WO2011120339A1

WO2011120339A1 - Microemulsion formulation containing arctigenin

Info

Publication number: WO2011120339A1
Application number: PCT/CN2011/000574
Authority: WO
Inventors: 赵志全
Original assignee: 鲁南制药集团股份有限公司
Priority date: 2010-04-03
Filing date: 2011-04-01
Publication date: 2011-10-06
Also published as: CN102210653A; CN102210653B

Abstract

A microemulsion formulation contains arctigenin and microemulsion carriers, said microemulsion carriers comprise oil phase and emulsifier, and the microemulsion has an average particle size of 10-90 nm.

Description

Microemulsion preparation of arctigenin

The invention belongs to the field of medicine and relates to a microemulsion preparation. In particular, the present invention relates to a microemulsion formulation of arctigenin. Background technique

Burdock is a dry and mature fruit of the burdock of the Compositae. It is a commonly used traditional Chinese medicine. It has the functions of evacuating wind and heat, venting the lungs, and detoxifying and pharynx. It is used for wind-heat, cold, cough, measles, rubella, sore throat. Itchy erysipelas, swollen sores. The traditional Chinese medicine contains lignan compounds, mainly arctiin and arctigenin. Although the content of burdock aglycone (also known as arctigenin) is very low in burdock, its precursor burdock is high in Chinese burdock and burdock fruit, so a large number of burdock can be obtained by transformation of burdock glycosides. Aglycone. According to reports in the literature, burdock is decomposed into burdock aglycon in the body and produces numerous pharmacological effects, and burdock aglycone has stronger biological activity than burdock, such as significant antibacterial, antiviral, antitumor, anti-PAF receptors and Calcium antagonistic activity.

The burdock aglycone is a white powder or a colorless block crystal. It is easily soluble in an organic solvent such as chloroform, methanol or ethanol. It is poorly soluble in petroleum ether and has a melting point of 111-112 °C. It is not volatile, it is not easily oxidized in the air, and its physical and chemical properties are relatively stable. However, the burdock aglycone is difficult to dissolve in water, and its bioavailability is low, which limits its application as a new drug to some extent. The molecular structure of bovine glycosides is as follows:

Bovine aglycone (Arctigenin)

The pharmacokinetic characteristics of arctigenin in mice and rabbits have been reported. The pharmacokinetics of burdock in mice, the gastrointestinal tract absorption of rabbits, the intravenous injection of rabbits, and the distribution of burdock aglycone in rats showed that: (1) burdock aglycone in the gastrointestinal tract Stable, less conversion, less damage. (2) When rabbits were injected with different doses of arctigenin, the main kinetic parameters such as half-life T _{1/2 of} each dose were very close, and the area under the drug-time curve (AUC) increased proportionally with the dose; The elimination of aglycones is linear kinetics; (3) rabbit gavage The absolute bioavailability of burdock aglycone was 9.5%. (4) The burdock aglycone was widely distributed in rats and distributed in liver and lung, followed by heart, spleen and kidney. Plasma protein binding rate experiments showed that the average binding rate of arctigenin to rat serum polyprotein was 78.3 %.

The bioavailability of intragastric administration in rabbits was only 9.5%. The main reason for the analysis was that the burdock aglycone was a fat-soluble compound with poor water solubility, which led to low oral bioavailability. The invention patent application publication CN 101036644A discloses a pharmaceutical composition containing arctigenin, which comprises an anthraquinone in a cyclodextrin derivative to solve the poor water solubility and bioavailability of arctigenin. Low problem. The invention patent application publication CN101036643A also discloses a pharmaceutical composition containing arctigenin, which comprises an alloin, an oil, an emulsifier and water, and specifically provides an oral or intravenous emulsion. The average particle size of the emulsion is more than 100 nm, and the emulsion contains 0.05-2.0 g of burdock aglycone, 5-20 g of oil, 0.5-5.0 g of emulsifier, 0.02-0.2 g of oleic acid, and the balance is water.

The microemulsion drug delivery system is a mixture of a drug, an oil phase (ie, an oil phase component), water, an emulsifier, and a co-emulsifier in an appropriate ratio, and has an average particle diameter of 10 to 100 nm, which is thermodynamically stable in vitro. The system is stabilized by an emulsifier and a co-emulsifier; it has good solubility for water-soluble, fat-soluble and poorly soluble drugs, and has high physical stability; it is easy to penetrate the stomach and intestine due to low surface tension. The hydration layer of the wall, the drug can directly contact with the gastrointestinal epithelial cells, promote drug absorption, improve bioavailability; can be absorbed by lymphatics after oral administration, overcoming the first-pass effect and obstacles when the macromolecule passes through the gastrointestinal epithelial cell membrane .

However, the preparation of microemulsions also has many difficulties. Since a large amount of emulsifier is required in the microemulsion, the emulsifier content increases and its accumulation in the body tends to produce certain toxicity, and the co-emulsifier, the oil phase and the ratio between them will all affect the microemulsion. The diameter and its stability have a major impact. Summary of the invention

In order to improve the water solubility of burdock aglycone and its oral bioavailability in vivo, the present invention provides a novel microemulsion preparation of arctigenin. The microemulsion of the present invention has a uniform particle size, generally 10-100 nm. The invention prepares the burdock aglycone microemulsion concentrate by using the microemulsion as a pharmaceutical carrier of the burdock aglycone, and can also prepare the microemulsion preparation containing the burdock aglycone by adding an excipient, including the solid micro Milk preparations and liquid microemulsion preparations.

The microemulsion preparation of the arctigenin of the present invention comprises an arctigenin microemulsion carrier comprising an oil phase and an emulsifier, and the microemulsion preparation has an average particle diameter of 10 to 90 nm. The burdock aglycone and the microemulsion carrier constitute a microemulsion concentrate of burdock aglycone. A microemulsion preparation of arctigenin according to the present invention, wherein the microemulsion carrier may further contain an excipient. The burdock aglycone microemulsion concentrate of the present invention may be liquid, solid or semi-solid at room temperature. The microemulsion preparation of the present invention comprises an arctigenin and a microemulsion carrier, the microemulsion carrier comprising an oil phase and an emulsifier, and may further comprise a co-emulsifier, preferably, the bovine aglycone in the microemulsion preparation of the present invention, The weight ratio of the oil phase, the emulsifier and the co-emulsifier is: 1-10 parts of burdock aglycone, 5-85 parts of an oil phase, 20-60 parts of an emulsifier, and 5-60 parts of a co-emulsifier. The present invention also preferably optimizes the oil phase, the emulsifier and the co-emulsifier, respectively.

Oil is one of the essential components relative to the burdock aglycone microemulsion preparation. The oil phase can promote the transport of drugs in the body, enhance the absorption of the body, and improve the bioavailability of the drug. Within a certain range, the larger the molecular volume of the oil phase, the stronger the solubility of the drug, but the molecular volume of the oil phase is too large to form a microemulsion. In order to increase the solubility of the drug and increase the area of microemulsion formation, the oil phase compatible with the drug should be selected.

Preferably, the oil phase of the above microemulsion preparation is a solid or semi-solid oil phase component selected from one or more of a fatty acid glyceride, an alcohol ester, a fatty alcohol, and a polysaccharide-derived saturated glyceride. Further preferably, the fatty acid glyceride is selected from one or more of hydrogenated cocoglyceride, hydrogenated palm oil PEG-6 ester and hydrogenated palm oil PEG-6 ester, and the alcohol ester is selected from propylene glycol stearate. One or more of PEG-2-stearate and cetyl palmitate, the fatty alcohol is myristyl alcohol, and the polysaccharide-saturated glyceride is lauroyl polyethylene glycol-32- Glyceride.

Preferably, the oil phase of the above microemulsion preparation is a liquid oil phase component selected from one or more of glycerides, natural vegetable oils and their fractions, essential oils, volatile oils and synthetic oils. The glyceride is a mixture of a monoglyceride and a diglyceride, a caprylic acid glyceride, a medium chain fatty acid glyceride, a monolinoleic acid glyceride or a glyceryl caprylate, and the natural vegetable oil and the fraction thereof are safflower oil. , sesame oil, corn oil, castor oil, coconut oil, cottonseed oil, soybean oil, lemon oil, peppermint oil, menthol, caraway, thymol or any mixture thereof, volatile oils are spearmint oil, clove oil, lemon The oil, peppermint oil or any mixture thereof, the synthetic oil is triacetin, ethyl butyrate, ethyl oleate, ethyl oleate, isopropyl myristate, ethyl octanoate or any mixture thereof.

The preference for the above oil phase is mainly to increase the solubility of the burdock aglycone in the oil phase and to increase the area in which the microemulsion is formed. All of the above "preferred" means that the oil phase having a larger area of microemulsion formation is selected as much as possible while ensuring greater solubility of the arctigenin. The preferred component has a stronger solubility of arctigenin. Ability and ability to form a 3⁄4 ^ large microemulsion area.

