WO2016008437A1 - 化合物1-棕榈酸-2-亚油酸-3-油酸甘油酯、制剂、制备方法及其应用 - Google Patents

化合物1-棕榈酸-2-亚油酸-3-油酸甘油酯、制剂、制备方法及其应用 Download PDF

Info

Publication number
WO2016008437A1
WO2016008437A1 PCT/CN2015/084290 CN2015084290W WO2016008437A1 WO 2016008437 A1 WO2016008437 A1 WO 2016008437A1 CN 2015084290 W CN2015084290 W CN 2015084290W WO 2016008437 A1 WO2016008437 A1 WO 2016008437A1
Authority
WO
WIPO (PCT)
Prior art keywords
preparation
injection
oil
acid
coix seed
Prior art date
Application number
PCT/CN2015/084290
Other languages
English (en)
French (fr)
Inventor
李大鹏
Original Assignee
浙江康莱特集团有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 浙江康莱特集团有限公司 filed Critical 浙江康莱特集团有限公司
Publication of WO2016008437A1 publication Critical patent/WO2016008437A1/zh

Links

Images

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
    • A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
    • A61K31/215—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids
    • A61K31/22—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acyclic acids, e.g. pravastatin
    • A61K31/23—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acyclic acids, e.g. pravastatin of acids having a carboxyl group bound to a chain of seven or more carbon atoms
    • A61K31/231—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acyclic acids, e.g. pravastatin of acids having a carboxyl group bound to a chain of seven or more carbon atoms having one or two double bonds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/44—Oils, fats or waxes according to two or more groups of A61K47/02-A61K47/42; Natural or modified natural oils, fats or waxes, e.g. castor oil, polyethoxylated castor oil, montan wax, lignite, shellac, rosin, beeswax or lanolin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0053—Mouth and digestive tract, i.e. intraoral and peroral administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/4841—Filling excipients; Inactive ingredients
    • A61K9/4858—Organic compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/4883—Capsule finishing, e.g. dyeing, aromatising, polishing
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/10—Selective adsorption, e.g. chromatography characterised by constructional or operational features
    • B01D15/16—Selective adsorption, e.g. chromatography characterised by constructional or operational features relating to the conditioning of the fluid carrier
    • B01D15/166—Fluid composition conditioning, e.g. gradient
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/42—Selective adsorption, e.g. chromatography characterised by the development mode, e.g. by displacement or by elution
    • B01D15/424—Elution mode
    • B01D15/426—Specific type of solvent
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
    • C11B1/00—Production of fats or fatty oils from raw materials
    • C11B1/10—Production of fats or fatty oils from raw materials by extracting
    • C11B1/104—Production of fats or fatty oils from raw materials by extracting using super critical gases or vapours
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
    • C11B1/00—Production of fats or fatty oils from raw materials
    • C11B1/10—Production of fats or fatty oils from raw materials by extracting
    • C11B1/106—Production of fats or fatty oils from raw materials by extracting using ultra-sounds
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
    • C11B3/00—Refining fats or fatty oils
    • C11B3/02—Refining fats or fatty oils by chemical reaction
    • C11B3/06—Refining fats or fatty oils by chemical reaction with bases
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
    • C11B3/00—Refining fats or fatty oils
    • C11B3/10—Refining fats or fatty oils by adsorption

