WO2019137349A1 - 一种红花多糖、其制备方法及在抗肿瘤药物中的应用 - Google Patents

一种红花多糖、其制备方法及在抗肿瘤药物中的应用 Download PDF

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WO2019137349A1
WO2019137349A1 PCT/CN2019/070761 CN2019070761W WO2019137349A1 WO 2019137349 A1 WO2019137349 A1 WO 2019137349A1 CN 2019070761 W CN2019070761 W CN 2019070761W WO 2019137349 A1 WO2019137349 A1 WO 2019137349A1
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safflower
polysaccharide
safflower polysaccharide
tumor
extraction
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French (fr)
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丁侃
王培培
姚艳丽
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中国科学院上海药物研究所
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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
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • 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
    • 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
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof

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  • the invention belongs to the technical field of polysaccharides, and particularly relates to a safflower polysaccharide HH1-1, a preparation method thereof and the use thereof in preparing an antitumor medicine.
  • a polysaccharide is a polymeric sugar polymer carbohydrate composed of a sugar chain bound by a glycosidic bond and having at least 10 monosaccharides.
  • Polysaccharides are widely found in the biological world and are one of the four basic substances that constitute life; they exist as physiologically active substances of animals, bacteria, higher plants, fungi and algae. With the deep development of sugar chemistry and glycobiology, plant polysaccharides as an important biologically active substance have been shown to have various biological activities such as immune regulation, antiviral, anti-oxidation and anti-infection, and the body The toxic side effects are small. Therefore, biologically active polysaccharides have received increasing attention.
  • the polysaccharide drugs that have been put on the market in China mainly include: Lentinus edodes polysaccharide injection, Polyporus polysaccharide injection, Yunzhi polysaccharide capsule, Astragalus polysaccharide, Yunzhi Gantai, and Hushuhua capsule. Foreign studies on polysaccharides have also paid great attention to it. At present, a variety of plant polysaccharides have been used in clinical treatment for adjuvant treatment of tumors and to reduce the side effects of chemotherapy (Schepetkin IA, Quinn MT. Botanical polysaccharides: Macrophage immunomodulation and therapeutic potential [J].
  • Polysaccharides can exert anti-tumor effects through various ways, for example, improve immune function, inhibit tumor cell proliferation, induce differentiation, induce tumor cell apoptosis, and scavenge free radicals.
  • Tumor is one of the malignant diseases that endanger human health. Many anticancer drugs also damage certain cells of the human body while killing cancer cells, especially the immune system of the human body, causing various complications. On the other hand, tumor anti-cancer drugs gradually produce multi-drug resistance, which is also an important reason for poor therapeutic effect of cancer.
  • the results of polysaccharide research show that many polysaccharides have immune enhancement, and combined with radiotherapy and chemotherapy drugs can enhance anti-cancer effect. To improve the physical condition, it is a good anti-cancer adjuvant treatment.
  • Carthamus tinctorius L. also known as safflower, safflower, red crocus, crocus.
  • the main producing area is Xinjiang, and the secondary producing areas are Henan, Zhejiang, Sichuan, Yunnan, Qinghai, Cambodia and other places.
  • Safflower is a traditional Chinese medicine in China. It has the effect of promoting blood circulation and relieving pain.
  • the chemical constituents of safflower are mainly concentrated on small molecular compounds such as Saffloryellow and Carthamin.
  • Another object of the present invention is to provide a method of extracting said safflower polysaccharide from safflower.
  • Another object of the present invention is to provide a pharmaceutical composition comprising the safflower polysaccharide.
  • Another object of the present invention is to provide a use of a safflower polysaccharide or a composition containing the safflower polysaccharide for the preparation of a medicament for preventing and/or treating a tumor.
  • Another object of the present invention is to provide use of the safflower polysaccharide or a composition containing the safflower polysaccharide for the preparation of a medicament for preventing and/or treating tumor metastasis.
  • a safflower polysaccharide HH1-1 having a weight average molecular weight of 10 to 500 kDa, preferably 20 to 200 kDa; more preferably 20 to 100 kDa, which is composed of arabinose and half.
  • the weight ratio of lactose to arabinose and galactose is about 1: (1.3 to 1.5).
  • the safflower polysaccharide HH1-1 has a relative molecular mass of 70.9 kDa.
  • the safflower polysaccharide HH1-1 mainly contains galactose (59.2% by weight) and arabinose (40.8% by weight).
  • a method for preparing safflower polysaccharide HH1-1 comprises the steps of: taking safflower as a raw material, extracting by water, concentrating, dialysis, and adding ethanol to obtain a crude polysaccharide of safflower; Further, by column chromatography, safflower polysaccharide HH1-1 was obtained.
  • the safflower polysaccharide HH1-1 of the present invention is specifically prepared by the following method: using safflower as a raw material, boiling water extraction, and the amount of boiling water is about the total mass of safflower per extraction. 5 to 20 times, each extraction time is about 1.5 to 5 hours, concentrated, dialyzed, and ethanol precipitated to obtain crude safflower polysaccharide; crude polysaccharide of safflower is taken, dissolved in water, centrifuged, and the supernatant is passed through a DEAE cellulose anion column.