The emulsifier is one of the essential components of the burdock aglycone microemulsion preparation. The emulsifier is generally a surface active material and a mixture thereof, and may be an ionic, nonionic or zwitterionic surfactant having a reduced interfacial tension to form an interfacial film to promote microemulsion formation. Not all surfactants are suitable as components of the microemulsions of the present invention.

The emulsification agent used in the present invention is preferably a nonylphenol ethoxylate, Polyoxyethylene fatty acid ester, polyoxyethylene castor oil, polyoxyethylene hydrogenated castor oil, sorbitan derivative, polyoxyethylene-polyoxypropylene copolymer, polyoxyethylene nonyl ether, polyglycerin fatty acid ester and sub One or more of the mercapto polyol esters. The polyoxyethylene fatty acid ester is polyoxyethylene stearate, PEG-40-stearate or a mixture thereof, and the sorbitan derivatives are Tween-20, Tween-40, Tween-60, and spit. Wen-80, or a mixture thereof, the polyoxyethylene-polyoxypropylene copolymer is poloxamer, the polyoxyethylene decyl ether is polyoxyethylene-2-cetyl ether, polyoxyethylene-10-whale wax Ether, polyoxyethylene-20-cetyl ether, polyoxyethylene-23-lauryl ether, polyoxyethylene-20-oleyl ether, polyoxyethylene-2-stearyl ether, polyoxyethylene-10 - stearyl ether, polyoxyethylene-20-stearyl ether or a mixture thereof, polyglycerol fatty acid ester is decaglyceryl monostearate, hexaglycerol monostearate, tetraglyceryl monostearate Or a mixture thereof, the alkylene polyol ester is stearyl, lauroyl polyethylene glycol-32-glyceride or a mixture thereof.

Preferably, the above polyoxyethylene alkyl ether is polyoxyethylene-23-lauryl ether or polyoxyethylene-20-oleyl ether.

Self-emulsifying drug delivery systems (SEDDS) require self-emulsifying and maintaining the state of the emulsion in the gastrointestinal tract. A large amount of emulsifier is required in the formulation, but a large emulsifier may irritate the gastrointestinal tract, so the safety of the emulsion should be fully considered. Therefore, it is preferred for the emulsifier to combine pharmaceutics and pharmacological toxicology studies to select an emulsifier which is less toxic, irritating and hemolytic, and which has a stronger ability to lower the interfacial tension. The "preferred" of the above emulsifiers indicates the screening of the emulsifier safety and emulsifying ability. Preferred components have greater safety and emulsifying capabilities.

In a preferred embodiment, the burdock aglycone microemulsion formulation of the invention further comprises a co-emulsifier. Co-emulsifiers are generally surface active materials. The co-emulsifier can not only increase the flexibility of the film, but also facilitate the formation of the microemulsion droplet interface film, increase the dissolution of the emulsifier, further reduce the interfacial tension, and is beneficial to the stability of the microemulsion. The emulsifier used in the present invention is a medium chain length alcohol or a nonionic surfactant polar organic substance having a suitable HLB value.

The co-emulsifier of the present invention is one of the following components or any mixture thereof: short-chain alcohol, organic ammonia, mercapto acid, glyceryl mono-dimercaptoate, and polyoxyethylene fatty acid ester. Preferably, the co-emulsifier is a short chain alcohol selected from one or more of polyethylene glycol, n-butanol, n-hexanol, ethanol, ethylene glycol, propylene glycol, and glycerin. Further preferably, the co-emulsifier is ethanol, glycerin, polyethylene glycol 200, polyethylene glycol 300, polyethylene glycol 400 or 1, 2-propanediol.

The preferred preference for the co-emulsifier is to consider the effect of the addition of the co-emulsifier on the stability and emulsification of the microemulsion. The length of the co-emulsifier chain has a certain influence on the auxiliary emulsification effect. The linear chain is superior to the branched chain, and the long chain is superior to the short chain. When the cosurfactant chain length reaches the surfactant carbon chain length, It works best. The "preferred" of the above co-emulsifiers means the screening of the emulsifying ability and stability of the co-emulsifier. The preferred components are more powerful in ensuring microemulsion stability and emulsifying ability to the drug.

After the oil phase, emulsifier and co-emulsifier are determined, the microemulsion region can be identified by the ternary phase diagram to determine it. Their usage. At the same time, it is necessary to consider the safety of the emulsifier, the stability of the co-emulsifier to the microemulsion and their solubilizing effect on the arctiin. Minimize the amount of emulsifier in the case of ensuring the preparation of microemulsions to ensure the lowest possible toxicity. Therefore, the weight ratio of the oil phase, the emulsifier and the co-emulsifier of the microemulsion is preferred, and the weight ratio of the oil phase, the emulsifier and the co-emulsifier is: 5-85 parts of the oil phase, 20-60 of the emulsifier 5 to 60 parts of co-emulsifier. Further preferably, the weight ratio of the oil phase, the emulsifier, and the co-emulsifier is: 20-40 parts of the oil phase, 35-55 parts of the emulsifier, and 15-45 parts of the co-emulsifier. '

Most of the microemulsion concentrate of the bovine aglycone prepared above has a liquid state, and in order to make the above microemulsion concentrate solid or semi-solid at room temperature, a hydrophilic component is added to the above microemulsion concentrate. The hydrophilic component is preferably polyethylene glycol, polyethylene oxide or a mixture thereof. The polyethylene glycol is polyethylene glycol 200, polyethylene glycol 300, polyethylene glycol 400 or a mixture thereof.

Thus, in one embodiment, the microemulsion concentrate formed in accordance with the present invention comprises components in the following weight ratios: 1-10 parts of arctigenin, 5-85 parts of oil phase, 20-60 parts of emulsifier, co-emulsifier 5-60 parts, hydrophilic component 20-70 parts. Preferably, the microemulsion concentrate comprises components in the following weight ratio: 1-10 parts of burdock aglycone, 20-40 parts of oil phase, 35-55 parts of emulsifier, 15-45 parts of co-emulsifier, hydrophilic group 30-50 parts. It is preferred for the kind and weight of the hydrophilic component to indicate that the crystal structure of the preferred hydrophilic component is not affected or less affected.

The above microemulsion preparation can be used as a self-emulsifying drug delivery system, and the microemulsion concentrate containing the medicine enters the gastrointestinal tract and automatically forms microemulsion after contact with the water in the stomach and intestine for absorption by the human body. It is also possible to prepare the microemulsion preparation by adding the microemulsion concentrate to the excipient. To increase the patient's choice of drug route and dosage form preferences for the drug microemulsion formulation. The excipient is a preservative, a filler, a pH adjuster, an isotonicity adjuster, an acidulant, water or a mixture thereof. The excipients range from 0.05% to 80% by weight of the microemulsion.

Preferably, the above preservative is ascorbic acid and its derivatives, tocopherol and its derivatives, butylated hydroxyanisole, butylhydroxytoluene or any mixture thereof. Fillers are microcrystalline cellulose, silica, starch and its derivatives, lactose, dicalcium dihydrate, mannitol or any mixture thereof. The acidifying agent is citric acid, succinic acid, fumaric acid, ascorbic acid, phosphoric acid, citric acid, oleic acid, cellulose acetate, acetic acid-1,2,4-benzenetricarboxylic acid cellulose, hydroxypropyl methylcellulose Acid ester, carboxymethyl ethyl cellulose, carbomer or any mixture thereof. The pH adjusting agent is a buffer solution of phosphate, sodium hydroxide, hydrochloric acid or any mixture thereof. The isotonicity adjusting agent is a sodium chloride solution, a potassium chloride solution or a mixture thereof.

The above microemulsion concentrate of bovine aglycone and an excipient can be prepared into an oral microemulsion preparation and a intravenous microemulsion preparation. Oral microemulsion formulations include tablets, granules and oral solutions. Due to the natural formation and thermodynamic stability, the preparation process of the burdock aglycone microemulsion preparation is simple. The burdock aglycone microemulsion preparation can be prepared into a water-in-oil type (W/0 type) or an oil-in-water type (0/W type), and the present invention is preferably prepared into a 0/W type microemulsion preparation. The preparation process of the burdock aglycone microemulsion preparation comprises the following steps:

(1) using a pseudo-ternary phase diagram to screen the prescription, determining the oil phase, emulsifier, co-emulsifier type of the burdock aglycone microemulsion preparation and the ratio between them, and determining the area where the microemulsion can be formed, and Finalizing the formulation of the oil phase, emulsifier and co-emulsifier of the burdock aglycone microemulsion;

(2) (a) dissolving the emulsifier in the oil phase, then adding the co-emulsifier and the burdock aglycone; or

(b) directly mixing the burdock aglycone, the oil phase, the emulsifier and the co-emulsifier;

Thereby obtaining a microemulsion concentrate of arctigenin; and/or

(3) The above microemulsion concentrate of burdock aglycone is added to the above excipient to prepare a microemulsion oral preparation and a microemulsion intravenous preparation.