Definitions

  • the present invention relates to the field of organic chemistry and pharmacy.
  • the present invention relates to a compound 1-palmitic acid-2-linoleic acid-3-oleic acid glyceride, a preparation, a preparation method thereof and use thereof in the preparation of an antitumor drug.
  • Coix seed is a mature dry seed of Coix lacryma-jobi L.var ma-yuen (Roman.) Stapf. It is a diuretic drug for a long time. Modern research has found that Coix seed has pharmacological effects such as analgesic, anti-inflammatory, immune regulation, anti-ulcer, hypolipidemic and weight loss. In recent years, domestic and foreign scholars have studied the chemical constituents of Coix seed by TLC, HPLC-MS, GC and other methods, and found that the main active ingredients include esters, unsaturated fatty acids, sugars and lactams. Among them, esters are the first to be found to have anti-tumor activity, and are also the most reported chemical components.
  • the present invention separates one of the above-mentioned hazelnut oil triglyceride components, palmitic acid linoleic acid oleic acid glyceride, and firstly prepares a compound having the structure of formula (I), namely 1-palmitic acid-2.
  • a compound having the structure of formula (I) namely 1-palmitic acid-2.
  • - Linoleic acid-3-oleic acid glyceride there have been no reports on the use of this compound and its pharmaceutical preparations for the treatment of tumors.
  • the object of the present invention is to provide a compound of the formula (I), namely 1-palmitic acid-2-linoleic acid-3-oleic acid glyceride:
  • a further object of the present invention is to provide a pharmaceutical composition comprising a compound of the formula (I), which may further comprise a pharmaceutically acceptable carrier.
  • the preparation method of the coix seed oil in the step (1) is as follows:
  • the coix seed with water content ⁇ 10% is pulverized into 10 to 300 mesh, and the crude oil of coix seed is separated by supercritical fluid extraction; the petroleum ether with the weight of coix seed crude oil is added 40% to 58%, and the alkali solution is alkalized. After the liquid separation, the organic phase is taken, neutral alumina and/or kaolin is added, filtered, dried at 150-180 ° C under reduced pressure, and cooled and filtered to obtain coix seed oil;
  • the supercritical fluid extraction solvent is carbon dioxide
  • the extraction temperature is 30-60 ° C
  • the extraction pressure is 19-24 Mpa
  • the separation temperature is 30-60 ° C
  • the separation pressure is 6-15 Mpa
  • the carbon dioxide flow rate is 10-5000 L/h
  • the continuous extraction is 1-4 hours.
  • the alkali solution may be an aqueous solution of 0.5% to 3% of potassium hydroxide or sodium hydroxide;
  • the process of adding neutral alumina and/or kaolin is: firstly adding activated neutral alumina with oil weight of 3% to 8%, the filtrate is heated to 40-50 ° C, and the oil weight is added by 2% to 6%. The activated kaolin is filtered, the solvent is removed under reduced pressure, and then filtered with an oil weight of 8% to 12% activated neutral alumina.
  • the coix seed with water content ⁇ 10% is pulverized into 10 ⁇ 300 mesh, extracted by supercritical carbon dioxide extraction device, the coix seed powder is charged into the extraction kettle, and the carbon dioxide preheater, the extraction kettle and the separation column are heated to make the extraction temperature reach 30 ⁇ 60 ° C, extraction pressure 19 ⁇ 24Mpa, separation temperature of 30 ⁇ 60 ° C, separation pressure 6 ⁇ 15Mpa, extraction of coix seed oil after the supercritical carbon dioxide fluid into the analytical tank analysis cycle, the analytical kettle can be set according to needs one or more, control Analytical temperature 30 ⁇ 60 ° C, analytical pressure 2 ⁇ 6Mpa, the entire cycle to maintain carbon dioxide flow 10 ⁇ 5000L / h, continuous extraction 1 ⁇ 4h, separated from the lower end of the separation column to obtain coix seed crude oil;
  • the above crude oil of coix seed is added to petroleum ether with oil weight of 40% to 58%, and then added with appropriate amount of 2% NaOH solution for alkali refining. After liquid separation, the organic phase is washed with purified water, and the organic phase is added with an appropriate amount of acetone. After the milk, the upper oil solution is taken, and the activated neutral alumina with oil weight of 3% to 8% is added for filtration, and the filtrate is heated to 40 to 50 ° C, and then filtered by activated kaolin with an oil weight of 2% to 6%. After the filtrate is recovered under reduced pressure, the oil is filtered by adding 8% to 12% by weight to activate the neutral alumina. The filtered oil is then sterilized by dry heat at 150-180 ° C for 1 to 3 hours, and after cooling for ⁇ 0.2 ⁇ m. The microporous membrane is filtered to obtain coix seed oil.
  • step (2) The preliminary separation method of 1-palmitic acid-2-linoleic acid-3-oleic acid glyceride in step (2) is:
  • test sample using mobile phase B to prepare a 0.1 g/ml hazelnut oil solution
  • the chromatographic peak fraction was collected for a retention time of approximately 17.3 min, and the solvent was drained under reduced pressure at a low temperature.
  • the second separation method of 1-palmitic acid-2-linoleic acid-3-oleic acid glyceride in step (3) is:
  • the compounds of the present invention have been confirmed by physical and chemical properties, mass spectrometry, nuclear magnetic, ultraviolet and infrared spectroscopy, and the structures are as follows:
  • Mass spectrometer Macromass GCT time-of-flight mass spectrometry (TOF) (Waters Corporation, USA);
  • Liquid chromatography mass spectrometer Finnigan LCQ advantage MAX (American Thermoelectric Corporation);
  • HPLC 20 mg of pure product in 1 ml of dichloromethane solution, Agilent 1100 HPLC, column: Agilent Zorbax SB-C18 (250 mm ⁇ 4.6 mm, id 5 ⁇ m), dichloromethane-acetonitrile (35:65, v/v) Mobile phase, injection volume 10 ⁇ l.
  • LC-MS Determined on a Finnigan LCQ advantage MAX in APCI positive ion mode according to liquid phase conditions.
  • the compound of the invention is a pale yellow oily liquid at room temperature.
  • the invention also includes pharmaceutical compositions containing a compound of the invention.
  • the pharmaceutical composition of the present invention is preferably in the form of a unit dose of a pharmaceutical preparation, which may contain the pharmaceutically active substance of the present invention in an amount of 0.1 mg to 1000 mg, the balance being a pharmaceutically acceptable carrier, and the pharmaceutically acceptable carrier may be used by weight. It is from 0.01% to 99.99% of the total weight of the formulation.
  • It is also an object of the present invention to provide a pharmaceutical preparation comprising at least one active ingredient together with one or more pharmaceutically acceptable carriers, wherein the active ingredient comprises 1-palmitic acid-2-linoleic acid-3- Oleic acid glyceride.
  • the 1-palmitic acid-2-linoleic acid-3-oleic acid glyceride has the structure of the formula (I).
  • the pharmaceutically acceptable carrier includes conventional diluents, excipients, fillers, emulsifiers, binders, lubricants, absorption enhancers, surfactants, disintegrants, lubricants, stabilizers in the pharmaceutical field. Etc., if necessary, flavoring agents, sweeteners, preservatives, and coloring agents may also be added.
  • the pharmaceutically acceptable carrier may be selected from the group consisting of: mannitol, sorbitol, sodium metabisulfite, sodium hydrogen sulfite, sodium thiosulfate, cysteine hydrochloride, thioglycolic acid, methionine, soybean phospholipid, vitamin C.
  • vitamin E disodium EDTA, calcium EDTA, monovalent alkali metal carbonate, acetate, phosphate or its aqueous solution, hydrochloric acid, acetic acid, sulfuric acid, phosphoric acid, amino acid, sodium chloride, potassium chloride, sodium lactate , hydroxyethyl ester solution, benzoic acid, potassium sorbate, chlorhexidine acetate, xylitol, maltose, glucose, fructose, dextran, glycine, starch, sucrose, lactose, mannitol, silicon derivatives, fiber And its derivatives, alginate, gelatin, polyvinylpyrrolidone, glycerin, Tween 80, agar, calcium carbonate, calcium bicarbonate, surfactant, polyethylene glycol, cyclodextrin, ⁇ -cyclodextrin, Phospholipid materials, kaolin, talc, calcium stearate, magnesium stearate, and
  • the pharmaceutical preparation of the present invention may be an oral solid preparation, an oral liquid preparation or an injection.
  • the oral solid preparation is selected from the group consisting of a capsule, a tablet, a dropping pill, a granule, and a concentrated pill;
  • the oral liquid preparation is selected from aqueous or oily suspensions, solutions, emulsions, syrups or elixirs, or a dry product which can be formulated with water or other suitable carrier before use;
  • the injection is selected from the group consisting of an injection solution, a lyophilized powder injection, and an aqueous injection.
  • the pharmaceutical preparation of the present invention is preferably an injection comprising the following ingredients:
  • the preparation method of the above injection comprises the following steps:
  • Another method is to take 1-palmitic acid-2-linoleic acid-3-oleic acid glyceride, and the weighed glyceride and the aqueous phase are separately heated to 60-70 ° C, and then placed in a high-pressure homogenizer for high-pressure emulsification.
  • the homogenizer has a low pressure of 5 to 12 MPa, a high pressure of 25 to 50 MPa, a recycling homogenization of 3 to 6 times, and a particle size of not more than 95% to 2 ⁇ m or less. Particles of 5 ⁇ m or more are not detected, and pH is adjusted with NaOH or HCl if necessary.
  • the uniform emulsion nitrogen gas is pressurized and filtered through a microporous filter filter of ⁇ 3 ⁇ m, sterilized by nitrogen filling, and cooled.
  • the pharmaceutical preparation of the present invention is preferably a capsule comprising the following ingredients:
  • the preparation method of the above capsule comprises the following steps:
  • Preparation of glue Weigh the appropriate amount of gelatin, purified water, glycerin and preservative according to the weight ratio of 1:0.6 ⁇ 1.2:0.3 ⁇ 0.8:0.0001 ⁇ 0.01; add glycerin, purified water and preservative to the plasticization tank in turn, heat After the temperature reaches 70 ° C ⁇ 90 ° C, add gelatin and stir constantly, vacuum until the gelatin is completely dissolved, filter the glue, store at 56 ⁇ 62 ° C, and set aside;
  • Preparation of the drug solution adding a formula amount of 1-palmitic acid-2-linoleic acid-3-oleic acid glyceride, an antioxidant and/or an emulsifier into the batching tank, and stirring constantly until the mixture is uniform;
  • Pressure capsule Select the appropriate pellet mold according to the size of the capsule, dry it at 15 ⁇ 30°C and relative humidity ⁇ 35%, then remove the size pellets, then wash the pellets with 95% medicinal ethanol and continue to dry. To the water content of less than 12%, visual inspection to remove the unqualified capsules, printing, packaging is available.
  • the preservative is selected from the group consisting of 10% hydroxyethyl ester solution, benzoic acid, potassium sorbate and chlorhexidine acetate;
  • the antioxidant is vitamin E and the emulsifier is Tween 80.
  • the invention shows that 1-palmitic acid-2-linoleic acid-3-oleic acid glyceride and its preparation have different degrees of inhibitory effects on seven human tumor cell lines, and can be used as a medicine for treating tumor diseases.
  • Cell lines PANC-1 (human pancreatic cancer cells), SKOV3 (human ovarian cancer cells), MCF-7 (human breast cancer cells), SMMC-7721 (human liver cancer cells), A549 (human lung cancer cells), BGC-823 (human gastric cancer cells), COLO205 (human colon cancer cells).
  • PANC-1 human pancreatic cancer cells
  • SKOV3 human ovarian cancer cells
  • MCF-7 human breast cancer cells
  • SMMC-7721 human liver cancer cells
  • A549 human lung cancer cells
  • BGC-823 human gastric cancer cells
  • COLO205 human colon cancer cells