  • the concentration of the NaCl solution is about 0.1M.
  • the extraction may be performed one or more times, for example 1, 2, 3 or more times.
  • the dialyzed dialysis bag has a molecular weight cut-off of 3500 Da.
  • the method of drying includes one or more of rotary evaporation, spray drying, and vacuum freeze drying, preferably vacuum freeze drying.
  • compositions comprising a therapeutically effective amount of the above-described safflower polysaccharide HH1-1 as an active ingredient, the composition may further comprise a pharmaceutically acceptable pharmaceutical excipient, such as a carrier, Excipients, adjuvants and/or diluents, and the like.
  • a pharmaceutically acceptable pharmaceutical excipient such as a carrier, Excipients, adjuvants and/or diluents, and the like.
  • the use of the above-described safflower polysaccharide HH1-1 or a pharmaceutical composition containing the same for the preparation of a medicament for treating and/or preventing a tumor is provided.
  • the tumor refers to a new organism formed by the local tissue cell proliferation under the action of various tumorigenic factors, because the new organism is mostly a occupying block, also called a scorpion.
  • the present invention screens 9 common tumors including breast cancer, pancreatic cancer, glioma, leukemia, liver cancer, colon cancer, cervical cancer, melanoma, lung cancer, and found that safflower polysaccharide HH1-1 can specifically inhibit pancreatic cancer cell proliferation. .
  • the safflower polysaccharide HH1-1 of the invention may be administered alone or in combination with other pharmaceutically acceptable therapeutic agents, particularly in combination with other drugs for the prevention or treatment of tumors or cancers.
  • therapeutic agents include, but are not limited to, nitrogen mustard, chlorambucil, cyclophosphamide, ifosfamide, melphalan, thiotepa, carmustine, semustine, busulfan, Cisplatin, carboplatin, platinum oxalate, mitomycin.
  • a method of treating and/or preventing a tumor comprising administering to a subject in need of such treatment a therapeutically effective amount of the above-described safflower polysaccharide HH1-1 or a pharmaceutical composition containing the same Things.
  • the safflower polysaccharide of the present invention can inhibit tumor growth by inhibiting tumor cell proliferation, inhibiting tumor cell cloning ability, and inhibiting growth of transplanted tumor in nude mice in vitro.
  • Example 1 is a 13 C NMR spectrum of safflower polysaccharide HH1-1 prepared in Example 1;
  • FIG. 2 is a schematic diagram showing the growth inhibition effect of safflower polysaccharide HH1-1 prepared in Example 1 on various cancer cells and human normal cells;
  • FIG. 3 is a schematic diagram showing the inhibitory effect of safflower polysaccharide HH1-1 prepared in Example 1 on the formation of human pancreatic cancer cell BxPC-3 plate clone;
  • FIG. 4 is a schematic view showing the inhibitory effect of safflower polysaccharide HH1-1 prepared in Example 1 on transplanted tumor in nude mice;
  • Fig. 5 is a view showing the inhibitory effect of safflower polysaccharide HH1-1 prepared in Example 1 on transplanted tumor of a nude mouse patient with pancreatic cancer.
  • HPLC High performance liquid chromatography
  • Nuclear magnetic resonance analysis was performed using a Brucker AM-500 nuclear magnetic resonance spectrometer (Brucker, Germany);
  • the cell proliferation assay was performed using a (SPECTRAmax 190) tunable wavelength microplate reader (Molecular Device, USA).
  • the dried safflower was degreased with 95% ethanol for 1 week and then naturally dried at room temperature.
  • 5 kg of dried safflower was extracted 5 times with boiling water (deionized water) 50 L, extracted 3 to 3 times for the first 1-3 times, and 4 to 6 times, each extraction for 6 hours.
  • Each extract was combined, filtered, and the extract was concentrated by heating to about 5 L under normal pressure, and dialyzed against running water for 3 days.
  • the liquid in the dialysis bag was concentrated to about 5 L, and the concentrated liquid was centrifuged.
  • the supernatant was added to a volume of 95% ethanol under stirring, and the mixture was allowed to stand, and the precipitate was collected by centrifugation.
  • the precipitate was washed twice with absolute ethanol and acetone, and then placed.
  • the mixture was dried at 50 ° C in a vacuum desiccator to obtain crude safflower polysaccharide HH (yield 5.73%).
  • the safflower crude polysaccharide HH10g prepared above was dissolved in 100 mL of water, and the insoluble matter was removed by centrifugation, and the supernatant was subjected to preliminary separation through a Cl-type DEAE-cellulose anion column. The mixture was washed with distilled water and 0.1 M NaCl, and then detected by sulfuric acid-phenol. The combined 0.1 M NaCl eluate was collected, concentrated, dialyzed, and lyophilized in vacuo to obtain safflower polysaccharide HH1-1 0.5 g.
