering effect, and clinical trials have also proved.
  • Ravikumer et al. found that diosgenin in Yunnan Baiyao has anticancer activity.
  • diosgenin and its glycosides also have anti-arthritic effects.
  • Diosgenin isolated from D. nipponicc from D. sylvestris has significant cardiovascular, antitumor and antifungal activity.
  • dioscin has good cytotoxicity against tumor cells such as lung cancer, breast cancer, gastric cancer, leukemia, colon cancer, cervical cancer, and melanin.
  • tumor cells such as lung cancer, breast cancer, gastric cancer, leukemia, colon cancer, cervical cancer, and melanin.
  • Dioscorea in China are used as raw materials to extract their total saponins as clinical hypolipidemic drugs, which have been produced in several factories.
  • D. zingibernsis is a successful new drug.
  • Xinning clinical treatment of coronary heart disease, angina pectoris has a certain effect.
  • Another object of the present invention is to provide a use of glucosinolate in the preparation of a medicament for treating or preventing cancer.
  • the C3 position links the glycosyl moiety, the glycosyl moiety contains three molecules of glucose and one molecule of rhamnose, and the C2 and C4 sites of the inner glucose are linked to one molecule of rhamnose and two molecules of glucose, respectively.
  • the invention develops a method for extracting and separating hydrophobic steroidal saponins from the medicinal plant Dioscorea Zingiberensis CH Wright.
  • the main component structure is determined, and the anti-tumor activity of the glucosides of turmeric is studied in vivo and in vitro.
  • the development of turmeric and scutellaria sinensis is used to treat malignant tumors such as colon cancer, lung cancer, breast cancer, cervical cancer and ovarian cancer.
  • the structure was identified by mass spectrometry (HR-ESI-MS), infrared spectroscopy (IR), one-dimensional nuclear magnetic resonance (1D-NMR) and two-dimensional nuclear magnetic resonance (2D-NMR) analysis.
  • the method comprises the following steps: using a tuber of turmeric as a raw material, extracting one or more solvents selected from water and CM-fatty alcohol, concentrating under reduced pressure, and lyophilizing to obtain a total saponin component of turmeric, using normal phase silica gel column chromatography, Separation and purification by gel chromatography and recrystallization, and the glucosides of turmeric.
  • the method for preparing the glucosinolate of the turmeric comprises the following steps: (1) drying and pulverizing the medicinal plant turmeric tubers, adding 70-100% according to the weight of the medicinal plant powder 2-3 times Product ratio) ethanol leaching for 10-24 hours, solid-liquid separation;
  • solvent extraction is performed by infiltration extraction at a temperature of 20 to 50 ° C or reflux extraction.
  • the normal phase silica gel column chromatography is atmospheric pressure or pressurized silica gel column chromatography, which is selected from the group consisting of benzene: acetone (for example, 3:1), trichloromethane: acetone (for example, 3:7), and dichlorocarb. ⁇ : methanol (eg 2:8), ethyl acetate: methanol (eg 6:4), benzene: acetone: water (eg 1:7:3), chloroform: methanolic ice (eg 6:2:2) Equivalent or gradient elution of one or more eluents in ethyl acetate:methanol:water (eg 4:3:3).
  • benzene acetone (for example, 3:1), trichloromethane: acetone (for example, 3:7), and dichlorocarb. ⁇ : methanol (eg 2:8), ethyl acetate: methanol (eg 6
  • the eluent of the gel chromatography is selected from isocratic or gradient elution of one or more eluents of chloroform/methanol, methanol/water, ethanol/water.
  • the recrystallization step is to recrystallize the product by dissolving it in methanol.
  • the present invention also provides the use of the genus turmeric, having the structure of the formula, for the preparation of a medicament for treating or preventing cancer
  • the cancer is selected from the group consisting of colon cancer, lung cancer, breast cancer, cervical cancer, and ovarian cancer.
  • the method for extracting glucosinolates comprises the following steps:
  • the solid phase obtained by the solid phase obtained in (5) is freeze-dried to obtain a turmeric total saponin component having high anticancer activity.
  • the method of the invention has the characteristics of simple operation, good reproducibility, and easy linear amplification.
  • the glucosinolate of the present invention can be used as a medicament, for example, in the form of a pharmaceutical composition.
  • the pharmaceutical compositions may be administered orally, for example, in the form of tablets, coated tablets, dragees, hard and soft gelatin capsules, solutions, emulsions or suspensions. However, administration is also carried out parenterally, for example, in the form of an injection.
  • the pharmaceutical composition of the present invention may contain a preservative, a solubilizer, a stabilizer, a wetting agent, an emulsifier, a sweetener, a coloring agent, a flavoring agent, a salt for changing the osmotic pressure, a buffer, a masking agent or an antioxidant. They may also contain other therapeutically valuable substances.
  • the dosage will depend on various factors, such as the mode of administration, species, age and/or individual health. In general, the dose may range from 20 mg/kg to 40 mg/kg.
  • Figure 1 shows the effect of glucosinolates on the growth and proliferation of tumor cells.
  • the concentration of glucosides in turmeric is 10 mg/L
  • the control group is the negative control group.
  • Figure 2 shows a phase contrast microscope for the observation of colon cancer cell lines treated with turmeric. Morphological changes of colon cancer cell lines were observed by phase contrast microscopy. Among them, A was negative control group; B was 10 mg/L shield foliin treatment group, the treatment time was 48 h, and the eyepiece magnification was 200 times. Morphological observations revealed that scutellarin can induce apoptosis in colon cancer cell lines.
  • Figure 3 shows the effect of turmeric on the tumor volume of colon cancer in C26 mice. detailed description:
  • 5CTC is dried and smashed.
  • Combine the filtrate with the soaking solution distill it under reduced pressure to a substantially ethanol-free taste, recover the ethanol, and then dissolve the soluble saponin by ultrasonic wave to dissolve the soluble saponin by 6-10 times the weight of the extract and add about 2000 mL of water.
  • the suspension is centrifuged (4000-6000r/min, 10 -20 min), the aqueous solution and the precipitate were separated, and the precipitate was freeze-dried at -65 ° C for 36 h to obtain 40 g of hydrophobic steroidal total saponin.
  • the hydrophobic saponin total saponin 16g was firstly separated by normal phase silica gel column chromatography.
  • the eluent was mixed solvent chloroform-methanol (V/V), gradient elution, gradient 100: 10 (600mL)-90 : 10 (1000mL)-80:20 (800mL)-70:30 (500mL), TLC tracking detection, 10% sulfuric acid ethanol color development, combined with the same components of Rf value, collecting 5 different components, and then passing through gel chromatography Sephade X LH20, eluent chloroform-methanol (8:1) (volume ratio), recrystallized repeatedly in methanol to obtain monomeric steroidal saponin, purity greater than 98% by HPLC, high resolution mass spectrometry HR -ESI-MS, IR spectroscopy, nuclear magnetic resonance spectroscopy, including 1H-NMR, 13 C-NMR, 135 DEPT, 1H-1HCOSY, HMBC, HMQC, NOECOSY
  • Colon cancer C26, lung cancer A549, breast cancer MCF-7, cervical cancer HeLa and ovarian cancer tumor SKOV3 cells were cultured in 10% inactivated fetal bovine serum, 100 U/mL penicillin, 100 g/mL streptomycin in RPMI 1640 or DMEM. Base, 37 ° C 10% CO2 incubator and culture under saturated humidity conditions, pass 3-4 days.
