CN103690580B - The microemulsion extracting method of Herba Andrographis and extract thereof - Google Patents

Abstract

The invention discloses a microemulsion extraction method of Andrographis paniculata, which is characterized in that it comprises that the medicinal material of Andrographis paniculata is fully contacted with a biocompatible microemulsion for a period of time, for example, 3 days, and then suction filtered to obtain a filtrate; wherein the biocompatible microemulsion Milk includes lecithin, cholesterol, bile salts and water in a weight ratio of 1:0.013:1:18. The invention also discloses the microemulsion extract of Andrographis paniculata extracted according to the microemulsion extraction method and the medicine, health food, nutritional food or cosmetic composition containing the microemulsion extract.

Description

The microemulsion extraction method of andrographis paniculata and its extract

technical field

The invention relates to a microemulsion extraction method of andrographis paniculata and its extract, especially a biocompatible microemulsion extraction method and its extract.

Background technique

Microemulsion (ME) is a low-viscosity, isotropic and thermodynamically stable colloidal dispersion system spontaneously formed by water phase, oil phase, surfactant and co-surfactant in appropriate proportions, with a transparent or semi-transparent appearance. It is transparent and the particle size is generally between 10-100nm. As a drug carrier, it can solve various problems such as poor stability, poor solubility, irritation, low bioavailability, and fast drug release. In particular, it can increase the solubility of insoluble drugs, and is an ideal carrier for insoluble drugs.

As a new type of drug carrier, microemulsion has great application potential. It mainly has the following advantages:

①Microemulsion preparation can improve the solubility of poorly soluble drugs, and can simultaneously solubilize drugs of different polarities.

② The microemulsion preparation has a small particle size, which can effectively and quickly pass through the epithelial cells of the gastrointestinal tract, greatly improving the bioavailability of the drug.

③Microemulsion preparations can be made into various dosage forms, including oral preparations, injection preparations and transdermal preparations.

④ Once the prescription is determined, the preparation method is simple.

Although microemulsions are unique in improving bioavailability, their problems cannot be ignored: microemulsions contain a large number of surfactants and co-surfactants, most of which are irritating to the gastrointestinal mucosa, Long-term use may cause systemic chronic toxicity. Therefore, efforts to find high-efficiency and low-toxic surfactants and co-surfactants are an effective way for the wide application of microemulsions.

Biocompatible pharmaceutical excipients:

Because pharmaceutical excipients work together with drugs, their safety has always been an important issue affecting their application in pharmaceutical preparations. Ideal pharmaceutical excipients should have the following characteristics: good formability, small dosage, strong effect, high physiological safety, no biological activity, stable chemical properties, etc. Among them, pharmaceutical excipients with biodegradability and tissue compatibility (biocompatible pharmaceutical excipients) are the most ideal. Pharmaceutical excipients with biocompatibility do not react and reject with the body, and are less likely to produce harmful reactions; and have good solubility in the body, which can reduce the dosage and promote the absorption and utilization of drugs. Endogenous substances refer to components that come from the body and are closely related to human survival and health. With the continuous development of molecular biology and biochemistry, more and more endogenous bioactive substances will show their medicinal value, and are widely used in the fields of medicine, health food and cosmetics, and endogenous substances as medicinal The application of excipients is less. At present, endogenous substances that can be used as pharmaceutical excipients include lecithin, bile salts, cholesterol, etc.

Lecithin is the basic substance of life, which exists in every cell, and more concentrated in the brain and nervous system, blood circulation system, immune system, liver, heart, kidney and other important organs. Lecithin is a compound whose constituents include phosphoric acid, choline, fatty acids, glycerol, glycolipids, triglycerides, and phospholipids. Lecithin is known as the "third nutrient" alongside protein and vitamins. Human life is inseparable from its nourishment and protection from beginning to end. It has the functions of regulating blood sugar, promoting neurodevelopment and delaying aging.