  • Trypsin purchased from Invitrogen, stored at -20 °C.
  • PBS Phosphate buffer
  • MTT (AMRESCO) solution A solution of 5 mg/ml was prepared with PBS.
  • Dissolving solution 10 ml of SDS, 5 ml of isobutanol and 0.1 ml of concentrated hydrochloric acid per 100 ml of deionized double distilled water.
  • the inhibitory effect of the compound of the present invention on the above cell strain was measured by the MTT method. Specific steps are as follows:
  • Cell culture 1 The cells were taken out from liquid nitrogen, rapidly thawed in a 37 ° C water bath, and the cells were transferred into a 10 ml sterile centrifuge tube in a sterile table to add 6 ml of cell culture medium, and the hearts were centrifuged at 1000 rpm for 5 minutes. Discard the supernatant, add 5-6 ml of cell culture medium to the pellet, pipette it to suspend it, transfer it to the cell culture flask, and place it in a 37 °C cell culture incubator.
  • the cells are trypsinized and washed, suspended in a medium containing 10% calf serum, counted by the trypan blue staining method, and the cell suspension is adjusted. Density to 2 x 10 5 cells/ml.
  • Figure 1 EI mass spectrum of 1-palmitic acid-2-linoleic acid-3-oleic acid glyceride.
  • Figure 2 APCI mass spectrum of 1-palmitic acid-2-linoleic acid-3-oleic acid glyceride.
  • Figure 3 is an infrared spectrum of 1-palmitic acid-2-linoleic acid-3-oleic acid glyceride.
  • Figure 4 1 H-NMR spectrum of 1 -palmitic acid-2-linoleic acid-3-oleic acid glyceride.
  • Figure 5 is a 13 C-NMR chart of 1-palmitic acid-2-linoleic acid-3-oleic acid glyceride.
  • Figure 7 HMBC spectrum of 1-palmitic acid-2-linoleic acid-3-oleic acid glyceride.
  • 1000g of coix seed with water content ⁇ 10% is pulverized into 100 mesh, extracted by supercritical carbon dioxide extraction device, and the coix seed powder is charged into the extraction kettle, and the carbon dioxide preheater, the extraction kettle and the separation column are heated to make the extraction temperature reach 60 ° C.
  • the extraction pressure is 24Mpa
  • the separation temperature reaches 60°C
  • the separation pressure is 15Mpa
  • the supercritical carbon dioxide fluid after the extraction of coix seed oil enters the analytical reactor analysis cycle.
  • the analytical kettle can be set up according to the need of 5, the analytical temperature is 50°C, and the analytical pressure is 6Mpa.
  • the carbon dioxide flow rate was maintained at 3000 L/h, and the extraction was continued for 4 hours.
  • the crude oil of the coix seed was separated from the lower end of the separation column;
  • the above-mentioned coix seed crude oil is added to petroleum oil with an oil weight of 58%, and then an appropriate amount of 2% NaOH solution is added for alkali refining.
  • the organic phase is washed with purified water, and after washing with water, the organic phase is added with an appropriate amount of acetone to break the emulsion.
  • the upper oil solution is taken, and the activated neutral alumina is added with an oil weight of 3%-8%, and the filtrate is heated to 46 ° C, and then filtered with activated oil of 6% by weight, and the filtrate is decompressed to recover the solvent, and then the oil is added.
  • the oil was filtered by 12% activated neutral alumina, and the filtered oil was further sterilized by dry heat at 160 ° C for 2 h. After cooling, it was filtered through a ⁇ 0.2 ⁇ m microporous membrane to obtain coix seed oil.
  • 1000g of glutinous rice with water content ⁇ 10% is pulverized into 300 mesh, extracted by supercritical carbon dioxide extraction device, and the barley powder is charged into the extraction kettle, and the carbon dioxide preheater, the extraction kettle and the separation column are heated to make the extraction temperature reach 50 °C.
  • the extraction pressure is 20Mpa
  • the separation temperature reaches 45°C
  • the separation pressure is 12Mpa
  • the supercritical carbon dioxide fluid after the extraction of coix seed oil enters the analytical reactor analysis cycle.
  • the analytical kettle can be set up according to requirements, the analytical temperature is 45°C, and the analytical pressure is 5Mpa.
  • the carbon dioxide flow rate was maintained at 2000 L/h, and the extraction was continued for 3 hours.
  • the crude oil of the coix seed was separated from the lower end of the separation column;
  • test sample using mobile phase B to prepare a 0.1 g/ml hazelnut oil solution
  • the chromatographic peak fraction was collected for a retention time of about 17.3 min, and the solvent was drained under reduced pressure at a low temperature to obtain a crude product of 1-palmitic acid-2-linoleic acid-3-oleic acid glyceride.
  • Soybean phospholipid for injection 25g
  • soy lecithin for injection add appropriate amount of water for injection, disperse it to a block-free and granular solid with a high-shear dispersing emulsifier, add glycerin for injection, which is weighed according to the formula, and add water for injection. To the specified amount, stir evenly;
  • Another method is to take 1-palmitic acid-2-linoleic acid-3-oleic acid glyceride, and the weighed oil ester and water phase are separately heated to 65 ° C, and then placed in a high-pressure homogenizer for high-pressure emulsification, homogenization during emulsification.
  • Machine low pressure is 9MPa
  • high pressure is 35MPa
  • recycling is homogeneous 4 times, to 2 ⁇ m below particles should be not less than 95%, particles above 5 ⁇ m should not be detected, if necessary, adjust pH to 6.8 with NaOH or HCl;
  • the uniform emulsion nitrogen gas is pressurized and filtered through a microporous filter filter of ⁇ 3 ⁇ m, sterilized by nitrogen filling, and cooled.
  • Preparation of glue Weigh the appropriate amount of gelatin, purified water, glycerin and potassium sorbate according to the weight ratio of 1:0.9:0.6:0.005; add glycerin, purified water and potassium sorbate to the gelatin tank in turn, and heat to 80 °C. Then add gelatin and stir constantly, vacuum until the gelatin is completely dissolved, filter the glue, store at 60 ° C, and set aside;
  • Preparation of the drug solution adding a formula amount of 1-palmitic acid-2-linoleic acid-3-oleic acid glyceride and vitamin E into the ingredient tank, and stirring continuously until evenly mixed;
  • Pressure capsule select the appropriate pellet mold according to the size of the capsule, dry it at 25 ° C, relative humidity ⁇ 35%, and then remove the size pellets, then wash the pellets with 95% medicinal ethanol, and continue to dry until The amount of water is less than 12%.
  • the unqualified capsules are removed by visual inspection, printed and packaged.
  • soy lecithin for injection add appropriate amount of water for injection, disperse it to a block-free and granular solid with a high-shear dispersing emulsifier, add glycerin for injection, which is weighed according to the formula, and add water for injection. To the specified amount, stir evenly;
  • Another method is to take 1-palmitic acid-2-linoleic acid-3-oleic acid glyceride, and the weighed oil ester and water phase are separately heated to 60 ° C, and then placed in a high pressure homogenizer for high pressure emulsification, homogenization during emulsification.
  • the machine has a low pressure of 7 MPa, a high pressure of 30 MPa, and a homogenization of 6 times.
  • the particles below 2 ⁇ m should be not less than 95%. Particles above 5 ⁇ m should not be detected. If necessary, adjust the pH to 4.8 with NaOH or HCl.
  • the uniform emulsion nitrogen gas is pressurized and filtered through a microporous filter filter of ⁇ 3 ⁇ m, sterilized by nitrogen filling, and cooled.
  • soy lecithin for injection add appropriate amount of water for injection, disperse it to a block-free and granular solid with a high-shear dispersing emulsifier, add glycerin for injection, which is weighed according to the formula, and add water for injection. To the specified amount, stir evenly;
  • the uniform emulsion nitrogen gas is pressurized and filtered through a microporous filter filter of ⁇ 3 ⁇ m, and the nitrogen is filled and sterilized, and the cooling is performed. Got it.
  • soy lecithin for injection add appropriate amount of water for injection, disperse it to a block-free and granular solid with a high-shear dispersing emulsifier, add glycerin for injection, which is weighed according to the formula, and add water for injection. To the specified amount, stir evenly;
  • Another method is to take 1-palmitic acid-2-linoleic acid-3-oleic acid glyceride, and the weighed oil ester and water phase are separately heated to 68 ° C, and then placed in a high-pressure homogenizer for high-pressure emulsification, homogenization during emulsification.
  • Machine low pressure is 7MPa
  • high pressure is 30MPa
  • recycling is homogeneous 5 times, to 2 ⁇ m or less particles should be not less than 95%, particles above 5 ⁇ m should not be detected, if necessary, adjust the pH to 7.1 with NaOH or HCl;
  • the uniform emulsion nitrogen gas is pressurized and filtered through a microporous filter filter of ⁇ 3 ⁇ m, sterilized by nitrogen filling, and cooled.
  • Preparation of glue Weigh the appropriate amount of gelatin, purified water, glycerin and 10% hydroxyethyl ester solution according to the weight ratio of 1:1.2:0.8:0.01; add glycerin, purified water and 10% hydroxyethyl ester solution to the rubber In the tank, after heating to 70 ° C, add gelatin and stir constantly, vacuum until the gelatin is completely dissolved, filter the glue, store at 62 ° C, and set aside;
  • Preparation of the drug solution adding a formula amount of 1-palmitic acid-2-linoleic acid-3-oleic acid glyceride and vitamin E into the ingredient tank, and stirring continuously until evenly mixed;
  • Pressure capsule select the appropriate pellet mold according to the size of the capsule, and then dry it at 15 °C and relative humidity ⁇ 35%, then remove the size pellets, then wash the pellets with 95% medicinal ethanol, and continue to dry until The amount of water is less than 12%.
  • the unqualified capsules are removed by visual inspection, printed and packaged.
  • Preparation of glue Weigh the appropriate amount of gelatin, purified water, glycerin and benzoic acid at a weight ratio of 1:1.2:0.8:0.01; add glycerin, purified water and benzoic acid to the gelatin tank in turn, heat to 90 °C, then Add gelatin and stir constantly, vacuum until the gelatin is completely dissolved, filter the glue, store at 56 ° C, and set aside;
  • Pressure capsule select the appropriate pellet mold according to the size of the capsule, dry it at 30 ° C, relative humidity ⁇ 35%, and then remove the size pellets, then wash the pellets with 95% medicinal ethanol, and continue to dry until The amount of water is less than 12%.
  • the unqualified capsules are removed by visual inspection, printed and packaged.
  • Preparation of glue Weigh the appropriate amount of gelatin, purified water, glycerin and chlorhexidine acetate according to the weight ratio of 1:1.0:0.5:0.008; add glycerin, purified water and chlorhexidine acetate to the gelatin tank in turn, and heat to 85 After °C, add gelatin and stir constantly, vacuum until the gelatin is completely dissolved, filter the glue, store at 58 °C, and set aside;
  • Pressure capsule select the appropriate pellet mold according to the size of the capsule, dry it at 25 ° C, relative humidity ⁇ 35%, and then remove the size pellets, then wash the pellets with 95% medicinal ethanol, and continue to dry until The amount of water is less than 12%.
  • the unqualified capsules are removed by visual inspection, printed and packaged.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Epidemiology (AREA)
  • Oil, Petroleum & Natural Gas (AREA)
  • Organic Chemistry (AREA)
  • Wood Science & Technology (AREA)
  • Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Analytical Chemistry (AREA)
  • General Chemical & Material Sciences (AREA)
  • Dermatology (AREA)
  • Emergency Medicine (AREA)
  • Dispersion Chemistry (AREA)
  • Nutrition Science (AREA)
  • Physiology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)
  • Medicinal Preparation (AREA)
  • Medicines Containing Plant Substances (AREA)