  • MTT assay was used to determine the effect of different concentrations of glucosinolates on the growth of tumor cell lines. The results are shown in Figure 1.
  • phase cancer microscope was used to observe the colon cancer cell line treated with turmeric, and the results are shown in Fig. 2. Morphological observation revealed that scutellarin can induce apoptosis in colon cancer cell lines. Second, in vivo anti-tumor activity research Experimental material
  • mice 60 Kunming mice, 7-8 weeks, half male and half female, weighing 18-20g
  • Tween Tween (TW-80).
  • glucosinolate has significant antitumor activity against malignant tumors such as colon cancer, lung cancer, breast cancer, cervical cancer and ovarian cancer, and it has broad application prospects as a new anti-tumor drug.

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Abstract

The preparative method of zingiberensis saponin with structure as followed (I) extracted from Dioscorea zingiberensis comprises steps as followed: extracting the tuber of Dioscorea zingiberensis C. H. Wright. as raw material using one or more solvents selected from water and C1-4 fatty alcohol, reduced pressure condensing, freeze- drying to obtain total steroidal saponins from Dioscorea zingiberensis, then separating and purifying through normal phase silica gel column chromatograph, gel chromatograph and recrystallization, receiving zingiberensis saponin extracted from Dioscorea zingiberensis. The use of zingiberensis saponin with structure as followed extracted from Dioscorea zingiberensis in the preparation of medicaments for treating or preventing cancers. The cancers are selected from colon cancer, lung cancer, mammary cancer, cervical cancer and ovary cancer.

Description

具有抗癌活性的黄姜盾叶新苷的制备方法和用途 技术领域  Preparation method and use of turmeric scutellaria glucoside with anticancer activity
本发明以药用植物一盾叶薯蓣 ( )/ί« ^α ζ/^/ «^ϊ。.Η. Wright ) 即黄姜为原 料, 采用溶剂提取总皂 柱色谱等方法分离疏水性皂苷组分。黄姜盾叶新苷进行了体内 外抗肿瘤活性研究, 对结肠癌、 肺癌、 乳腺癌、 宫颈癌、 卵巢癌等肿瘤具有明显地抑 制活性。 背景技术  The invention uses a medicinal plant, Dioscorea zingiberensis ( ) / ί« ^α ζ/^/ «^ϊ. . Wright ) That is, turmeric is the raw material, and the hydrophobic saponin component is separated by solvent extraction total soap column chromatography. The anti-tumor activity of turmeric and glucoside has been significantly inhibited against colon cancer, lung cancer, breast cancer, cervical cancer, ovarian cancer and other tumors. Background technique
肿瘤是危害人类健康和生命的主要疾病。全世界每年约有 500万人死于恶性肿瘤。 近年来, 由于环境的恶化, 以及现代人的工作压力, 疾病的传播, 恶性肿瘤的发病率 呈上升趋势, 并成为继心血管疾病之后的第二大致死病因。 因此, 全球每年投入大量 的人力物力用于研究开发防治癌症的新药物。近年来, 药物学专家除了研究西药以外, 也对中药进行了很多研究, 对恶性肿瘤形成的机理也进行探讨, 组合化学、 基因工程 等先进技术的发明和应用加速了对恶性肿瘤药物开发进程。 特别是目前以天然活性成 分为先导化合物寻找新药有广阔的发展前景。 迄今为止已评估了 4万余种高等植物的 抗肿瘤活性。 据国外资料介绍, 全世界己经开发了几百种抗癌新药, 其中有很多属于 天然植物提取物, 近年来, 研究人员从传统中药进行的广泛的筛选, 发现了大量的抗 肿瘤植物, 如红豆杉、 银杏、 喜树、 灵芝等。 但上述植物中, 人们对红豆杉、 喜树碱 的研究较多, 很多植物由于生长缓慢, 资源贫乏, 很难满足市场需求。  Tumors are the main diseases that endanger human health and life. About 5 million people worldwide die from malignant tumors each year. In recent years, due to the deterioration of the environment, the pressure of modern people's work, the spread of diseases, the incidence of malignant tumors is on the rise, and it is the second leading cause of death after cardiovascular disease. Therefore, the world invests a lot of manpower and resources every year to research and develop new drugs for cancer prevention. In recent years, in addition to studying western medicine, pharmacologists have also conducted many studies on traditional Chinese medicine, and also explored the mechanism of malignant tumor formation. The invention and application of advanced technologies such as combinatorial chemistry and genetic engineering have accelerated the development of malignant tumor drugs. In particular, the current search for new drugs with natural active components has broad prospects for development. The antitumor activity of more than 40,000 higher plants has been evaluated to date. According to foreign data, hundreds of new anticancer drugs have been developed in the world, many of which are natural plant extracts. In recent years, researchers have extensively screened traditional Chinese medicines and found a large number of anti-tumor plants, such as Yew, Ginkgo, Hibiscus, Ganoderma Lucidum, etc. However, among the above plants, there are many studies on yew and camptothecin. Many plants are difficult to meet market demand due to slow growth and lack of resources.
