WO2020253689A1 - 一种绿原酸自乳化组合物及其用途 - Google Patents

一种绿原酸自乳化组合物及其用途 Download PDF

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WO2020253689A1
WO2020253689A1 PCT/CN2020/096427 CN2020096427W WO2020253689A1 WO 2020253689 A1 WO2020253689 A1 WO 2020253689A1 CN 2020096427 W CN2020096427 W CN 2020096427W WO 2020253689 A1 WO2020253689 A1 WO 2020253689A1
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self
chlorogenic acid
preparation
acid
emulsifier
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French (fr)
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刘玉玲
陈晓光
张洁
杨艳芳
叶军
季鸣
高越
李燕
金晶
刘东东
冯遇
廖恒峰
周君卓
姬勋
杨华蓉
黄望
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四川九章生物科技有限公司
博睿先锋(北京)生物科技有限公司
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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
    • A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
    • A61K31/215—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids
    • A61K31/216—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acids having aromatic rings, e.g. benactizyne, clofibrate
    • 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
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/14—Esters of carboxylic acids, e.g. fatty acid monoglycerides, medium-chain triglycerides, parabens or PEG fatty acid esters
    • 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
    • A61K9/107—Emulsions ; Emulsion preconcentrates; Micelles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/06—Antihyperlipidemics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • 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
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P39/00—General protective or antinoxious agents
    • A61P39/06—Free radical scavengers or antioxidants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/12—Antihypertensives

Definitions

  • the invention relates to a chlorogenic acid self-emulsifying composition and a preparation method and application thereof, and belongs to the technical field of medicine preparation.
  • Chlorogenic acid also known as coffee tannic acid (CA)
  • CA coffee tannic acid
  • It has antioxidant, Antibacterial, antiviral, hypoglycemic, lipid-lowering, blood pressure and immune regulation and other pharmacological effects.
  • the immunomodulatory effect of chlorogenic acid makes it one of the most popular natural products in tumor immunotherapy in recent years. Xue et al. found that chlorogenic acid can reverse the transition from M2 to M1 in tumor-associated macrophages, thereby playing an anti-tumor effect.
  • chlorogenic acid ice dry powder injection for injection which has poor compliance with long-term intramuscular injection. Due to the poor pharmacological activity of oral administration of chlorogenic acid, no oral preparation of chlorogenic acid has been used for clinical treatment.
  • patent CN 1048566954 B discloses a chlorogenic acid microemulsion and its preparation process and application. Its core content is to use the characteristics of chlorogenic acid that is strong in hydrophilicity and poor in lipophilicity.
  • W/O water-in-oil
  • chlorogenic acid is a polyphenol Similar to the chemical structure, it is prone to chemical degradation when stored in an aqueous solution. Therefore, the preparation of microemulsions containing water phase has defects.
  • SEDDS Self-emulsifying drug delivery system
  • oil phase emulsifier and co-emulsifier
  • SEDDS composition does not contain an aqueous phase, so there is an essential difference in composition, which is more advantageous for the inclusion of chemically unstable drugs.
  • preparation of SEDDS has extremely high requirements on the physical and chemical properties of the drug. Lipophilic drugs are more suitable for encapsulation, while the hydrophilic chlorogenic acid is restricted.
  • the three are a whole, any one of the components changes, or the composition ratio of the three changes, will directly affect the appearance of SEDDS (if it can be Liquid, semi-solid or solid), physical stability (such as a homogeneous solution, or layering or even precipitation), chemical stability (different degree of drug degradation), and emulsified particle size (in contact with water to form nano Grade or micron grade emulsion).
  • the ideal drug-loaded self-emulsifying composition should meet the following conditions: 1) Long-term storage under refrigeration or room temperature conditions, it should be a clear and transparent liquid with uniform appearance, and no delamination; 2) Long-term storage under refrigeration or room temperature conditions It should be a clear and transparent liquid with uniform appearance, and should not be solidified or precipitated.
  • the emulsion droplets formed by spontaneous emulsification in the gastrointestinal tract should be nano-sized (ie 1-1000nm) , Pharmaceutics believe that 1-1000nm belong to the nanometer range), and should not be micron (ie >1 ⁇ m, pharmacy believes that more than 1 ⁇ m belongs to the micron level).
  • the self-microemulsion solution with a drug dosage of 60mg/g showed that the prepared chlorogenic acid self-microemulsion concentrate showed obvious stratification.
  • the layered state will increase.
  • Patent CN104826118A discloses a phospholipid modification of chlorogenic acid and its derivatives and a preparation method thereof, which applies the phospholipid modification to the preparation of "nano drug delivery system or micro drug delivery system".
  • this patent also has obvious defects; 1) when used in the preparation of microemulsion or/self-microemulsion drug delivery system, the result is a micron drug delivery system instead of a nano drug delivery system; 2) it is used for microemulsion or/self-microemulsion drug delivery system
  • the emulsifier used is one or more mixtures of polysorbate 80, polyoxyethylene oleate, liquid lecithin, and polyoxyethylene castor oil. The verification shows that the prepared self- The microemulsion mixture is clearly stratified, and the upper layer is a solid gel with no fluidity.
  • the patent of the present invention discloses a self-emulsifying composition of a chlorogenic acid nano drug delivery system and its preparation method and application on the basis of system research.
  • the present invention provides a self-emulsifying composition of a chlorogenic acid nano drug delivery system, which is composed of a chlorogenic acid lipid material compound, an oil phase, an emulsifier and a co-emulsifier; the emulsifier is polyethylene Any one or more of alcohol glycerides and their derivatives; the chlorogenic acid lipid material complex is made by compounding chlorogenic acid and lipid materials.
  • the composite rate of the chlorogenic acid lipid material complex is greater than 70%, preferably greater than 80%, more preferably greater than 90%.
  • the content of chlorogenic acid in the composition is 5-200 mg/g.
  • the content of chlorogenic acid in the composition is 10-150 mg/g.
  • the molar ratio of chlorogenic acid to lipid material in the chlorogenic acid lipid material composite is 1:0.25 to 1:8, preferably 1:0.25 to 1:4.
  • the lipid material is selected from cholesterol, phospholipids or bile salts; preferably, phospholipids.
  • the phospholipids are soybean phospholipids, egg yolk phospholipids, phosphatidylglycerides, distearoylphosphatidylcholine, dipalmitoylphosphatidylcholine, dipalmitoylphosphatidylethanolamine, dimyristoylphosphatidylcholine Any one or more of choline and ceramide; preferably, soybean phospholipid and/or egg yolk phospholipid.
  • the mass percentages of the oil phase, emulsifier and co-emulsifier are based on the total of 100% of the three, the oil phase is 10-50%, the emulsifier is 30-60%, and the co-emulsifier is 20-60%.
  • polyethylene glycol glyceride derivative is caprylic acid capric acid polyethylene glycol glyceride, oleic acid polyethylene glycol glyceride or linoleic acid polyethylene glycol glyceride.
  • the oil phase is soybean oil, corn oil, olive oil, coconut oil, peanut oil, camellia oil, castor oil, sorbitol oleate, glyceryl oleate, glyceryl linoleate, labrafil 1944cs, Maisine 35-1 , Ethyl oleate, ethyl linoleate, C 8 /C 10 monoglycerides, coconut oil C 8 /C 10 diglycerides, coconut oil C 8 /C 10 triglycerides, caprylic triglycerides, caprylic glycerol Diester, caprylic acid monoglyceride, capric acid monoglyceride, capric acid diglyceride, capric acid triglyceride, caprylic acid monoglyceride, caprylic acid glyceride, caprylic acid triglyceride, myristic acid iso Propyl ester, linoleic acid polyethylene glycol glyceride, Ge
  • the co-emulsifier is propylene carbonate, ethylene glycol monoethyl ether, glycerol furfural, dimethyl isosorbide, diethylene glycol monoethyl ether, PEG400, glycerin, labraosol, benzyl alcohol Any one or more of; preferably, any one or more of dimethyl isosorbide, diethylene glycol monoethyl ether, glycerin, and labrosol.
  • the antioxidant is any one or more of vitamin E, vitamin E ester derivatives or dibutyl hydroxytoluene.
  • the present invention also provides a method for preparing a self-emulsifying composition of the chlorogenic acid nano drug delivery system, which includes the following steps:
  • step (1) Add the compound of step (1) together with the oil phase, emulsifier and co-emulsifier according to the ratio, stir and mix evenly, and get it;
  • step (1) first dissolve and mix the compound of step (1) with any one of the oil phase, emulsifier or co-emulsifier, and then add the other two components and mix them to obtain.
  • the organic solvent in step (1) is a low-molecular alcohol; the low-molecular alcohol is any one or more of ethanol, anhydrous ethanol, and tert-butanol; preferably, anhydrous ethanol.
  • the dissolution in step (3) is shaking dissolution, with a shaking speed of 210 rpm and a temperature of 25°C.
  • the present invention also provides a chlorogenic acid preparation, which is a preparation composed of the aforementioned chlorogenic acid self-emulsifying composition and a pharmaceutically acceptable carrier.