  • the relative molecular mass of safflower polysaccharide HH1-1 was determined by high performance gel permeation chromatography (HPGPC) to be 70.9 kDa.
  • HPGPC high performance gel permeation chromatography
  • the monosaccharide composition analysis was carried out, that is, the safflower polysaccharide HH1-1 was completely hydrolyzed, reduced, acetylated, extracted, concentrated, and sent to gas chromatography and GC analysis.
  • the monosaccharide composition analysis showed that the safflower polysaccharide HH1-1 mainly contained galactose (59.2% by weight) and arabinose (40.8% by weight).
  • the terminal carbon signals at ⁇ 110- ⁇ 108 are the C1 signals of terminal arabinose, 1,3-arabinose and 1,3,5-arabinose; respectively, located at ⁇ 106- ⁇ 104
  • the terminal carbon signals are the C1 signals of terminal galactose, 1,3-galactose, 1,6-galactose and 1,3,6-galactose, respectively.
  • the signal peak of rhamnose methyl carbon at ⁇ 17.7 is the signal peak of galacturonic acid carboxy carbon at ⁇ 176.7. From the above results, it was found that safflower polysaccharide HH1-1 is an arabinogalactan.
  • Example 2 Safflower polysaccharide HH1-1 inhibits pancreatic cancer tumor activity
  • human breast cancer cell line MDA-MB-231 human glioma cell line U87, human myeloid leukemia K652, human liver cancer cell BEL-7402, human colon cancer cell SW116, human cervical cancer cell line Hela, human malignant melanin Tumor cell A375, human non-small cell lung cancer A549, human pancreatic cancer cell line BxPC-3, Panc-1 and AsPC-1, and human normal liver cell line LO2 and human normal pancreatic ductal epithelial cell HPDE6-C7 (typical culture of Chinese Academy of Sciences) The Cell Bank of the Depository Committee, Cell Resource Center, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences) was cultured in DMEM or 1640 medium (HyClone) containing 10% fetal bovine serum (Gibco), 100 U/mL penicillin and 100 U/mL streptomycin. in. The cells were cultured in a 5% CO 2 incubator at 37 °C.
  • the cells were inoculated with logarithmic growth phase, trypsinized, and counted in a single cell suspension.
  • the cells were seeded at 3000-4000 cells/well in 96-well culture plates, and blank control wells were added. base. After being cultured overnight in an incubator containing 5% CO 2 and saturated humidity at 37 ° C, the cells were attached to the cells, and then treated with different concentrations of safflower polysaccharide HH1-1 to obtain a final concentration of from 1 mg/mL to 0.015625 mg/mL.
  • the final volume per well is l00 ⁇ L, and each group has 3 duplicate wells.
  • safflower polysaccharide HH1-1 specifically inhibited the proliferation of pancreatic cancer cells, and the inhibition was in a concentration- and time-dependent manner, but had no obvious inhibitory effect on other tumor cells. And when the concentration of safflower polysaccharide HH1-1 is as high as 1 mg/mL, there is still almost no toxicity to normal pancreatic cells and hepatocytes.
  • the cells in the logarithmic growth phase were trypsinized and pipetted into individual cells, and the cells were counted by making a single cell suspension.
  • the cell suspension was inoculated into a 35 mm cell culture dish with 100 cells per dish, and gently rotated to uniformly disperse the cells, and safflower polysaccharide HH1-1 was added thereto to have a final concentration of 0 mg/mL, 0.5 mg/mL and 1 mg. /mL.
  • the cells were cultured in a cell incubator at 37 ° C, 5% CO 2 and saturated humidity for 2 to 3 weeks. It is often observed that when macroscopic clones appear in the culture dish, the culture is terminated.
  • the supernatant was discarded and carefully immersed twice with phosphate buffered saline (PBS).
  • PBS phosphate buffered saline
  • the cells were fixed with 4% paraformaldehyde for 15 minutes, and the fixing solution was added.
  • the appropriate amount of GIMSA purchased from Sigma-Aldrich
  • safflower polysaccharide HH1-1 could inhibit the colony forming ability of pancreatic cancer cells.
  • Panc-1 and BxPC-3 cells were incubated with different concentrations of safflower polysaccharide HH1-1 for two weeks. The results showed that safflower polysaccharide HH1-1 can significantly inhibit the number of clones of pancreatic cancer cells and the size of clones.
  • Example 3 Safflower polysaccharide HH1-1 inhibits xenograft activity in nude mice
  • mice Female BALB/c nu/nu mice (6-8 weeks old, weight 18-20 g) were purchased from Shanghai Experimental Animal Resource Center (Shanghai Xipuer-Beikai Experimental Animal Co., Ltd.) at Shanghai Institute of Drugs Experimental Animal Center , constant temperature (24-26 ° C), Specific Pathogen Free (SPF) conditions, cages, litter, drinking water and feed are treated with high pressure and UV disinfection, aseptic operation, the experiment follows the laboratory animal of Shanghai Institute of Materia Medica, Chinese Academy of Sciences Use and management committee regulations.