近年来人们发现 体皂苷具有广泛的抗肿瘤活性, 是一种高效, 低毒的较理想的 抗癌药物。 甾体皂苷(Steroidal saponins )是一类螺甾垸(spirosanes )化合物衍生物的 寡糖苷。 主要分布在薯蓣科、 百合科、 玄参科、 菝葜科、 龙舌兰科等植物中, 其他科 如石蒜科、 豆科以及鼠李科的一些植物中也含有 体皂苷。 常用中药知母、 天门冬、 麦门冬、 七叶一枝花等都均有大量 体皂苷。 体皂苷经水解脱糖而得的苷元, 除因 作为天然人工半合成 ¾体激素类药物和避孕用药的基础而著名外, 其皂苷本身的药用 价值近十几年来也得到较快发展。 研究发现, 某些皂苷具有降血脂、 降血糖、 抗癌、 抗病毒、 抗菌抗感染、 治疗冠心病及调节生理机能等活性。 菝葜 illin 具有显著 的抗霉菌作用。 蒺藜 buius terresris ) 总皂苷不但用于治疗冠心病, 还可以防止肺 心病、 降血压、 增强性功能与抗衰老等作用。 以黄山药 Dioscorea panthaiapraia et Burkill ) 为原料提取而得的甾体总皂苷制成的地奥心血康, 用于治疗冠心病、 心绞痛、 气短、 心悸、 胸闷、 胸痛等症状, 总有效率达 90%以上。 可见; 陣着现代分离技术的 进步, 研究手段的发展, 使得极性较大, 糖链复杂的皂苷也有突破性发展, 这将促进 甾体皂苷自身药用价值的发展和利用。 In recent years, it has been found that saponins have a wide range of anti-tumor activities, and are an ideal anticancer drug with high efficiency and low toxicity. Steroidal saponins are oligosaccharides of a class of spirosanes compound derivatives. It is mainly distributed in plants such as Dioscorea, Liliaceae, Scrophulariaceae, Polygonaceae, Agaveaceae, and other plants such as Amaryllidaceae, Leguminosae and Rhamnaceae also contain saponins. Commonly used traditional Chinese medicine Zhimu, Aspartame, Maimendong, Qiyeyihuahua, etc. all have a large number of saponins. The aglycons obtained by hydrolysis and de-sugarization of saponins are well-known as the basis of natural artificial semi-synthetic steroids and contraceptives, and the medicinal value of saponins itself has also developed rapidly in the past decade. Studies have found that certain saponins have activities such as lowering blood fat, lowering blood sugar, anti-cancer, anti-virus, anti-bacterial and anti-infective, treating coronary heart disease and regulating physiological functions.菝葜illin has a significant anti-fungal effect.蒺藜buius terresris) Total saponins are not only used to treat coronary heart disease, but also prevent pulmonary heart disease, lower blood pressure, enhance sexual function and anti-aging. Yellow yam Dioscorea panthaiapraia et Burkill) is a kind of saponin total saponin extracted from raw materials. It is used to treat coronary heart disease, angina pectoris, shortness of breath, palpitations, chest tightness, chest pain and other symptoms. The total effective rate is over 90%. It can be seen that the development of modern separation technology and the development of research methods have made the saponins with large polarity and complex sugar chains also have a breakthrough development, which will promote the development and utilization of the medicinal value of steroidal saponins.
薯蓣皂苷 (dioscin)是 体皂苷中的重要成分, 薯蓣皂苷具有较好的生物活性。前苏 联的科研人员发现高加索薯蓣中的皂苷提取物有降胆固醇的作用,临床实验也有证明。 80 年代 Ravikumer 等发现云南白药中的薯蓣皂苷有抗癌活性。 另外薯蓣皂苷元 ( diosgenin) 及其苷还有抗关节炎作用。 从重楼、 穿龙薯蓣 D. nipponicc 中分离得 到的薯蓣皂苷具有明显的心血管、 抗肿瘤和抗真菌的活性。 最近几年的研究发现薯蓣 皂苷 (dioscin) 对肺癌、 乳癌、 胃癌、 白血病、 结肠癌、 宫颈癌、 黑色素等肿瘤细胞都 有很好的细胞毒性。 目前, 国内以不同种的薯蓣属植物为原料, 提取其总 体皂苷作 为临床上的降血脂药已有数家工厂生产, 其中用盾叶薯蓣 (D. zingibernsis) 为原料研 制成功的新药 "盾叶冠.心宁", 临床治疗冠心病、 心绞痛有一定疗效。  Dioscin is an important component of saponins, and dioscin has good biological activity. The former Soviet Union researchers found that the saponin extract in the Caucasian yam has a cholesterol-lowering effect, and clinical trials have also proved. In the 1980s, Ravikumer et al. found that diosgenin in Yunnan Baiyao has anticancer activity. In addition, diosgenin and its glycosides also have anti-arthritic effects. Diosgenin isolated from D. nipponicc from D. sylvestris has significant cardiovascular, antitumor and antifungal activity. In recent years, studies have found that dioscin has good cytotoxicity against tumor cells such as lung cancer, breast cancer, gastric cancer, leukemia, colon cancer, cervical cancer, and melanin. At present, different kinds of Dioscorea in China are used as raw materials to extract their total saponins as clinical hypolipidemic drugs, which have been produced in several factories. Among them, D. zingibernsis is a successful new drug. . Xinning", clinical treatment of coronary heart disease, angina pectoris has a certain effect.
经文献检索发现, 在临床上, 体皂苷主要运用于降血脂, 治疗心血管疾病等。 工业上, 盾叶薯蓣作为原料主要用于生产皂素, 作为合成 体激素类药物和避孕药的 原料。 同时, 在临床上主要应用于治疗心血管疾病, 但其 体皂苷的成分并不十分清 楚, 且目前的研究并未涉及抗肿瘤活性研究。 目前, 在甾体皂苷抗肿瘤活性研究中, 薯蓣皂苷 (dioscin), 甾体皂苷元 (diosgenin)等报道较多, 盾叶新苷 (zingiberensis saponin) 并没有文献报道。 发明内容  According to literature search, in the clinical, saponins are mainly used to lower blood fat and treat cardiovascular diseases. Industrially, Dioscorea zingiberensis is mainly used as a raw material for the production of saponin, as a raw material for synthetic hormone drugs and contraceptives. At the same time, it is mainly used in the treatment of cardiovascular diseases, but the composition of its saponins is not very clear, and the current research does not involve anti-tumor activity research. At present, in the study of anti-tumor activity of steroidal saponins, dioscin and diosgenin have been reported, and zingiberensis saponin has not been reported in the literature. Summary of the invention
本发明的目的在于提供一种制备黄姜盾叶新苷的方法。  It is an object of the present invention to provide a method for preparing turmeric.
本发明的另一个目的在于提供黄姜盾叶新苷在制备用于治疗或预防癌症的药物中 的用途。  Another object of the present invention is to provide a use of glucosinolate in the preparation of a medicament for treating or preventing cancer.
黄姜盾叶新苷 (zingiberensis saponin ) , 其结构特征为: The zingiberensis saponin is characterized by:
Figure imgf000005_0001
Figure imgf000005_0001
Zingiberensis saponin (C51H82O22, 1046)  Zingiberensis saponin (C51H82O22, 1046)
3-O- -D-吡喃糖 -[ ( 1—2 ) - -L-吡喃鼠李糖 ]-[ ( 1—4 ) - -D -吡喃葡萄糖- ( 1— 3 ) - ^ -D-吡喃葡萄糖] -薯蓣皂苷元 ( 3-O-9-Z)-glucopyranosyl(l→3)-g-/)-glucopyranosyl ( 14)- [«-Z-rhamnopyranosyl( 1→2)] -^-D-glucopyranoside-diosgenin ), 属于螺甾院阜苷,3-O--D-pyranose-[(1-2)--L-pyran rhamnose]-[(1-4)--D-glucopyranose-(1-3)-^- D-glucopyranose] - Diosgenin ( 3-O-9-Z)-glucopyranosyl (l→3)-g-/)-glucopyranosyl ( 1 4)- [«-Z-rhamnopyranosyl ( 1→2) ] -^-D-glucopyranoside-diosgenin ), belonging to the scorpion scorpion glycosides,
C3位链接糖基部分,糖基部分含有三分子葡萄糖和一分子鼠李糖,内侧葡萄糖的 C2 和 C4位分别连有一分子鼠李糖和两分子葡萄糖。 The C3 position links the glycosyl moiety, the glycosyl moiety contains three molecules of glucose and one molecule of rhamnose, and the C2 and C4 sites of the inner glucose are linked to one molecule of rhamnose and two molecules of glucose, respectively.