In one embodiment, the present invention provides a process for the preparation of the burdock aglycone microemulsion formulation of the present invention, the method comprising the steps of:

(1) dissolving the emulsifier in the oil phase, then adding the co-emulsifier, mixing uniformly to obtain a mixture;

(2) adding the burdock aglycone to the above mixture to obtain a microemulsion concentrate of the burdock aglycone;

(3) adding water to the above microemulsion concentrate in a weight ratio of 10-20:1 to obtain a microemulsion;

(4) The above microemulsion is added to an excipient to prepare a microemulsion preparation.

In another embodiment, the present invention provides a process for the preparation of the burdock aglycone microemulsion formulation of the present invention, the method comprising the steps of:

(2) adding burdock aglycone to the above mixture, and ultrasonically treating to obtain a microemulsion concentrate of burdock aglycone;

In another embodiment, the method for preparing the burdock aglycone microemulsion preparation of the present invention, the method comprising the steps of:

(1) mixing an emulsifier, a sputum, a co-emulsifier and an burdock aglycone to obtain a microemulsion concentrate of burdock aglycone;

(2) adding water to the above microemulsion concentrate in a weight ratio of 10-20:1, and licking to the microemulsion;

(3) The above microemulsion is added to an excipient to prepare a microemulsion preparation.

In another embodiment, the present invention provides a method of preparing the burdock aglycone microemulsion preparation of the present invention, the method Including the following steps:

(1) mixing an emulsifier, an oil phase, a co-emulsifier and an burdock aglycone, and ultrasonically treating to obtain a microemulsion concentrate of arctigenin;

(2) adding water to the above microemulsion concentrate in a weight ratio of 10-20:1 to obtain a microemulsion;

The preparation process of the burdock aglycone microemulsion preparation may further comprise diluting the microemulsion concentrate into an appropriate amount of an aqueous medium to prepare a naturally dispersible microemulsion clarified concentrate, thereby preparing a microemulsion oral solution which becomes bovine aglycone. When diluted with an aqueous medium to a dilution of 1: 1 to 300, preferably 1 : 10-70, more preferably 1: 10, the naturally dispersible microemulsion preconcentrate forms a 0/W microemulsion.

The microemulsion concentrate of the burdock aglycone of the present invention may also be mixed and heated with the above excipients and stirred until melted. It may be prepared into granules, or the obtained granules may be further refined and then placed in a capsule shell to form a soft capsule or a hard capsule. When the granules or capsules are contacted with an aqueous medium, including gastric juice, the bovine aglycone microemulsion concentrate can be diluted into naturally dispersible microemulsions.

In another aspect, the invention provides a method for treating a condition or disease, such as a viral infection, a bacterial infection, a tumor, etc., the method comprising administering to a subject in need thereof an effective amount of an aglycone of the invention Microemulsion preparation. Such viral infections include, but are not limited to, influenza viruses, parainfluenza viruses, herpes viruses, mumps viruses, and the like. Such bacterial infections include, but are not limited to, Staphylococcus aureus, pneumococci, streptococci, and the like. The tumor includes sarcoma and cancer, such as lung cancer, liver cancer, esophageal cancer, gastric cancer, breast cancer and the like. Further, the burdock aglycone microemulsion preparation of the present invention can also be used for the treatment of cardiovascular diseases and senile dementia and the like.

For these applications, the burdock aglycone microemulsion preparation of the present invention may be combined with other drugs which enhance its pharmacological effects, and examples of the other drugs include, but are not limited to, antiviral drugs, antibiotics, antineoplastic agents, immunopotentiators, and the like. Therapeutic agent.

According to a further embodiment of the present invention, the present invention provides the use of the burdock aglycone microemulsion preparation of the present invention for the preparation of a medicament for treating a viral infection, a bacterial infection, a tumor or the like.

The invention describes the burdock aglycone microemulsion preparation of the invention and the preparation method thereof by the embodiment 1-30, and all the microemulsion preparations diluted by the microemulsion preparation or the microemulsion concentrate of the invention meet the quality standard of the microemulsion preparation. . After the centrifugation test, the microemulsion was still clear and transparent, and no delamination or turbidity occurred. The results of particle size and particle size distribution of the burdock aglycone microemulsion concentrate and diluent showed that the average particle size was 10-90 nm, which did not exceed 100 nm, and the distribution range was narrow and uniform, which was consistent with the distribution requirements of particle size and particle size of microemulsion. .

The present invention investigates the oral bioavailability of the burdock aglycone microemulsion preparation, and as a result, finds the burdock glycosides provided by the present invention. The micro-milk preparation (including Example 1-31) significantly increased the oral bioavailability of the bovine glycoside emulsion disclosed in CN101036643A by 1.39-4.73 times. The invention also finds that the microemulsion preparation of the burdock aglycone provided by the invention has the effect of significantly improving the anti-mouse S180 tumor of the burdock aglycone emulsion disclosed by the invention according to CN101036643A. Yuan's microemulsion preparation has significant advantages over the bovine glucoside aglycone emulsion disclosed in CN101036643A in inhibiting H22 hepatocarcinoma and mouse Lewis lung cancer solid tumor; in combination with the burdock aglycone emulsion and tegafur at the same dose In contrast, the combination of the burdock aglycone microemulsion preparation of the present invention and tegafur has a significant advantage in inhibiting H22 liver cancer solid tumors and mouse Lewis lung cancer solid tumors, and the tumor inhibition rate is significantly improved.

The above experimental results further demonstrate that the microemulsion preparation of the burdock aglycone of the invention has better antitumor effect than the emulsifier of the burdock aglycone, which may be improved with the emulsifier of the burdock aglycone. Oral availability, increased drug absorption. '

In summary, microemulsions improve drug delivery, which increases drug loading, enhances permeability, reduces particle size, improves particle size uniformity, increases drug dissolution rates, and increases bioavailability compared to emulsions and conventional formulations. Reduce inter- and intra-individual differences in pharmacokinetics. When administered orally, the microemulsion formulations of the present invention exhibit advantageous properties such as high levels of bioavailability obtained in standard bioavailability assays. detailed description

The invention is further described below by way of specific embodiments, which are not limited to the following examples. Part I Microemulsion concentrate and microemulsion preparation of arctigenin Example 1 Preparation and quality evaluation of arctigenin microemulsion

1. Formulation of burdock aglycone microemulsion concentrate

Component Weight /g

Angerin 5

Caprylic acid glyceride 15

Nonylphenol ethoxylate 50

1,2-propanediol 12.5 Anhydrous ethanol 12.5

2. Preparation process of burdock aglycone microemulsion

Weigh the prescribed amount of caprylic acid glyceryl ester, nonylphenol ethoxylate, 1,2-propanediol, absolute ethanol, mix and stir evenly, then add the burdock aglycone, sonicate to accelerate the dissolution, to obtain a clear concentrate, It is the burdock aglycone microemulsion concentrate. The microemulsion concentrate obtained above was diluted with water to a clear solution in a weight ratio of 1:10-20 to obtain a microemulsion.

3. Physical and chemical properties of burdock aglycone microemulsion and its quality evaluation method

3.1 Appearance traits

Observing the appearance of burdock aglycone microemulsion

3.2 Identification of microemulsion types

A small amount of burdock aglycone microemulsion concentrate was added to the methylene blue and Sudan red solution respectively to observe the diffusion rate of the two different color solutions in the microemulsion.

3.3 Centrifugation experiment

The burdock aglycone microemulsion was centrifuged at 30 mii in a DT5-3 centrifuge to observe the phenomenon and examine its physical stability. 3.4 Study on particle size and particle size distribution

Take the appropriate amount of burdock aglycone microemulsion, determine its particle size and particle size distribution by laser granulometry; take appropriate amount of burdock aglycone microemulsion, dilute ₅₀ times with water; determine the particle size and particle size distribution of the diluent by laser granulometry .

3.5 Establishment of determination method of burdock aglycon content

3.5.1 Chromatographic conditions

Column: Diamonsil C ₁₈ (size: 5 n}, -150 mm x 4.6 mm) _; mobile phase: methanol-acetonitrile-water (40:

20:40); Flow rate: 0.8 ml/min; UV detection wavelength: 280 nm; Column temperature: room temperature.

3.5.2 Standard curve

Accurately weigh the burdock aglycone control ^ 0.1562g (equivalent to arctalin aglycon 0.1381g), placed in a 25ml brown volumetric flask, add methanol to the mark, shake well; take 1ml in a 25ml brown volumetric flask, add methanol to dilute To the scale, shake well; take the diluent 1, 2, 3, 4, 5, 6ml separately into a 10ml brown bottle, add methanol to the mark, shake well, and obtain the concentration of 22.1, 44.2, 66.3, 88.4, 110.5, A series of 132.6 g/ml standard solutions were injected according to the chromatographic conditions.

3.5.3 Precision and sample stability

Take the bovine aglycone standard solution 66.3 g / ml, repeat the determination 6 times, calculate the peak area RSD; respectively in the 0, 2 4, 6, 8, 12, 24h were injected separately to calculate the peak area RSD.