Abstract

本发明涉及一种具有式（Ⅰ）结构的化合物，即1-棕榈酸-2-亚油酸-3-油酸甘油酯，本发明还包括以薏苡仁粉为原料经过薏苡仁油的制备、1-棕榈酸-2-亚油酸-3-油酸甘油酯的初步分离、二次分离得到本发明化合物的方法，本发明还涉及含有式（Ⅰ）结构的化合物的药物制剂，其制备方法，及上述化合物和制剂在抗肿瘤药物中的应用。

Description

化合物1-棕榈酸-2-亚油酸-3-油酸甘油酯、制剂、制备方法及其应用 技术领域
本发明涉及有机化学和药学领域，具体而言，本发明涉及化合物1-棕榈酸-2-亚油酸-3-油酸甘油酯、制剂、制备方法及其在制备抗肿瘤药物中的应用。
背景技术
薏苡仁是禾本科薏苡属植物薏苡Coix lacryma-jobi L.var ma-yuen(Roman.)Stapf的成熟干燥种仁，为利水渗湿药，很久以来为药食两用品种。现代研究发现薏苡仁具有镇痛抗炎、免疫调节、抗溃疡、降血脂和减肥等药理作用。近年来，国内外学者通过TLC、HPLC-MS、GC等方法对薏苡仁的化学成分进行了研究，发现其主要的活性成分包括酯类、不饱和脂肪酸类、糖类及内酰胺类等。其中，酯类是首先被发现的具有抗肿瘤活性的成分，也是报道最多最受关注的化学成分，在中国临床上已经得到普遍应用的康莱特注射液的有效成分便是提自薏苡仁中的酯类。杨玲等[食品科学,2001,22(5):59-62]研究表明，薏苡仁油脂中甘油三酯的摩尔百分比在87.64％-97.66％之间，其次为甘油一酯、甘油二酯和脂肪酸酯。同时，进一步研究表明，薏苡仁油甘油三酯中，相对含量不低于3％的的成分多达8种，如三亚油酸甘油酯(1)、二亚油酸油酸甘油酯(2)、棕榈酸二亚油酸甘油酯(3)、亚油酸二油酸甘油酯(4)、棕榈酸亚油酸油酸甘油酯(5)、二棕榈酸亚油酸甘油酯(6)、三油酸甘油酯(7)和棕榈酸二油酸甘油酯，约占甘油三酯总量的90％以上。由此可见，薏苡仁油脂成分的复杂性，这必然会使实际生产过程中的质量控制和临床用药安全面临很大的挑战。
因此，开发一种安全、有效、可控的治疗肿瘤的药物即成为本发明关注的热点。本发明对上述薏苡仁油甘油三酯成分之一——棕榈酸亚油酸油酸甘油酯进行了分离，首次制备得到了一种具有式(Ⅰ)结构的化合物，即1-棕榈酸-2-亚油酸-3-油酸甘油酯，目前尚未见关于此化合物及其药物制剂用于治疗肿瘤的相关报道。
发明内容：
本发明的目的在于提供一种具有式(Ⅰ)结构化合物，即1-棕榈酸-2-亚油酸-3-油酸甘油酯：
Figure PCTCN2015084290-appb-000001
本发明的又一目的在于提供一种含有式(Ⅰ)结构化合物的药物组合物，其还可含有药学上可接受的载体。
本发明的目的还在于提供一种具有式(Ⅰ)结构化合物的制备方法，其包括如下步骤：
(1)薏苡仁油的制备；
(2)1-棕榈酸-2-亚油酸-3-油酸甘油酯的初步分离；
(3)1-棕榈酸-2-亚油酸-3-油酸甘油酯的二次分离；
其中，
步骤(1)中薏苡仁油的制备方法为：
取水分≤10％的薏苡仁粉碎成10～300目，采用超临界流体萃取，分离得到薏苡仁粗油；加入薏苡仁粗油重量40％～58％的石油醚，及适量碱溶液碱炼，分液后取有机相，加入中性氧化铝和/或高岭土，过滤，150～180℃减压干热灭菌，冷却过滤，得薏苡仁油；
优选的，
所述超临界流体萃取溶媒为二氧化碳，萃取温度30～60℃，萃取压力19～24Mpa，分离温度30～60℃，分离压力6～15Mpa，二氧化碳流量10～5000L/h，连续萃取1～4小时；
所述碱溶液可以为0.5％～3％的氢氧化钾、氢氧化钠的水溶液；
所述加入中性氧化铝和/或高岭土的过程为：首先加入油重3％～8％的活化中性氧化铝过滤，滤液加热至40～50℃，又加入油重2％～6％经活化的高岭土过滤，减压除溶剂，再加入油重8％～12％活化中性氧化铝过滤即可。
更优选的，
取水分≤10％的薏苡仁粉碎成10～300目，采用超临界二氧化碳萃取装置萃取，将薏苡仁粉装入萃取釜，加热二氧化碳预热器、萃取釜和分离柱，使萃取温度达到30～60℃，萃取压力19～24Mpa，分离温度达到30～60℃，分离压力6～15Mpa，萃取薏苡仁油后的超临界二氧化碳流体进入解析釜解析循环，解析釜可根据需要设一至多个，控制解析温度30～60℃，解析压力2～6Mpa，整个循环保持二氧化碳流量10～5000L/h，连续萃取1～4h，自分离柱下端分离得到薏苡仁粗油；
将上述薏苡仁粗油加入油重量40％～58％的石油醚，再加入适量的2％NaOH溶液碱炼，分液后取有机相用纯化水进行水洗，取水洗后有机相加适量丙酮破乳后，取上层油溶液，加入油重3％～8％的活化中性氧化铝过滤，取滤液加热到40～50℃后，加入油重2％～6％经活化的高岭土过滤。滤液减压回收溶剂后，再加油重8％～12％活化中性氧化铝过滤得油，过滤后的油再经150～180℃减压干热灭菌1～3h，冷却后经≤0.2μm微孔滤膜过滤，得薏苡仁油。
其中，
步骤(2)中1-棕榈酸-2-亚油酸-3-油酸甘油酯的初步分离方法为：
采用Gilson制备型高效液相色谱仪和蒸发光散射检测器联用；制备型色谱柱为Agilent Zorbax SB-C18(250mm×21.2mm,7μm)；流动相为流动相A(乙腈)和流动相B(四氢呋喃：乙腈＝1:1)，采用梯度条件洗脱：流动相B：0～27min，50％→60％，27～35min，60％→90％，35～45min，90％→100％；流速：18ml/min，进样体积为200～300μl；收集响应设置：peak level min＝30mV；
供试品配制：用流动相B配制成0.1g/ml的薏苡仁油溶液；
收集保留时间约为17.3min色谱峰流分，低温减压抽干溶剂。
其中，
步骤(3)中1-棕榈酸-2-亚油酸-3-油酸甘油酯的二次分离方法为：
一次制备条件为：Agilent 1100分析型高效液相色谱仪，DAD检测器，，色谱柱：Venusil XBP C18(L)(10*250mm，5μm，
Figure PCTCN2015084290-appb-000002
)，流动相，乙腈：四氢呋喃＝75:25，流速：4mL/min；检测波长：205nm；供试品配制：取1-棕榈酸-2-亚油酸-3-油酸甘油酯的初步分离样品，加入适量正己烷，超声促溶后备用。进样量：60μl，收集保留时间约为27.3min的色谱峰流分，将此流分氮吹浓缩。
二次制备条件为：色谱柱，Venusil CA(10*250mm，5μm，
Figure PCTCN2015084290-appb-000003
)，流动相，正己烷：乙醇＝95:5，紫外检测波长：205nm，流速：4mL/min；供试品配制：取一次制备的流分样品若干，加入适量正己烷，超声促溶后备用；进样量：60μl，收集保留时间约为2.5min～5min色谱峰，氮吹浓缩、低温冻存。
本发明化合物经过理化性质、质谱、核磁、紫外和红外光谱的鉴定，确证了其结构，具体如下：
1.实验仪器
高效液相色谱仪：Agilent 1100(DAD,ELSD)(美国安捷伦公司)；
核磁共振波谱仪：BRUKER AVANCE 500MHz(美国布鲁克公司)；
质谱仪：Macromass GCT飞行时间质谱(TOF)(美国沃特世公司)；
Finnigan TRACE DSQ-MS(美国热电公司)；
液相质谱联用仪：Finnigan LCQ advantage MAX(美国热电公司)；
红外光谱仪：Nicolet 6700(美国热电公司)；
旋光仪：Jasco P-1030(日本JASCO公司)。
2.实验方法
HPLC：取纯品20mg于1ml二氯甲烷溶液中，Agilent 1100HPLC，色谱柱：Agilent Zorbax SB-C18(250mm×4.6mm，i.d.5μm)，二氯甲烷-乙腈(35:65，v/v)为流动相，进样量10μl。
LC-MS：按液相条件，在Finnigan LCQ advantage MAX上以APCI正离子模式测定。
IR：将所得纯品分别涂膜于溴化钾晶体表面，在红外光谱仪Nicolet 6700上测定。
HR-EI与Qu.-EI：取所得纯品5μg，涂于进样池中，于Macromass GCT飞行时间质谱(TOF)上，以Source Temp:200℃；Sample Temp:400℃；DIRECT，70eV测定。同样以Source Temp:250℃；Sample Temp:250℃；DIRECT，70eV条件在Finnigan TRACE DSQ-MS上测定。
NMR：将所得纯品取30～40mg，置于核磁管中，加0.5ml氘代氯仿(CDCl3)，注入高纯氩气，在氩气保护下用酒精喷灯将核磁管熔融密闭，于BRUKER 500M核磁共振波谱仪上测定1H-NMR，13C-NMR，HSQC，HMBC。
比旋光度：取纯品适量溶于1.50ml氯仿中，摇匀，于JASCO P-1030上测定。
3.谱图解析
室温下，本发明化合物为淡黄色油状液体。
HR-EI-MS：m/z＝856.7519(理论值＝856.7520，C55H100O6)，不饱和度＝6(见图1)。
APCI-MS：准分子离子信号[M+H]+＝857.5，同时APCI+出现[M+H-棕榈酸]+＝601.3,[M+H-亚油酸]+＝577.1，[M+H-油酸]+＝575.3碎片离子，推断其有棕榈酸，油酸，亚油酸3种脂肪酰链(见图2)。
IR(KBr flim)：1747，1164，1098；2925，2854，723；3008，1655cm-1(弱)(见图3)。
1H-NMR：δ5.27(m,1H)，4.30(dd,J＝4.29Hz，J＝11.9Hz,2H)，4.15(dd,J＝5.9Hz,J＝11.9Hz,2H)，2.31(m，6H)，2.77(t，2H)，5.35(m，6H)，0.88(m，9H)。
13C-NMR：δ14.12，14.07，22.69，22.58，22.71，24.85，24.87，24.88，25.64，27.18，27.21，27.23，29.06，29.78，31.54，31.92，31.94，34.04，34.06，34.2，62.11，68.91，127.9，128.09，129.71，129.98，130.01，130.22，172.86，173.28，173.32(见图5)。
本发明还包括，含有本发明化合物的药物组合物。
本发明的药物组合物，优选的是单位剂量的药物制剂形式，其可含有本发明的药物活性物质0.1mg-1000mg，其余为药学上可接受的载体，药学上可接受的载体以重量计可以是制剂总重量的0.01％-99.99％。