本发明开发了药用植物盾叶薯蓣 Dioscorea Zingiberensis C.H. Wright. ) 疏水性 甾体皂苷的提取分离方法, 确定了主要组分结构, 同时对于黄姜盾叶新苷进行了体内 外抗肿瘤活性研究, 最后将黄姜盾叶新苷开发运用于治疗结肠癌、 肺癌、 乳腺癌、 宫 颈癌、 卵巢癌等恶性肿瘤。 通过质谱 (HR-ESI-MS )、 红外光谱 (IR)、 一维核磁共振 (1D-NMR)和二维核磁共振 (2D-NMR)分析鉴定其结构。  The invention develops a method for extracting and separating hydrophobic steroidal saponins from the medicinal plant Dioscorea Zingiberensis CH Wright. The main component structure is determined, and the anti-tumor activity of the glucosides of turmeric is studied in vivo and in vitro. The development of turmeric and scutellaria sinensis is used to treat malignant tumors such as colon cancer, lung cancer, breast cancer, cervical cancer and ovarian cancer. The structure was identified by mass spectrometry (HR-ESI-MS), infrared spectroscopy (IR), one-dimensional nuclear magnetic resonance (1D-NMR) and two-dimensional nuclear magnetic resonance (2D-NMR) analysis.
本 一种制备具有下式结构的黄姜盾叶新苷的方法,  a method for preparing a glucoside of turmeric, which has the structure of the following formula,
Figure imgf000005_0002
Figure imgf000005_0002
包括以下步骤: 以黄姜的块茎为原料, 采用一种或多种选自水和 CM-脂肪醇的溶 剂提取, 减压浓缩、 冷冻干燥得到黄姜 体总皂苷组分, 采用正相硅胶柱色谱、 凝胶 色谱、 重结晶分离纯化, 得到黄姜盾叶新苷。  The method comprises the following steps: using a tuber of turmeric as a raw material, extracting one or more solvents selected from water and CM-fatty alcohol, concentrating under reduced pressure, and lyophilizing to obtain a total saponin component of turmeric, using normal phase silica gel column chromatography, Separation and purification by gel chromatography and recrystallization, and the glucosides of turmeric.
本发明的制备黄姜盾叶新苷的方法, 具体包括以下步骤- (1) 将药用植物黄姜块茎烘干, 粉碎, 按药用植物粉末重量的 2-3倍加入 70-100% (体 积比)乙醇浸提 10-24小时, 固液分离; The method for preparing the glucosinolate of the turmeric, comprises the following steps: (1) drying and pulverizing the medicinal plant turmeric tubers, adding 70-100% according to the weight of the medicinal plant powder 2-3 times Product ratio) ethanol leaching for 10-24 hours, solid-liquid separation;
(2) 过滤分离的固相按药用植物重量的 3-5倍加入 70-100% (体积比)乙醇回流 2-5小时, 趁热过滤, 重复回流 3-5次;  (2) The solid phase separated by filtration is added 3-5 times the weight of the medicinal plant to 70-100% (by volume) ethanol reflux for 2-5 hours, filtered while hot, and refluxed 3-5 times;
(3)将浸泡的液相与热回流提取的液相合并后, 减压蒸馏回收乙醇, 得褐色浸膏; (3) combining the soaked liquid phase with the liquid phase extracted by hot reflux, and recovering the ethanol by distillation under reduced pressure to obtain a brown extract;
(4)将褐色浸膏按重量的 6-10倍加水超声波溶解; (4) ultrasonically dissolve the brown extract by ultrasonic wave 6-10 times by weight;
(5)将 (4)得到的混合物离心, 分离水相与固相;  (5) centrifuging the mixture obtained in (4) to separate the aqueous phase from the solid phase;
(6)将 (5)得到的固相得到的固相冷冻干燥得到黄姜甾体总皂苷组分;  (6) lyophilizing the solid phase obtained by the solid phase obtained in (5) to obtain a total saponin component of turmeric carcass;
(7) 采用正相硅胶柱色谱、 凝胶色谱、 重结晶分离纯化, 得到黄姜盾叶新苷。  (7) Separation and purification by normal phase silica gel column chromatography, gel chromatography and recrystallization, and the glucosides of turmeric.
在本发明中, 溶剂提取在 20至 50°C的温度下浸润提取, 或回流提取。  In the present invention, solvent extraction is performed by infiltration extraction at a temperature of 20 to 50 ° C or reflux extraction.
根据本发明,所述正相硅胶柱色谱为常压或加压硅胶柱色谱,采用选自苯:丙酮(例 如 3: 1 )、 三氯甲垸:丙酮(例如 3:7)、 二氯甲垸:甲醇(例如 2:8)、 乙酸乙酯:甲醇(例 如 6:4)、苯:丙酮:水(例如 1 :7:3 )、三氯甲烷:甲醇冰 (例如 6:2:2)、 乙酸乙酯:甲醇:水 (例 如 4:3:3)中的一种或多种洗脱剂等度洗脱或梯度洗脱。  According to the present invention, the normal phase silica gel column chromatography is atmospheric pressure or pressurized silica gel column chromatography, which is selected from the group consisting of benzene: acetone (for example, 3:1), trichloromethane: acetone (for example, 3:7), and dichlorocarb.垸: methanol (eg 2:8), ethyl acetate: methanol (eg 6:4), benzene: acetone: water (eg 1:7:3), chloroform: methanolic ice (eg 6:2:2) Equivalent or gradient elution of one or more eluents in ethyl acetate:methanol:water (eg 4:3:3).
在本发明中, 凝胶色谱的洗脱剂选自氯仿 /甲醇、 甲醇 /水、 乙醇 /水的一种或多种 洗脱剂等度洗脱或梯度洗脱。  In the present invention, the eluent of the gel chromatography is selected from isocratic or gradient elution of one or more eluents of chloroform/methanol, methanol/water, ethanol/water.
根据本发明, 重结晶步骤是将产物溶解在甲醇中重结晶。  According to the invention, the recrystallization step is to recrystallize the product by dissolving it in methanol.
本发明还提供了具有下式结构的黄姜盾叶新苷在制备用于治疗或预防癌症的药物 中的用途  The present invention also provides the use of the genus turmeric, having the structure of the formula, for the preparation of a medicament for treating or preventing cancer
Figure imgf000006_0001
优选地, 所述癌症选自结肠癌、 肺癌、 乳腺癌、 宫颈癌和卵巢癌。
Figure imgf000006_0001
Preferably, the cancer is selected from the group consisting of colon cancer, lung cancer, breast cancer, cervical cancer, and ovarian cancer.
