WO2007124625A1 - Polysaccharides de limace, procédé de production et utilisation de ceux-ci - Google Patents

Polysaccharides de limace, procédé de production et utilisation de ceux-ci Download PDF

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Publication number
WO2007124625A1
WO2007124625A1 PCT/CN2006/002813 CN2006002813W WO2007124625A1 WO 2007124625 A1 WO2007124625 A1 WO 2007124625A1 CN 2006002813 W CN2006002813 W CN 2006002813W WO 2007124625 A1 WO2007124625 A1 WO 2007124625A1
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WIPO (PCT)
Prior art keywords
supernatant
polysaccharide
preparation
ammonium sulfate
lung cancer
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PCT/CN2006/002813
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English (en)
Chinese (zh)
Inventor
Jinkui Xie
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Guangzhou Konzern Pharmaceutical Co., Ltd
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Publication of WO2007124625A1 publication Critical patent/WO2007124625A1/fr

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    • 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
    • C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the invention relates to a traditional Chinese medicine extract, a preparation method thereof and a use thereof, in particular to an anthraquinone polysaccharide for treating lung cancer and bronchial asthma, a preparation method thereof and use thereof.
  • This malignant tumor of lung cancer has become the number one killer of human health.
  • the incidence of lung cancer has increased by 11.90% in the past 10 years with the increase of environmental pollution.
  • the number of new malignant tumors in the world has exceeded 10 million, and 6.2 million deaths have occurred, including 1,239,000 new cases of lung cancer, 12.32% of total cancer cases, and 11.03 million cases of lung cancer deaths. 17.77% of the total number of people ranked first.
  • 1.5 million to 1.8 million people die of lung cancer every year in the world.
  • the risk of lung cancer in China is even more serious, calculated at an incidence rate of 83.43 per 100,000 people.
  • There are no more than 1 million new lung cancer patients in China every year, and 600,000 people die of lung cancer every year! Lung cancer, a malignant tumor has become the number one killer of human health. Therefore, research and development of new special drugs for the prevention and treatment of lung cancer has become an urgent task, focus and hot spot in the pharmaceutical industry at home and abroad.
  • lung cancer There are many methods and related drugs for the treatment of lung cancer, but there are certain limitations.
  • traditional methods for treating lung cancer include surgical resection, radiation therapy and chemotherapy (drug therapy).
  • chemotherapy drug therapy
  • 803 ⁇ 4 patients lose the opportunity for surgical resection; radiotherapy has local limitations and radiation damage to normal tissues; chemical (drug) treatment has universal cytotoxicity and its dose Restrictive; particularly serious side effects on liver, kidney, bone marrow and digestive system, greatly restricting the therapeutic effect of lung cancer.
  • the dosage form includes external preparations such as injection, oral administration and acupoint application. Oral dosage forms involve a variety of granules, powders, pills, ointments, decoctions, oral liquids, and the like.
  • the research content includes clinical trials, animal experiments, and studies on the mechanism of action of drugs at the cellular and molecular levels.
  • polysaccharides having a righting anti-tumor effect include swine fever Polysaccharide, Lentinus edodes polysaccharide, Lycium barbarum polysaccharide, Ganoderma lucidum polysaccharide, Rehmannia polysaccharide, etc.; Saponin medicinal ginseng total saponin, Gynostemma total soap ring, Ginsenoside Rbl, Rgl, Re, Rhl, Rg3 and yinyang yang;
  • the medicines include Han Fang A, tea pigment, coix seed ester, ⁇ -carotene, allicin, dihydroteranol, diterpenoid bF in lobelia and brucea javanica extract.
  • the ⁇ PE-40 glycoprotein developed by the applicant not only directly inhibits small cell lung cancer and non-small cell lung cancer cells, but also has synergistic effects on chemotherapeutic drugs such as DDP and 5-FU and cytokine TNF-a, and does not have any Toxic side effects;
  • the overall effect is superior to other traditional Chinese medicines, but due to the presence of protein components, there is a problem that sensitization and stability are difficult to control, which limits the scope of adaptation to some extent.
  • the present invention is directed to solving the problem of removing protein, extracting a polysaccharide, and maintaining the effect of the drug on the basis of an effective glycoprotein which is effective for lung cancer. Therefore, the present invention is an intensive improvement of the former invention.