本发明的目的还在于提供一种药物制剂，含有至少一种活性成分以及一种或几种药学上可接受的载体，其中所述活性成分含有1-棕榈酸-2-亚油酸-3-油酸甘油酯。
其中，
所述1-棕榈酸-2-亚油酸-3-油酸甘油酯的结构如式(Ⅰ)。
Figure PCTCN2015084290-appb-000004
所述药学上可接受的载体包括药学领域常规的稀释剂、赋形剂、填充剂、乳化剂、粘合剂、润滑剂、吸收促进剂、表面活性剂、崩解剂、润滑剂、稳定剂等，必要时还可以加入香味剂、甜味剂、防腐剂及着色剂等。
具体的，所述药学上可接受的载体可以选自：甘露醇、山梨醇、焦亚硫酸钠、亚硫酸氢钠、硫代硫酸钠、盐酸半胱氨酸、巯基乙酸、蛋氨酸、大豆磷脂、维生素C、维生素E、EDTA二钠、EDTA钙钠，一价碱金属的碳酸盐、醋酸盐、磷酸盐或其水溶液、盐酸、醋酸、硫酸、磷酸、氨基酸、氯化钠、氯化钾、乳酸钠、羟苯乙酯溶液、苯甲酸、山梨酸钾、醋酸氯己定、木糖醇、麦芽糖、葡萄糖、果糖、右旋糖苷、甘氨酸、淀粉、蔗糖、乳糖、甘露糖醇、硅衍生物、纤维素及其衍生物、藻酸盐、明胶、聚乙烯吡咯烷酮、甘油、吐温80、琼脂、碳酸钙、碳酸氢钙、表面活性剂、聚乙二醇、环糊精、β-环糊精、磷脂类材料、高岭土、滑石粉、硬脂酸钙、硬脂酸镁等。
优选的，
本发明所述药物制剂可以是口服固体制剂、口服液体制剂或注射剂。
其中，
所述口服固体制剂选自胶囊剂、片剂、滴丸、颗粒剂、浓缩丸中的一种；
所述口服液体制剂选自水性或油性悬浮液、溶液、乳剂、糖浆剂或酏剂，或是一种在使用前可用水或其它适宜的载体复配的干燥产品；
所述注射剂选自注射液、冻干粉针剂和水针剂中的一种。
其中，
本发明所述药物制剂优选为注射剂，包括如下成分：
1-棕榈酸-2-亚油酸-3-油酸甘油酯  100-300g
注射用大豆磷脂或可供注射用大豆磷脂  10-40g
注射用甘油或可供注射用甘油  15-50g
注射用水加至  1000ml
上述注射剂的制备方法包括如下步骤：
称取处方配制量的注射用大豆磷脂或可供注射用大豆磷脂，加适量的注射用水后，用高剪切分散乳化机分散至无块状及颗粒状固体，加入按配方量称取的注射用甘油或可供注射用甘油，并加注射用水至规定量，搅拌均匀备用；
另称取1-棕榈酸-2-亚油酸-3-油酸甘油酯，将称取的甘油酯与水相分别加热至60～70℃后，置高压均质机进行高压乳化，乳化时均质机低压为5～12MPa，高压为25～50MPa，再循环均质3～6遍，至2μm以下颗粒应不少于95％，5μm以上颗粒不得检出，必要时用NaOH或HCl调节pH至4.8～8.5，优选6.8～7.0，最优选为6.8；
将制得均匀乳剂氮气加压通过≤3μm的微孔滤芯过滤器过滤，充氮灌装灭菌，冷却即得。
其中，
本发明所述药物制剂优选为胶囊剂，包括如下成分：
1-棕榈酸-2-亚油酸-3-油酸甘油酯  200-800g
抗氧化剂和/或乳化剂  0.20-0.60g
制成  1000粒
上述胶囊剂的制备方法包括如下步骤：
胶液配制：按重量比为1:0.6～1.2:0.3～0.8:0.0001～0.01称取适量明胶、纯化水、甘油和防腐剂；依次将甘油、纯化水、防腐剂加入化胶罐内，加热至70℃～90℃后，再加入明胶不断搅拌、抽真空，直至明胶完全溶解，将胶液过滤，在56～62℃下存放，备用；
药液配制：将配方量的1-棕榈酸-2-亚油酸-3-油酸甘油酯、抗氧化剂和/或乳化剂加入配料罐内，不断搅拌，直至混合均匀；
压胶囊：根据胶囊大小选择合适的压丸模具，在15～30℃、相对湿度<35％条件下进行压丸定型后干燥，剔除大小丸，再用95％药用乙醇洗丸后，继续干燥至含水量小于12％，目检剔除不合格胶囊，印字，包装即得。
其中，
所述防腐剂选自10％羟苯乙酯溶液、苯甲酸、山梨酸钾和醋酸氯己定中的一种；
所述抗氧化剂为维生素E，乳化剂为吐温80。
本发明通过药效实验表明，1-棕榈酸-2-亚油酸-3-油酸甘油酯及其制剂对7种人肿瘤细胞株均有不同程度的抑制作用，可作为治疗肿瘤疾病药物。
因此，本发明的又一目的在于提供具有式(Ⅰ)结构的化合物及其制剂在制备抗肿瘤药物中的应用，尤其是在治疗乳腺癌、胰腺癌、卵巢癌、肝癌、肺癌、胃癌和结肠癌中的应用。
以下通过实验数据说明本发明所述化合物及其制剂在抗肿瘤方面的有益效果。
1、实验目的
在体外用MTT法测定本发明人化合物及其制剂对7种人肿瘤细胞株的抑制作用。
2、实验材料及配制
(1)细胞株：PANC-1(人胰腺癌细胞)、SKOV3(人卵巢癌细胞)、MCF-7(人乳腺癌细胞)、SMMC-7721(人肝癌细胞)、A549(人肺癌细胞)、BGC-823(人胃癌细胞)、COLO205(人结肠癌细胞)。上述细胞株由上海医药工业研究药理评价研究中心保存，传代维持。
(2)DMEM完全培养基：添加10％新生牛血清(GIBCO BRL)、双抗；
(3)0.25％胰蛋白酶溶液(Trypsin)：购自Invitrogen公司，-20℃保存。
(4)磷酸缓冲液(PBS)：NaCl 8g，KCl 0.2g，Na2HPO41.15g，KH2PO40.2g，溶于1L双蒸水，121℃高压消毒20min，4℃保存。
(5)MTT(AMRESCO)溶液：用PBS配成5mg/ml溶液。
(6)溶解液：每100ml去离子双蒸水含SDS 10g，异丁醇5ml，浓盐酸0.1ml。
3、实验方法
本发明化合物对上述细胞株的抑制作用通过MTT法测得。具体步骤如下：
(1)细胞培养：①将细胞从液氮中取出，在37℃水浴中迅速解冻，细胞在无菌操作台中移入10ml无菌离心管中加6ml细胞培养基，1000转/分离心5分钟。弃去上清液，沉淀中加入5～6ml细胞培养基，滴管吹打使其悬浮后移入细胞培养瓶中，置37℃细胞培养箱内。②次日，自培养箱中取出细胞，弃去细胞瓶中细胞培养基，加入5～6ml细胞培养基，置37℃细胞培养箱内。③隔日，自培养箱中取出细胞，弃去细胞瓶中细胞培养基，加入PBS(PH7.4)2～3ml晃动清洗，倒掉PBS溶液后再重复一次清洗。在培养瓶中加入3～5滴0.25％胰蛋白酶溶液晃动均匀，加盖置于37℃细胞培养箱内3分钟左右，于显微镜下观察发现细胞自培养瓶壁上脱离，加细胞培养基2ml，滴管吹打使细胞完全脱离瓶壁后，分别移入2个干净培养瓶中，加入细胞培养基5～6ml吹打均匀，置于37℃细胞培养箱内。④隔日，重复③步骤。在整个培养过程中，贴壁细胞不允许生长过密，悬浮细胞始终保持对数生长期。
(2)样品及对照品制备：称取适量本发明化合物样品溶解于DMSO中，得到浓度为100mg/ml的溶液。再用PBS作梯度稀释，得到浓度10mg/ml、5000μg/ml、2500μg/ml、1250μg/ml、625μg/ml、312.5μg/ml的稀释样品。
(3)将稀释好的样品加入平底96孔板中，每孔10μl，每点作两个平行测试。将DMSO相应作梯度稀释后加入板中，作为对照。
(4)取处于对数生长期的细胞，细胞经胰酶消化并洗涤后悬浮于含10％小牛血清的培养基中，经苔盼蓝染色排除法计活细胞数，并调节细胞悬浮液密度至2×105细胞/ml。
(5)将加入细胞的平底96孔板在37℃、5％CO2细胞培养箱中培养48小时。
(6)每孔中加入20μl 5mg/ml MTT溶液，继续在培养箱中保温3～4小时。
(7)每孔加入100μl溶解液，继续在培养箱中保温过夜，使生成的甲臢晶体充分溶解。测定570nm光吸收值。
(8)根据光吸收值计算各样品浓度组的细胞生长抑制率。计算公式如下：
(1－实验孔平均光吸收值/对照孔平均光吸收值)×100％
4、实验结果
表1.不同浓度的本发明化合物对7种细胞株的生长抑制率(％)
Figure PCTCN2015084290-appb-000005
表2.本发明化合物在体外对7种细胞株的IC50(μg/ml)
Figure PCTCN2015084290-appb-000006
5、结论
实验结果表明，不同浓度的本发明化合物及其制剂，对上述7种癌细胞株均具有显著的抑制作用，可作为抗肿瘤药物应用。并且，由于本发明所述药物制剂中活性成分组成简单、含量明确，因此临床用药更为安全，给药剂量较小，生产过程质量可控。避免了现有技术中直接以提取出的薏苡仁油入药时，成分复杂所带来的较多副作用等缺陷。
附图说明
图1 1-棕榈酸-2-亚油酸-3-油酸甘油酯的EI质谱图。
图2 1-棕榈酸-2-亚油酸-3-油酸甘油酯的APCI质谱图。
图3 1-棕榈酸-2-亚油酸-3-油酸甘油酯的红外光谱图。
图4 1-棕榈酸-2-亚油酸-3-油酸甘油酯的1H-NMR谱图。
图5 1-棕榈酸-2-亚油酸-3-油酸甘油酯的13C-NMR谱图。
图6 1-棕榈酸-2-亚油酸-3-油酸甘油酯的HSQC谱图。
图7 1-棕榈酸-2-亚油酸-3-油酸甘油酯的HMBC谱图。
具体实施方式：
以下通过实施例进一步说明本发明，但不作为对本发明的限制。
实施例1薏苡仁油的制备
取水分≤10％的薏苡仁1000g粉碎成10目，采用超临界二氧化碳萃取，萃取温度40℃，萃取压力20Mpa，分离温度30℃，分离压力6Mpa，二氧化碳流量50L/h，连续萃取2h，分离得到薏苡仁粗油；加入薏苡仁粗油重量40％的石油醚，及0.5％的氢氧化钾的水溶液碱炼；分液后取有机相，首先加入油重3％的活化中性氧化铝过滤，滤液加热至45℃，又加入油重2％经活化的高岭土过滤，减压除溶剂，再加入油重8％活化中性氧化铝，过滤，170℃减压干热灭菌，冷却过滤，得薏苡仁油；
实施例2薏苡仁油的制备
取水分≤10％的薏苡仁1000g粉碎成100目，采用超临界二氧化碳萃取，萃取温度50℃，萃取压力22Mpa，分离温度40℃，分离压力8Mpa，二氧化碳流量500L/h，连续萃取3h，分离得到薏苡仁粗油；加入薏苡仁粗油重量46％的石油醚和1％的氢氧化钠的水溶液碱炼；分液后取有机相，首先加入油重5％的活化中性氧化铝过滤，滤液加热至40℃，又加入油重4％经活化的高岭土过滤，减压除溶剂，再加入油重10％活化中性氧化铝，过滤，180℃减压干热灭菌，冷却过滤，得薏苡仁油；
实施例3薏苡仁油的制备
取水分≤10％的薏苡仁1000g粉碎成200目，采用超临界二氧化碳萃取，萃取温度60℃，萃取压力24Mpa，分离温度50℃，分离压力15Mpa，二氧化碳流量1500L/h，连续萃取1.5h，分离得到薏苡仁粗油；加入薏苡仁粗油重量50％的石油醚，及2％的氢氧化钾的水溶液碱炼；分液后取有机相，首先加入油重6％的活化中性氧化铝过滤，滤液加热至50℃，又加入油重5％经活化的高岭土过滤，减压除溶剂，再加入油重10％活化中性氧化铝，过滤，150℃减压干热灭菌，冷却过滤，得薏苡仁油；
实施例4薏苡仁油的制备