在本发明的一个优选实施方案中, 黄姜盾叶新苷的提取方法包括以下步骤: In a preferred embodiment of the invention, the method for extracting glucosinolates comprises the following steps:
(1) 将药用植物黄姜块茎 50 'C烘干, 粉碎, 按药用植物粉末重量的 2-3 倍加入 70-100% (体积比)乙醇浸提 10-24小时, 固液分离; (1) Drying and pulverizing the medicinal plant turmeric tubers 50 'C, adding 2-3 times of the weight of the medicinal plant powder to 70-100% (by volume) ethanol for 10-24 hours, solid-liquid separation;
(2) 过滤分离的固相按药用植物重量的 3-5倍加入 70-100% (体积比)乙醇回流 2-5小时, 3-5次, 趁热过滤; (2) The solid phase separated by filtration is added to 70-100% (by volume) ethanol for 3-5 times by weight of medicinal plants for 2-5 hours. 3-5 times, hot filtered;
(3) 将浸泡的液相与热回流提取的液相合并后, 减压蒸馏回收乙醇至基本无乙醇味, 得褐色浸膏;  (3) After combining the soaked liquid phase with the liquid phase extracted by hot reflux, the ethanol is distilled off under reduced pressure to obtain substantially no ethanol flavor, and a brown extract is obtained;
(4)将褐色浸膏按重量的 6-10倍加水超声波溶解;  (4) ultrasonically dissolve the brown extract by ultrasonic wave 6-10 times by weight;
(5) 将 (4)得到的混合物 4000-6000r/min离心 15-20分钟, 分离水相与固相;  (5) Centrifuge the mixture obtained in (4) at 4000-6000r/min for 15-20 minutes to separate the aqueous phase from the solid phase;
(6) 将 (5)得到的固相得到的固相冷冻干燥得到一种具有高效抗癌活性的黄姜 体总皂 苷组分。  (6) The solid phase obtained by the solid phase obtained in (5) is freeze-dried to obtain a turmeric total saponin component having high anticancer activity.
(7) 将 (6)得到总皂苷采用正相硅胶柱色谱分离, 采用洗脱剂梯度洗脱, 合并 Rf值相同 组分。  (7) The total saponin obtained in (6) was separated by normal phase silica gel column chromatography, eluted with an eluent gradient, and the same Rf value was combined.
(8)将 (7)得到 Rf值相同组分采用凝胶色谱分离, 采用洗脱剂洗脱, 再反复重结晶后得 盾叶新苷。  (8) The same components with the Rf value obtained by (7) were separated by gel chromatography, eluted with an eluent, and repeatedly recrystallized to obtain a new glucoside.
本发明的方法与现有技术方法比较, 有操作较简单, 重现性好, 很容易线性放大 等特点。  Compared with the prior art method, the method of the invention has the characteristics of simple operation, good reproducibility, and easy linear amplification.
本发明的黄姜盾叶新苷可以用作药物, 例如以药物组合物的形式。 药物组合物可 以口服施用, 例如以片剂、 包衣片剂、 糖锭剂、 硬和软明胶胶囊、 溶液、 乳剂或混悬 剂的形式。 然而, 给药还例如以注射液的形式肠胃外进行。  The glucosinolate of the present invention can be used as a medicament, for example, in the form of a pharmaceutical composition. The pharmaceutical compositions may be administered orally, for example, in the form of tablets, coated tablets, dragees, hard and soft gelatin capsules, solutions, emulsions or suspensions. However, administration is also carried out parenterally, for example, in the form of an injection.
本发明的药物组合物可以含有防腐剂, 增溶剂, 稳定剂, 润湿剂, 乳化剂, 甜味 剂, 着色剂, 调味剂, 改变渗透压的盐, 缓冲液, 掩蔽剂或抗氧化剂。 它们也可以还 含有其它的在治疗上有价值的物质。  The pharmaceutical composition of the present invention may contain a preservative, a solubilizer, a stabilizer, a wetting agent, an emulsifier, a sweetener, a coloring agent, a flavoring agent, a salt for changing the osmotic pressure, a buffer, a masking agent or an antioxidant. They may also contain other therapeutically valuable substances.
剂量取决于各种因素而定, 例如, 给药方式, 物种, 年龄和 /或个体健康状况。 一 般情况下, 剂量可以为 20mg/kg-40mg/kg。  The dosage will depend on various factors, such as the mode of administration, species, age and/or individual health. In general, the dose may range from 20 mg/kg to 40 mg/kg.
附图说明 DRAWINGS
图 1显示了黄姜盾叶新苷对肿瘤细胞生长增殖影响, 图中黄姜盾叶新苷药物浓度 均为 10mg/L, 其中控制组 (control ), 即阴性对照组。  Figure 1 shows the effect of glucosinolates on the growth and proliferation of tumor cells. In the figure, the concentration of glucosides in turmeric is 10 mg/L, and the control group is the negative control group.
图 2显示了相差显微镜观察黄姜盾叶新苷处理的结肠癌细胞株。 相差显微镜观察 盾叶新苷处理结肠癌细胞系形态学变化, 其中: A为阴性对照组; B为 10mg/L盾叶新 苷处理组, 处理时间为 48h,目镜放大倍数为 200倍。 形态学观察发现, 盾叶新苷能诱 导结肠癌细胞系凋亡。  Figure 2 shows a phase contrast microscope for the observation of colon cancer cell lines treated with turmeric. Morphological changes of colon cancer cell lines were observed by phase contrast microscopy. Among them, A was negative control group; B was 10 mg/L shield foliin treatment group, the treatment time was 48 h, and the eyepiece magnification was 200 times. Morphological observations revealed that scutellarin can induce apoptosis in colon cancer cell lines.
图 3显示了黄姜盾叶新苷对 C26小鼠结肠癌肿瘤体积影响。 具体实施方式: Figure 3 shows the effect of turmeric on the tumor volume of colon cancer in C26 mice. detailed description:
实施例 1 Example 1
新鲜盾叶薯蕷块茎清洗干净后, 切成块状, 5CTC烘干, 粉碎。 称取约 600g黄姜粉 末于 5000mL烧杯中, 加入约 2000mL95%乙醇浸泡过夜, 抽滤, 分离固相与液相, 固 相转入 5L回流瓶中, 加入 1200-2000mL乙醇, 78 °C回流 2h-5h, 趁热过滤, 重复回流 提取 3次。 将滤液与浸泡液合并, 减压蒸馏至基本无乙醇味, 回收乙醇, 再按浸膏重 量的 6-10倍加水约 2000mL水超声波溶解可溶性皂苷, 悬浊液离心 (4000-6000r/min, 10-20min) , 分离水溶液与沉淀, 再将沉淀 -65 °C低温冷冻干燥 36h, 即得疏水性甾体总 皂苷 40g。  After the fresh stalk potato tubers are cleaned, cut into pieces, 5CTC is dried and smashed. Weigh about 600g of turmeric powder in a 5000mL beaker, add about 2000mL of 95% ethanol soaked overnight, filter by suction, separate the solid phase and the liquid phase, transfer the solid phase to a 5L reflux bottle, add 1200-2000mL ethanol, reflux at 78 °C for 2h- 5h, hot filtered, repeated reflux extraction 3 times. Combine the filtrate with the soaking solution, distill it under reduced pressure to a substantially ethanol-free taste, recover the ethanol, and then dissolve the soluble saponin by ultrasonic wave to dissolve the soluble saponin by 6-10 times the weight of the extract and add about 2000 mL of water. The suspension is centrifuged (4000-6000r/min, 10 -20 min), the aqueous solution and the precipitate were separated, and the precipitate was freeze-dried at -65 ° C for 36 h to obtain 40 g of hydrophobic steroidal total saponin.