  • the preparation is an oral preparation, a mucosal administration preparation, a transdermal administration preparation, an enteral administration preparation or a spray inhalation preparation.
  • the oral preparations are tablets, granules, capsules, oral liquids, drops or sprays;
  • the mucosal or transdermal preparations are paints, sprays or other suitable dosage forms;
  • the enteral administration preparation is a liquid preparation or dissolved in an oily base to prepare a suppository.
  • the tablets, granules or capsules are prepared by dispersing the self-emulsifying composition of chlorogenic acid on micronized silica gel or other absorbent solid materials, and adding pharmaceutically acceptable auxiliary materials.
  • the preparation for nebulization inhalation is the self-emulsifying composition of chlorogenic acid is divided into a concentrated solution for nebulization inhalation, and it is diluted with nebulization device or by adding water, physiological saline or glucose injection before use.
  • the present invention finally provides a use of a chlorogenic acid self-emulsifying composition or a chlorogenic acid preparation in the preparation of a medicine for treating and/or preventing malignant tumors, immunocompromised, bacterial virus infection and/or antioxidant.
  • the present invention attempts to make chlorogenic acid into a chlorogenic acid/phospholipid complex and then mix it with the oil phase, emulsifier and/or co-emulsifier to make self-microemulsion.
  • the quality conditions are as follows:
  • chlorogenic acid raw material drug and the phospholipid complex of chlorogenic acid (the mixture of chlorogenic acid and soybean lecithin 1:1, dissolved in absolute ethanol, and dried) was added to the above blank self-microemulsion, dissolved , Make the concentration of chlorogenic acid 60mg/ml, leave it for one week, and observe whether there is stratification; Take two kinds of self-loaded microemulsions, add 100 times of water to dilute, shake, within 2 minutes, the laser particle size analyzer measures the droplets Particle size.
  • the self-microemulsion containing chlorogenic acid is prepared with small particle size after dilution, but it is stored in cold storage or at room temperature for 3 days The phenomenon of stratification occurs; the prepared self-microemulsion containing the chlorogenic acid phospholipid complex has a particle size of micrometers after dilution, and solidification occurs in cold storage, and obvious stratification occurs when stored at room temperature.
  • the existing reports of chlorogenic acid self-microemulsion itself are not stable, and the defects of unstable state or large particle size appear after being placed.
  • the self-microemulsion prepared according to its oil phase, emulsifier and co-emulsifier composition is still unstable.
  • the self-microemulsion system shows layered, semi-gelatinous and semi-gelatinous to varying degrees. Solidify three states.
  • the self-emulsifying composition of the chlorogenic acid nano drug delivery system of the present invention uses a chlorogenic acid/lipid complex as an intermediate carrier, and the emulsifier adopts one or more mixtures of polyethylene glycol glycerides and derivatives thereof.
  • the prepared self-emulsifying composition placed at room temperature and 2-8°C for at least 6 months, will not coagulate or delaminate, and exhibit uniform liquid properties, which can be quickly emulsified to form nano-scale emulsion after dilution with water
  • the particle size of the emulsion droplet is ⁇ 500nm.
  • soybean lecithin as the lipid material, feed chlorogenic acid and soybean lecithin according to the drug-to-lipid ratio in the table below, and put them into a 1L round-bottom flask together, and use absolute ethanol as the solvent to control the drug concentration at 60mg /ml, after the chlorogenic acid and soybean lecithin are all dissolved, place at room temperature for 15 minutes, then use a rotary evaporator at 100rpm and 40°C to dry under reduced pressure to remove anhydrous ethanol. After the ethanol is evaporated, continue to reduce pressure at room temperature at 100rpm Dry for 1h to remove ethanol.
  • Reference solution Weigh 25mg of chlorogenic acid standard into a 25ml volumetric flask, dilute the volume with ethanol solution, accurately measure 1ml to 50ml volumetric flask, dilute the volume with 30% ethanol water, as the reference solution;
  • natural phospholipids synthetic phospholipids, cholesterol, and cholate can all meet the requirements, but natural phospholipids are preferred.
  • the chlorogenic acid self-emulsifying composition is prepared; take an appropriate amount of the drug-loaded self-emulsifying solution, add water 100 times to dilute, and investigate the self-emulsification efficiency and the particle size after emulsification (the particle size detection method: Pipette precisely 20 ⁇ L of chlorogenic acid self-emulsifying composition, add it to a 5ml EP tube containing 2ml Wahaha deionized water, shake it gently, transfer to a cuvette, and then measure its particle size with a laser scattering particle size analyzer ). The results are shown in Table 5.
  • the molar ratio of chlorogenic acid to lipid material is 1:0.25 ⁇ 1:8 can meet the requirements, but it is preferably 1:0.25 to 1:4; the composite ratio of chlorogenic acid and lipid material is more than 70% to meet the requirements, but preferably more than 90%.
  • Vitamin E its esters, and BHT can all be used.
  • the compound of chlorogenic acid and lipid material can be added together with the oil phase, emulsifier and co-emulsifier, stirred and mixed uniformly to obtain a self-emulsified composition; or Dissolve and mix the compound and any one of the oil phase, emulsifier or co-emulsifier first, and then add the other two ingredients and mix well to obtain.
  • the self-emulsifying composition of the chlorogenic acid nano drug delivery system disclosed in the patent of the present invention can be used to prepare a preparation suitable for oral administration, such as directly filling the self-emulsifying concentrate into soft capsules, hard capsules, and oral liquids. , Drops, sprays, etc., of which soft capsules and drops are preferred.
  • the self-emulsifying composition concentrate can also be dispersed on micronized silica gel or other absorbent solid materials, and added with auxiliary materials commonly used in pharmaceuticals to make tablets, capsules, granules or granules for oral use.
  • Oral preparations are taken directly without diluting and emulsifying with water before taking. Utilizing the self-emulsifying ability of the self-emulsifying composition, it can quickly form nano-sized emulsion droplets in the gastrointestinal fluid environment and gastrointestinal peristalsis conditions.
  • the self-emulsifying composition of the present invention can also be divided into concentrated solutions for atomized inhalation, diluted with water, physiological saline or glucose injection before use, and atomized inhalation using an atomized inhalation device.
  • the self-emulsifying composition of the patent of the present invention has been used in the treatment of lewis lung cancer and glioma successively, and the dosage is within the range of 20-80mg/kg by intragastric administration, and both show good therapeutic effects, compared with injection administration There is no obvious difference.
  • the molar ratio of chlorogenic acid to lipid material is 1:0.25 ⁇ 1:8, preferably 1:0.25 ⁇ 1:4; the oil phase, emulsifier and co-emulsifier are calculated as 100% of the total.
  • the composition is made into different preparations for anti-tumor, anti-inflammatory immunity and anti-viral therapy, and has significant effects and has practical application promotion value.
  • Example 2 A self-emulsifying composition of chlorogenic acid prepared by using CHAPC 3 as an intermediate and using different oil phases: emulsifier: co-emulsifier
  • blade self-emulsifying concentrate use ethyl oleate as the oil phase, labrasol as the emulsifier, and transcutol HP as the co-emulsifier, according to the mass ratio of oil phase: emulsifier: co-emulsifier in Table 7 Take a certain amount of ethyl oleate, labrasol and transcutol HP, stir and mix evenly to obtain a blank self-emulsifying concentrate;
  • blade self-emulsifying concentrate use ethyl oleate as the oil phase, labrasol as the emulsifier, transcutol HP as the co-emulsifier, and weigh the ethyl oleate and labrasol in a mass ratio of 2:5:3 With transcutol HP, stir and mix evenly to obtain a blank self-emulsifying concentrate;
  • blade self-emulsifying concentrate use ethyl oleate as the oil phase, labrasol as the emulsifier, and transcutol HP as the co-emulsifier.
  • the mass ratio of oil phase: emulsifier: co-emulsifier is 2: 5:3
  • Example 6 Chlorogenic acid self-emulsifying composition is used for oral soft capsule preparation or atomized inhalation administration
  • blade self-emulsifying concentrate use ethyl oleate as the oil phase, labrasol as the emulsifier, and transcutol HP as the co-emulsifier.
  • the mass ratio of oil phase: emulsifier: co-emulsifier is 2: 5:3
  • the chlorogenic acid self-emulsifying composition can be diluted with water, physiological saline or glucose injection, and connected to an atomized inhalation device for atomized inhalation and administration.
  • Test example 1 HPLC content determination method research
  • Chlorogenic acid has a polyphenolic structure and is easily chemically degraded in solution. If the chromatographic conditions cannot effectively separate the main peak from the degradation products, it will directly affect the accuracy of the stability study results. To this end, a self-emulsified sample with accelerated destruction at high temperature was used to demulsify with absolute ethanol, diluted with water to a test solution with a chlorogenic acid concentration of about 0.1 mg/ml, and the following two conditions were investigated for determination.
  • Chromatographic condition 2 Gradient elution is used, and the mobile phase conditions are as follows.