  • Shanghai Experimental Animal Resource Center Shanghai Xipuer-Beikai Experimental Animal Co., Ltd.
  • SPF Specific Pathogen Free
  • the cells were suspended in PBS, and the cell suspension concentration was adjusted to be inoculated subcutaneously in the right axilla of the nude mice at a dose of 5 ⁇ 10 6 cells.
  • the tumor volume reached 20-100 mm 3
  • the animals were randomly divided into control group, low dose group (0.5 mg/kg), medium dose group (5 mg/kg), and high dose group (50 mg/). Kg) and gemcitabine group (GEM, 40 mg/kg).
  • mice Female SCID nu/nu mice (6-8 weeks old, body weight 18-20 g), and female BALB/c nu/nu mice (6-8 weeks old, body weight 18-20 g) were purchased from Shanghai test animals.
  • Resource Center (Shanghai Xipuer-Beikai Experimental Animal Co., Ltd.) is kept at the Shanghai Institute of Drugs Laboratory Animals, constant temperature (24-26 ° C), no specific pathogen (SPF), cages, litter, Drinking water and feed are treated with high pressure and ultraviolet disinfection, aseptic operation, and the experiment is carried out in accordance with the regulations of the Laboratory Animal Use and Management Committee of the Shanghai Institute of Materia Medica, Chinese Academy of Sciences.
  • the patient's specimen of surgically resected pancreatic cancer was taken from Changhai Hospital affiliated to the Second Military Medical University. The operation was carried out in accordance with the regulations of the Ethics and Ethics Committee of Changhai Hospital of the Second Military Medical University. The patient signed the informed consent form.
  • Surgical resected human pancreatic cancer tumor specimens, the pathological report showed moderately differentiated ductal adenocarcinoma, and the excised tumor tissue was cut into 3 cm ⁇ 3 cm and placed in RPMI-1640 medium containing penicillin and streptomycin double antibody, and transferred to ice.
  • Transplantation was carried out in the barrier zone of the animal room, the tumor tissue was transferred to a sterile culture dish, and the saline was washed 3 times, and the tumor tissue was cut into small pieces of about 10-30 mm 3 in size, and the plug was transplanted to the SCID with severe combined immunodeficiency.