3.5.4 Determination of content

Take 1 ml of burdock aglycone microemulsion, place it in a 100 ml volumetric flask, dilute to the mark with methanol, and measure the content of arctigenin. 4. Investigation of physical and chemical properties of burdock aglycones and its quality evaluation results

4.1 Appearance traits

4.2 microemulsion type

Red diffuses faster in the microemulsion than in blue, indicating that the microemulsion is a 0/W microemulsion.

4.3 Centrifugal test

The microemulsion is still clear and transparent, no precipitation occurs, and no delamination or turbidity occurs. It shows that its physical stability is good, which can indirectly indicate that it can maintain good stability after standing for a long time.

4.4 particle size and particle size distribution

The results of particle size and particle size distribution of the burdock aglycone microemulsion concentrate and diluent showed that the average particle size of the prepared bovine aglycone microemulsion was 25 nm, and the polydispersity coefficient was 0.105. The average particle size of the burdock aglycone microemulsion dilution solution 22nm, the polydispersity coefficient is 0.078; It can be seen that the prepared dilution of the burdock aglycone microemulsion and the microemulsion has a small particle size and a narrow and uniform distribution range. It meets the requirements for the distribution of particle size and particle size of microemulsion.

4.5 Determination of the content of arctigenin

In 1.916xl (T ⁴ -4.79xl (r^g/ml, the peak area of the chromatogram and the injection amount of arctigenin ^g) showed a good linear relationship. The regression equation is: Y=1119986X+1508.8, r=0.9993 Example 2 Microemulsion concentrate

Preparation process: Weigh the prescription amount of caprylic acid glyceride, polyoxyethylene castor oil EL-40, 1, 2-propanediol, mix and stir evenly, then add the burdock aglycone to dissolve, sonicate to accelerate the dissolution, to obtain a clear concentrate, ie is arctigenin _ώ microemulsion concentrate obtained above microemulsion concentrate with water in a 1: 10-20 ratio by weight was diluted to a clear solution to obtain a microemulsion. The particle size was measured by a laser granulometer, and the average particle diameter was 15 nm. Example 3 Microemulsion concentrate

The preparation process was the same as in Example 2. Laser particle size analyzer using a particle diameter, an average particle diameter of 35nm _£ microemulsion concentrate of Example 4

The preparation process was the same as in Example 2. The particle size of the laser granulometer was measured, and the average particle size was lOnmc. Example 5 Microemulsion concentrate

The preparation process was the same as in Example 2. The particle size was measured by a laser granulometer, and the average particle diameter was 19 nm. EXAMPLES ό Microemulsion concentrate

Preparation process: Weigh the prescribed amount of octanoic acid ethyl oleate, Tween-80, 1, 2-propanediol, polyethylene glycol 3350, mix and mix evenly, then add the burdock aglycone dissolved, sonicated to accelerate the dissolution, to clarify The concentrate is the burdock aglycone microemulsion concentrate. The microemulsion concentrate obtained above was diluted with water to a clear solution in a weight ratio of 1:10-20 to obtain a microemulsion. The particle size was measured by a laser granulometer, and the average particle diameter was 40 nm.

Example 7 Microemulsion concentrate

The preparation process was the same as in Example 6. The particle size was measured by a laser granulometer, and the average particle diameter was 35 nm.

Example 8 Microemulsion concentrate

The preparation process was the same as in Example 6. The particle size was measured by a laser granulometer, and the average particle diameter was 40 nm.

Example 9 Microemulsion concentrate

The preparation process was the same as in Example 6. The particle size was measured by a laser granulometer, and the average particle size was 48 nn^ Example 10 Microemulsion concentrate

The preparation process was the same as in Example 6. The particle size was measured by a laser granulometer, and the average particle diameter was 44 nm. Example 11 Microemulsion concentrate

The preparation process was the same as in Example 2. The particle size was measured by a laser granulometer, and the average particle diameter was 46 nmc. Example 12 Microemulsion concentrate

The preparation process is the same as that of the implementation 2. Laser particle size analyzer using a particle diameter, an average particle diameter of 13 micro Example embodiments 45nm _t) milk concentrate

The preparation process was the same as in Example 2. The particle size was measured by a laser granulometer, and the average particle diameter was 36 nm. Example 14 Microemulsion concentrate

The preparation process was the same as in Example 2. The particle size was measured by a laser granulometer, and the average particle diameter was 26 nm. Example 15 Microemulsion concentrate

The preparation process was the same as in Example 2. The particle size was measured by a laser granulometer, and the average particle diameter was 16 nm. Example 16 Microemulsion concentrate

Example 17 Microemulsion concentrate

The preparation process was the same as in Example 2. The particle size was measured by a laser granulometer, and the average particle diameter was 56 nmc. Example 18 Microemulsion concentrate

The preparation process was the same as in Example 2. Laser particle size analyzer using a particle diameter, an average particle diameter of 27nm _t microemulsion concentrate of Example 19

The preparation process was the same as in Example 2. The particle size was measured by a laser granulometer, and the average particle diameter was 82 η^^ Example 20 microemulsion concentrate

The preparation process was the same as in Example 2. The particle size was measured by a laser granulometer, and the average particle diameter was 68 nm. Example 21 Microemulsion concentrate

The preparation process was the same as in Example 2. The particle size was measured by a laser granulometer, and the average particle diameter was 54 nm. Example 22 Microemulsion concentrate

The preparation process was the same as in Example 2. The particle size was measured by a laser granulometer, and the average particle diameter was 63 nmc. Example 24 Microemulsion concentrate

Example laser particle size analyzer using the same preparation process embodiment the particle size, the average particle diameter of 25 Example embodiment microemulsion concentrate 80nm _t Component weight / g

Angerin 5

Caprylic acid glyceride 30

Hydrogenated cocoglyceride 50

1-butanol 20

Ascorbic acid 0.05

The preparation process was the same as in Example 2. The particle size was measured by a laser granulometer, and the average particle diameter was 66 nm. Example 26 Microemulsion concentrate

The preparation process was the same as in Example 2. The particle size was measured by a laser granulometer, and the average particle diameter was 86 nmc. Example 27 Microemulsion concentrate

The preparation process was the same as in Example 2. The particle size was measured by a laser granulometer, and the average particle diameter was 74 nm. Example 28 Microemulsion injection of arctigenin.

Normal saline added to 100ml

Preparation Process:

Weigh the prescribed amount of lauroyl polyethylene glycol-32-glyceride, hydrogenated coconut glyceryl ester, 1, 2-propanediol, mix well, stir evenly, then add the burdock aglycone to dissolve, sonicate to accelerate the dissolution, to obtain a clear concentrate , slowly add an appropriate amount of physiological saline with stirring. At the same time, sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution was added to adjust the pH to 5.0-7.0.

0.2μιη filter membrane filter sterilization, nitrogen filling 5ml potting, that is, the burdock aglycone microemulsion injection. The particle size was measured by a laser granulometer, and the average particle diameter was 54 nm.

Example 29 Microemulsion oral solution of arctigenin

Preparation Process:

Weigh the prescribed amount of peppermint oil, hydrogenated cocoglyceride, 1, 2-propanediol, mix well, stir evenly, then add the burdock aglycone to dissolve, sonicate to accelerate the dissolution, get the clarified concentrate, slowly add the appropriate amount of distilled water under stirring, The prescribed amount of lactose and 5% benzalkonium bromide solution, that is, the bovine aglycone microemulsion oral solution. The particle diameter was measured by a laser granulometer, and the average particle diameter was 57 nm.

Example 30 Capsules containing burdock aglycone microemulsion

Preparation Process:

Weigh the prescribed amount of ethyl oleate, hydrogenated cocoglyceride, 1, 2-propanediol, mix well, stir evenly, then add the burdock aglycone to dissolve, sonicate to accelerate the dissolution, get the clarified concentrate, slowly add after stirring The prescribed amount of PEG3350 and silica, after thorough mixing, the mixture is added to the capsule shell to obtain the burdock aglycone microemulsion capsule. The particle size was measured by a laser granulometer, and the average particle diameter was 90 nm.

Example 31 Microemulsion concentrate

Preparation process: Weigh the prescribed amount of hydrogenated coconut glyceride, polyoxyethylene castor oil EL-40, 1, 2-propanediol, mix and stir evenly, then add the burdock aglycone to dissolve, ultrasonic treatment to accelerate the dissolution, to obtain a clear concentrate , that is, the burdock aglycone microemulsion concentrate. The microemulsion concentrate obtained above was diluted with water to a clear solution in a weight ratio of 1:10-20 to obtain a microemulsion. The particle size was measured by a laser granulometer, and the average particle diameter was 80 nm.