取水分≤10％的薏苡仁1000g粉碎成100目，采用超临界二氧化碳萃取装置萃取，将薏苡仁粉装入萃取釜，加热二氧化碳预热器、萃取釜和分离柱，使萃取温度达到60℃，萃取压力24Mpa，分离温度达到60℃，分离压力15Mpa，萃取薏苡仁油后的超临界二氧化碳流体进入解析釜解析循环，解析釜可根据需要设5个，控制解析温度50℃，解析压力6Mpa，整个循环保持二氧化碳流量3000L/h，连续萃取4h，自分离柱下端分离得到薏苡仁粗油；
将上述薏苡仁粗油加入油重量58％的石油醚，再加入适量的2％NaOH溶液碱炼，分液后取有机相用纯化水进行水洗，取水洗后有机相加适量丙酮破乳后，取上层油溶液，加入油重3％-8％的活化中性氧化铝过滤，取滤液加热到46℃后，加入油重6％经活化的高岭土过滤，滤液减压回收溶剂后，再加入油重12％活化中性氧化铝过滤得油，过滤后的油再经160℃减压干热灭菌2h，冷却后经≤0.2μm微孔滤膜过滤，得薏苡仁油。
实施例5薏苡仁油的制备
取水分≤10％的薏苡仁1000g粉碎成300目，采用超临界二氧化碳萃取装置萃取，将薏苡仁粉装入萃取釜，加热二氧化碳预热器、萃取釜和分离柱，使萃取温度达到50℃，萃取压力20Mpa，分离温度达到45℃，分离压力12Mpa，萃取薏苡仁油后的超临界二氧化碳流体进入解析釜解析循环，解析釜可根据需要设3个，控制解析温度45℃，解析压力5Mpa，整个循环保持二氧化碳流量2000L/h，连续萃取3h，自分离柱下端分离得到薏苡仁粗油；
将上述薏苡仁粗油加入油重量48％的石油醚，再加入适量的2％NaOH溶液碱炼，分液 后取有机相用纯化水进行水洗，取水洗后有机相加适量丙酮破乳后，取上层油溶液，加入油重6％的活化中性氧化铝过滤，取滤液加热到48℃后，加入油重4％经活化的高岭土过滤，滤液减压回收溶剂后，再加油重9％活化中性氧化铝过滤得油，过滤后的油再经175℃减压干热灭菌3h，冷却后经≤0.2μm微孔滤膜过滤，得薏苡仁油。
实施例6 1-棕榈酸-2-亚油酸-3-油酸甘油酯的分离
采用Gilson制备型高效液相色谱仪和蒸发光散射检测器联用；制备型色谱柱为Agilent Zorbax SB-C18(250mm×21.2mm,7μm)；流动相为流动相A(乙腈)和流动相B(四氢呋喃：乙腈＝1:1)，采用梯度条件洗脱：流动相B：0～27min，50％→60％，27～35min，60％→90％，35～45min，90％→100％；流速：18ml/min，进样体积为200～300μl；收集响应设置：peak level min＝30mV；
供试品配制：用流动相B配制成0.1g/ml的薏苡仁油溶液；
收集保留时间约为17.3min色谱峰流分，低温减压抽干溶剂，得1-棕榈酸-2-亚油酸-3-油酸甘油酯粗品。
实施例7 1-棕榈酸-2-亚油酸-3-油酸甘油酯的二次分离
一次制备条件为：Agilent 1100分析型高效液相色谱仪，DAD检测器，，色谱柱：Venusil XBP C18(L)(10*250mm，5μm，
Figure PCTCN2015084290-appb-000007
)，流动相，乙腈：四氢呋喃＝75:25，流速：4mL/min；检测波长：205nm；供试品配制：取实施例6中制备的1-棕榈酸-2-亚油酸-3-油酸甘油酯粗品若干，加入适量正己烷，超声促溶后备用。进样量：60μl，收集保留时间约为27.3min的色谱峰流分，将此流分氮吹浓缩。
二次制备条件为：色谱柱，Venusil CA(10*250mm，5μm，
Figure PCTCN2015084290-appb-000008
)，流动相，正己烷：乙醇＝95:5，紫外检测波长：205nm，流速：4mL/min；供试品配制：取一次制备的流分样品若干，加入适量正己烷，超声促溶后备用；进样量：60μl，收集保留时间约为2.5min～5min色谱峰，氮吹浓缩、低温冻存，即得1-棕榈酸-2-亚油酸-3-油酸甘油酯。
实施例8 1-棕榈酸-2-亚油酸-3-油酸甘油酯注射液的制备
处方：
1-棕榈酸-2-亚油酸-3-油酸甘油酯200g
注射用大豆磷脂  25g
可供注射用甘油  30g
注射用水加至  1000ml
工艺：
称取处方配制量的注射用大豆磷脂，加适量的注射用水后，用高剪切分散乳化机分散至无块状及颗粒状固体，加入按配方量称取的注射用甘油，并加注射用水至规定量，搅拌均匀备用；
另称取1-棕榈酸-2-亚油酸-3-油酸甘油酯，将称取的油酯与水相分别加热至65℃后，置高压均质机进行高压乳化，乳化时均质机低压为9MPa，高压为35MPa，再循环均质4遍，至2μm以下颗粒应不少于95％，5μm以上颗粒不得检出,必要时用NaOH或HCl调节pH至6.8；
将制得均匀乳剂氮气加压通过≤3μm的微孔滤芯过滤器过滤，充氮灌装灭菌，冷却即得。
实施例9 1-棕榈酸-2-亚油酸-3-油酸甘油酯胶囊剂的制备
处方：
1-棕榈酸-2-亚油酸-3-油酸甘油酯  500g
维生素E  0.40g
制成  1000粒
工艺：
胶液配制：按重量比为1:0.9:0.6:0.005称取适量明胶、纯化水、甘油和山梨酸钾；依次将甘油、纯化水、山梨酸钾加入化胶罐内，加热至80℃后，再加入明胶不断搅拌、抽真空，直至明胶完全溶解，将胶液过滤，在60℃下存放，备用；
药液配制：将配方量的1-棕榈酸-2-亚油酸-3-油酸甘油酯、维生素E加入配料罐内，不断搅拌，直至混合均匀；
压胶囊：根据胶囊大小选择合适的压丸模具，在25℃、相对湿度<35％条件下进行压丸定型后干燥，剔除大小丸，再用95％药用乙醇洗丸后，继续干燥至含水量小于12％，目检剔除不合格胶囊，印字，包装即得。
实施例10 1-棕榈酸-2-亚油酸-3-油酸甘油酯注射液的制备
处方：
1-棕榈酸-2-亚油酸-3-油酸甘油酯  100g
注射用大豆磷脂  10g
注射用甘油  15g
注射用水加至  1000ml
工艺：
称取处方配制量的注射用大豆磷脂，加适量的注射用水后，用高剪切分散乳化机分散至无块状及颗粒状固体，加入按配方量称取的注射用甘油，并加注射用水至规定量，搅拌均匀备用；
另称取1-棕榈酸-2-亚油酸-3-油酸甘油酯，将称取的油酯与水相分别加热至60℃后，置高压均质机进行高压乳化，乳化时均质机低压为7MPa，高压为30MPa，再循环均质6遍，至2μm以下颗粒应不少于95％，5μm以上颗粒不得检出,必要时用NaOH或HCl调节pH至4.8；
将制得均匀乳剂氮气加压通过≤3μm的微孔滤芯过滤器过滤，充氮灌装灭菌，冷却即得。
实施例11 1-棕榈酸-2-亚油酸-3-油酸甘油酯注射液的制备
处方：
1-棕榈酸-2-亚油酸-3-油酸甘油酯  300g
可供注射用大豆磷脂  40g
可供注射用甘油  50g
注射用水加至  1000ml
工艺：
称取处方配制量的注射用大豆磷脂，加适量的注射用水后，用高剪切分散乳化机分散至无块状及颗粒状固体，加入按配方量称取的注射用甘油，并加注射用水至规定量，搅拌均匀备用；
另称取1-棕榈酸-2-亚油酸-3-油酸甘油酯，将称取的油酯与水相分别加热至70℃后，置高压均质机进行高压乳化，乳化时均质机低压为11MPa，高压为50MPa，再循环均质3遍，至2μm以下颗粒应不少于95％，5μm以上颗粒不得检出,必要时用NaOH或HCl调节pH至8.5；
将制得均匀乳剂氮气加压通过≤3μm的微孔滤芯过滤器过滤，充氮灌装灭菌，冷却即 得。
实施例12 1-棕榈酸-2-亚油酸-3-油酸甘油酯注射液的制备
处方：
1-棕榈酸-2-亚油酸-3-油酸甘油酯  150g
可供注射用大豆磷脂  35g
注射用甘油或  30g
注射用水加至  1000ml
工艺：
称取处方配制量的注射用大豆磷脂，加适量的注射用水后，用高剪切分散乳化机分散至无块状及颗粒状固体，加入按配方量称取的注射用甘油，并加注射用水至规定量，搅拌均匀备用；
另称取1-棕榈酸-2-亚油酸-3-油酸甘油酯，将称取的油酯与水相分别加热至68℃后，置高压均质机进行高压乳化，乳化时均质机低压为7MPa，高压为30MPa，再循环均质5遍，至2μm以下颗粒应不少于95％，5μm以上颗粒不得检出,必要时用NaOH或HCl调节pH至7.1；
将制得均匀乳剂氮气加压通过≤3μm的微孔滤芯过滤器过滤，充氮灌装灭菌，冷却即得。
实施例13 1-棕榈酸-2-亚油酸-3-油酸甘油酯胶囊剂的制备
处方：
1-棕榈酸-2-亚油酸-3-油酸甘油酯  200g
维生素E  0.20g
制成  1000粒
工艺：
胶液配制：按重量比为1:1.2:0.8:0.01称取适量明胶、纯化水、甘油和10％羟苯乙酯溶液；依次将甘油、纯化水、10％羟苯乙酯溶液加入化胶罐内，加热至70℃后，再加入明胶不断搅拌、抽真空，直至明胶完全溶解，将胶液过滤，在62℃下存放，备用；
药液配制：将配方量的1-棕榈酸-2-亚油酸-3-油酸甘油酯、维生素E加入配料罐内，不断搅拌，直至混合均匀；
压胶囊：根据胶囊大小选择合适的压丸模具，在15℃、相对湿度<35％条件下进行压丸定型后干燥，剔除大小丸，再用95％药用乙醇洗丸后，继续干燥至含水量小于12％，目检剔除不合格胶囊，印字，包装即得。
实施例14 1-棕榈酸-2-亚油酸-3-油酸甘油酯胶囊剂的制备
处方：
1-棕榈酸-2-亚油酸-3-油酸甘油酯  800g
吐温80  0.60g
制成  1000粒
工艺：
胶液配制：按重量比为1:1.2:0.8:0.01称取适量明胶、纯化水、甘油和苯甲酸；依次将甘油、纯化水、苯甲酸加入化胶罐内，加热至90℃后，再加入明胶不断搅拌、抽真空，直至明胶完全溶解，将胶液过滤，在56℃下存放，备用；
药液配制：将配方量的1-棕榈酸-2-亚油酸-3-油酸甘油酯、吐温80加入配料罐内，不断搅拌，直至混合均匀；
压胶囊：根据胶囊大小选择合适的压丸模具，在30℃、相对湿度<35％条件下进行压丸定型后干燥，剔除大小丸，再用95％药用乙醇洗丸后，继续干燥至含水量小于12％，目检剔除不合格胶囊，印字，包装即得。
实施例15 1-棕榈酸-2-亚油酸-3-油酸甘油酯胶囊剂的制备
处方：
1-棕榈酸-2-亚油酸-3-油酸甘油酯  600g
吐温80  0.3g
制成  1000粒
工艺：
胶液配制：按重量比为1:1.0:0.5:0.008称取适量明胶、纯化水、甘油和醋酸氯己定；依次将甘油、纯化水、醋酸氯己定加入化胶罐内，加热至85℃后，再加入明胶不断搅拌、抽真空，直至明胶完全溶解，将胶液过滤，在58℃下存放，备用；
药液配制：将配方量的1-棕榈酸-2-亚油酸-3-油酸甘油酯、吐温80加入配料罐内，不断搅拌，直至混合均匀；
压胶囊：根据胶囊大小选择合适的压丸模具，在25℃、相对湿度<35％条件下进行压丸定型后干燥，剔除大小丸，再用95％药用乙醇洗丸后，继续干燥至含水量小于12％，目检剔除不合格胶囊，印字，包装即得。