  • Appearance traits 9 groups of samples were placed at 4°C and room temperature for 7 months, and they were all clear and transparent uniform solutions without precipitation, indicating good physical stability.
  • Test Example 3 Investigation of the anti-tumor effect of chlorogenic acid self-emulsifying composition on subcutaneous ICR mice bearing Lewis lung cancer
  • chlorogenic acid self-microemulsion was administered to tumor-bearing mice at the oral doses of 35 mg/kg and 30 mg/kg respectively (the preparation method is the same as that in the literature Li Chen, Chang-shun Liu, Qing -zhen Chen,SenWang,Yong-ai Xiong,JingJing,Jia-jia Lv.Characterization,pharmacokinetics and tissue distribution of chlorogenic acid-loaded self-microemulsifying drug delivery system.European Journal of 100 Pharmaceutical, 2017, Science 108 , Abbreviated as chlorogenic acid self-microemulsion) and chlorogenic acid self-emulsifying composition (sample number 1 in the example).
  • Oral normal saline was taken as a control.
  • the animals were administered continuously for 13 days, during which the state and body weight of the animals were observed.
  • the mice were sacrificed by cervical dislocation, the tumor tissue was stripped and weighed, and compared with the tumor weight of the control group as the evaluation of tumor suppression efficiency. The results are shown in Figure 3.
  • Figure 3 shows that the chlorogenic acid self-emulsifying composition disclosed in the present invention has a tumor inhibition rate of 88.57 ⁇ 0.17% for Lewis lung cancer, while the tumor inhibition rate of chlorogenic acid self-microemulsion is only 50.24 ⁇ 9.07%.
  • the tumor suppressive efficiency of the chlorogenic acid self-emulsified composition is 1.76 times that of the chlorogenic acid self-microemulsion.
  • Test Example 4 Investigation of the anti-tumor effect of chlorogenic acid self-emulsifying composition on subcutaneous ICR mice bearing glioma
  • ICR mice inoculated with G422 subcutaneously were used to evaluate the tumor suppressive effect of the chlorogenic acid self-emulsifying composition on glioma.
  • the positive control drug cyclophosphamide (CTX, dose) and chlorogenic acid self-emulsifying composition (sample in the example) were administered to the tumor-bearing mice by gavage at the oral administration dose of 30 mg/kg on the second day after the tumor. No. 1, the doses are: 20mg/kg, CHASME 20 for short; 40mg/kg, CHASME 40 for short; 60mg/kg, CHASME 60 for short).
  • Oral normal saline is taken as a control, and it is administered continuously for 13 days. .
  • Figure 4 shows that the self-emulsifying composition of chlorogenic acid (CHA SME 20, CHA SME 40 and CHA SME 60) disclosed in the present invention has a tumor inhibition rate of 30.71 ⁇ 6.42% and 20.17 ⁇ 8.94 to ICR mice inoculated with G422 subcutaneously. % And 33.46 ⁇ 5.97%, the effect is equivalent to the intraperitoneal injection group of chlorogenic acid raw material solution (CHA 10: 31.7 ⁇ 8.07%; CHA 20: 27.82 ⁇ 9.0%; CHA 40: 23.82 ⁇ 4.92%).
  • Test Example 5 Investigation of the tumor-inhibiting effect of chlorogenic acid self-emulsifying composition on ICR mice with in situ glioma
  • ICR mice inoculated with G422 in situ were used to evaluate the tumor suppressive effect of the chlorogenic acid self-emulsifying composition on glioma.
  • the positive control drug temozolomide (TMZ, 50mg/kg) and chlorogenic acid self-emulsifying composition were administered to the tumor-bearing mice on the second day after the tumor.
  • TTZ positive control drug
  • chlorogenic acid self-emulsifying composition examples number 1 in the example, the dose was 20mg/kg respectively
  • 35mg/kg abbreviated as: CHA SME 20mg/kg and CHA SME 35mg/kg).
  • Oral normal saline was taken as a control, and the rats were given by intragastric administration for 13 days.