  • the primary nude mice xenografts were P0 generation, and the successfully established transplanted tumors were transplanted again to BA1/c nude mice for P1 generation, and so on.
  • safflower polysaccharide HH1-1 can inhibit the growth of xenografts in nude mice derived from pancreatic cancer patients, and its inhibition is dose-dependent.
  • the results showed that the safflower polysaccharide HH1-1 treatment group significantly reduced the tumor weight, the relative inhibition rate was 40.45% in the low dose group (0.5mg/kg), and the relative inhibition rate was 45.27% in the middle dose group (5mg/kg).
  • the relative inhibition rate (50 mg/kg) was 58.78%.
  • safflower polysaccharide HH1-1 had no significant effect on body weight and signs of nude mice.
  • the above experiments show that safflower polysaccharide HH1-1 can significantly inhibit tumor growth.

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Abstract

一种红花多糖HH1-1、其制备方法及在制备抗肿瘤药物中的用途。制备方法包括：红花经沸水提取、浓缩、透析、醇沉得粗多糖，进一步经离子交换柱层析得红花多糖HH1-1。该多糖可显著抑制癌细胞增殖及克隆，体内实验表明该多糖可显著抑制裸鼠移植瘤生长，具有良好的抑制肿瘤作用，有望成为治疗肿瘤潜在多糖药物。

Description

一种红花多糖、其制备方法及在抗肿瘤药物中的应用 技术领域
本发明属于多糖技术领域，具体涉及一种红花多糖HH1-1、其制备方法及在制备抗肿瘤药物中的用途。
背景技术
多糖是由糖苷键结合的糖链，至少要超过10个的单糖组成的聚合糖高分子碳水化合物。多糖在生物界广泛存在，是构成生命的四大基本物质之一；作为动物、细菌、高等植物、真菌和藻类的生理活性物质存在。随着糖化学与糖生物学的深入发展，植物多糖作为一类重要的生物活性物质，经大量研究已表明其具有免疫调节、抗病毒、抗氧化及抗感染等多种生物活性，且对机体的毒副作用小。因此，具有生物活性的多糖已日益受到重视。自1969年，Nature杂志文章首次报道香菇多糖具有抗肿瘤功效以来(ChiharaG,MaedaY,Hamuro J,Sasaki T,Fukuoka F.Inhibition of mouse sarcoma 180by polysaccharides from Lentinus edodes(Berk.)sing.Nature.1969May 17；222(5194):687-8.)，多糖的抗肿瘤活性受到越来越多关注。目前，在我国已投放市场的多糖药物主要有：香菇多糖注射液、猪苓多糖注射液、云芝多糖胶囊、黄芪多糖、云芝肝泰、灰树花胶囊等。国外对多糖的研究也十分重视，目前有多种植物多糖已经在临床治疗上用于肿瘤的辅助治疗和减轻化疗的副作用(Schepetkin IA,Quinn MT.Botanical polysaccharides:Macrophage immunomodulation and therapeutic potential[J].Int Immunopharmacol,2006,6(3):317-333.)。多糖可以通过多种途径发挥抗肿瘤作用，例如，提高机体免疫功能，抑制肿瘤细胞增殖、诱导分化，诱导肿瘤细胞凋亡，清除自由基等。
肿瘤是危害人类健康的恶性疾病之一，很多抗癌药在杀灭癌细胞的同时对人体正常细胞也有一定的损害，尤其是损害人体的免疫系统，造成多种并发症，另一方面，在治疗的同时，肿瘤对抗癌药逐渐产生多药耐药性，也是肿瘤治疗效果不佳的重要原因，多糖的研究结果表明，很多多糖有免疫增强作用，与放化疗药合用可增强抗癌效果，改善身体状况，是很好的抗癌辅助治疗药物。
红花(Carthamus tinctoriusL.)，又称草红花、刺红花、杜红花、金红花。主产地为新疆，次产地为河南、浙江、四川、云南、青藏、西藏等地。红花是我国传统中药，具有活血通经，去瘀止痛的功效。目前对于红花的化学成分研究，主要集中在红花黄色素 (Saffloryellow)及红花甙(Carthamin)等小分子化合物。而对于红花多糖的提取分离结构及药理活性的研究报道很少。目前尚无以红花为原料提取多糖，并用于治疗肿瘤药物的研究。