Claims (13)

  1. 一种具有式(Ⅰ)结构的化合物，即1-棕榈酸-2-亚油酸-3-油酸甘油酯：
    Figure PCTCN2015084290-appb-100001
  2. 根据权利要求1所述化合物的制备方法，其特征在于包括如下步骤：
    (1)薏苡仁油的制备；
    (2)1-棕榈酸-2-亚油酸-3-油酸甘油酯的初步分离；
    (3)1-棕榈酸-2-亚油酸-3-油酸甘油酯的二次分离；
  3. 根据权利要求2所述方法，其特征在于步骤(1)中薏苡仁油的制备具体为：
    取水分≤10％的薏苡仁粉碎成10～300目，采用超临界流体萃取，分离得到薏苡仁粗油；加入薏苡仁粗油重量40％～58％的石油醚，及适量碱溶液碱炼，分液后取有机相，加入中性氧化铝和/或高岭土，过滤，150～180℃减压干热灭菌，冷却过滤，得薏苡仁油；
    优选的，
    所述超临界流体萃取溶媒为二氧化碳，萃取温度30～60℃，萃取压力19～24Mpa，分离温度30～60℃，分离压力6～15Mpa，二氧化碳流量10～5000L/h，连续萃取1～4h；
    所述碱溶液可以为0.5％～3％的氢氧化钾、氢氧化钠的水溶液；
    所述加入中性氧化铝和/或高岭土的过程为：首先加入油重3％～8％的活化中性氧化铝过滤，滤液加热至40～50℃，又加入油重2％～6％经活化的高岭土过滤，减压除溶剂，再加入油重8％～12％活化中性氧化铝过滤即可。
  4. 根据权利要求2所述方法，其特征在于步骤(1)中薏苡仁油的制备具体为：
    取水分≤10％的薏苡仁粉碎成10～300目，采用超临界二氧化碳萃取装置萃取，将薏苡仁粉装入萃取釜，加热二氧化碳预热器、萃取釜和分离柱，使萃取温度达到30～60℃，萃取压力19～24Mpa，分离温度达到30～60℃，分离压力6～15Mpa，萃取薏苡仁油后的超临界二氧化碳流体进入解析釜解析循环，解析釜可根据需要设一至多个，控制解析温度30～60℃，解析压力2～6Mpa，整个循环保持二氧化碳流量10～5000L/h，连续萃取1～4h，自分离柱下端分离得到薏苡仁粗油；
    加入薏苡仁粗重量40％～58％的石油醚，再加入适量的2％NaOH溶液碱炼，分液后取有机相用纯化水进行水洗，取水洗后有机相加适量丙酮破乳后，取上层油溶液，加入油重3％～8％的活化中性氧化铝过滤，取滤液加热到40～50℃后，加入油重2％～6％经活化的高岭土过滤，滤液减压回收溶剂后，再加油重8％～12％活化中性氧化铝过滤得油，过滤后的油再经150～180℃减压干热灭菌1～3h，冷却后经≤0.2μm微孔滤膜过滤，得薏苡仁油。
  5. 根据权利要求2所述方法，其特征在于步骤(2)中1-棕榈酸-2-亚油酸-3-油酸甘油酯的初步分离具体为：
    采用制备型高效液相色谱仪和蒸发光散射检测器联用，制备型色谱柱为Agilent Zorbax SB-C18(250mm×21.2mm,7μm)，流动相为A(乙腈)和B(四氢呋喃：乙腈＝1:1)，梯度洗脱：流动相B：0～27min，50％→60％，27～35min，60％→90％，35～45min，90％→100％，流速：18ml/min；进样体积为200～300μl；收集响应设置：peak level min＝30mV；
    供试品配制：用流动相B配制成0.1g/ml的薏苡仁油溶液；
    收集保留时间约为17.3min色谱峰流分，低温减压抽干溶剂。
  6. 根据权利要求2所述方法，其特征在于步骤(3)中1-棕榈酸-2-亚油酸-3-油酸甘油酯的二次分离为：
    一次制备：分析型高效液相色谱仪，DAD检测器，色谱柱：Venusil XBP C18(L)(10*250mm，5μm，
    Figure PCTCN2015084290-appb-100002
    )，流动相，乙腈：四氢呋喃＝75:25，流速：4mL/min；检测波长：205nm；供试品配制：取1-棕榈酸-2-亚油酸-3-油酸甘油酯的初步分离样品，加入适量正己烷，超声促溶后备用，进样量：60μl，收集保留时间约为27.3min的色谱峰流分，将此流分氮吹浓缩；
    二次制备：色谱柱，Venusil CA(10*250mm，5μm，
    Figure PCTCN2015084290-appb-100003
    )，流动相，正己烷：乙醇＝95:5，流速：4mL/min，紫外检测波长：205nm；供试品配制：取一次制备的流分样品若干，加入适量正己烷，超声促溶后备用；进样量：60μl，收集保留时间约为2.5min～5min的色谱峰，氮吹浓缩、低温冻存。
  7. 一种药物制剂，含有权利要求1所述化合物以及一种或几种药学上可接受的载体；
    优选的，所述药学上可接受的载体包括药学领域常规的稀释剂、赋形剂、填充剂、乳化剂、粘合剂、润滑剂、吸收促进剂、表面活性剂、崩解剂、润滑剂、稳定剂等，必要时还可以加入香味剂、甜味剂、防腐剂及着色剂等；
    更优选的，所述药学上可接受的载体可以选自：甘露醇、山梨醇、焦亚硫酸钠、亚硫酸氢钠、硫代硫酸钠、盐酸半胱氨酸、巯基乙酸、蛋氨酸、大豆磷脂、维生素C、维生素E、EDTA二钠、EDTA钙钠，一价碱金属的碳酸盐、醋酸盐、磷酸盐或其水溶液、盐酸、醋酸、硫酸、磷酸、氨基酸、氯化钠、氯化钾、乳酸钠、羟苯乙酯溶液、苯甲酸、山梨酸钾、醋酸氯己定、木糖醇、麦芽糖、葡萄糖、果糖、右旋糖苷、甘氨酸、淀粉、蔗糖、乳糖、甘露糖醇、硅衍生物、纤维素及其衍生物、藻酸盐、明胶、聚乙烯吡咯烷酮、甘油、吐温80、琼脂、碳酸钙、碳酸氢钙、表面活性剂、聚乙二醇、环糊精、β-环糊精、磷脂类材料、高岭土、滑石粉、硬脂酸钙、硬脂酸镁等。
  8. 根据权利要求7所述药物制剂，其可以是口服固体制剂、口服液体制剂或注射剂；
    优选的，
    所述口服固体制剂选自胶囊剂、片剂、滴丸、颗粒剂、浓缩丸中的一种；
    所述口服液体制剂选自水性或油性悬浮液、溶液、乳剂、糖浆剂或酏剂，或是一种在使用前可用水或其它适宜的载体复配的干燥产品；
    所述注射剂选自注射液、冻干粉针剂和水针剂中的一种。
  9. 根据权利要求8所述药物制剂，其特征在于所述注射剂包括如下成分：
    1-棕榈酸-2-亚油酸-3-油酸甘油酯 100-300g
    注射用大豆磷脂或可供注射用大豆磷脂 10-40g
    注射用甘油或可供注射用甘油 15-50g
    注射用水加至 1000ml
  10. 根据权利要求9所述注射剂的制备方法，其特征在于包括如下步骤：
    称取处方配制量的注射用大豆磷脂或可供注射用大豆磷脂，加适量的注射用水后，用高剪切分散乳化机分散至无块状及颗粒状固体，加入按配方量称取的注射用甘油或可供注射用甘油，并加注射用水至规定量，搅拌均匀备用；
    另称取1-棕榈酸-2-亚油酸-3-油酸甘油酯，将称取的油酯与水相分别加热至60～70℃后，置高压均质机进行高压乳化，乳化时均质机低压为5～12MPa，高压为25～50MPa，再循环均质3～6遍，至2μm以下颗粒应不少于95％，5μm以上颗粒不得检出，必要时用NaOH或HCl调节pH至4.8～8.5，优选6.8～7.0，最优选为6.8；
    将制得均匀乳剂氮气加压通过≤3μm的微孔滤芯过滤器过滤，充氮灌装灭菌，冷却即 得。
  11. 根据权利要求8所述药物制剂，其特征在于所述胶囊剂包括如下成分：
    1-棕榈酸-2-亚油酸-3-油酸甘油酯 200-800g
    抗氧化剂和/或乳化剂 0.20-0.60g
    制成 1000粒
  12. 根据权利要求11所述胶囊剂的制备方法，其特征在于包括如下步骤：
    胶液配制：按重量比为1:0.6～1.2:0.3～0.8:0.0001～0.01称取适量明胶、纯化水、甘油和防腐剂；依次将甘油、纯化水、防腐剂加入化胶罐内，加热至70℃～90℃后，再加入明胶不断搅拌、抽真空，直至明胶完全溶解，将胶液过滤，在56～62℃下存放，备用；
    药液配制：将配方量的1-棕榈酸-2-亚油酸-3-油酸甘油酯、抗氧化剂和/或乳化剂加入配料罐内，不断搅拌，直至混合均匀；
    压胶囊：根据胶囊大小选择合适的压丸模具，在15～30℃、相对湿度<35％条件下进行压丸定型后干燥，剔除大小丸，再用95％药用乙醇洗丸后，继续干燥至含水量小于12％，目检剔除不合格胶囊，印字，包装即得；
    优选的，
    所述防腐剂选自10％羟苯乙酯溶液、苯甲酸、山梨酸钾或醋酸氯己定中的一种；
    所述抗氧化剂为维生素E，乳化剂为吐温80。
  13. 根据权利要求1或7所述化合物或药物制剂在制备抗肿瘤药物中的应用；
    优选的，所述肿瘤为乳腺癌、胰腺癌、卵巢癌、肝癌、肺癌、胃癌或结肠癌。
PCT/CN2015/084290 2014-07-18 2015-07-17 化合物1-棕榈酸-2-亚油酸-3-油酸甘油酯、制剂、制备方法及其应用 WO2016008437A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201410343081.5A CN105294438A (zh) 2014-07-18 2014-07-18 化合物1-棕榈酸-2-亚油酸-3-油酸甘油酯、制剂、制备方法及其应用
CN201410343081.5 2014-07-18