  • Figure 5 shows that the self-emulsifying composition of chlorogenic acid (CHA SME 35mg/kg) disclosed in the present invention has a tumor inhibition rate of 54.8% for ICR mice inoculated with G422 in situ, which is significantly better than intraperitoneal injection of chlorogenic acid raw materials.
  • Group (CHA 10mg/kg, 27.1%; CHA 20mg/kg, 45.6%).
  • the self-emulsifying composition of the chlorogenic acid nano drug delivery system of the present invention will not cause solidification or delamination when placed at room temperature and 2-8°C, and can be used for oral administration, mucosal administration, and transdermal administration.
  • the composition is diluted with 10-1000 times the volume of water, different pH buffers, artificial gastric juice or artificial intestinal juice. After slight shaking, it can quickly form an average particle size below 500nm Nano-scale drug delivery system.
  • the self-emulsifying composition and its preparation of the present invention can be used for anti-tumor, anti-inflammatory immunity and anti-viral therapy.

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Abstract

涉及一种绿原酸纳米级给药系统的自乳化组合物及其用途，该纳米级给药系统的自乳化组合物含有绿原酸-脂质材料形成的复合率>70％的复合物，特定的油相、乳化剂和助乳化剂，但不含水相，可用于抗肿瘤、抗炎免疫和抗病毒的治疗。

Description

一种绿原酸自乳化组合物及其用途 技术领域
本发明涉及一种绿原酸自乳化组合物及其制备方法与用途，属于医药制备的技术领域。
背景技术
绿原酸又名咖啡鞣酸(Chlorogenic acid，简称CA)，是广泛存在于金银花、杜仲科植物杜仲的叶、葵花籽、茵陈和蓝盆花等植物中的一种有机酸，具有抗氧化、抗菌、抗病毒、降糖、降脂、降血压和免疫调节等多种药理作用。在多种生理活性中，绿原酸所具有的免疫调节作用使得其在肿瘤免疫治疗方面成为近年来研究热门的天然产物之一。Xue等研究发现，绿原酸可以逆转肿瘤相关巨噬细胞中的M2型向M1型转变，从而起到抗肿瘤的效果。
目前临床上使用的为注射用绿原酸冰干粉针，长期肌肉注射顺应性差。由于绿原酸口服给药的药理活性较差，至今未见绿原酸口服制剂用于临床治疗。
对于免疫调节类药物，并非是药物浓度越高药效越好，因此，单纯提高生物利用度不一定能获得理想的口服治疗效果。什么样的组合物能够突破绿原酸口服给药用于临床治疗的限制，无法预测。
为了开发适用于口服给药的制剂，专利CN 1048566954 B公开了一种绿原酸微乳及其制备工艺和应用，其核心内容，是利用绿原酸亲水性强而亲脂性差的特点，制备了由水相、油相、乳化剂和助乳化剂形成的油包水(W/O)微乳，其目的是为了提高生物利用度、改善口服治疗效果，但由于绿原酸为多酚类化学结构，在水溶液中贮存极易发生化学降解，因此，制备含有水相的微乳存在缺陷。
自乳化给药系统(SEDDS)是由油相、乳化剂和助乳化剂形成的均一液体、半固体或固体，可用于药物的包载。与微乳相比，SEDDS组合物不含水相，因此组成上有着本质的区别，这对于化学性质不稳定药物的包载，更具优势。但SEDDS的制备对药物理化性质有着 极高要求，亲脂性药物更适用于包载，而对于亲水性的绿原酸则受到限制。
影响载药SEDDS特性的两大因素：1)被包载药物的性质。脂溶性药物更有利于SEDDS的包载，而对于极性较大的亲水性药物，通常载药困难，或放置后易发生分层甚至沉淀。2)油相、乳化剂和助乳化剂的类别及组成比例：三者是一个整体，其中任一成分发生改变，或者三者的组成比例发生变化，都会直接影响SEDDS的外观性状(如可以是液体，也可能是半固体或固体)、物理稳定性(如可表现为均一溶液，也可能分层甚至沉淀)、化学稳定性(药物降解程度的不同)以及乳化后粒径(遇水形成纳米级或微米级乳滴)。理想的载药自乳化组合物，应满足以下条件：1)在冷藏或室温条件下长期贮存，应为外观均一的澄清透明液体，不应发生分层；2)在冷藏或室温条件下长期贮存，应为外观均一的澄清透明液体，不应发生凝固或沉淀，避免使用前“将药物混合物加热融化”的处理环节，便于服用，且减少药物降解；3)自乳化效率高，模拟胃肠环境，加一定量的水稀释后，可快速自发乳化，形成纳米级的乳滴；4)自乳化混合物直接口服后，在胃肠中自发乳化形成的乳滴，应为纳米级(即1-1000nm，药剂学上认为，1-1000nm均属于纳米范筹),而不应为微米级(即>1μm，药剂学上认为，超过1μm则属于微米级)。
但现有的关于绿原酸自乳化系统的研究文献，均存在明显的缺陷。
陈莉等，在《绿原酸自微乳给药系统的处方设计与体外评价》中以油酸乙酯为油相，吐温80为乳化剂，甘油为助化剂，制备了包载绿原酸的自微乳，载药量可达60mg/g，加100倍水稀释，在37度和50rpm条件下搅拌，可乳化为粒径约16nm的乳滴。但参照文献方法，以油酸乙酯为油相，吐温80为乳化剂，甘油为助化剂，按照油相：乳化剂：助乳化剂＝1:3:6的质量比，制备了载药量为60mg/g的自微乳溶液，结果显示，所制备的绿原酸自微乳浓缩液显示明显的分层现象。在室温以及4℃放置，分层状态加重。
专利CN104826118A公开了一种绿原酸及其衍生物的磷脂修饰物及其制备方法，其将磷脂修饰物应用于“纳米给药系统或微米给药 系统”的制备。但该专利同样存在明显缺陷；1)用于微乳或/自微乳给药系统制备时，得到的为微米给药系统而不是纳米给药系统；2)用于微乳或/自微乳给药系统制备时，采用的乳化剂为聚山梨酯80、聚氧乙烯油酸酯、液体卵磷脂和聚氧乙烯蓖麻油中的一种或一种以上混合物，经验证，表明制得的自微乳混合物明显分层，且上层为无流动性的固态凝胶。
发明内容
鉴于现有研究存在的缺陷，本发明专利在系统研究基础上，公开了一种绿原酸纳米给药系统的自乳化组合物及其制备方法和用途。
本发明提供了一种绿原酸纳米给药系统的自乳化组合物，它是由绿原酸脂质材料复合物、油相、乳化剂和助乳化剂组成；所述乳化剂为聚乙二醇甘油酯及其衍生物的任意一种或几种；所述绿原酸脂质材料复合物是绿原酸与脂质材料复合制成。
进一步地，所述绿原酸脂质材料复合物的复合率大于70％，优选大于80％，更优选大于90％。
进一步地，所述组合物中绿原酸含量为5－200mg/g。
更进一步地，所述组合物中绿原酸含量为10－150mg/g。
进一步地，所述绿原酸脂质材料复合物中绿原酸与脂质材料的摩尔比为1:0.25～1:8，优选1:0.25～1:4。
进一步地，所述脂质材料选自胆固醇、磷脂类或胆酸盐类；优选，磷脂类。
更进一步地，所述磷脂类为大豆磷脂、蛋黄磷脂、磷脂酰甘油酯、二硬脂酰磷脂酰胆碱、二棕榈酰磷脂酰胆碱、二棕榈酰磷脂酰乙醇胺、二肉蔻酰磷脂酰胆碱、神经酰胺中的任意一种或几种；优选，大豆磷脂和/或蛋黄磷脂。
进一步地，所述油相、乳化剂与助乳化剂质量百分比，按三者总和100％计，油相为10-50％，乳化剂为30-60％，助乳化剂为20-60％。
进一步地，所述聚乙二醇甘油酯衍生物为辛酸癸酸聚乙二醇甘油酯、油酸聚乙二醇甘油酯或亚油酸聚乙二醇甘油酯。