The microemulsion concentrate or microemulsion preparation of the arctigenin prepared in Example 2-31 was repeated, and the "investigation and quality evaluation of the physicochemical properties of the burdock aglycone microemulsion" in Example 1 was repeated, and all the microemulsion preparations were found or The microemulsion preparations diluted by the microemulsion concentrates all meet the quality standards of the microemulsion preparations. After the centrifugation test, the microemulsion was still clear and transparent, and no delamination or turbidity occurred. The results of particle size distribution and particle size distribution of the burdock aglycone microemulsion concentrate and the diluent showed that the average particle size of the burdock aglycone microemulsion prepared in Example 2-31 was 10-90 nm, and the distribution range was not more than 100 nm, and the distribution range was narrow and uniform. It meets the requirements for the distribution of particle size and particle size of microemulsion.

The microemulsion concentrate or microemulsion preparation of the arctigenin prepared in Example 1-31 was subjected to an accelerated test at 40 ° C under 75% RH, and sampled at 1, 2, 3, and 6 months, using laser particle size. The analyzer was used to detect the particle size, and the results are shown in Table 1 (bit nm). It can be seen from Table 1 that the burdock aglycone microemulsion prepared by the invention has good stability, especially the stability of the burdock aglycone microemulsion concentrate or the microemulsion preparation prepared in Examples 1-18 is more The path is smaller.

Table 1 Results of microemulsion particle size change of arctigenin

The second part of the main pharmacokinetics and pharmacodynamics of the burdock aglycone microemulsion

Test Example 1 Oral bioavailability of bovine aglycone microemulsion preparation

1. Experimental materials

1.1 Drugs and reagents

The burdock aglycone emulsion: the formulation and the preparation process are the same as the embodiment 3 of the CN101036643A specification, and the formulation of the burdock aglycone emulsion: burdock aglycone 1.0 g + egg yolk phospholipid 3.0 g + medium chain oil 15 g + oleic acid 0.08 g + glycerol 3.0 g. Bork aglycone microemulsion: the microemulsion prepared in Example 1 of the present invention;

Anthraquinone reference substance (purity 99.5%); heparin sodium, normal saline, methanol, acetonitrile (all chromatographic reagents), others are analytical reagents.

1.2 Experimental instruments

LC-10A high performance liquid chromatography; SPD-10AV UV detector: ShimazuClass-vp Version 5.03 working station; KQ-100 ultrasonic cleaner; desktop centrifuge TGL-16C; manual homogenizer.

1.3 Experimental animals

New Zealand rabbits, weighing 1400-1500g, both male and female, are provided by Shandong New Age Pharmaceutical Animal Center.

2, the experimental method

2.1 Determination of drug concentration in plasma

2.1.1 Preparation of reference solution

Accurately weighed 9.58 mg of the burdock aglycone reference substance, placed in a 10 ml volumetric flask, and diluted to the mark with methanol to obtain a reference solution of 0.958 mg/ml.

2.1.2 Plasma sample pretreatment

0.5 ml of the sample was placed in a separatory funnel, 5 ml of dichloromethane was added, shaken, and extracted twice. The dichloromethane layer was separated and combined, and the dichloromethane solution was evaporated to dryness in a 40-inch water bath. The residue was dissolved in 0.5 ml of acetonitrile, and used as a test solution, placed in a refrigerator (-20 ° C) to be tested, and 10 μl was injected. .

2.1.3 Establishment of a method for determination of drug content in biological samples

2.1.3.1 Chromatographic conditions

Column: Diamonsil C ₁₈ (size: 5μηι, 150mmx4.6mm); Mobile phase: acetonitrile-τΚ (35:

65); Flow rate: 0.8 ml/min; UV detection wavelength: 280 nm; Column temperature: room temperature.

2.1.3.2 Method specificity investigation

The plasma of the experimental animals was treated as described above, and then injected into a high performance liquid chromatograph. The blank plasma sample was processed in the same way. As a result, under the above chromatographic conditions, the peak of bovine aglycone has a good separation from other peaks, and the resolution of the adjacent peak is R: 2.5, and the number of theoretical plates is 3566 pieces/meter. The blank plasma has no interference with its analysis.

2.1.3.3 Determination of the minimum detection concentration

The low detection of arctigenin in plasma was 9.58x10 g/ml based on the peak height as a baseline noise of 3 times.

2.1.3.4 Linear range

Take a blank biological sample and add different concentrations of arctigenin standard solution to make the sample concentration: 1.9ΐ6χ1 (Τ ² , 4.79xl 0 ^-2 , 9.58xl (T ² , 4.79><10 people 9.58x 10"^ 2.874, 4.79 g/ml, each injection 10μ1, with injection volume ^g) as the abscissa, with chromatographic peak area On the ordinate, the standard curve was drawn.Results: Within the range of 1.916><10 ⁴ -4.79><10 _§ , the peak area of the chromatogram showed a good linear relationship with the injection amount of arctigenin ^g). The regression equation is: Y=11 '19986X+1508.8, r=0.9993.

2.1.3.5 Recovery and precision

Recovery rate: Take a blank biological sample ^, prepare 4.79x 1 (T g / ml, 4.79x lO" Vg mK 4.79 g / ml three concentrations of biological samples and the same concentration of arctigenin standard solution, respectively in the above HPLC chromatographic conditions The next measurement was performed 5 times, and the measured results were compared to calculate the absolute recovery. The results showed that the recovery rate of the method at the low, medium and high levels was 75.6%, which could meet the test requirements of biological samples.

Precision: Take 4.79x l0 g/ml, 4.79 lO"Vg/mK 4.79 g/ml three concentrations of plasma in the same day for five measurements, calculate the intraday relative standard deviation, precision RSD were 2.22%, 1.67 %, 0.98%. The intra- and inter-day relative standard deviations of other biological samples were determined to be less than 5%, which met the test requirements of biological samples.

2.1.4 Selection of biological sample pretreatment methods such as blood paddle

Plasma samples need to be removed from impurities such as proteins before injection into the column to prevent clogging of the column, resulting in reduced column efficiency. Generally, a water-soluble organic solvent such as methanol, acetonitrile, or a strong acid such as 10% trichloroacetic acid is added to remove the protein. Or extract with an organic solvent. In this experiment, a biological sample was extracted from dichloromethane with good solubility of arctigenin. Considering the high volatility of dichloromethane, the dichloromethane solution was evaporated and then stabilized with acetonitrile to dissolve. Injection.

2.2 grouping and administration

2.2.1 Burdock aglycone microemulsion group

Ten rabbits who were fasted for 12 hours and were free to drink water were given 1 ml of blank blood and then given 30 mg/kg of burdock aglycone microemulsion for 0.5 hour, 1 hour, 1.5 hours, 2.0 after administration. Hour, 2.5 hours, 3.0 hours, 4.0' hours, 5.0 hours, 6.0 hours, 1 ml of blood from the ear vein was placed in a heparin-coated centrifuge tube, centrifuged (3O00 rpm) for 10 min, and 0.5 ml of plasma was taken and stored at -20 ° ( In the refrigerator, according to the above biological sample preparation method, and prepared, preserved in a -20 Γ refrigerator, and the 血μΙ injection analysis is performed when the blood concentration is measured.

2.2.2 burdock aglycone emulsion group

Ten rabbits who were fasted for 12 hours and were free to drink water were given 1 ml of sputum white blood and then given a 30 mg/kg burdock aglycone emulsion for 0.5 hour, 1 hour, 1.5 hours, 2.0 hours, 2.5 hours after administration. , 3.0 hours, 4.0 hours, 5.0 hours, 6.0 small 吋 ear vein blood lml, placed in a heparin-coated centrifuge tube, centrifuge (3000rpm) lOmin, take 0.5ml of plasma, stored in a refrigerator at -20 ° C, press The aforementioned biological sample preparation method is processed, prepared, and stored in In the -20 °C refrigerator, when the blood concentration was measured, ΙΟμΙ injection analysis was performed.

2.3 Data Processing

The blood drug concentration-time curve was processed by the 3ρ87 practical pharmacokinetics program prepared by the Chinese Pharmaceutical Association's Committee of Mathematics and Pharmacology, and the curve was fitted by computer to obtain the main pharmacokinetic parameters.

2.4 Experimental results

The results are shown in Table 1. It can be seen that after the preparation of the burdock aglycone into a microemulsion preparation, the oral bioavailability (F) is more than twice as high as that of the emulsion.