盾叶薯蓣疏水性的甾体总皂苷 16g, 首先采用正相硅胶柱色谱分离, 洗脱剂为混 合溶剂氯仿 -甲醇 (V/V ), 梯度洗脱, 梯度为 100: 10 (600mL)-90: 10(1000mL)-80:20 (800mL)-70:30(500mL), TLC跟踪检测, 10%硫酸乙醇显色, 合并 Rf值相同组分, 收 集 5个不同组分, 再过凝胶色谱 SephadeX LH20, 洗脱剂氯仿 -甲醇 (8: 1 ) (体积比), 再反复在甲醇中重结晶, 得单体甾体皂苷, 经 HPLC检测纯度均大于 98%, 经高分辨 率质谱 HR-ESI-MS , 红外光谱 IR, 核磁共振谱, 包括 1H-NMR、 13C-NMR、 135DEPT、 1H-1HCOSY、 HMBC、 HMQC、 NOECOSY等光谱方法, 其中化合物 5鉴定其为盾叶 新苷 (30mg)。 部分光谱分析的数据如下: The hydrophobic saponin total saponin 16g was firstly separated by normal phase silica gel column chromatography. The eluent was mixed solvent chloroform-methanol (V/V), gradient elution, gradient 100: 10 (600mL)-90 : 10 (1000mL)-80:20 (800mL)-70:30 (500mL), TLC tracking detection, 10% sulfuric acid ethanol color development, combined with the same components of Rf value, collecting 5 different components, and then passing through gel chromatography Sephade X LH20, eluent chloroform-methanol (8:1) (volume ratio), recrystallized repeatedly in methanol to obtain monomeric steroidal saponin, purity greater than 98% by HPLC, high resolution mass spectrometry HR -ESI-MS, IR spectroscopy, nuclear magnetic resonance spectroscopy, including 1H-NMR, 13 C-NMR, 135 DEPT, 1H-1HCOSY, HMBC, HMQC, NOECOSY, etc., in which compound 5 is identified as Shieldin ( 30mg). The data of some spectral analyses are as follows:
无定型粉末,易溶于吡啶,可溶于甲醇,难溶于水,分子式 C51H82022。 ESI-MS m/z 1045.2[M-H]" , 883.4[Μ-Η-162]- , 721.4[Μ-Η-162-162]- , 574.7[Μ-Η- 162- 162-146]"; IR(KBr)vmax: 3418 (νΟΗ), 1637(vc=c)» 919 , 900(900919) cm" ; 1H-NMR (600MHz, C5D5N): δ0.69(3Η, d, J=5.4Hz, H-27), 0.82(3H, s, H-18), 1.04(3H, s, H-19), 1.13(3H, d, J=6.6Hz, H-21), 5.29(1H, d, J=4.8Hz, H-6), 4.90(1H, d, J=7.8Hz, H-l -Glc'), 5.03(1H, d, J=7.8Hz , H-1-Glc〃), 5.22(1H , d, J=7.8Hz, H-l-Glc'") , 6.18(1H, s, H-l-Rha), 1.75(3H, d, J=6.0Hz, H-6-Rha); 13C-NMR (150MHz, C5D5N): 16.4(C-18), 19.4(C-19) , 15.0(C-21) , 109.7(C-22) 17.3(C-27) , lOO. l (C-l-Glc'), 77.7(C-2-Glc'), 81.9(C-4-Glc'), 104.7(C-l-Glc〃), 88.2(C-3-Glc〃), 106.0(C-l-Glc"'), 101.8(C-l -Rha), 18.7(C-6-Rha)。 结合氢谱和碳谱数据, 与参考文献对照, 两者基本一致, 故鉴定为盾 叶新昔 ( zingiberensis saponin) 。 活性实施例: Amorphous powder, soluble in pyridine, soluble in methanol, insoluble in water, molecular formula C 51 H 82 0 22 . ESI-MS m/z 1045.2[MH]" , 883.4[Μ-Η-162]- , 721.4[Μ-Η-162-162]- , 574.7[Μ-Η- 162- 162-146]"; IR( KBr)v max : 3418 (ν ΟΗ ), 1637(v c =c)» 919 , 900(900919) cm"; 1H-NMR (600MHz, C 5 D 5 N): δ0.69(3Η, d, J =5.4Hz, H-27), 0.82(3H, s, H-18), 1.04(3H, s, H-19), 1.13(3H, d, J=6.6Hz, H-21), 5.29(1H , d, J=4.8Hz, H-6), 4.90(1H, d, J=7.8Hz, Hl -Glc'), 5.03(1H, d, J=7.8Hz, H-1-Glc〃), 5.22 (1H, d, J=7.8Hz, Hl-Glc'"), 6.18(1H, s, Hl-Rha), 1.75(3H, d, J=6.0Hz, H-6-Rha); 13 C-NMR (150MHz, C 5 D 5 N): 16.4(C-18), 19.4(C-19) , 15.0(C-21) , 109.7(C-22) 17.3(C-27) , lOO. l (Cl- Glc'), 77.7(C-2-Glc'), 81.9(C-4-Glc'), 104.7(Cl-Glc〃), 88.2(C-3-Glc〃), 106.0(Cl-Glc"') , 101.8 (Cl -Rha), 18.7 (C-6-Rha). Combined with the hydrogen spectrum and carbon spectrum data, compared with the reference, the two are basically the same, so it was identified as zingiberensis saponin. Active example:
黄姜盾叶新苷抑制恶性肿瘤活性研究 Study on the inhibition of malignant tumor activity by turmeric
一、 体外抗肿瘤活性研究 I. Antitumor activity in vitro
1. 细胞株和培养  Cell line and culture
将结肠癌 C26、肺癌 A549、 乳腺癌 MCF-7、 宫颈癌 HeLa和卵巢癌肿瘤 SKOV3细 胞培养于 10%灭活胎牛血清, 100U/mL青霉素, 100 g/mL链霉素的 RPMI1640或 DMEM培养基中, 37°C 10%CO2培养箱及饱和湿度条件下培养, 3-4天传代一次。 Colon cancer C26, lung cancer A549, breast cancer MCF-7, cervical cancer HeLa and ovarian cancer tumor SKOV3 cells were cultured in 10% inactivated fetal bovine serum, 100 U/mL penicillin, 100 g/mL streptomycin in RPMI 1640 or DMEM. Base, 37 ° C 10% CO2 incubator and culture under saturated humidity conditions, pass 3-4 days.