Pure lecithin (phosphatidylcholine) is a colorless, odorless white solid at room temperature, and is light yellow to brown due to oxidation due to different preparation or refining methods and storage conditions. Lecithin content below 55% is mostly used in health food, nutritious food, and can also be used as medical excipients. 60%-80% are mostly used in cosmetics and pharmaceutical excipients, and more than 90% are mainly used in the pharmaceutical industry. At present, the raw material of "fat emulsion" used clinically in our country is lecithin, which is essentially a product of lecithin and water emulsification, which has good safety and can be used for injection.

Bile salts are sodium or potassium salts formed by combining bile acids secreted by liver cells with glycine or taurine. It is the main component of bile involved in fat digestion and absorption. Bile salts in bile have many functions: emulsifying fat, promoting fat digestion; combining with fatty acids, promoting the absorption of fatty acids; promoting the absorption of fat-soluble vitamins; inhibiting the growth of intestinal bacteria; promoting the dissolution of cholesterol.

Cholesterol is widely present in animals, especially in brain and nerve tissue, and is also high in kidney, spleen, skin, liver and bile. Cholesterol is an indispensable and important substance in animal tissue cells. It not only participates in the formation of cell membranes, but also is a raw material for the synthesis of bile acids, vitamin D and steroid hormones. Cholesterol can be used as an emulsifier to reduce interfacial tension and form a firm interfacial film.

The traditional Chinese medicine Andrographis paniculata has anti-inflammatory and analgesic activities. At present, water extraction, alcohol extraction and alkali extraction and acid precipitation are mostly used to extract the active ingredients in Andrographis paniculata for medicinal purposes, but these methods have certain disadvantages. Water extraction can generally extract water-soluble components such as flavonoids and polysaccharides, but due to the low water solubility of andrographolide, the extraction rate of water extraction is not high. The content of andrographolide in the alcohol extraction solution is relatively high, and moderate concentration of alcohol is also suitable for extracting flavonoid glycosides, but the extraction time of alcohol extraction is long, the amount of solvent is large, the operation is troublesome, and the yield is low, which is not conducive to the full utilization of resources. Alkaline water extraction method, the ring-opening product of andrographolide is easily soluble in water, and can still be reduced to lactone after adjusting the pH, so the extraction rate of the two lactones is acceptable, but the extraction rate of total flavonoids is significantly reduced in an alkaline environment . It can be seen that due to the large difference in polarity of the active ingredients of Andrographis paniculata, it is difficult to extract the active ingredients more comprehensively with a single extraction solvent. Therefore, finding a suitable solvent that can simultaneously solubilize multiple polar components to extract Andrographis paniculata is the key to ensure comprehensive and efficient extraction of active ingredients from Andrographis paniculata.

Contents of the invention

One aspect of the present invention provides a biocompatible microemulsion and a preparation method thereof.

Another aspect of the present invention provides a biocompatible microemulsion extraction method of Andrographis paniculata and its extract.

The applicant has conducted extensive and in-depth research on biocompatible microemulsions and methods for extracting Andrographis paniculata paniculata from biocompatible microemulsions, thus completing the present invention.

The present invention provides:

1. A biocompatible microemulsion, characterized in that, comprises lecithin, cholesterol, bile salts and water, and their weight ratio is: 1:0.013:1:18.

2. The microemulsion according to item 1, which also includes isopropyl myristate,

Wherein the weight ratio of lecithin, cholesterol, bile salt, isopropyl myristate and water is: 1:0.013:1:0.3:18.

3. Microemulsion according to item 1, which also includes polyoxyethylene hydrogenated castor oil,

Wherein the weight ratio of lecithin, cholesterol, bile salt, polyoxyethylene hydrogenated castor oil and water is: 1:0.013:1:1:18.

4. The microemulsion according to item 1, which also includes polyoxyethylene hydrogenated castor oil and isopropyl myristate, wherein lecithin, cholesterol, bile salts, polyoxyethylene hydrogenated castor oil, isopropyl myristate and water The weight ratio is: 1:0.013:1:1:0.3:18.

5. The microemulsion according to item 4, which also includes 95% ethanol, wherein the weight ratio of lecithin, cholesterol, bile salts, polyoxyethylene hydrogenated castor oil, isopropyl myristate, 95% ethanol, and water is: 1 :0.013:1:1:0.3:8:18.