进一步地，所述油相为大豆油、玉米油、橄榄油、椰子油、花生油、山茶油、蓖麻油、油酸山梨醇酯、油酸甘油酯，亚油酸甘油酯、labrafil1944cs、Maisine35-1、油酸乙酯、亚油酸乙酯、C 8/C 10甘油单酯、椰子油C 8/C 10甘油双酯、椰子油C 8/C 10甘油三酯、辛酸甘油三酯、辛酸甘油二酯、辛酸甘油单酯、癸酸甘油单酯、癸酸甘油二酯、 癸酸甘油三酯、辛癸酸甘油单酯、辛癸酸甘油酯、辛癸酸甘油三酯、肉豆蔻酸异丙酯、亚油酸聚乙二醇甘油酯、Gelucire、Capryol 90中的任意一种或几种；优选，油酸乙酯、油酸山梨醇酯、油酸甘油酯，亚油酸甘油酯、labrafil1944cs、Maisine35-1、亚油酸乙酯、C 8/C 10甘油单酯、椰子油C 8/C 10甘油双酯、椰子油C 8/C 10甘油三酯、辛酸甘油三酯、辛酸甘油二酯、辛酸甘油单酯、癸酸甘油单酯、癸酸甘油二酯、癸酸甘油三酯、辛癸酸甘油单酯、辛癸酸甘油酯、辛癸酸甘油三酯、肉豆蔻酸异丙酯、亚油酸聚乙二醇甘油酯、Gelucire、Capryol 90中的任意一种或几种。
进一步地，所述助乳化剂为碳酸丙二酯、乙二醇单乙基醚、甘油糠醛、二甲基异山梨酯、二乙二醇单乙基醚、PEG400、甘油、labraosol、苯甲醇中的任意一种或几种；优选，二甲基异山梨酯、二乙二醇单乙基醚、甘油、labraosol中的任意一种或几种。
进一步地，它还包括抗氧剂；所述抗氧剂为维生素E、维生素E酯类衍生物或二丁基羟基甲苯任意一种或几种。
本发明还提供了一种制备绿原酸纳米给药系统的自乳化组合物方法，它包括如下步骤：
(1)制备绿原酸与脂质材料的复合物：按配比称取绿原酸与脂质材料，溶于有机溶剂中，混匀，静置15-30min，旋转蒸发或喷雾干燥去除有机溶剂，再减压干燥，即得；
(2)制备空白自乳化浓缩液：按配比称取油相、乳化剂和助乳化剂，混合均匀，即得；
(3)制备绿原酸自乳化组合物：将步骤(1)复合物加入步骤(2)浓缩液中，溶解完全，即得；
或：
按配比将步骤(1)复合物与油相、乳化剂和助乳化剂一同加入，搅拌混合均匀，即得；
或：
按配比先将步骤(1)复合物与油相、乳化剂或助乳化剂中任意一种溶解混匀，再加入另外两种成分混匀，即得。
进一步地，步骤(1)所述有机溶剂为低分子醇类；所述低分子醇类为乙醇、无水乙醇、叔丁醇中的任意一种或几种；优选，无水乙醇。
进一步地，步骤(3)所述溶解为振摇溶解，振摇速度210rpm， 温度25℃。
本发明还提供了一种绿原酸制剂，它是由前述绿原酸自乳化组合物，加上药学上可接受的载体组成的制剂。
进一步地，所述制剂为口服制剂、粘膜给药制剂、经皮给药制剂、肠道给药制剂或雾化吸入用制剂。
更进一步地，所述口服制剂为片剂、颗粒剂、胶囊剂、口服液、滴剂或喷剂；所述粘膜给药制剂或经皮给药制剂为涂剂、喷剂或其它适宜剂型；所述肠道给药制剂为液体制剂或溶于油性基质制成栓剂。
更进一步地，所述片剂、颗粒剂或胶囊剂是将绿原酸自乳化组合物分散在微粉硅胶或其他吸附性固体材料上，再加上药剂上可接受的辅料制成。
更进一步地，所述雾化吸入用制剂是将绿原酸自乳化组合物分装制成供雾化吸入用的浓溶液，临用时用雾化装置或加水、生理盐水或葡萄糖注射液稀释后用雾化吸入装置进行雾化吸入。
本发明最后提供了一种绿原酸自乳化组合物或者绿原酸制剂在制备治疗和/或预防恶性肿瘤、免疫低下、细菌病毒感染和/或抗氧化的药物中的用途。
本发明在公开报道的绿原酸自微乳的基础上，尝试将绿原酸制成绿原酸/磷脂复合物后再与其油相、乳化剂和/或助乳剂混合制成自微乳，质量情况具体如下：
在陈莉等《绿原酸自微乳给药系统的处方设计与体外评价》研究基础上，尝试先将绿原酸制成绿原酸/磷脂复合物(摩尔比为1:1,质量比为1:2.2)，以改善药物亲脂性，并以复合物为中间载体，再按同样的油相、乳化剂和助化剂处方制备60mg/ml(以绿原酸计)自微乳，但制备过程中发现，所得到的绿原酸自乳化组合物仍显示分层，且上层为固态凝胶，无流动性。于37℃放置1天后，仍呈现上层为固体凝胶状态的分层状态，无明显改善。进一步，采用“聚氧乙烯蓖麻油”代替“吐温80”进行自微乳制备，结果发现，所制备的绿原酸自乳化组合物初始性状仍呈现分层状态。由此提示，当采用绿原酸/磷脂复合物为中间载体制备自微乳时，无论采用“吐温80”为乳化剂还是采用“聚氧乙烯蓖麻油”为乳化剂，均无法获得外观为均一液体的自乳化混合物。
参照专利CN104826118A公开的一种绿原酸及其衍生物的磷脂 修饰物及其制备方法、以油酸乙酯为油相，吐温80、聚氧乙烯蓖麻油和RH40氢化聚氧乙烯蓖麻油为乳化剂，二乙二醇单乙基醚、丙二醇和聚乙二醇为助乳化剂，绿原酸磷脂复合物为中间载体，按照油相：乳化剂：助乳化剂质量比为2:5:3制备了绿原酸磷脂复合物自微乳(载药量60mg/g)。结果显示，所制备的自微乳系统呈现分层、半凝胶状和凝固三个状态，且室温或4℃放置的情况下仍然呈现上述三种状态，具体结果描述请见表1。
表1 参照公开专利的绿原酸磷脂复合物自微乳制备情况
Figure PCTCN2020096427-appb-000001
Figure PCTCN2020096427-appb-000002
参考目前常用的油相、乳化剂和助乳化剂，并将其用于绿原酸自乳化组合物的制备与粒径检测。具体步骤：以油酸乙酯为油相，吐温80为乳化剂，甘油为助乳化剂，三者比例按1:3:6混合(质量比)，得空白自微乳溶液；
另分别取绿原酸原料药及绿原酸的磷脂复合物(绿原酸与大豆卵磷脂1：1混合后，用无水乙醇溶解，干燥制得)加入到上述空白自微乳中，溶解，使绿原酸载药浓度为60mg/ml，放置一周，观察是否分层；取两种载药自微乳，加入100倍的水稀释，振摇，在2min内，激光粒度仪测定乳滴粒径。
结果如下表2：
表2:吐温80为乳化剂的自乳化组合物考察结果
Figure PCTCN2020096427-appb-000003
结果表明，油酸乙酯为油相，吐温80为乳化剂，甘油为助乳化剂，制得的包载绿原酸的自微乳，稀释后粒径小，但冷藏或室温放置3天即出现分层现象；制得的包载绿原酸磷脂复合物的自微乳，稀释后粒径为微米级，且冷藏出现凝固现象，室温贮存出现明显的分层。
综上，现有报道的绿原酸自微乳本身并不稳定，放置后出现不 稳定状态或粒径大的缺陷。将绿原酸与脂质材料复合后，再按照其油相、乳化剂和助乳剂组成制备的自微乳仍然不稳定，放置后自微乳系统不同程度的呈现分层、半凝胶状和凝固三个状态。
而本发明绿原酸纳米给药系统的自乳化组合物以绿原酸/脂质复合物为中间载体，乳化剂采用聚乙二醇甘油酯及其衍生物一种或一种以上的混合，制成的自乳化组合物，在室温及2-8℃条件下放置至少6个月，均不会出现凝固或分层现象，表现为均一的液体性状，加水稀释后可快速乳化形成纳米级乳滴，乳滴粒径<500nm。
本发明具体实验如下：
1、关于绿原酸/脂质复合物CHAPC为中间载体的制备与检测
以大豆卵磷脂为脂质材料，将绿原酸和大豆卵磷脂按照下表的药脂比进行投料，一同加入至1L的圆底烧瓶中，以无水乙醇为溶剂，使药物浓度控制在60mg/ml,待绿原酸和大豆卵磷脂全部溶解后,室温放置15min，随后于旋转蒸发仪100rpm、40℃旋蒸减压干燥除去无水乙醇，待乙醇蒸干后，于室温继续100rpm减压干燥1h将乙醇除干净。
取制备的系列复合物粉末，照如下方法测定复合率，结果见表3。
(1)对照品溶液：称取绿原酸标准品25mg至25ml容量瓶中，用乙醇溶液定容，精密量取1ml至50ml容量瓶中，用30％乙醇水定容，作为对照品溶液；
(2)W 总：称取80mg绿原酸复合物至25ml容量瓶中，用无水乙醇定容摇匀，精密量取1ml至50ml容量瓶中，用30％乙醇水溶液定容摇匀，作为供试品W 总溶液；
(3)W 复合：称取80mg绿原酸复合物至25ml容量瓶中，用氯仿溶解后定容，用0.22um滤膜过滤，精密量取续滤液1ml，氮吹挥干溶剂(氯仿)后，用30％乙醇水准确稀释至50mL，作为供试品W 复合溶液。
在制得以上样品的基础上，采用以下HPLC条件进行测定。
流动相：0.4％磷酸：乙腈＝82:18
检测波长：328nm
流速：1.0mL/min
柱温：25℃
进样量：10uL
Stop Time：8min
表3 无水乙醇为溶剂，不同药脂比复合物(CHAPC)及复合率
Figure PCTCN2020096427-appb-000004
结果表明，药脂比(摩尔比)为1:0.25至1:8，制得的复合物均复合完全，复合率在99％以上。
按绿原酸/大豆卵磷脂的药脂比(摩尔比)为1:1投料，采用不同溶剂制备复合物，并考察复合率，结果如下表4。
表4 不同溶剂对复合物(CHAPC)制备结果的影响
Figure PCTCN2020096427-appb-000005
结果表明，药脂比(摩尔比)为1:1时，甲醇、四氢呋喃、氯仿、二氯甲烷中的复合率均较低，叔丁醇、乙酸乙酯中的复合率在70％以上，乙醇(95％浓度)的复合率在80％以上。
以蛋黄卵磷脂、磷脂酰甘油酯、二硬脂酰磷脂酰胆碱、胆固醇、胆酸盐为脂质材料，无水乙醇为溶剂，按药脂比(摩尔比)为1:1，同上法制备复合物，编号依次为CHAPC 15、CHAPC 16、CHAPC 17、CHAPC18、CHAPC 19,测定复合率，结果表明，复合率均在92％以上。
关于脂质材料的种类，天然磷脂、合成磷脂、胆固醇和胆酸盐均 可满足要求，但优选天然磷脂。
2、本发明专利的自乳化组合物的组合比例研究结果