Relative bioavailability of burdock aglycone emulsion and microemulsion for intragastric administration

Group dose: (mg/kg) AUC g'mr ¹ '!!) AUC/DF (%) Emulsion group 30 1.4978 0.0499 14.56

Microemulsion group 30 4.6520 0.1550 45.21

The microemulsion concentrate or microemulsion preparation of the arctigenin prepared in Examples 2 to 31 was substituted for the microemulsion preparation of the bovine aglycone of Example 1 used in Test Example 1, and the description of the examples 1 to 7 was replaced with the test example of CN101036643A. The emulsion in 1 was repeated in Test Example 1, and as a result, it was found that the oral bioavailability of the emulsion of the Example 1 to 7 burdock aglycone disclosed in the specification of CN101036643A was 9.60% to 16.23%, and the oral bioavailability of the microemulsion preparation of the burdock aglycone of the present invention. From 38.80% to 55.02%, the oral bioavailability of the emulsion of bovine aglycone was significantly increased by 1.39~4.73 times. Test Example 2 Experiment of transplantation of arctigenin against S180 sarcoma cells in mice

1. Test drugs and experimental animals

Kunming mice 18~22 g, male and female, a total of 50. Mouse sarcoma S180 cells, tegafur, burdock aglycone emulsion (burdock aglycone emulsion: prescription and preparation process same as CN101036643A manual page 4 Example 3, ie the formulation of burdock aglycone emulsion: burdock aglycone 1.0g + egg yolk phospholipid 3.0 g + medium chain oil 15 g + oleic acid 0.08 g + glycerol 3.0 g), burdock aglycone microemulsion (burdox aglycone microemulsion prepared in Example 1 of the present invention).

2. Establishment of a transplanted tumor model of S180 sarcoma cells in mice

S180 cells were cultured in 1640 medium, n. C, under 5% C0 ₂ were cultured, passaged on average once every 2 days to the preparation with physiological saline to a density of logarithmic growth phase was 3.0xl0 ⁷ / mL single cell suspension, under sterile conditions in mice inoculated intraperitoneally Within (about 5 mice, both male and female), the mice were obviously swollen in the abdominal cavity about 10 days after inoculation. At this time, the mice were sacrificed by cervical dislocation and soaked in a beaker containing 75% ethanol for 2 to 3 minutes. Place the sterilized mice on the ultra-clean workbench, use the tweezers to clip the skin in the middle of the abdomen, cut the skin with the surgical scissors in the lower abdomen, expose the abdomen, and pass through the sterile syringe. Ascites was taken from the abdominal muscles and placed in a sterile reagent bottle for later use.

The above ascites fluid extracted by trypan blue exclusion, using tumor cells> 95% of the graft was diluted with physiological saline to adjust the cell number 2χ10 ^⁷ ~6χ 10 ⁷ / ml, and obtained tumor cell suspension.

One person fixed the mouse, the other hand pinched the right anterior axillary skin of the mouse, and the lmL syringe needle was inserted into the skin under the pleats, and each mouse was inoculated with 0.2 mL of the tumor cell suspension.

3, grouping and drug delivery

The rats were weighed the next day after inoculation. The healthy mice were randomly divided into 4 groups, 1Q each, male and female, and each weighing was recorded. The cells were inoculated 24 hours after inoculation of the tumor cells. 0.2 mL each, once a day for 14 days.

Table 3 group of dosing regimens

4, measurement indicators

After the last administration, fasting for 24 h, the mice were sacrificed by cervical dislocation, weighed, and the tumor pieces were removed and weighed. The tumor inhibition rate (%) = (the average tumor weight of the negative control group - the average tumor weight of the experimental group) / the average tumor weight of the negative control group χΐοο%, and the tumor was taken for pathological examination.

5. Results

According to the pathological section experiment, the excised tissue is tumor tissue, and the tissue type is less. The inhibition rate of the burdock aglycone emulsion group was only 17.0%, while the inhibition rate of the burdock aglycone microemulsion group was 46.4%. This indicates that the effect of the burdock aglycone microemulsion formulation against the mouse S180 tumor was significantly improved relative to the emulsion group.

Table 4 Body mass control and tumor inhibition rate before and after administration

The burdock aglycone emulsion group 27.3 37.6 3.4 17.0 The microemulsion concentrate or microemulsion preparation of 6 bursin aglycone was prepared in Example 2 to 31 instead of the microemulsion preparation of the burdock aglycone of Example 1 used in Test Example 2, using CN101036643A The specification discloses the use of the emulsion of Example 2 to 7 in the replacement of Example 33, and the test example 2 was repeated. As a result, it was found that the microemulsion preparation of the arctiin of the present invention has a tumor inhibition rate of 35.60% to 53.82%, and CN101036643A discloses the embodiment 2 The tumor inhibition rate of the ~7 emulsion was 10.5% to 18.3%, which indicates that the effect of the emulsion of the burdock aglycone microemulsion provided by the present invention is significantly higher than that of the emulsion disclosed by CN101036643A against the mouse S180 tumor. Test Example 3 Inhibition test of burdock aglycone on KM mouse H22 liver cancer

1 Research purposes

To investigate the inhibitory effect of arctigenin on solid tumors in KM mice implanted with H22 liver cancer.

2 for test and reference

2.1 Test sample

Burdock aglycone microemulsion, refrigerated, sealed

2.2 reference substance

Tegafur, white granules, room temperature, sealed in the dark

2.3 Preparation of test materials and reference materials

2.3.1 Preparation of test samples:

Arctigenin 5mg

Caprylic acid glyceride 30mg

Nonylphenol ethoxylate 50mg

Propylene glycol 20mg

The burdock aglycone microemulsion was weighed with caprylic acid glyceryl ester, nonylphenol ethoxylate and propylene glycol according to the prescription amount, and evenly mixed. The prescription amount of burdock aglycone was ultrasonicated to dissolve, and then the burdock aglycone microemulsion concentrate was obtained. . Autoclave treatment is required before use. Determination of burdock aglycone microemulsion: Take the burdock aglycone microemulsion lg and add it to 100g of water, stir until the emulsification is complete, and measure the particle size, the average is 25nm, which meets the requirement of microemulsion (less than 100nm).

2.3.2 Control substance: burdock aglycone emulsion (prescription and preparation process are the same as the embodiment of page 4 of CN101036643A specification)

1) 3 Experimental animal grouping and identification methods

60 SPM KM mice, certificate number: 0008062, female 3⁄4 half, 4 weeks old, 20

3.1 Grouping method: Grouping according to the completely random grouping method;

3.2 Identification method: Using 3% picric acid staining method, the label between groups is shown in Table 4.

Inter-group marker

, mouse group 1 2 3 4 5 6 stained part 'white head back tail left front left middle

3.3 Animal wording environmental conditions

Ordinary environment, 20~25 °C, daily temperature difference ≤3 °C; humidity 40%~70%; animal illuminance: 15~20 1x _; working illuminance: 150 k or more; thoroughly test the room before and after the test Clean and disinfect. Clean and disinfect after all work is done every day.

3.4 wording conditions

Cage Name and Specifications: Cage Name and Specifications: Squirrel Cage (LxWxH 1450 mmx400 mmx l500 mm) Mouse Cartridge (LxWxH 300 mmx lOOmmx 150mm)

Word density: 5 / cage

Frequency of replacement: The mouse case is replaced every two days.

Cleaning and disinfection: The cage is wet-disinfected every day. The disinfectant is 0.1% chlorpheniramine and 75% ethanol solution. The disinfectant is used alternately every week.

3.5 vocabulary, litter and drinking water

SPF Rat, Irradiated Feed (Batch No.: 10063011), Regular Words, Free Feed, Free Drinking Water;

4 test methods

4.1 Dosage design: The dosage design of the drug administration group is shown in Table 5.

The inoculated mice were randomly divided into 6 groups, 10 in each group, half male and half female.

Table 6 dose and mode of administration of mice

Group name group administration dose administration method

1 model group one

2 low dose of emulsion 15 mg/kg

3 emulsion high dose group 45 mg / kg gavage 4 microemulsion low dose group 15 mg / kg gavage

5 microemulsion high dose group 45 mg/kg gavage

6 tegafur group 10 mg / m ² dimensional

濯冃

7 emulsion high dose + tegafur group 45 mg / kg +10 mg / m ² dimensional

濯冃

8 microemulsion high dose + tegafur group 45 mg / kg +10 mg / m ² gavage

4.2 Test method:

Mouse H22 cells were cultured in 1640 medium, routinely cultured at 37 ° C under 5 % CO ₂ , subcultured every two days, and prepared to a density of 3.0 χ ^{10 7} /ml single cells with physiological saline in the logarithmic growth phase. The suspension was injected into the abdominal cavity of the mice under aseptic conditions. The abdominal cavity of the mice was swollen 7 days after inoculation. At this time, the neck was sacrificed and placed in a beaker containing 75% ethanol for 2 3 minutes. The sterilized mice were placed in a clean bench, exposed to the abdomen, and ascites was taken with a sterile syringe and placed in a sterile reagent bottle for use. The ascites was counted with trypan blue, diluted with physiological saline, and the number of cells was adjusted to Ι.ΟχΙΟ ⁷ /ml, and inoculated into the right axilla of the mouse, each 0.2 ml.

The animals were administered on the fourth day after inoculation of the tumor, and were administered once a day according to Table 6 (administered every morning) at a dose of 10 ml 'kg ^-1 ; the model group was given an equal amount of physiological saline. Continuous administration for 10 days.

5 indicator detection

5.1 weight

Detection frequency: Weighed animals were weighed before and before anatomy.