发明内容
本发明的一个目的是提供一种红花多糖。
本发明的另一目的是提供一种从红花中提取所述的红花多糖的方法。
本发明的另一目的是提供一种包含所述的红花多糖的药物组合物。
本发明的另一目的在于提供红花多糖或含有所述红花多糖的组合物在制备用于预防和/或治疗肿瘤的药物中的用途。
本发明的另一目的是提供所述红花多糖或含有所述红花多糖的组合物在制备用于预防和/或治疗肿瘤转移的药物中的用途。
本发明的一个方面，提供了一种红花多糖HH1-1，所述红花多糖的重均分子量为10～500kDa，优选为20～200kDa；更优选为20～100kDa，其由阿拉伯糖和半乳糖组成，阿拉伯糖和半乳糖的重量比约为1:(1.3～1.5)。
在本发明的一个实施例中，所述的红花多糖HH1-1的相对分子质量为70.9kDa。
所述的红花多糖HH1-1主要含有半乳糖(59.2wt％)，阿拉伯糖(40.8wt％)。
本发明的另一个方面，提供了一种红花多糖HH1-1的制备方法，包括以下步骤：以红花为原料，经水提取、浓缩、透析、加乙醇沉淀，得红花粗多糖后，进一步经柱层析，得到红花多糖HH1-1。
本发明的一种优选的实施方式，本发明所述的红花多糖HH1-1具体通过以下方法制备：以红花为原料，沸水提取，每次提取时沸水的用量约为红花总质量的5～20倍，每次提取时间约为1.5～5h，浓缩、透析、加乙醇沉淀，得红花粗多糖；取红花粗多糖，用水溶解，离心，上清液通过DEAE纤维素阴离子柱进行分离，依次以蒸馏水和NaCl溶液洗脱，硫酸-苯酚检测，收取合并NaCl溶液洗脱液，浓缩，透析，干燥得所述红花多糖HH1-1。
优选地，所述NaCl溶液的浓度约为0.1M。
所述的提取可以进行一次或多次，例如1、2、3次或3次以上。
所述透析的透析袋的截留分子量为3500Da。
所述的干燥的方法包括旋转蒸发、喷雾干燥和真空冷冻干燥中的一种或多种，优选为真空冷冻干燥。
本发明的另一方面提供了一种药物组合物，其包含治疗有效量的上述的红花多糖 HH1-1作为活性成分，该组合物可以进一步包括药剂学上可接受的药物辅料，例如载体、赋形剂、佐剂和/或稀释剂等。
本发明的又一方面，提供了上述的红花多糖HH1-1或含有其的药物组合物在制备用于治疗和/或预防肿瘤的药物中用途。所述的肿瘤是指机体在各种致瘤因子作用下，局部组织细胞增生所形成的新生物，因为这种新生物多呈占位性块状突起，也称赘生物。本发明筛选了9种常见肿瘤包括乳腺癌、胰腺癌、神经胶质瘤、白血病、肝癌、大肠癌、宫颈癌、黑色素瘤、肺癌，发现红花多糖HH1-1可特异性抑制胰腺癌细胞增殖。
发明所述的红花多糖HH1-1可以单独给药，或者与其他药学上可接受的治疗剂联合给药，特别是与其他用于预防或治疗肿瘤或癌症药物组合。所述的治疗剂包括但不限于：氮芥、苯丁酸氮芥、环磷酰胺、异环磷酰胺、美法仑、塞替派、卡莫司汀、司莫司汀、白消安、顺铂、卡铂、草酸铂、丝裂霉素。
本发明的又一方面，还提供了上述的红花多糖HH1-1或含有其的药物组合物在制备用于预防和/或治疗肿瘤转移的药物中的用途。
本发明的又一方面，还提供了一种治疗和/或预防肿瘤的方法，所述方法包括向需要该治疗的对象给药治疗有效量的上述红花多糖HH1-1或含有其的药物组合物。
经实验验证，本发明的红花多糖可以通过体外抑制肿瘤细胞增殖、抑制肿瘤细胞克隆能力、体内抑制裸鼠移植瘤生长，从而起到抑制肿瘤的功效。
附图说明
图1为实施例1制备的红花多糖HH1-1的 13C NMR谱图；
图2为实施例1制备的红花多糖HH1-1对多种癌细胞以及人正常细胞生长抑制作用随浓度变化的示意图；
图3为实施例1制备的红花多糖HH1-1对人胰腺癌细胞BxPC-3平板克隆形成抑制作用的示意图；
图4为实施例1制备的红花多糖HH1-1对裸鼠移植瘤的抑制作用的示意图；
图5为实施例1制备的红花多糖HH1-1对胰腺癌患者的裸鼠移植瘤的抑制作用的示意图。
具体实施方式
本发明通过以下附图和实施例作进一步阐述，但并不限制本发明的内容。
高效凝胶渗透色谱采用Ultrahydrogel TM 2000(25cm×0.75cm，美国Waters公司)；和Ultrahydrogel TM 500(25cm×0.75cm，美国Waters公司)串联柱，以用不同分子量的T-系列标准葡聚糖(Dextran)制作标准曲线；
高效液相色谱(HPLC)采用Agilent 1260 Seri高效液相系统测定(美国Agilent公司)；
红外分析采用Perkin-Elmer 599B型红外分光光度计测定(美国Perkin-Elmer公司)；
核磁共振分析采用Brucker AM-500型核磁共振仪测定(德国Brucker公司)；
细胞增殖分析采用(SPECTRAmax190)可调波长式微孔酶标仪(美国Molecular Device公司)。
实施例1：红花多糖HH1-1的制备
a.多糖提取：
干燥的红花，用95％的乙醇脱脂1周，然后室温自然干燥。干燥后的红花5kg用沸水(去离子水)50L提取5次，第1-3次每次提取3h，第4-6次，每次提取6h。将每次的提取液合并，过滤，提取液常压下加热浓缩至约5L，对流动水透析3天。透析袋内液浓缩至约5L，浓缩液离心，取上清液在搅拌下加入4倍体积95％乙醇，静置，离心收集沉淀，沉淀经无水乙醇和丙酮依次各洗涤2次后，置真空干燥器中50℃下干燥，得到红花粗多糖HH(得率5.73％)。