Publications (1)

Publication Number Publication Date
WO2016008437A1 true WO2016008437A1 (zh) 2016-01-21

Family

ID=55073636

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2015/084290 WO2016008437A1 (zh) 2014-07-18 2015-07-17 化合物1-棕榈酸-2-亚油酸-3-油酸甘油酯、制剂、制备方法及其应用

Country Status (3)

Country Link
US (1) US20160015670A1 (zh)
CN (1) CN105294438A (zh)
WO (1) WO2016008437A1 (zh)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106978254A (zh) * 2016-12-20 2017-07-25 烟台固特丽生物科技股份有限公司 一种高效率dha油脂脱色方法

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101940738A (zh) * 2010-07-27 2011-01-12 辽宁中医药大学 薏苡仁抗癌中药及其制备方法

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1778380A (zh) * 2004-11-23 2006-05-31 乔志亚生技股份有限公司 薏仁油萃取的方法及组成分及其疗效
US20110077291A1 (en) * 2009-09-30 2011-03-31 Jianming Chen Preparations of Taxanes for Intravenous Administration and the Preparation Method Thereof
CN103131533A (zh) * 2013-03-11 2013-06-05 浙江康莱特集团有限公司 一种超临界co2萃取、精馏、过滤提取薏苡仁油的方法

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101940738A (zh) * 2010-07-27 2011-01-12 辽宁中医药大学 薏苡仁抗癌中药及其制备方法

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
YU , F. ET AL.: "Inhibition of Coix Seed Extract on Fatty Acid Synthase, a Novel Target for Anticancer Activity", JOURNAL OF ETHNOPHARMACOLOGY, vol. 119, no. 2, 23 July 2008 (2008-07-23), pages 252 - 258, XP025399344, ISSN: 0378-8741, DOI: doi:10.1016/j.jep.2008.07.015 *
YU , F. ET AL.: "Inhibition of Coix Seed Extract on Fatty Acid Synthase, a Novel Target for Anticancer Activity", JOURNAL OF ETHNOPHARMACOLOGY, vol. 119, no. 2, 23 July 2008 (2008-07-23), XP025399344, ISSN: 0378-8741, DOI: doi:10.1016/j.jep.2008.07.015 *

Also Published As

Publication number Publication date
US20160015670A1 (en) 2016-01-21
CN105294438A (zh) 2016-02-03

Similar Documents

Publication Publication Date Title
WO2016008439A1 (zh) 含有8种甘油三酯的薏苡仁油、制剂及其应用
KR101919386B1 (ko) 11가지 트리글리세라이드를 포함한 의이인오일, 제제 및 그 응용
WO2016008436A1 (zh) 化合物1,3-二油酸-2-亚油酸甘油酯、制剂、制备方法及其应用
KR101919387B1 (ko) 16가지 글리세리드를 포함한 의이인오일, 제제 및 그 응용
WO2016008437A1 (zh) 化合物1-棕榈酸-2-亚油酸-3-油酸甘油酯、制剂、制备方法及其应用
EP3153164B1 (en) Coix seed oil comprising 13 glycerides, formulation and application thereof
CN117551161A (zh) 一类五环三萜类化合物在治疗神经退行性疾病中的用途
WO2016008441A1 (zh) 含有13种甘油酯的药物组合物、制剂及其应用
WO2016008438A1 (zh) 含有8种甘油三酯的药物组合物、制剂及其应用

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 15822458

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 15822458

Country of ref document: EP

Kind code of ref document: A1