  • the lycium polysaccharide provided by the present invention achieves therapeutic effects by interfering with the normal metabolism of DNA and RNA in cancer cells and promoting the apoptosis of cancer cells.
  • the present invention has a definite therapeutic effect on bronchial asthma.
  • Conventional drugs for treating bronchial asthma such as aminophylline and ⁇ -receptor agonists, play an important role in clinical treatment, but these drugs have strong side effects and short duration of drug efficacy.
  • the present invention uses natural Chinese medicine as a raw material. The polysaccharide was extracted, and animal experiments showed that the extract had little side effects. Long-lasting efficacy, can achieve the effect of radical bronchial asthma, providing a better choice for the clinical treatment of bronchial asthma. Summary of the invention
  • the object of the present invention is to provide a Chinese medicine extract-one polysaccharide.
  • Another object of the present invention is to provide a process for the preparation of lycium polysaccharide.
  • Another object of the present invention is to provide a medical use of Lycium barbarum polysaccharides.
  • the lycium polysaccharide provided by the present invention is extracted from glycoprotein which is extracted from sputum.
  • the preparation method of scorpion polysaccharide includes: two steps of extracting sucrose protein by segmental salting out method and extracting scorpion polysaccharide by Sevag deproteinization method.
  • the segmented salting-out method comprises the following steps: (1) taking materials: taking the whole worm, adding the pre-cooled PDB solution, homogenizing the slurry at high speed, and taking the supernatant by centrifugation; (2) salting out: adding ammonium sulfate to the supernatant (3) Re-salting out: Centrifugate the supernatant and add ammonium sulfate; (4) Dialysis: Collect the precipitate, dissolve in PBS, dialyze with pre-cooled PBS, remove ammonia ion, negative for naphthalene reagent, centrifuge The supernatant was taken to obtain a glycoprotein solution.
  • the Sevag deproteinization method comprises the following steps: (1) taking a material: a glycoprotein solution, heating with water, taking a supernatant by centrifugation, and storing at 4 ° C for 1-3 hours; (2) deproteinizing: adjusting the pH of trichloroacetic acid to 3-5, ice bath for 2-4 hours, centrifuge to take the supernatant; adjust the pH to 8.2, add trypsin reaction, centrifuge to remove the supernatant; (3) precipitated polysaccharide: add 3 times the volume of the supernatant 95% ethanol, After standing overnight, the supernatant was centrifuged, and the precipitate was washed once with absolute ethanol and acetone. ' (4) Dialysis: Dissolve the precipitate with deionized water, dialyze, heat the dialyzate, centrifuge to remove the supernatant, and obtain the polysaccharide.
  • the percent saturation of ammonium sulfate is from 45% to 100%.
  • the percent saturation of ammonium sulfate is preferably 55%.
  • the percent saturation of ammonium sulfate is preferably 85%.
  • lycium polysaccharide of the invention in the preparation of a medicament for treating lung cancer.
  • lycium polysaccharide of the present invention for the preparation of a medicament for treating bronchial asthma.
  • the lycium polysaccharide of the present invention together with a pharmaceutically acceptable carrier, can be prepared into capsules, granules, tablets, pills, pills, powders, syrups, aerosols, injections, lyophilized powders.
  • Lycium barbarum polysaccharide has great advantages in the treatment of lung cancer and bronchial asthma diseases. Long-lasting, exact, no damage to normal organs and tissues. Moreover, the present invention is based on the glycoprotein, which removes the protein, extracts the polysaccharide therein, and eliminates the problem that the sensitization and stability caused by the glycoprotein containing the protein component are difficult to control, but maintains The same effect.
  • FIG. 1 is a graph showing changes in latency of guinea pig asthma attack
  • FIG. 2 is a morphological observation view of lung tissue eosinophil infiltration in a normal control group (x 200 );
  • FIG 3 is a lung in a model group ( ⁇ 200 ) Morphological observation of tissue eosinophil infiltration;
  • Figure 4 is a morphological observation of the eosinophil infiltration of the lung tissue in the aminophylline treatment group ( ⁇ 200);
  • Figure 5 is the low dose treatment group ( x Morphological observation of eosinophil infiltration in lung tissue of 200);
  • Figure 6 is a morphological observation of eosinophil infiltration in lung tissue of the sputum treatment group ( ⁇ 200); Morphological observation of the eosinophil infiltration of lung tissue in the treatment group ( ⁇ 200 ).