b.多糖纯化：
取上述制备的红花粗多糖HH10g，100mL水溶解，离心除去不溶物，上清液通过Cl-型DEAE-纤维素阴离子柱进行初步分离。依次以蒸馏水和0.1M NaCl洗脱，硫酸-苯酚检测，收取合并0.1M NaCl洗脱液，浓缩，透析，真空冷冻干燥得红花多糖HH1-1 0.5g。
c.多糖结构鉴定：
经高效凝胶渗透色谱法(HPGPC)测定红花多糖HH1-1相对分子质量为70.9kDa。将其进行单糖组成分析，即将红花多糖HH1-1完全水解、还原、乙酰化、萃取、浓缩后送入气相色谱GC分析。
单糖组成分析结果显示，红花多糖HH1-1主要含有半乳糖(59.2wt％)，阿拉伯糖(40.8wt％)。
13C NMR谱中(图1)，位于δ110-δ108的端基碳信号，分别为末端阿拉伯糖、1,3-阿拉伯糖及1,3,5-阿拉伯糖的C1信号；位于δ106-δ104的端基碳信号，分别为末端半乳糖、1,3-半乳糖、1,6-半乳糖及1,3,6-半乳糖的C1信号。此外，在δ17.7处为鼠李糖甲基碳的信号峰，在δ176.7处为半乳糖醛酸羧基碳的信号峰。从上述结果可以发现红花多糖HH1-1为一阿拉伯半乳聚糖。
实施例2：红花多糖HH1-1抑制胰腺癌肿瘤活性
a.MTT(四氮唑盐法)实验
实验采用人乳腺癌细胞株MDA-MB-231，人神经胶质瘤细胞株U87，人髓系白血病K652，人肝癌细胞BEL-7402，人大肠癌细胞SW116，人宫颈癌细胞Hela，人恶性黑色素瘤细胞A375，人非小细胞肺癌A549，人胰腺癌细胞株BxPC-3、Panc-1和AsPC-1以及人正常肝细胞株LO2和人正常胰腺导管上皮细胞HPDE6-C7(中国科学院典型培养物保藏委员会细胞库，中国科学院上海生命科学研究院细胞资源中心)培养于含有10％胎牛血清(Gibco公司)、100U/mL青霉素和100U/mL链霉素的DMEM或1640培养基(HyClone公司)中。细胞在37℃含5％CO 2培养箱中培养。
接种细胞取对数生长期细胞，胰酶消化，用培养基制成单细胞悬液后计数，以3000-4000个细胞/孔接种于96孔培养板，另外设空白对照孔，只加完全培养基。在37℃、含5％CO 2、饱和湿度的培养箱内常规培养过夜待细胞贴壁后，再使用不同浓度红花多糖HH1-1处理，使终浓度从1mg/mL至0.015625mg/mL，每孔终体积为l00μL，每组设3个复孔。药物处理72小时后，加入MTT液(5mg/mL购自Sigma-Aldrich)20μL每孔，继续培养4小时。小心吸尽板中的培养液，各孔加l50μLDMSO溶液，将平板置于微孔板振荡器上振荡10min，使结晶物甲躜充分溶解。用酶标仪570nm处测定各孔的光密度OD值，各孔均需减去空白对照孔的OD值，记录结果。
MTT实验结果显示(图2)，红花多糖HH1-1可特异性抑制胰腺癌细胞增殖，该抑制作用呈现明显的浓度依赖性和时间依赖性，而对其他肿瘤细胞没有明显的抑制作用。并且当红花多糖HH1-1浓度高达1mg/mL时，对正常胰腺细胞和肝细胞仍然几乎没有毒性。
b.平板克隆形成实验
取对数生长期的细胞，胰酶消化并吹打成单个细胞，用培养基制成单细胞悬液后计数。将细胞悬液以每皿100个细胞接种于35mm细胞培养皿，轻轻转动，使细胞分散均匀，加入红花多糖HH1-1处理，使其终浓度为0mg/mL，0.5mg/mL和1mg/mL。置于37℃5％CO 2及饱和湿度的细胞培养箱中培养2～3周。经常观察，当培养皿中出现肉眼可见的克隆时，终止培养。弃去上清液，用磷酸缓冲盐溶液(PBS)小心浸洗2次。加4％多聚甲醛固定细胞15分钟，去固定液，加适量GIMSA(购自Sigma-Aldrich)应用染色液染10～30分钟，然后用流水缓慢洗去染色液，空气干燥。将平皿倒置并叠加一张带网格的透明胶片，用肉眼直接计数克隆，或在显微镜(低倍镜)计数大于10个细胞的克隆数。
平板克隆形成实验结果显示(图3)，红花多糖HH1-1能够抑制胰腺癌细胞克隆形成能力Panc-1和BxPC-3细胞与不同浓度红花多糖HH1-1共孵育两周，平板克隆实验结果显示红花多糖HH1-1能明显抑制胰腺癌细胞克隆数量以及克隆大小。
实施例3：红花多糖HH1-1抑制裸鼠移植瘤活性
a.BxPC-3裸鼠移植瘤模型
实验用雌性BALB/c nu/nu小鼠(6-8周龄，体重18-20g)，购自上海试验动物资源中心(上海西普尔-必凯实验动物有限公司)在上海药物所实验动物中心，恒温(24-26℃)，Specific Pathogen Free(SPF)条件下饲养，笼具、垫料、饮水及饲料均做高压和紫外线消毒处理，无菌操作，实验遵照中国科学院上海药物研究所实验动物使用与管理委员会规定进行。收获对数生长期的BxPC-3细胞计数后将细胞悬于PBS，调整细胞悬液浓度，以5×10 6个细胞量接种在裸鼠右侧腋下皮下。在肿瘤体积达到20-100mm 3时，将动物进行随机分组，分为空白对照组(control)，低剂量组(0.5mg/kg)，中剂量组(5mg/kg)，高剂量组(50mg/kg)和吉西他滨组(GEM，40mg/kg)。其中低中高红花处理组均每日口服灌胃红花多糖HH1-1，空白组每日口服灌胃等量PBS，吉西他滨组每周两次给以使用腹腔注射。实验期间每周测定动物体重和肿瘤大小。给药结束时，拍照记录肿瘤大小。取肿瘤组织，称重，固定于4％多聚甲醛或取蛋白或冻存于液氮中。肿瘤体积(Tumor Volume,TV)的计算公式为:TV＝(L×W 2)/2。其中，L、W分别代表肿瘤测量长和宽。