以油酸乙酯为油相，辛酸癸酸聚乙二醇甘油酯(labrasol)为乳化剂，transcutol HP为助乳化剂，三者质量比为2:3：5，混合均匀，制备空白自乳化溶液；另取绿原酸的磷脂复合物CHAPC 3，溶于上述空白自乳化溶液中，使绿原酸的载药量控制在10-200mg/ml，温度25℃，转速210rpm。待复合物溶解完全后即制得所述绿原酸自乳化组合物；取载药自乳化溶液适量，加水100倍进行稀释，考察自乳化效率及乳化后粒径(其中粒径检测方法：用移液枪精密移取20μL绿原酸自乳化组合物，加入装有2ml娃哈哈去离子水的5ml EP管中，轻轻摇匀，转移至比色皿，随后采用激光散射粒度仪测定其粒径)。结果如表5。
表5:辛酸癸酸聚乙二醇甘油酯为乳化剂的自乳化考察结果
Figure PCTCN2020096427-appb-000006
提示在10mg/g－150mg/g(药物/组合物)载药量范围内，均可获 得良好的自乳化纳米给药系统。
在表5的基础上，又开展了系列的研究，包括：
(1)使用不同编号的绿原酸/磷脂复合物进行自乳化组合物制备，发现CHAPC 1至CHAPC 5,CHAPC 15至CHAPC 19,均可获得良好的结果，外观为均一溶液，且乳化后粒径在200nm以下，其次为CHAPC 6、CHAPC 7、CHAPC 9、CHAPC 10，乳化后粒径在500nm以下。其他复合物效果较差，乳化粒径为微米级。
由此提示，对于本发明专利的绿原酸纳米给药系统的自乳化组合物，其中的“绿原酸/脂质复合物”，绿原酸与脂质材料的摩尔比在1:0.25～1:8均可满足要求，但优选的是1:0.25～1:4；绿原酸与脂质材料的复合率在70％以上可满足要求，但优选90％以上。
(2)尝试采用不同的聚乙二醇甘油酯衍生物代替辛酸癸酸聚乙二醇甘油酯(labrasol)，制备自乳化组合物，发现油酸聚乙二醇甘油酯(
Figure PCTCN2020096427-appb-000007
1944cs)、亚油酸聚乙二醇甘油酯(
Figure PCTCN2020096427-appb-000008
M2125CS)的任何一种或一种以上任意比例的混合，均可满足要求。
(3)尝试采用不同的油相代替油酸乙酯，制备自乳化组合物，发现大豆油、玉米油、橄榄油、椰子油、花生油、山茶油、蓖麻油、油酸山梨醇酯、油酸甘油酯，亚油酸甘油酯、labrafil1944cs、Maisine35-1、亚油酸乙酯、C8/C10甘油单酯、椰子油C8/C10甘油双酯、椰子油C8/C10甘油三酯、辛酸甘油三酯、辛酸甘油二酯、辛酸甘油单酯、癸酸甘油单酯、癸酸甘油二酯、癸酸甘油三酯、辛癸酸甘油单酯、辛癸酸甘油酯、辛癸酸甘油三酯、肉豆蔻酸异丙酯、亚油酸聚乙二醇甘油酯(
Figure PCTCN2020096427-appb-000009
M2125CS)、Gelucire、Capryol 90均可用于油相，且基本满足自乳化组合物制备的要求，其中，以油酸乙酯、油酸山梨醇酯、油酸甘油酯，亚油酸甘油酯、labrafil1944cs、Maisine35-1、亚油酸乙酯、C8/C10甘油单酯、椰子油C8/C10甘油双酯、椰子油C8/C10甘油三酯、辛酸甘油三酯、辛酸甘油二酯、辛酸甘油单酯、癸酸甘油单酯、癸酸甘油二酯、癸酸甘油三酯、辛癸酸甘油单酯、辛癸酸甘油酯、辛癸酸甘油三酯、肉豆蔻酸异丙酯、亚油酸聚乙二醇甘油酯(
Figure PCTCN2020096427-appb-000010
M2125CS)、Gelucire、Capryol 90中的一种或一种以上任意比例的混合效果更好，制备的自乳化组合物，稀释后的乳滴粒径更小。
(4)尝试了用不同的助乳化剂代替二乙二醇单乙基醚(
Figure PCTCN2020096427-appb-000011
HP)，制备自乳化组合物，结果表明，碳酸丙二酯、甘 油糠醛、二甲基异山梨酯、PEG400、甘油、labraosol、苯甲醇中的一种或两种以上任意比例的混合，均可用于乳化剂，其中，以二甲基异山梨酯、二乙二醇单乙基醚(
Figure PCTCN2020096427-appb-000012
HP)、甘油、labraosol中的一种或一种以上任意比例的混合，效果更好，而其中又以二乙二醇单乙基醚(
Figure PCTCN2020096427-appb-000013
HP)的效果最优。
(5)调整油相、乳化剂与助乳化剂三者的组成质量比，进行自乳化组合物制备。按三者总和为100％计，当油相为10-50％，乳化剂为30-60％，助乳化剂20-60％，可获得良好自乳化组合物。
(6)此外，为防止油脂类成分长期贮存的酸败，还偿试了在自乳化组合物中加入抗氧剂，维生素E及其酯类、BHT均可使用。
3、本发明专利的自乳化组合物的制备方法
(1)“绿原酸与脂质材料的复合物”的制备：称取绿原酸与脂质材料，溶于极性适中的有机溶剂中，混匀，放置一定时间(15-30min)，旋转蒸发或喷雾干燥去除有机溶剂，减压干燥，即得；
(2)“空白自乳化浓缩液”的制备：按比例称取油相、乳化剂和助乳化剂，搅拌混合均匀，即得；
(3)“绿原酸纳米给药系统的自乳化组合物”的制备：将“绿原酸与脂质材料的复合物”加入至“空白自乳化浓缩液”中，并将其置于空气浴振荡器中，温度25℃，转速210rpm或相当条件振荡，待“绿原酸与脂质材料的复合物”溶解完全后，即制得所述绿原酸自乳化组合物。性状为均一澄清的液体。
上述方法的3个步聚，任选地，还可将绿原酸与脂质材料的复合物与油相、乳化剂和助乳化剂一同加入，搅拌混合均匀，制得自乳化组合物；或将复合物与油相、乳化剂或助乳化剂的任一项中先溶解混匀，再加入另外两种成分，混匀，即得。
4、本发明专利的自乳化组合物的应用
本发明专利公开的一种绿原酸纳米给药系统的自乳化组合物，可用于制成适于口服给药的制剂，如将自乳化浓缩液直接分装成软胶囊、硬胶囊、口服液、滴剂、喷剂等,其中，优选软胶囊和滴剂。此外，还可将自乳化组合物浓缩液分散在微粉硅胶或其他吸附性固体材料上，加上药剂上常用的辅料，制成口服用的片剂、胶囊剂、颗粒剂或冲剂 等。
口服制剂直接服用，无需先用水稀释乳化后再服用。利用自乳化组合物的自乳化能力，可在胃肠液环境及胃肠蠕动条件下快速形成纳米级的乳滴。
除口服制剂外，本发明的自乳化组合物，还可分装制成供雾化吸入用的浓溶液，临用时加水、生理盐水或葡萄糖注射液稀释，采用雾化吸入装置进行雾化吸入。
将本发明专利的自乳化组合物先后用于lewis肺癌和胶质瘤治疗，采用灌胃给药，剂量在20-80mg/kg范围内，均显示了良好的治疗效果，与注射给药相比无明显差异。
综上，本发明在绿原酸与脂质材料摩尔比1:0.25～1:8，优选1:0.25～1:4；油相、乳化剂与助乳化剂三者按总和100％计，油相10-50％，乳化剂30-60％，助乳化剂20-60％，其中乳化剂采用聚乙二醇甘油酯及其衍生物一种或一种以上的混合；载药量10mg/g－150mg/g(药物/组合物)，制得的绿原酸纳米给药系统的自乳化组合物，稳定性好且与水混合后粒径小。将该组合物制成不同制剂用于抗肿瘤、抗炎免疫和抗病毒治疗，有显著的效果，具备实际应用推广价值。
显然，根据本发明的上述内容，按照本领域的普通技术知识和惯用手段，在不脱离本发明上述基本技术思想前提下，还可以做出其它多种形式的修改、替换或变更。
以下通过实施例形式的具体实施方式，对本发明的上述内容再作进一步的详细说明。但不应将此理解为本发明上述主题的范围仅限于以下的实例。凡基于本发明上述内容所实现的技术均属于本发明的范围。
附图说明
图1:试验例1中色谱条件一的分离检测HPLC图谱
图2:试验例1中色谱条件二的分离检测HPLC图谱
图3：本专利的绿原酸自乳化组合物和绿原酸自微乳对lewis肺癌的抑瘤效果
图4:绿原酸自乳化组合物对皮下胶质瘤的抑瘤效果
图5：绿原酸自乳化组合物对原位脑胶质瘤的抑瘤效果
具体实施方式
实施例1 以CHAPC 3为中间体采用不同的乳化剂制备绿原酸自乳化组合物
(1)“绿原酸与脂质材料的复合物”的制备：按照上述CHAPC 3的制备方法称取绿原酸与脂质材料，溶于无水乙醇中，混匀，放置15min，旋转蒸发去除有机溶剂，减压干燥，即得CHAPC 3；
(2)“空白自乳化浓缩液”的制备：按质量比2:5:3称取油酸乙酯、乳化剂(按照表6中乳化剂的种类进行变化)和transcutol HP，搅拌混合均匀，即得；
(3)“绿原酸纳米给药系统的自乳化组合物”的制备：按照表6中载药量计算称取CHAPC3与空白乳，混合后置于空气浴振荡器中，温度25℃，转速210rpm。待CHAPC 3溶解完全后即制得所述绿原酸自乳化组合物。并考察其初始性状、加水100倍稀释后粒径、2-8℃放置6个月性状、室温放置6个月性状。
表6 不同乳化剂制备的绿原酸自乳化组合物
Figure PCTCN2020096427-appb-000014
实施例2 以CHAPC 3为中间体采用不同的油相：乳化剂：助乳化剂制备的绿原酸自乳化组合物
(1)“绿原酸与脂质材料的复合物”的制备：按照上述CHAPC 3的制备方法称取绿原酸与脂质材料，溶于无水乙醇中，混匀，放置15min，旋转蒸发去除有机溶剂，减压干燥，即得CHAPC 3；