  • the lycium polysaccharide of the present invention is prepared by a stepwise salting-out method for extracting sucrose protein and a Sevag method for deproteinizing ruthenium polysaccharide.
  • the preparation method of one preparation method is as follows:
  • Sevag method for protein extraction of lycium polysaccharide (1) Material: Take 15ml of the glycoprotein solution obtained in the above step, heat in an 80 ⁇ water bath for 2 hours, .3000 rpm, centrifuge for 5 minutes, take the supernatant, 4 °C save 2 hours; (2) to a protein: 10% trichloroacetic acid adjusted to PH 4, set in an ice retained for 3 hours and 3000 rev / min, centrifuged for 5 minutes, the supernatant, adjusted PH to 82, was added. Trypsin 0.
  • Lycium barbarum polysaccharide can be used as a medicine for treating lung cancer and bronchial asthma.
  • the present invention proves that the polysaccharide has obvious curative effect in treating lung cancer and bronchial asthma, and has great advantages compared with existing clinical therapeutic drugs and methods. It has small toxic and side effects, long-lasting and exact curative effect, and no damage to normal organs and tissues of the human body.
  • the solid ammonium sulphate is added, and the percent saturation is 0%, 25%, 45%, 55%, respectively. 65%, 85%, 100%, separately packed into 1'-7, in the segmentation salt extraction step (3), after salting out, the precipitates 2, - 7, are separately packed into 8 '-Number 13.
  • the lycium polysaccharides were labeled as No. 1-13. The following cytological experiments were carried out on samples No. 1-13:
  • the 13 protein dialysates obtained above were each taken to be 100 ⁇ l, diluted with water to 2 ml, treated with the acid-phenol method, and the absorbance was measured at a wavelength of 49 Onm. Substituting the measured 0D value into the regression equation yields the polysaccharide content.
  • component 4 (55% salting protein fraction) is the most potent drug part.
  • the preparation method is as follows: Take about 15kg of frozen mash, and after thawing at room temperature, add 30L of distiller water, homogenize with a high-speed tissue masher (temperature is kept at 4 °C), then centrifuge at 1200 rpm for 10 minutes, and take the supernatant. 351 g of solid 3 ⁇ 4 acid was added per 1 L to give a percent saturation of 55%. Allow to stand overnight at 4°. At 3000 rpm, centrifuge for 15 minutes, collect the precipitated PBS solution, and pre-cool the PBS dialyzed (1000 ml) to remove the ammonia ions by dialysis.
  • Example 2 Determination of proliferative activity of alfalfa polysaccharide against human lung cancer A549 cells in vitro
  • Example 3 Effect of Lycium barbarum polysaccharides on proliferation activity of human lung cancer cells in vitro.
  • the polysaccharide sample No. 4 was obtained as a tan powder, which was provided by the Department of Pharmacology, Nanhua University. 4 g of lycium polysaccharide was weighed into the penicillin vial by electronic analytical balance, and 100 ml of normal saline was added thereto. Prepare a 4% suspension, centrifuge at 25 ° C, 5 000 r / min for 30 min, remove the supernatant with a qualitative filter paper twice, filter the supernatant through 0.45 ⁇ filter membrane for pre-filtration Then, the filtrate was subjected to 0.22 ⁇ filter membrane filtration under aseptic conditions, and the obtained filtrate was sealed and placed in a microcentrifuge tube and placed in 4. C is stored in the refrigerator.
  • lycium barbarum polysaccharide on the proliferation of human lung cancer A549 cells in vitro was determined by MTT colorimetry.
  • the polysaccharide was 20 ⁇ 7 well, and the positive control group was added with DDP (2.0 jng/ml).
  • IC 5Q uses Sun's improved comprehensive calculation method.
  • Example 4 Determination of proliferation activity of NCI-446 cells against human lung cancer outside the extract of Lycium barbarum polysaccharide
  • Table 5 shows that the polysaccharides tested have different inhibitory effects on the proliferation of NCI-446 cells, and the polysaccharides obtained by 55% salting out have the strongest effect, IC 5 .
  • the value of the dilution is 1: 237, and the polysaccharide content is 26. 6 ⁇ g/ml.
  • Table 5 Determination of anti-cancer NC 1-44"6 ⁇ cell proliferation inhibition rate ( n - 4 ) in vitro by MTT assay Test product WE inhibition rate (%)
  • NCI-H446 human small cell lung cancer cells were purchased from the China Center for Type Culture Collection, cultured in a culture medium containing 10% calf serum RPMI1640, placed in a 37 ° C, 5% CO 2 incubator, routinely passaged. , take the logarithmic growth phase cells for experiments. 5. 1. 2 Take the sample No. 4 of the polysaccharide in the first example, in the form of a brown powder, provided by the Department of Pharmacology, Nanhua University.
  • the soft agar culture colony formation method was used to determine the effect of Lycium barbarum polysaccharides on the colony forming ability of human small cell lung cancer NCI-H446 cells.