BxPC-3裸鼠移植瘤模型结果显示(图4)，红花多糖HH1-1处理的各个组的肿瘤大小以及瘤重与空白对照组相比显著减少，并且其抑制肿瘤效果与阳性药吉西他滨处理组相当。经计算，红花多糖HH1-1的低、中、高剂量组和吉西他滨组的相对抑制率分别为55.95％、46.46％、46.80％和51.95％。实验过程中，红花多糖HH1-1对小鼠的体重及状态没有明显影响，以上结果表明红花多糖HH1-1体内也具有抑制胰腺癌细胞生长的作用。
b.胰腺癌患者来源的裸鼠移植瘤实验
实验用雌性SCID nu/nu小鼠(6-8周龄，体重18-20g)，以及雌性BALB/c nu/nu小鼠(6-8周龄，体重18-20g)均购自上海试验动物资源中心(上海西普尔-必凯实验动物有限公司)在上海药物所实验动物中心，恒温(24-26℃)，无特定病原(Specific Pathogen Free，SPF)条件下饲养，笼具、垫料、饮水及饲料均做高压和紫外线消毒处理，无菌操作，实验遵照中国科学院上海药物研究所实验动物使用与管理委员会规定进行。手术切除的胰腺癌病人样本取自第二军医大学附属长海医院，操作遵照第二军医大学长海医院道德伦理委员会规定进行，病人签署知情同意书。手术切除的人胰腺癌肿瘤标本，病理报告显示为中分化导管腺癌，切下的肿瘤组织切取3cm×3cm置于含有青霉素和链霉素双抗的RPMI-1640培养基中，冰上转移至动物房屏障区内进行移植，将肿瘤组织转移到无菌培养皿上，生理盐水冲洗3次，把肿瘤组织剪切成体积约10-30mm 3的小块，插块移植到重症联合免疫缺陷SCID裸鼠皮下，该样本原代裸鼠移植瘤为P0代，将成功建立的移植 瘤再次移植于BALB/c裸鼠为P1代，此后依次类推。在P3代肿瘤体积达到20-100mm 3时，将动物进行随机分组，分为空白对照组(control)，低剂量组(0.5mg/kg)，中剂量组(5mg/kg)和高剂量组(50mg/kg)，并开始口服灌胃给药。实验期间每周测定动物体重和肿瘤大小。每日观察记录临床症状。给药结束时，拍照记录肿瘤大小。取肿瘤组织，称重，固定于4％多聚甲醛或取蛋白或冻存于液氮中。肿瘤体积(Tumor Volume,TV)的计算公式为:TV＝(L×W 2)/2。
实验结果发现(图5)，红花多糖HH1-1能够抑制胰腺癌患者来源的裸鼠移植瘤生长，且其抑制作用呈剂量依赖性。结果显示红花多糖HH1-1处理组，瘤重明显减少，低剂量组(0.5mg/kg)相对抑制率为40.45％，中剂量组(5mg/kg)相对抑制率为45.27％，高剂量组(50mg/kg)相对抑制率为58.78％。实验过程中，红花多糖HH1-1对裸鼠体重以及体征并没有明显影响。以上实验表明，红花多糖HH1-1能够明显抑制肿瘤的生长。

Claims (10)

  1. 一种红花多糖HH1-1，其特征在于，所述的红花多糖HH1-1的重均分子量为10～500kDa，其由阿拉伯糖和半乳糖组成，所述的阿拉伯糖和半乳糖的重量比约为1:1.3～1.5。
  2. 根据权利要求1所述的红花多糖HH1-1，其特征在于，所述的红花多糖HH1-1的相对分子质量为70.9kDa；所述的红花多糖HH1-1主要含有59.2wt％半乳糖和40.8wt％阿拉伯糖。
  3. 一种制备红花多糖HH1-1的方法，其特征在于，所述的方法包括以下步骤：
    以红花为原料，经水提取、浓缩、透析、加乙醇沉淀，得红花粗多糖后，进一步经柱层析，得到红花多糖HH1-1。
  4. 根据权利要求3所述的制备红花多糖HH1-1的方法，其特征在于，所述的经水提取为沸水提取，每次提取时沸水的用量约为红花总质量的5～20倍，每次提取时间约为1.5～5小时。
  5. 根据权利要求3所述的制备红花多糖HH1-1的方法，其特征在于，所述的方法包括以下步骤：
    以红花为原料，沸水提取时，每次提取时沸水的用量约为红花总质量的5～20倍，每次提取时间约为1.5～5小时，浓缩、透析、加乙醇沉淀，得红花粗多糖；取红花粗多糖，用水溶解，离心，上清液通过DEAE纤维素阴离子柱进行分离，依次以蒸馏水和NaCl溶液洗脱，硫酸-苯酚检测，收取合并NaCl溶液洗脱液，浓缩，透析，干燥得所述红花多糖HH1-1。
  6. 根据权利要求5所述的制备红花多糖HH1-1的方法，其特征在于，
    所述NaCl溶液的浓度约为0.1M；和/或
    所述透析的透析袋的截留分子量为3500Da。
  7. 一种药物组合物，其包含权利要求1或2中所述的红花多糖HH1-1作为活性成分。
  8. 如权利要求7所述的药物组合物，其进一步包括药剂学上可接受的药物辅料。
  9. 权利要求1或2所述的红花多糖HH1-1或权利要求7或8所述的药物组合物在制备用于治疗和/或预防肿瘤、肿瘤转移的药物中的用途。
  10. 据权利要求9所述的用途，其中，所述肿瘤为胰腺癌。
PCT/CN2019/070761 2018-01-15 2019-01-08 一种红花多糖、其制备方法及在抗肿瘤药物中的应用 WO2019137349A1 (zh)

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