2. 药物抗肿瘤活性测定 2. Determination of antitumor activity of drugs
在 96孔培养板中每孔加入 200uL (含有 2-3 X 105个 /mL结肠癌、 肺癌、 乳腺癌、 宫颈癌和卵巢癌肿瘤细胞) 含 10%FBS的 RPMI1640或 DMEM 培养基的细胞悬液, 置 37°C 10%CO2培养箱中培养 12h后, 受试药设 6个剂量组, 每组 5个平行孔, 对照 组则加入等浓度等体积溶剂的培养液, 重复 3次实验。 96孔培养板置 rrc io%co2 培养箱中培养 48h后, 弃去上清液, 加入 200uL/孔新鲜配制的含 0.5mg/mL MTT的无 血清培养液, TTC 继续培养 4h。 弃去清夜, 加入 150uLDMSO, 震荡混匀后, 在酶标 仪上以波长为 570nm, 参比波长为 490nm 测定 OD值, 计算抑制率, 公式为: 细胞生 长抑制率= (对照组 OD值-实验组 OD值) /对照组 OD值 X 100%。 并由细胞生长抑制 率与对应的浓度, 通过软件计算 IC5Q值 (半数抑制率), IC50 (细胞生长抑制率为 50% 的药物浓度。 Add 200 uL (containing 2-3 X 105 /mL colon cancer, lung cancer, breast cancer, cervical cancer and ovarian cancer tumor cells) per well to a 96-well culture plate. Cell suspension containing RPMI1640 or DMEM medium containing 10% FBS. After incubating for 12 h in a 10 ° CO 2 incubator at 37 ° C, the test drug was set to 6 dose groups, 5 parallel wells in each group, and the control group was added with an equal volume of solvent in the same volume, and the experiment was repeated 3 times. After culturing for 96 hours in a rrc io%co 2 incubator, the supernatant was discarded, and 200 uL/well of freshly prepared serum-free medium containing 0.5 mg/mL MTT was added, and TTC was further cultured for 4 hours. Discard the night, add 150uL DMSO, mix and shake, and measure the OD value on the microplate reader with a wavelength of 570nm and a reference wavelength of 490nm. Calculate the inhibition rate. The formula is: Cell growth inhibition rate = (Control group OD value - experiment Group OD value) / control group OD value X 100%. The IC 5Q value (half inhibition rate), IC 50 (cell growth inhibition rate, 50% drug concentration) was calculated by software from the cell growth inhibition rate and the corresponding concentration.
3. 实验结果  3. Experimental results
MTT法测定不同浓度黄姜盾叶新苷对于肿瘤细胞株生长活性影响, 结果如图 1所 示。  MTT assay was used to determine the effect of different concentrations of glucosinolates on the growth of tumor cell lines. The results are shown in Figure 1.
黄姜盾叶新苷对各种肿瘤细胞体外生长抑制活性 IC50值如下表 1所示。  The inhibitory activity of glucosinolates on the growth of various tumor cells in vitro is shown in Table 1.
表 1 黄姜盾叶新苷对肿瘤细胞体外生长抑制活性  Table 1 Inhibitory activity of gingival glucosides on tumor cells in vitro
έΗ π,| 卵巢癌细胞 乳腺癌细胞 宫颈癌细胞 结肠癌细胞 肺癌细胞έ Η π,| ovarian cancer cells breast cancer cells cervical cancer cells colon cancer cells lung cancer cells
J (SK-OV-3) (MCF-7) (HeLa) (C26) (A549) J (SK-OV-3) (MCF-7) (HeLa) (C26) (A549)
IC50 ( μ Μ) 1. 5 ± 1. 4** 1. 2±0. 7** 0. 8±0. 5* 0. 75 + 0. 5** 1. 5 ± 0. 7** 与对照组比较 *P<0.05, **P<0.01 IC50 (μ Μ) 1. 5 ± 1. 4** 1. 2±0. 7** 0. 8±0. 5* 0. 75 + 0. 5** 1. 5 ± 0. 7** Comparison of the control group *P<0.05, **P<0.01
采用相差显微镜观察黄姜盾叶新苷处理的结肠癌细胞株, 结果如图 2所示。形态 学观察发现, 盾叶新苷能诱导结肠癌细胞系凋亡。 二、 体内抗肿瘤活性研究 1. 实验材料 The phase cancer microscope was used to observe the colon cancer cell line treated with turmeric, and the results are shown in Fig. 2. Morphological observation revealed that scutellarin can induce apoptosis in colon cancer cell lines. Second, in vivo anti-tumor activity research Experimental material
1. 1实验动物与瘤株  1. 1 experimental animals and tumor strains
昆明种小鼠 60只, 7-8周, 雌雄各半, 体重 18-20g 60 Kunming mice, 7-8 weeks, half male and half female, weighing 18-20g
C26鼠结肠癌细胞 C26 murine colon cancer cells
1. 2 药物 1. 2 drugs
黄姜盾叶新苷 (阳性对照) Dioscorea zingiberensis sinensis (positive control)
2. 实验步骤  2. Experimental steps
2. 1 动物造模与分组  2. 1 Animal modeling and grouping
健康昆明种小鼠 60只, 雌雄各半, 置于清洁动物室中, 自由摄食饮水, 适应两天后, 随机分 10只为正常对照组,剩余 50只造模。在小鼠右前腋接种 C26肿瘤细胞 5 X 106, 造 C26实体瘤模型, 两周后, 按质量随机分成黄姜盾叶新苷组。 Sixty healthy Kunming mice, half male and half female, were placed in a clean animal room and fed freely. After two days of adaptation, 10 were randomly divided into normal control group and the remaining 50 were modeled. C26 tumor cells 5 X 106 were inoculated into the right anterior iliac crest of mice, and a C26 solid tumor model was constructed. Two weeks later, they were randomly divided into turmeric and turmeric.
2. 2 给药方法  2. 2 method of administration
黄姜盾叶新苷用吐温 (TW-80 ) 分散。 药物组每只小鼠每次按 20mg/Kg灌胃给药, 一周两次, 对照组灌以无药 TW- 80。 测量肿瘤大小, 每周两次。 连续给药 10次后处死 小鼠分离瘤块,用电子天平称量瘤块重量。按下列公式计算抑瘤率:抑瘤率 = (吐温 -80 组瘤重-药物组瘤重) I TW-80组瘤重 X 100%。  Dioscorea zingiberensis leaves are dispersed by Tween (TW-80). Each group of mice in the drug group was intragastrically administered at 20 mg/kg twice a week, and the control group was administered with drug-free TW-80. Tumor size was measured twice a week. After 10 consecutive administrations, the mice were sacrificed and the tumor pieces were isolated, and the weight of the tumor pieces was weighed using an electronic balance. The tumor inhibition rate was calculated according to the following formula: tumor inhibition rate = (Tween-80 group tumor weight - drug group tumor weight) I TW-80 group tumor weight X 100%.