  • Colony inhibition rate [(1 - treatment group colony mean / blank control group colony mean) X 100%]
  • test mixture of Lycium barbarum L. has a significant inhibitory effect on the colony forming ability of human small cell lung cancer NCI-H446 cells in a dose-dependent manner, and its IC 5 .
  • the value is 18.0/ig / ml.
  • Example 6 Colorimetric determination of the effect of lycium barbarum polysaccharide on the proliferation of human lung cancer cell line NCI-H446 in vitro 6.1 Materials:
  • NCI- ⁇ 446 Human small cell lung cancer
  • the cells were purchased from the China Center for Type Culture Collection and cultured in a 37 ° C, 5% CO 2 incubator containing 10% calf serum RPMI 1640, routinely passaged. The logarithmic growth phase cells were taken for experiments.
  • 6.1.2 Take the No. 4 polysaccharide from the first sample in the first example, which is a brown powder. It is provided by the Department of Pharmacology, Nanhua University. 4 g of glycoprotein (LE-4) was weighed and extracted into a penicillin vial using an electronic analytical balance, and 100 ml of physiological saline was added thereto to prepare a 4% suspension, which was centrifuged at 25 ° C, 5000 r/min.
  • the supernatant is filtered twice with a qualitative filter paper, and the filtered supernatant is pre-filtered through a 0.45 ⁇ filter membrane, and then the filtrate is subjected to 0.22 ⁇ m filter membrane filtration under aseptic conditions, and the filtrate is sealed and packaged.
  • lycium barbarum polysaccharide on the proliferation of human lung cancer NCI-H446 cells was determined by MTT colorimetry.
  • Preparation of human small cell lung cancer (NCI-H446) cell single cell suspension adjusted to a concentration of 1.
  • Ox 10 5 / ml inoculated in 96-well plastic culture plate, 180 ⁇ 1 per well, cultured for 4 hours after the start of administration, respectively
  • Different concentrations of lycium polysaccharides were 20 ⁇ 7 wells, and the positive control group was added with DDP (2.0/ig/ml).
  • 1. 1 animal 48 healthy guinea pigs, weighing 250-300g male or female (purchased from the Department of Animals, Central South University)
  • each animal was intraperitoneally injected with 1 ml of 100 g/L aluminum hydroxide, 1% ovalbumin (Sigma) 0. 5 ml of the thigh muscle, and the blank control group replaced the aluminum hydroxide with physiological saline. Ovalbumin treatment of animals.
  • Groups D, E, and F were administered intragastrically one hour before the next day of induction.
  • Groups A and B were intragastrically administered with normal saline, and group C aminophylline (1 mg/kg/d) was to be determined.
  • Level 1 slight nose, trembling, vertical hair.
  • Level 2 coughing several times, having nose, trembling, vertical hair.
  • Level 3 Multiple or continuous cough, difficulty breathing, or convulsions, etc.
  • Level 4 sputum, convulsions, incontinence, shock and death.) 24 hours after 1% pentobarbital 1. 5ral intraperitoneal injection anesthesia.
  • the anti-asthma mechanism is related to inhibiting the infiltration of tissue inflammatory cells and reducing the release of inflammatory mediators. The mechanism needs further investigation. Further research on the pharmacological effects of extracting glycoproteins from sputum can not only provide a scientific theoretical basis for the study of new drugs, but also is expected to be developed as a new type of bronchial asthma drug.
  • Lycium barbarum polysaccharide can be used in the preparation of a medicament for treating lung cancer and bronchial asthma, and can be formulated into various dosage forms such as capsules, granules, tablets, pills, pills, powders, syrups, aerosols, freeze-dried powders. , injections, etc.
  • the following is a preparation method of a polysaccharide dry powder injection and an injection.
  • Other dosage forms can be prepared according to the Pharmacopoeia standard method, and the dosage can be referred to the following two dosage forms. Example VIII.
  • Formulations prepared mainly from lycium polysaccharides are prepared according to the following components and methods: lycium polysaccharide 20 g , mannitol 1000 g (formulating agent), and water for injection After adding to 1000ml, mix and adjust the pH to 5. 0-5. 5 , add 0.
  • the preparation method of the sputum polysaccharide injection is prepared according to the following components and methods: glutinous polysaccharide 20g, mannitol 1000 g (formation agent), add water for injection to 1000m, and then mix, adjust the ⁇ to 5. 0-5. 5, 0. 05% activated carbon was decolorized at 40-45 °C for 15 minutes, first coarse filtration with 0. 45 um microporous membrane, and then filtered with 0.222 um microporous membrane to clarify, retest content and PH Value, aseptically filled into the 5ml ampoule for injection, routine packaging, labeling, sterilization, storage. An injection of 100 mg/ampoules was obtained.