Alternatives to polyoxyethylene hydrogenated castor oil and/or isopropyl myristate may also be included in the microemulsions of the present invention and are known to those skilled in the art.

In a specific embodiment of the present invention, microemulsion of the present invention is only made up of lecithin, cholesterol, bile salt, polyoxyethylene hydrogenated castor oil, isopropyl myristate, 95% ethanol, water, and their weight ratio For: 1:0.013:1:1:0.3:8:18.

As a mixed surfactant, bile salt/lecithin has a good solubilizing effect on insoluble oil phase cholesterol, and bile salt/lecithin and its degradation products are components that exist under the physiological conditions of the body and can be completely absorbed by the body. It has a high degree of safety, and when bile salts are used in combination with lecithin, it can significantly reduce the hemolytic toxicity of lecithin degradation products (lysophospholipids). So the microemulsion formed by bile salt, lecithin and cholesterol is a good drug carrier.

The biocompatible microemulsion drug-loading system of the present invention is based on human endogenous substances (bile salts, cholesterol, lecithin) and has a low dosage. The advantages of the biocompatible microemulsion are: 1. It can effectively solve the Adverse reactions caused by excessive surfactant content (the content of microemulsion surfactants is generally more than 30%, commonly used pharmaceutical excipients have certain toxicity, and often cause a series of adverse reactions, which also limits the wide application of microemulsions main reason). 2. The formula is composed of endogenous biocompatible materials of the human body, which is easily accepted by the human body, does not produce response and rejection, and is not easy to produce harmful effects. 3. Biocompatible materials have good solubility in the body and are easily absorbed by the body, which helps to improve the bioavailability of drugs.

The biocompatible microemulsion of the present invention can be used in health food and nutritious food, and can also be used as a medical auxiliary material. The biocompatible microemulsion of the present invention can also be used as "fat emulsion" and can be used for injection.

The present invention also provides:

6. A method for preparing a microemulsion described in any one of items 1-5, comprising the following steps:

(1) Weigh a certain amount of lecithin and optional polyoxyethylene hydrogenated castor oil and/or 95% ethanol according to the prescription ratio, and dissolve;

(2) Mix the prescribed amount of cholesterol and optional isopropyl myristate and dissolve;

(3) Add the prescribed amount of bile salt into water and dissolve;

(4) Add the solution of step (2) to the solution prepared in step (1), stir evenly, and then add the solution of step (3).

7. The preparation method according to item 6, wherein step (1) weighs a certain amount of lecithin and polyoxyethylene hydrogenated castor oil according to the prescription ratio, preferably lecithin, polyoxyethylene hydrogenated castor oil and 95% ethanol.

8. The preparation method according to item 6 or 7, wherein step (2) mixes the prescribed amount of cholesterol and isopropyl myristate.

9. The preparation method according to any one of items 6-8, wherein the dissolution in steps (1), (2) and (3) is carried out in a 37°C water bath.

The present invention also provides:

10. A method for extracting Andrographis paniculata microemulsion, which is characterized in that, comprising the Andrographis paniculata medicinal material fully contacted with the biocompatible microemulsion for a period of time such as 3 days, and then suction filtration to obtain the filtrate; wherein the biocompatible microemulsion includes Lecithin, cholesterol, bile salts and water, their weight ratio is: 1:0.013:1:18.

11. The microemulsion extraction method according to item 10, wherein said biocompatible microemulsion also includes isopropyl myristate,

Wherein the weight ratio of lecithin, cholesterol, bile salt, isopropyl myristate and water is: 1:0.013:1:0.3:18.

12. The microemulsion extraction method according to item 10, wherein said biocompatible microemulsion further comprises polyoxyethylene hydrogenated castor oil,

Wherein the weight ratio of lecithin, cholesterol, bile salt, polyoxyethylene hydrogenated castor oil and water is: 1:0.013:1:1:18.