(2)“空白自乳化浓缩液”的制备：以油酸乙酯作为油相、labrasol为乳化剂、transcutol HP作为助乳化剂，按照表7中油相：乳化剂：助乳化剂的质量比称取一定量的油酸乙酯、labrasol和transcutol HP，搅拌混合均匀，即得空白自乳化浓缩液；
(3)“绿原酸纳米给药系统的自乳化组合物”的制备：按照表7中载药量计算称取CHAPC3与空白乳，混合后置于空气浴振荡器中，温度25℃，转速210rpm。待CHAPC 3溶解完全后即制得所述绿原酸自乳化组合物。并考察其初始性状、加水100倍稀释后粒径、2-8℃放置6个月性状、室温放置6个月性状。
表7 不同质量比的油相：乳化剂：助乳化剂制备的绿原酸自乳化组合物
Figure PCTCN2020096427-appb-000015
实施例3 以不同的CHAPC为中间体制备绿原酸自乳化组合物
1)“绿原酸与脂质材料的复合物”的制备：按照上述CHAPC的制备 方法称取绿原酸与脂质材料，溶于无水乙醇中，混匀，放置15min，旋转蒸发去除有机溶剂，减压干燥，即得不同的绿原酸和磷脂复合物CHAPC(见表8)；
(2)“空白自乳化浓缩液”的制备：以油酸乙酯作为油相、labrasol为乳化剂、transcutol HP作为助乳化剂，按质量比2:5:3称取油酸乙酯、labrasol和transcutol HP，搅拌混合均匀，即得空白自乳化浓缩液；
(3)“绿原酸纳米给药系统的自乳化组合物”的制备：按照表8中载药量计算称取CHAPC与空白乳，混合后置于空气浴振荡器中，温度25℃，转速210rpm。待CHAPC溶解完全后即制得所述绿原酸自乳化组合物。并考察其初始性状、加水100倍稀释后粒径、2-8℃放置6个月性状、室温放置6个月性状。
表8 不同CHAPC为中间体制备的绿原酸自乳化组合物
Figure PCTCN2020096427-appb-000016
实施例4 不同CHAPC：空白自微乳比例制备绿原酸自乳化组合物
(1)“绿原酸与脂质材料的复合物”的制备：按照上述CHAPC 3的制备方法称取绿原酸与脂质材料，溶于无水乙醇中，混匀，放置15min， 旋转蒸发去除有机溶剂，减压干燥，即得CHAPC 3；
(2)“空白自乳化浓缩液”的制备：以油酸乙酯作为油相、labrasol为乳化剂、transcutol HP作为助乳化剂，按照油相：乳化剂：助乳化剂的质量比为2:5:3称取一定量的油酸乙酯、labrasol和transcutol HP，搅拌混合均匀，即得得空白自乳化浓缩液；
(3)“绿原酸纳米给药系统的自乳化组合物”的制备：按照表9中CHAPC与空白乳的质量比称取CHAPC 3和空白乳剂，混合后得到不同载药量的混合物，并将其置于空气浴振荡器中，温度25℃，转速210rpm。待CHAPC 3溶解完全后即制得所述绿原酸自乳化组合物。并考察其初始性状、加水100倍稀释后粒径、2-8℃放置6个月性状、室温放置6个月性状。
表9 不同CHAPC为中间体制备的绿原酸自乳化组合物
Figure PCTCN2020096427-appb-000017
实施例5 加入不同抗氧剂的绿原酸自乳化组合物的稳定性考察
(1)“绿原酸与脂质材料的复合物”的制备：按照上述CHAPC 3的制备方法称取绿原酸与脂质材料，溶于无水乙醇中，混匀，放置15min， 旋转蒸发去除有机溶剂，减压干燥，即得CHAPC 3；
(2)“空白自乳化浓缩液”的制备：以油酸乙酯作为油相、labrasol为乳化剂、transcutol HP作为助乳化剂，按照油相：乳化剂：助乳化剂的质量比为2:5:3称取一定量的油酸乙酯、labrasol和transcutol HP，搅拌混合均匀，即得空白自乳化浓缩液。并在所得的空白自乳化浓缩液中加入抗氧剂(种类如表10所示)；
(3)“绿原酸纳米给药系统的自乳化组合物”的制备：按照CHAPC与空白乳的质量比1.2:5称取“绿原酸与脂质材料的复合物CHAPC 3”加入至“空白自乳化浓缩液”中，并将其置于空气浴振荡器中，温度25℃，转速210rpm。待CHAPC 3溶解完全后即制得所述绿原酸自乳化组合物。并考察其初始性状、加水100倍稀释后粒径、2-8℃放置6个月性状和室温放置6个月性状。
表10 加入不同抗氧剂的绿原酸自乳化组合物
Figure PCTCN2020096427-appb-000018
Figure PCTCN2020096427-appb-000019
实施例6 绿原酸自乳化组合物用于口服用软胶囊制剂或雾化吸入给药
(1)“绿原酸与脂质材料的复合物”的制备：按照上述CHAPC 3的制备方法称取绿原酸与脂质材料，溶于无水乙醇中，混匀，放置15min，旋转蒸发去除有机溶剂，减压干燥，即得CHAPC 3；
(2)“空白自乳化浓缩液”的制备：以油酸乙酯作为油相、labrasol为乳化剂、transcutol HP作为助乳化剂，按照油相：乳化剂：助乳化剂的质量比为2:5:3称取一定量的油酸乙酯、labrasol和transcutol HP，搅拌混合均匀，即得空白自乳化浓缩液；
(3)“绿原酸纳米给药系统的自乳化组合物”的制备：按照CHAPC与空白乳的质量比1.2:5称取“绿原酸与脂质材料的复合物CHAPC 3”加入至“空白自乳化浓缩液”中，并将其置于空气浴振荡器中，温度25℃，转速210rpm。待CHAPC 3溶解完全后即制得所述绿原酸自乳化组合物。
(4)将该绿原酸自乳化组合物装入软胶囊，制备口服用软胶囊制剂。此外还可以将该绿原酸自乳化组合物加水、生理盐水或葡萄糖注射液稀释，连接雾化吸入装置进行雾化吸入给药。
以下通过试验例进一步说明本发明的有益效果：
试验例1：HPLC含量测定方法研究
绿原酸为多酚类结构，在溶液状态下极易发生化学降解，若色谱条件不能使主峰与降解产物有效分离，则会直接影响稳定性考察结果的准确性。为此，采用高温加速破坏的自乳化样品，用无水乙醇破乳，加水稀释至绿原酸浓度约为0.1mg/ml的供试液，考察下述两种条件进行测定。
色谱条件一：等度洗脱，流动相为0.4％磷酸：乙腈＝82:18；流速：1mL/min；检测波长：328nm；柱温：25℃；进样体积：10μL。
色谱图见附图1。
色谱条件二：采用梯度洗脱，流动相条件如下。
Figure PCTCN2020096427-appb-000020
柱温：25℃；流速：1.0mL/min；波长：328nm进样体积：10μL
色谱图见图2。
图1和图2比较发现，在设定的测定浓度条件下，两种色谱条件测得的绿原酸主峰面积差别不大，但对杂质的分离检测能力，色谱条件二明显优于色谱条件一。因此，后续采用色谱条件二进行自乳化样品的稳定性考察。
试验例2：绿原酸自乳化组合物的稳定性考察
取实施例1及实施例2的9组不同编号的自乳化组合物样品，于4℃和室温条件下放置7个月，取出，进行外观性状和化学稳定性考察。
外观性状：9组样品经4℃和室温条件下放置7个月，均为澄清透明的均一溶液，无沉淀，表明物理稳定性良好。
含量：取稳定性样品，精密称定，用无水乙醇破乳，加水稀释至绿原酸浓度约为0.1mg/ml的供试溶液，另取绿原酸原料药，配制相当浓度的对照溶液；取供试溶液与对照溶液各10μL注入液相色谱仪，照试验例1中的色谱条件二(梯度洗脱)依法检测，外标法计算药物浓度，并根据理化载药量计算标示含量。
结果表明，9组自乳化组合物样品，于4℃和室温条件下放置7个月，标示含量均稳定在95-105％范围内，与初始相比，含量及杂质无明显变化。
试验例3：绿原酸自乳化组合物对皮下荷lewis肺癌的ICR小鼠 的抑制肿瘤效果考察
采用皮下接种lewis肺癌的C57BL/6小鼠进行绿原酸自乳化组合物的肿瘤抑制效果评价。在荷瘤后的第二天分别按照35mg/kg和30mg/kg的口服给药剂量对荷瘤小鼠灌胃给予绿原酸自微乳(制备方法同文献Li Chen,Chang-shun Liu,Qing-zhen Chen,SenWang,Yong-ai Xiong,Jing Jing,Jia-jia Lv.Characterization,pharmacokinetics and tissue distribution of chlorogenic acid-loaded self-microemulsifying drug delivery system.European Journal of Pharmaceutical Sciences,2017,100:102–108，简称绿原酸自微乳)和绿原酸自乳化组合物(实施例中样品编号1)，灌胃口服生理盐水作为对照，连续灌胃给药13天，期间观察动物的状态及体重，给药结束后第二天断颈处死小鼠，剥取肿瘤组织进行称重，并与对照组的瘤重相比较，作为肿瘤抑制效率评价。结果见附图3。
结果显示，在整个给药期间，所有实验动物均状态良好，体重未出现明显下降。图3结果显示，本发明公开的绿原酸自乳化组合物对lewis肺癌的抑瘤率达到88.57±0.17％，而绿原酸自微乳的抑瘤率只有50.24±9.07％。在给药剂量小于绿原酸自微乳的情况下，绿原酸自乳化组合物的肿瘤抑制效率是绿原酸自微乳的1.76倍。
