WO2012061954A1 - Procédé de préparation et d'utilisation de saponine de zingiberensis extraite de dioscorea zingiberensis à activité antitumorale - Google Patents

Procédé de préparation et d'utilisation de saponine de zingiberensis extraite de dioscorea zingiberensis à activité antitumorale Download PDF

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Publication number
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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WIPO (PCT)
Prior art keywords
cancer
turmeric
methanol
zingiberensis
water
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PCT/CN2010/001801
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English (en)
Chinese (zh)
Inventor
黄文�
吴晓华
倪晋仁
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北京大学
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Priority to PCT/CN2010/001801 priority Critical patent/WO2012061954A1/fr
Publication of WO2012061954A1 publication Critical patent/WO2012061954A1/fr

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Classifications

    • 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
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7048—Compounds having saccharide radicals and heterocyclic rings having oxygen as a ring hetero atom, e.g. leucoglucosan, hesperidin, erythromycin, nystatin, digitoxin or digoxin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/88—Liliopsida (monocotyledons)
    • A61K36/894—Dioscoreaceae (Yam family)
    • A61K36/8945—Dioscorea, e.g. yam, Chinese yam or water yam
    • 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
    • A61P35/04—Antineoplastic agents specific for metastasis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J71/00—Steroids in which the cyclopenta(a)hydrophenanthrene skeleton is condensed with a heterocyclic ring
    • C07J71/0005—Oxygen-containing hetero ring

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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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Natural Medicines & Medicinal Plants (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Organic Chemistry (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Epidemiology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Alternative & Traditional Medicine (AREA)
  • Molecular Biology (AREA)
  • Engineering & Computer Science (AREA)
  • Oncology (AREA)
  • Biotechnology (AREA)
  • Botany (AREA)
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Abstract

L'invention concerne un procédé de préparation de saponine de zingiberensis dont la structure (I) est indiquée dans la description et qui est extraite de Dioscorea zingiberensis. Le procédé comprend les étapes suivantes qui consistent : à extraire la tubercule de Dioscorea zingiberensis C. H. Wright en tant que matière première au moyen d'un ou plusieurs solvants sélectionnés parmi eau et alcool gras C1-4, à condenser sous pression réduite, à lyophiliser pour obtenir des saponines stéroïdiennes totales à partir de Dioscorea zingiberensis; puis à séparer et purifier par chromatographie sur colonne à gel de silice en phase normale, chromatographie sur gel et recristallisation; et à obtenir la saponine de zingiberensis extraite de Dioscorea zingiberensis. L'invention concerne l'utilisation de la saponine de zingiberensis dont la structure est indiquée dans la description extraite de Dioscorea zingiberensis dans la préparation de médicaments pour traiter ou prévenir des cancers. Parmi les cancers concernés, on peut citer le cancer du côlon, le cancer du poumon, le cancer du sein, le cancer du col de l'utérus et le cancer des ovaires.
PCT/CN2010/001801 2010-11-10 2010-11-10 Procédé de préparation et d'utilisation de saponine de zingiberensis extraite de dioscorea zingiberensis à activité antitumorale WO2012061954A1 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014126486A1 (fr) 2013-02-15 2014-08-21 Leano Milagros B Indications pharmacologiques et utilisations de préparations de dioscorea hispida
CN105125408A (zh) * 2015-09-30 2015-12-09 河南行知专利服务有限公司 一种根茎类中药材免煎剂的制备方法及其应用
CN108840900A (zh) * 2018-07-25 2018-11-20 江苏黄河药业股份有限公司 一种提取盾叶新苷的方法

Citations (1)

* Cited by examiner, † Cited by third party
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CN101607981A (zh) * 2009-06-30 2009-12-23 中国地质大学（武汉） 集成柱色谱分离与纯化黄姜中甾体皂甙的方法

Patent Citations (1)

* Cited by examiner, † Cited by third party
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CN101607981A (zh) * 2009-06-30 2009-12-23 中国地质大学（武汉） 集成柱色谱分离与纯化黄姜中甾体皂甙的方法

Non-Patent Citations (4)

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Title
KANG A-LONG ET AL.: "TLCS determination of Zingiberensis Newsaponin and Dioscin in Dioscorea zingiberensis.", CHINESE JOURNAL OF PHARMACEUTICAL ANALYSIS (CHINESE)., vol. 23, no. 1, 2003, pages 59 - 60 *
WANG LIJUAN ET AL.: "The Antitumor Activity of Diosgenin in vivo and in vitro.", CHINA JOURNAL OF CHINESE MATERIA MEDICA (CHINESE)., vol. 27, no. 10, October 2002 (2002-10-01), pages 777 - 779 *
WANG, YINHONG ET AL.: "Study of steroidal saponins in Dioscorea Zingiberensis C. H. Wright.", JOURNAL OF NATURAL PRODUCTS (CHINESE)., vol. 2, 2009, pages 123 - 132 *
ZHAO QINGBING ET AL.: "Study on the water-insoluble steroidal saponins from Dioscorea zingiberensis and cell growth inhibitive effects of steroidal saponins against human hepatocellular carcinoma hepG2 cells.", HUAXI YAOXUE ZAZHI (CHINESE)., vol. 25, no. 5, October 2010 (2010-10-01), pages 527 - 529 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014126486A1 (fr) 2013-02-15 2014-08-21 Leano Milagros B Indications pharmacologiques et utilisations de préparations de dioscorea hispida
CN105125408A (zh) * 2015-09-30 2015-12-09 河南行知专利服务有限公司 一种根茎类中药材免煎剂的制备方法及其应用
CN108840900A (zh) * 2018-07-25 2018-11-20 江苏黄河药业股份有限公司 一种提取盾叶新苷的方法

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