Equipment: ACS-3A-a type electronic scale, Shanghai Dahua Electronic Scale Factory

5.2 tumor quality

Detection frequency: The test was terminated 10 days after administration, and the blood was sacrificed and the necropsy was performed.

Weighing the weight of the tissue and organs: the underarm tumor.

Anatomy: Intraperitoneal injection of 2% sodium pentobarbital (0.3 ml.kg) anesthesia, after the main abdominal vein was taken, the abdominal aorta was cut and sacrificed, and then the tumor was routinely removed.

Equipment: BS224S electronic balance, Beijing Sartorius Instrument System Co., Ltd.

ACS-6A electronic price scale, Shanghai Dahua Electronic Scale Factory

6 data statistics processing method

The test data is expressed in ± s and data processing was performed using SPSS 11.5 statistical software.

7 experimental results

The experimental results are shown (experimental results are shown in Table 6): (1) Compared with the high-dose emulsion group and the low-dose emulsion group, the tumor inhibition rate of the high-dose microemulsion group and the low-dose microemulsion group was significantly improved, and there was a significant difference;

(2) The present invention also investigates the combination of burdock aglycon and tegafur, and finds that, at the same dose, the burdock aglycone is compared with the combination of burdock aglycone and tegafur. ^ ^ The combination of preparation and tegafur has a significant advantage in inhibiting H22 solid tumors of liver cancer, and the tumor inhibition rate is significantly improved.

The above experimental results further demonstrate that the microemulsion preparation of burdock aglycone has a better antitumor effect than the emulsifier of bovine aglycone, which may be improved with the emulsifier of burdock aglycone. Oral availability, increased drug absorption.

Table 7 Effect of burdock aglycone on tumor weight and tumor inhibition rate in KM mice (s) s group dose tumor tumor inhibition rate model group - 2.05 ± 0.89 - emulsion low dose group 15 mg / kg 1.92 ± 1.55 6.3 emulsion high Dosage group 45 mg/kg 1.85±0.96 9.8 Microemulsion low dose group 15 mg/kg 1.69±1.55 ^AA 17.6 Microemulsion high dose group 45 mg/kg 1.56±0.96 ^AA 23.9

Tegafur group 10 mg/m ² 1.54±0.52 ^{Δ Α} 24.9 emulsion high dose + tegafur group 45 mg/kg +10 mg/m ² 1.48 ± 0.67 27.8 microemulsion high dose + tegafur group 45 mg/kg +10 Mg/m ² 1.23±0.51" ^ΔΑ 40.0 Note: ρ<0.05 compared with the low dose group of emulsion; ρ<0.05 compared with the high dose group of emulsion;

■ ρ < 0.05 compared with the high dose of emulsion + tegafur group. Test Example 4 Effect of burdock aglycone on tumor inhibition rate of transplanted Lewis lung cancer

1 Research purposes

To study the inhibitory effect of arctigenin on solid tumors in mice implanted with lung cancer Lewis tumor strain.

2 for test and reference

2.1 Test sample

Burdock aglycone microemulsion, refrigerated, sealed

2.2 reference substance

Tegafur, white granules, room temperature, sealed in the dark 2.3 Preparation of test materials and reference materials

2.3.1 Preparation of test samples:

Arctigenin 5mg

Caprylic acid glyceride 30mg

Nonylphenol ethoxylate 50mg

Propylene glycol 20mg

The glyceryl caprylate, nonylphenol ethoxylate and propylene glycol were weighed according to the prescription amount, and the mixture was mixed. The prescription amount of burdock aglycone was ultrasonicated to dissolve, and the burdock aglycone microemulsion concentrate was obtained. Autoclave treatment is required before use. Determination of burdock aglycone microemulsion: Take burdock aglycone microemulsion lg to 100g water, stir until the emulsification is complete, and measure the particle size, the average is 25nm, which meets the requirements of microemulsion (less than 100nm).

1)

3 Experimental animal grouping and identification methods

60 SPM KM mice, certificate number: 0008062, male and female, 4 weeks old, 20

Table 8 Marking between groups of mice

Mouse group 1 2 3 4 5 6 Dyeing site White head Back tail Left front Left middle

3.3 Animal feeding environment conditions

Ordinary environment, 20~25 °C, daily temperature difference ≤3 °C; humidity 40%~70%; animal illuminance: 15~20 1x _; working illuminance: 150 1x or more; thoroughly test the room before and after the test Clean and disinfect. Clean and disinfect after all work is done every day.

3.4 feeding conditions

Cage Name and Specifications: Cage Name and Specifications: Squirrel Cage (LxWxH 1450 mmx400 mmxl500 mm) Mouse Cartridge (LxWxH 300 mmx lOOmmx 150mm)

Feeding density: 5 / cage

Frequency of replacement: The mouse case is replaced every two days. Cleaning and disinfection: The cage is wet-disinfected every day. The disinfectant is 0.1% of benzalkonium chloride and 75% of ethanol solution. The disinfectant is used alternately every week.

3.5 word materials, litter and drinking water

SPF rat feed, irradiated feed (batch number: 10063011), conventional words, free feeding, free drinking water; test method

Table 9 dose and mode of administration of mice

Group name for the drug j

1 model group 3⁄4

One mg

冃

2 emulsion low dose group 15 mg/kg Mg

濯冃

3 emulsion high dose group 45 mg / kg gavage

4 microemulsion low dose group 15 mg/kg gavage

5 microemulsion high dose group 45 mg/kg gavage

6 tegafur group 10 mg / m ² dimensional

濯冃

7 emulsion high dose + tegafur group 45 mg / kg +10 mg / m ² dimensional

濯冃

8 microemulsion high dose + tegafur group 45 mg / kg +10 mg / m ² dimensional

濯冃

4.2 Test method:

Mouse lung cancer Lewis tumor cells were cultured in 1640 medium, routinely cultured at 37 ° C under 5 % CO ₂ , subcultured every two days, and prepared to a density of 3.0 χ ^{10 7} with physiological saline in the logarithmic growth phase. Ml single cell suspension was injected into the peritoneal cavity of mice under aseptic conditions. The abdominal cavity of the mice was swollen 7 days after inoculation. At this time, the neck was sacrificed and placed in a beaker containing 75% ethanol. 2 3 In minutes, the sterilized mice were placed in a clean bench, the abdomen was exposed, and ascites was taken with a sterile syringe and placed in a sterile reagent bottle for use. The above ascites was counted with trypan blue, diluted with physiological saline, and the number of cells was adjusted to 1.0×10 ⁷ cells/ml, and inoculated into the right axilla of the mouse, each 0.2 ml.

The animals were administered on the fourth day after the tumor was inoculated, and administered once a day according to Table 6 (administered every morning) at a dose of 10 ml 'kg - the same amount of normal saline was administered to the model group. Continuous administration for 10 days.

5 indicator detection

5.1 weight

Detection frequency: Weighed animals were weighed before and before anatomy. Equipment: ACS-3A-a type electronic scale, Shanghai Dahua Electronic Scale Factory

5.2 Tumor quality

Weighing the weight of the tissue: underarm tumor

Anatomy: Intraperitoneal injection of 2% sodium pentobarbital (0.3 ml * kg) anesthesia, after the main abdominal vein was taken, the abdominal aorta was cut and sacrificed, and then the tumor was routinely removed.

ACS-6A electronic price scale, Shanghai Dahua Electronic Scale Factory

6 data statistics processing method

The test data is represented by ^s s, and data processing is performed using SPSS 11.5 statistical software.

7 experimental results

The experimental results are shown (experiment results are shown in Table '7):

(1) Compared with the high-dose emulsion group and the low-dose emulsion group, the tumor inhibition rate of the high-dose microemulsion group and the low-dose microemulsion group was significantly improved, and there was a significant difference;

(2) The present invention also investigates the combination of burdock aglycon and tegafur, and finds that, at the same dose, the burdock aglycone is compared with the combination of the burdock aglycone and tegafur. The combination of a milk preparation and tegafur has a significant advantage in inhibiting Lewis solid tumors of lung cancer, and the tumor inhibition rate is significantly improved.

The above experimental results further illustrate that the microemulsion preparation of burdock aglycone has a better antitumor effect than the emulsifier of arctigenin, which may improve oral utilization with the emulsifier of burdock aglycone. Degree, increased drug absorption.

Table 10 Effect of arctigenin on tumor weight and tumor inhibition rate in mice (±s) Group - dose Tumor weight Tumor inhibition rate Model group - 3.86 ± 1.12 -

- Low dose of emulsion 15 mg/kg 3.52±1.55 8.8 High dose of emulsion 45 mg/kg 3.35±1.01 13.2 Low dose of 15 mg/kg 2.89±1.44 ^AA 25.1 High dose of microemulsion 45 mg/kg 2.60± 0.89 ^{Δ Α} 32.6

Fluorine group 10 mg/m ² 2.29±0.77 ^{Δ Α} 40.6 High dose of emulsion + tegafur group 45mg/kg+10mg/m ² 2.18±0.50 43.5 Microemulsion high dose + tegafur group 45mg/kg+10mg/m ² 1.82±0.5Γ ^ΔΑ 52.8 Note: Low dose group with emulsion Comparison, ρ<0.05; compared with the high-dose emulsion group, ρ<0.05;

"ρ<0.05 compared with the emulsion high dose + tegafur group.