试验例4：绿原酸自乳化组合物对皮下荷脑胶质瘤的ICR小鼠的抑制肿瘤效果考察
采用皮下接种G422的ICR小鼠进行绿原酸自乳化组合物对脑胶质瘤的肿瘤抑制效果评价。在荷瘤后的第二天分别按照30mg/kg的口服给药剂量对荷瘤小鼠灌胃给予阳性对照药环磷酰胺(CTX，剂量)和绿原酸自乳化组合物(实施例中样品编号1，剂量分别为：20mg/kg,简称CHA SME 20；40mg/kg,简称CHA SME 40；60mg/kg,简称CHA SME 60)，灌胃口服生理盐水作为对照，连续灌胃给药13天。同时腹腔注射绿原酸原料药水溶液(剂量分别为：10mg/kg，简称CHA 10；20mg/kg，简称CHA 20；40mg/kg，简称CHA 40.)作为对比。期间观察动物的状态及体重，给药结束后第二天断颈处死小鼠，剥取肿瘤组织进行称重，并与对照组的瘤重相比较，作为肿瘤抑制效率评价。结果见附图4。
结果显示，在整个给药期间，所有实验动物均状态良好，体重未出现明显下降。图4结果显示，本发明公开的绿原酸自乳化组合物(CHA SME 20、CHA SME 40和CHA SME 60)对皮下接种G422的ICR小鼠的抑瘤率达到30.71±6.42％、20.17±8.94％和33.46±5.97％，效果与绿原酸原料药溶液的腹腔注射组相当(CHA 10:31.7±8.07％；CHA 20:27.82±9.0％；CHA 40:23.82±4.92％)。
试验例5：绿原酸自乳化组合物对原位脑胶质瘤的ICR小鼠的抑制肿瘤效果考察
采用原位接种G422的ICR小鼠进行绿原酸自乳化组合物对脑胶质瘤的肿瘤抑制效果评价。在荷瘤后的第二天分别对荷瘤小鼠灌胃给予阳性对照药替莫唑胺(TMZ,50mg/kg)和绿原酸自乳化组合物(实施例中样品编号1,剂量分别为20mg/kg和35mg/kg，简称：CHA SME 20mg/kg和CHA SME 35mg/kg)，灌胃口服生理盐水作为对照，连续灌胃给药13天。此外，同时采用腹腔给药绿原酸原料药溶液(剂量分别为10mg/kg和20mg/kg，简称：CHA10mg/kg和CHA 20mg/kg)作为对照。期间观察动物的状态及体重，给药结束后量动物的肿瘤体积大小，作为肿瘤抑制效率评价。结果见附图5。
结果显示，在整个给药期间，所有实验动物均状态良好，体重未出现明显下降。图5结果显示，本发明公开的绿原酸自乳化组合物(CHA SME 35mg/kg)对原位接种G422的ICR小鼠的抑瘤率达到54.8％，明显优于绿原酸原料药腹腔注射组(CHA 10mg/kg,27.1％；CHA 20mg/kg,45.6％)。
综上，本发明绿原酸纳米给药系统的自乳化组合物，在室温及2-8℃条件下放置不会出现凝固或分层现象，可用于口服给药、粘膜给药、经皮给药、肠道给药或雾化吸入用制剂的制备。模拟口服给药后的胃肠环境，组合物用10-1000倍体积的水、不同pH缓冲液、人工胃液或人工肠液进行稀释，经轻微振摇，均可快速形成平均粒径在500nm以下的纳米级给药系统。本发明的自乳化组合物及其制剂，可用于抗肿瘤、抗炎免疫和抗病毒的治疗。

Claims (21)

  1. 一种绿原酸纳米给药系统的自乳化组合物，其特征在于，它是由绿原酸与脂质材料形成的复合物、油相、乳化剂和助乳化剂组成；所述乳化剂为聚乙二醇甘油酯及其衍生物的任意一种或几种。
  2. 根据权利要求1所述的自乳化组合物，其特征在于，所述绿原酸脂质材料复合物的复合率大于70％，优选大于80％，更优选大于90％。
  3. 根据权利要求1所述的自乳化组合物，其特征在于，所述组合物中绿原酸含量为5－200mg/g。
  4. 根据权利要求3所述的自乳化组合物，其特征在于，所述组合物中绿原酸含量为10－150mg/g。
  5. 根据权利要求1所述的绿原酸自乳化组合物，其特征在于，所述绿原酸脂质材料复合物中绿原酸与脂质材料的摩尔比为1:0.25～1:8，优选1:0.25～1:4。
  6. 根据权利要求1或5所述的绿原酸自乳化组合物，其特征在于，所述脂质材料选自胆固醇、磷脂类或胆酸盐类；优选，磷脂类。
  7. 根据权利要求6所述的绿原酸自乳化组合物，其特征在于，所述磷脂类为大豆磷脂、蛋黄磷脂、磷脂酰甘油酯、二硬脂酰磷脂酰胆碱、二棕榈酰磷脂酰胆碱、二棕榈酰磷脂酰乙醇胺、二肉蔻酰磷脂酰胆碱、神经酰胺中的任意一种或几种；优选，大豆磷脂和/或蛋黄磷脂。
  8. 根据权利要求1所述的自乳化组合物，其特征在于，所述油相、乳化剂与助乳化剂质量百分比，按三者总和100％计，油相为10-50％，乳化剂为30-60％，助乳化剂为20-60％。
  9. 根据权利要求1所述的绿原酸自乳化组合物，其特征在于，所述聚乙二醇甘油酯衍生物为辛酸癸酸聚乙二醇甘油酯、油酸聚乙二醇甘油酯或亚油酸聚乙二醇甘油酯。
  10. 根据权利要求1或8所述的绿原酸自乳化组合物，其特征在于，所述油相为大豆油、玉米油、橄榄油、椰子油、花生油、山茶油、蓖麻油、油酸山梨醇酯、油酸甘油酯，亚油酸甘油酯、labrafil1944cs、Maisine35-1、油酸乙酯、亚油酸乙酯、C 8/C 10甘油单酯、椰子油C 8/C 10甘油双酯、椰子油C 8/C 10甘油三酯、辛酸甘油三酯、辛酸甘油二酯、辛酸甘油单酯、癸酸甘油单酯、癸酸甘油二酯、癸酸甘油三酯、辛癸酸甘油单酯、辛癸酸甘油酯、辛癸酸甘油三酯、肉豆蔻酸异丙酯、亚 油酸聚乙二醇甘油酯、Gelucire、Capryol 90中的任意一种或几种；优选，油酸乙酯、油酸山梨醇酯、油酸甘油酯，亚油酸甘油酯、labrafil1944cs、Maisine35-1、亚油酸乙酯、C 8/C 10甘油单酯、椰子油C 8/C 10甘油双酯、椰子油C 8/C 10甘油三酯、辛酸甘油三酯、辛酸甘油二酯、辛酸甘油单酯、癸酸甘油单酯、癸酸甘油二酯、癸酸甘油三酯、辛癸酸甘油单酯、辛癸酸甘油酯、辛癸酸甘油三酯、肉豆蔻酸异丙酯、亚油酸聚乙二醇甘油酯、Gelucire、Capryol 90中的任意一种或几种。
  11. 根据权利要求1或8所述的绿原酸自乳化组合物，其特征在于，所述助乳化剂为碳酸丙二酯、乙二醇单乙基醚、甘油糠醛、二甲基异山梨酯、二乙二醇单乙基醚、PEG400、甘油、labraosol、苯甲醇中的任意一种或几种；优选，二甲基异山梨酯、二乙二醇单乙基醚、甘油、labraosol中的任意一种或几种。
  12. 根据权利要求1所述的绿原酸自乳化组合物，其特征在于，它还包括抗氧剂；所述抗氧剂为维生素E、维生素E酯类衍生物或二丁基羟基甲苯任意一种或几种。
  13. 一种制备权利要求1-12任一项所述的绿原酸纳米给药系统的自乳化组合物方法，其特征在于，它包括如下步骤：
    (1)制备绿原酸与脂质材料的复合物：按配比称取绿原酸与脂质材料，溶于有机溶剂中，混匀，静置15-30min，旋转蒸发或喷雾干燥去除有机溶剂，再减压干燥，即得；
    (2)制备空白自乳化浓缩液：按配比称取油相、乳化剂和助乳化剂，混合均匀，即得；
    (3)制备绿原酸自乳化组合物：将步骤(1)复合物加入步骤(2)浓缩液中，溶解完全，即得；
    或：
    按配比将步骤(1)复合物与油相、乳化剂和助乳化剂一同加入，搅拌混合均匀，即得；
    或：
    按配比先将步骤(1)复合物与油相、乳化剂或助乳化剂中任意一种溶解混匀，再加入另外两种成分混匀，即得。
  14. 根据权利要求13所述的制备方法，其特征在于，步骤(1)所述有机溶剂为低分子醇类；所述低分子醇类为乙醇、无水乙醇、叔丁醇中的任意一种或几种；优选，无水乙醇。
  15. 根据权利要求13所述的制备方法，其特征在于，步骤(3)所述溶解为振摇溶解，振摇温度为室温，速度210rpm。
  16. 一种绿原酸制剂，其特征在于，它是由权利要求1-12任意一项所述的绿原酸自乳化组合物，加上药学上可接受的载体组成的制剂。
  17. 根据权利要求16所述的制剂，其特征在于，所述制剂为口服制剂、粘膜给药制剂、经皮给药制剂、肠道给药制剂或雾化吸入用制剂。
  18. 根据权利要求17所述的制剂，其特征在于，所述口服制剂为片剂、颗粒剂、胶囊剂、口服液、滴剂或喷剂；所述粘膜给药制剂或经皮给药制剂为涂剂、喷剂或其它适宜剂型；所述肠道给药制剂为液体制剂或溶于油性基质制成栓剂。
  19. 根据权利要求18所述的制剂，其特征在于，所述片剂、颗粒剂或胶囊剂是将绿原酸自乳化组合物分散在微粉硅胶或其他吸附性固体材料上，再加上药剂上可接受的辅料制成。
  20. 根据权利要求17所述的制剂，其特征在于，所述雾化吸入用制剂是将绿原酸自乳化组合物分装制成供雾化吸入用的浓溶液。
  21. 权利要求1-12任意一项所述的绿原酸自乳化组合物或者权利要求16-20任意一项所述的绿原酸制剂在制备治疗和/或预防恶性肿瘤、免疫低下、细菌病毒感染和/或抗氧化的药物中的用途。
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