3. 实验结果  3. Experimental results
实验结果如表 2和图 3所示。  The experimental results are shown in Table 2 and Figure 3.
表 2 黄姜盾叶新苷对 C26小鼠结肠癌的抑瘤率 组别 N 瘤块重量 (g) 抑瘤率(%) 吐温对照组 10 2.45±0.24 ―  Table 2 Tumor inhibiting rate of scutellaria sinensis on colon cancer of C26 mice Group N Tumor weight (g) Tumor inhibition rate (%) Tween control group 10 2.45±0.24 ―
盾叶新苷组 10 0.81±0.13 * * 67.33% 与吐温对照组比较 * P<0. 05, * * P<0. 01 三、 结论  Shield leaf glucoside group 10 0.81±0.13 * * 67.33% compared with Tween control group * P<0. 05, * * P<0. 01 III. Conclusion
实验数据显示, 黄姜盾叶新苷对结肠癌、 肺癌、 乳腺癌、 宫颈癌、 卵巢癌等恶性 肿瘤肿瘤具有显著的抑瘤活性, 将其开发为抗肿瘤新药具有广泛的应用前景。  The experimental data show that glucosinolate has significant antitumor activity against malignant tumors such as colon cancer, lung cancer, breast cancer, cervical cancer and ovarian cancer, and it has broad application prospects as a new anti-tumor drug.

Claims

权 利 要 求 Rights request
1、 一 1 a
Figure imgf000011_0001
Figure imgf000011_0001
包括以下步骤: 以黄姜的块茎为原料, 采用一种或多种选自水和 CM-脂肪醇的溶 剂提取, 减压浓缩、 冷冻干燥得到黄姜 体总皂苷组分, 采用正相硅胶柱色谱、 凝胶 色谱、 重结晶分离纯化, 得到黄姜盾叶新苷。  The method comprises the following steps: using a tuber of turmeric as a raw material, extracting one or more solvents selected from water and CM-fatty alcohol, concentrating under reduced pressure, and lyophilizing to obtain a total saponin component of turmeric, using normal phase silica gel column chromatography, Separation and purification by gel chromatography and recrystallization, and the glucosides of turmeric.
2、 根据权利要求 1的方法, 包括以下步骤:  2. The method of claim 1 comprising the steps of:
(1) 将药用植物黄姜块茎烘干, 粉碎, 按药用植物粉末重量的 2-3倍加入 70-100% (体 积比)乙醇浸提 10-24小时, 固液分离;  (1) drying and pulverizing the medicinal plant turmeric tubers, adding 2-3 times the weight of the medicinal plant powder to 70-100% (volume ratio) ethanol for 10-24 hours, solid-liquid separation;
(2) 过滤分离的固相按药用植物重量的 3-5倍加入 70-100% (体积比)乙醇回流 2-5小时, 趁热过滤, 重复回流 3-5次;  (2) The solid phase separated by filtration is added 3-5 times the weight of the medicinal plant to 70-100% (by volume) ethanol reflux for 2-5 hours, filtered while hot, and refluxed 3-5 times;
(3)将浸泡的液相与热回流提取的液相合并后, 减压蒸馏回收乙醇, 得褐色浸膏; (3) combining the soaked liquid phase with the liquid phase extracted by hot reflux, and recovering the ethanol by distillation under reduced pressure to obtain a brown extract;
(4) 将褐色浸膏按重量的 6-10倍加水超声波溶解; (4) Dissolve the brown extract by ultrasonic wave 6-10 times the weight of water;
(5) 将 (4)得到的混合物离心, 分离水相与固相;  (5) centrifuging the mixture obtained in (4) to separate the aqueous phase from the solid phase;
(6) 将 (5)得到的固相得到的固相冷冻干燥得到黄姜甾体总皂苷组分;  (6) The solid phase obtained by the solid phase obtained in (5) is freeze-dried to obtain a total saponin component of the turmeric carcass;
(7)采用正相硅胶柱色谱、 凝胶色谱、 重结晶分离纯化, 得到黄姜盾叶新苷。  (7) Separation and purification by normal phase silica gel column chromatography, gel chromatography and recrystallization, and the glucosides of turmeric.
3、 根据权利要求 1的方法, 其中溶剂提取在室温至 78°C的温度下浸润提取, 或 回流提取。  The method according to claim 1, wherein the solvent extraction is infiltrated and extracted at a temperature of from room temperature to 78 ° C, or refluxed.
4、 根据权利要求 1的方法, 其中所述正相硅胶柱色谱为常压或加压硅胶柱色谱, 采用选自苯 /丙酮、 二氯甲烷 /丙酮、 三氯甲烷 /丙酮、 苯 /甲醇、 二氯甲烷 /甲醇、 三氯甲 垸 /甲醇、 乙酸乙酯 /甲醇、 苯 /丙酮 /水、 三氯甲焼 /甲醇冰、 二氯甲垸 /甲醇 /水或乙酸乙 酯 /甲醇 /水中的一种或多种洗脱剂等度洗脱或梯度洗脱。 4. The method according to claim 1, wherein the normal phase silica gel column chromatography is atmospheric pressure or pressurized silica gel column chromatography, and is selected from the group consisting of benzene/acetone, dichloromethane/acetone, chloroform/acetone, benzene/methanol, Methylene chloride/methanol, trichloromethane/methanol, ethyl acetate/methanol, benzene/acetone/water, trichloromethane/methanol ice, dichloromethane/methanol/water or ethyl acetate/methanol/water One or more eluents are isocratically or gradient eluted.
5、 根据权利要求 1的方法, 其中所述凝胶色谱的洗脱剂选自甲醇 /水, 乙醇 /水的 一种或多种洗脱剂等度洗脱或梯度洗脱。 5. A method according to claim 1 wherein said gel chromatography eluent is selected from the group consisting of methanol/water, one or more eluents of ethanol/water, isocratic or gradient elution.
6、 根据权利要求 1的方法, 其中所述重结晶步骤是将产物溶解在甲醇中重结晶。  6. A process according to claim 1 wherein said recrystallization step is the recrystallization of the product dissolved in methanol.
7、 具有下式结构的黄姜盾叶新苷在制备用于治疗或预防癌症的 的用途  7. Use of a glucoside of turmeric, having the following structure, for the preparation of a medicament for treating or preventing cancer
Figure imgf000012_0001
Figure imgf000012_0001
8、 根据权利要求 7的用途, 其中所述癌症选自结肠癌、 肺癌、 乳腺癌、 宫颈癌和 卵巢癌。 8. The use according to claim 7, wherein the cancer is selected from the group consisting of colon cancer, lung cancer, breast cancer, cervical cancer and ovarian cancer.
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