While the invention has been described with respect to the specific embodiments of the present invention, it will be understood that Accordingly, the present invention is intended to embrace all such modifications and alternatives

Claims

Rights request

A bovine aglycone microemulsion preparation comprising an arctigenin and a microemulsion carrier, wherein the microemulsion carrier comprises an oil phase and an emulsifier, and the microemulsion preparation has an average particle diameter of 10 to 90 nm. . . .

The microemulsion preparation according to claim 1, wherein the microemulsion carrier further comprises a co-emulsifier.

The microemulsion preparation according to claim 2, wherein the ratio of the components of the burdock aglycone to the microemulsion carrier is: 1-10 parts of burdock aglycone and 5-85 parts of an oil phase. 20-60 parts of emulsifier and 5-60 parts of emulsifier.

The microemulsion preparation according to any one of claims 1 to 3, wherein the oil phase is a solid or semi-solid oil phase component selected from the group consisting of fatty acid glycerides, alcohol esters, fatty alcohols, and polysaccharides. One or more of the saturated glycerides.

The microemulsion preparation according to claim 4, wherein the fatty acid glyceride is one or more selected from the group consisting of hydrogenated coconut glyceride, hydrogenated palm oil PEG-6 ester, and hydrogenated palm oil PEG-6 ester. The alcohol ester is selected from one or more of propylene glycol stearate, PEG-2-stearate, and cetyl palmitate, the fatty alcohol is myristyl alcohol, The polysaccharide-derived saturated glyceride is lauroyl polyethylene glycol-32-glyceride.

The microemulsion preparation according to any one of claims 1 to 3, wherein the oil phase is a liquid oil phase component selected from the group consisting of glycerides, natural vegetable oils and fractions thereof, essential oils, volatile oils, and One or more of synthetic oils.

The microemulsion preparation according to claim 6, wherein the glyceride is a mixture of a monoglyceride and a diglyceride, a caprylic acid glyceride, a medium chain fatty acid glyceride, a monolinoleic acid glyceride or a bismuth phthalate. Acid glyceride, the natural vegetable oil and the fraction thereof are safflower oil, sesame oil, corn oil, castor oil, coconut oil, cottonseed oil, soybean oil, lemon oil, peppermint oil, menthol, parsley, thyme a phenol or any mixture thereof, wherein the volatile oil is spearmint oil, clove oil, lemon oil, peppermint oil or any mixture thereof, and the synthetic oil is triacetin, ethyl butyrate or ethyl oleate Ethyl oleate, isopropyl myristate, ethyl octanoate or any mixture thereof.

The microemulsion preparation according to any one of claims 1 to 3, wherein the oil phase is selected from the group consisting of glyceryl caprylate, glyceryl caprylate, corn oil, soybean oil, ethyl oleate, and oil. Ethyl acetate, isopropyl myristate, ethyl octanoate or any mixture thereof.

The microemulsion preparation according to any one of claims 1 to 8, wherein the emulsifier is selected from the group consisting of nonylphenol ethoxylates, polyoxyethylene fatty acid esters, polyoxyethylene castor oil, polyoxygen One or more of ethylene hydrogenated castor oil, sorbitan derivative, polyoxyethylene-polyoxypropylene copolymer, polyoxyethylene nonyl ether, polyglycerin fatty acid ester, and fluorenyl polyol ester.

The microemulsion preparation according to claim 9, wherein the polyoxyethylene fatty acid ester is polyoxyethylene stearic acid. An ester, PEG-40-stearate or a mixture thereof, said sorbitan derivative being Tween-20, Tween-40, Tween-60, Tween-80 or a mixture thereof The polyoxyethylene-polyoxypropylene copolymer is a poloxamer, and the polyoxyethylene alkyl ether is polyoxyethylene-2-cetyl ether, polyoxyethylene-10-cetyl ether, polyoxygen Ethylene-20-cetyl ether, polyoxyethylene-23-lauryl ether, polyoxyethylene-20-oleyl ether, polyoxyethylene-2-stearyl ether, polyoxyethylene-10-stearyl Ether, polyoxyethylene-20-stearyl ether or a mixture thereof, said polyglycerol fatty acid ester being decaglyceryl monostearate, hexaglyceryl monostearate, tetraglyceryl monostearate or A mixture thereof, the alkylene polyol ester is stearyl, lauroyl polyethylene glycol-32-glyceride or a mixture thereof.

The microemulsion preparation according to claim 10, wherein the polyoxyethylene alkyl ether is polyoxyethylene-23-lauryl ether or polyoxyethylene-20-oleyl ether.

The microemulsion preparation according to any one of claims 1 to 8, wherein the emulsifier is selected from the group consisting of nonylphenol ethoxylate, polyoxyethylene castor oil, polyoxyethylene hydrogenated castor oil, and Tween -80, poloxamer, polyoxyethylene-23-lauryl ether or a mixture thereof.

The microemulsion preparation according to any one of claims 2 to 12, wherein the co-emulsifier is one of the following components or any mixture thereof: short-chain alcohol, organic ammonia, mercapto acid, Mono-dialkyl glycerides and polyoxyethylene fats

_ acid ester.

The microemulsion preparation according to claim 13, wherein the co-emulsifier is a short-chain alcohol selected from the group consisting of polyethylene glycol, ethanol, n-butanol, n-hexanol, ethylene glycol, propylene glycol and glycerin. One or several.

The microemulsion preparation according to claim 14, wherein the co-emulsifier is ethanol, glycerin, polyethylene glycol 200, polyethylene glycol 300, polyethylene glycol 400 or 1,2-propanediol.

The microemulsion preparation according to claim 3, wherein the weight ratio of the arctiin to the oil phase, the emulsifier, and the co-emulsifier _: ί: 1-10 parts of burdock, oil phase 20-40 Parts, emulsifier 35-55 parts, co-emulsifier 15-45 parts.

The microemulsion preparation according to claim 1, wherein the microemulsion carrier further comprises a hydrophilic component.

The microemulsion preparation according to claim 17, wherein the ratio of the components of the burdock aglycone and the microemulsion carrier is: 1-10 parts of burdock aglycone and 5-85 parts of an oil phase. 20-60 parts of emulsifier, 5-60 parts of co-emulsifier, and 20-70 parts of hydrophilic component.

The microemulsion preparation according to claim 18, wherein the ratio of the components of the burdock aglycone and the microemulsion carrier is: 1-10 parts of burdock aglycone, 20-40 parts of an oil phase. 35-55 parts of emulsifier, 15-45 parts of co-emulsifier, 30-50 parts of hydrophilic component.

The microemulsion preparation according to any one of claims 17 to 19, wherein the hydrophilic component is polyethylene glycol, Polyethylene epoxide or a mixture thereof.

The microemulsion preparation according to claim 20, wherein the polyethylene glycol is polyethylene glycol 200, polyethylene glycol 300, polyethylene glycol 400 or a mixture thereof.

The microemulsion formulation of burdock aglycone according to any one of claims 1 to 3, further comprising an excipient selected from one or more of the following: preservative, filling Agent, pH regulator, isotonicity regulator, acidifier and water.

The microemulsion preparation according to claim 22, wherein the preservative is ascorbic acid and a derivative thereof, tocopherol and a derivative thereof, butylated hydroxyanisole, butylhydroxytoluene or any mixture thereof. The fillers are microcrystalline cellulose, silica, starch and derivatives thereof, lactose, dicalcium phosphate, mannitol or any mixture thereof, and the acidifying agent is citric acid, succinic acid, fumaric acid, Ascorbic acid, phosphoric acid, citric acid, oleic acid, cellulose acetate, cellulose acetate-1,2,4-benzenetricarboxylate, hydroxypropylmethylcellulose phthalate, carboxymethylethylcellulose, Kappa Or a mixture thereof, the pH adjusting agent is a buffer solution of phosphate, sodium hydroxide, hydrochloric acid or any mixture thereof, and the isotonicity adjusting agent is a sodium chloride solution, a potassium chloride solution or mixture.

24. The microemulsion formulation of claim 22, wherein the excipient comprises from 0.05% to 80% by weight of the microemulsion formulation.

A method of preparing the microemulsion preparation according to any one of claims 1 to 24, the method comprising:

26. The method of claim 25, further comprising sonicating the burdock aglycone added to the mixture in step (2) to obtain a microemulsion concentrate of the burdock aglycone.

27. A method of treating an infection or a tumor, the method comprising administering to a patient having an infection or tumor using the microemulsion formulation of any one of claims 1-24.

28. The microemulsion formulation of any of claims 1-24 for use in treating an infection or tumor.

Use of the microemulsion formulation of any of claims 1-24 in the manufacture of a medicament for the treatment of an infection or tumor.