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Abstract

La présente invention concerne des polysaccharides de limace, ainsi que le procédé de production et l'utilisation de ceux-ci. Les polysaccharides de limace sont extraits de glycoprotéines de limace. Le procédé de production consiste à précipiter (salting-out) les protéines par morceaux et à les retirer au moyen de Sevag. Les polysaccharides de limace selon cette invention peuvent être utilisés pour produire des médicaments permettant de traiter le cancer du poumon, la bronchite et l'asthme.
PCT/CN2006/002813 2006-04-27 2006-10-23 Polysaccharides de limace, procédé de production et utilisation de ceux-ci WO2007124625A1 (fr)

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CNA2006100352025A CN1939346A (zh) 2006-04-27 2006-04-27 蛞蝓多糖及其生产工艺
CN200610035202.5 2006-04-27

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JP2011525550A (ja) * 2008-06-23 2011-09-22 広州康采恩医薬有限公司 肺癌治療用デキストラン
CN114853919A (zh) * 2022-05-30 2022-08-05 哈尔滨商业大学 一种能预防和抑制血栓的黑木耳超微粉多糖制备方法

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CN101298482B (zh) * 2008-06-23 2011-05-11 广州康采恩医药有限公司 一种葡聚糖
CN102697814A (zh) * 2011-03-28 2012-10-03 谢金魁 一种治疗乙型肝炎的新药及其提取分离方法
CN103610699B (zh) * 2013-12-06 2015-07-22 广州白云山汉方现代药业有限公司 一种蛞蝓的提取方法及其抗肺癌应用
WO2018059241A1 (fr) * 2016-09-27 2018-04-05 广西久福生物科技有限公司 Extrait à effet pharmaceutique de détoxication et son procédé de préparation

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Cited By (4)

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Publication number Priority date Publication date Assignee Title
JP2011525550A (ja) * 2008-06-23 2011-09-22 広州康采恩医薬有限公司 肺癌治療用デキストラン
US20110263842A1 (en) * 2008-06-23 2011-10-27 Guangzhou Kanzem Pharmaceuticla Co., Ltd. Dextran for treating lung cancer
US8519121B2 (en) * 2008-06-23 2013-08-27 Guangzhou Konzern Pharmaceutical Co., Ltd. Dextran for treating lung cancer
CN114853919A (zh) * 2022-05-30 2022-08-05 哈尔滨商业大学 一种能预防和抑制血栓的黑木耳超微粉多糖制备方法

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