13. The microemulsion extraction method according to item 10, wherein said biocompatible microemulsion also includes polyoxyethylene hydrogenated castor oil and isopropyl myristate, wherein lecithin, cholesterol, bile salt, polyoxyethylene hydrogenated castor oil The weight ratio of sesame oil, isopropyl myristate and water is: 1:0.013:1:1:0.3:18.

14. According to the microemulsion extraction method of item 13, wherein said biocompatible microemulsion also includes 95% ethanol, wherein lecithin, cholesterol, bile salt, polyoxyethylene hydrogenated castor oil, isopropyl myristate, 95 The weight ratio of % ethanol to water is: 1:0.013:1:1:0.3:8:18.

15. A microemulsion extraction method described in any one of items 10-14, wherein the biocompatible microemulsion is prepared by a method comprising the following steps:

(1) Weigh a certain amount of lecithin and optional polyoxyethylene hydrogenated castor oil and/or 95% ethanol according to the prescription ratio, and dissolve;

(2) Mix the prescribed amount of cholesterol and optional isopropyl myristate and dissolve;

(3) Add the prescribed amount of bile salt into water and dissolve;

(4) Add the solution of step (2) to the solution prepared in step (1), stir evenly, and then add the solution of step (3).

16. According to the microemulsion extraction method of item 15, wherein step (1) weighs a certain amount of lecithin and polyoxyethylene hydrogenated castor oil according to the prescription ratio, preferably lecithin, polyoxyethylene hydrogenated castor oil and 95% ethanol; step (1), (2) and (3) the dissolution is carried out in a 37 ° C water bath.

17. The microemulsion extraction method according to any one of items 10-14, wherein said sufficient contacting includes shaking or stirring.

18. A microemulsion extract of Andrographis paniculata, characterized in that it is extracted according to any one of the microemulsion extraction methods in items 10-17.

19. A drug, health food, nutraceutical or cosmetic composition, which includes the microemulsion extract described in item 18, preferably the composition is an oral preparation, a spray or an external application.

The microemulsion extract of Andrographis paniculata of the present invention can be administered orally as medicine, health food and nutritional food, can also be administered as medicine by oral cavity or nasal cavity spray, and can be applied externally as cosmetic.

Description of drawings

Fig. 1 andrographolide standard curve

Figure 2 Blood drug concentration-time curve

detailed description

The present invention will be described in detail below through specific examples, but it should not be construed as limiting the present invention in any way.

Instruments and materials

Instruments MD110-2 electronic analytical balance, 81B-2 constant temperature magnetic stirrer, nanometer particle size analyzer (Malvern), 8002 water bath. TGL-16G high-speed refrigerated centrifuge (Shanghai Anting Scientific Instrument Factory), MVS-1 vortex mixer (Beijing Dongfang Kaiwu Scientific Equipment Co., Ltd.), TP-300 ultrasonic cleaning machine (Tianpeng Electronic New Technology Co., Ltd.), HP1100 high performance liquid chromatography, SPD210AV detector.

Materials PC50 lecithin (Shanghai Taiwei Pharmaceutical, batch number 20120301), cholesterol (US SIGMA company, batch number MKBD9436V), pig bile salt (Beijing Shuangxuan Microbial Culture Media Products Factory, batch number 20120420), isopropyl myristate (USA SIGMA company, batch number 20120322), polyoxyethylene hydrogenated castor oil (Germany BASF company, batch number 512882), 95% ethanol (analytical grade), double distilled water.

Preparation Example 1

(1) According to the proportion of the prescription, weigh 10 grams of lecithin, put it in a beaker, dissolve it in a constant temperature hot water bath, stir it evenly with a magnetic force, and set it aside.

(2) Dissolve 0.13 g of cholesterol in a constant temperature hot water bath and set aside.

(3) Add 10 grams of bile salt into 18 grams of water, dissolve in a constant temperature hot water bath, and set aside.

(4) Slowly add the solution of step (2) to the solution prepared in step (1), stir evenly with a magnetic stirrer, and then add the solution of step (3). Promptly obtain the microemulsion of the present invention.

Preparation example 2

(1) According to the proportion of the microemulsion prescription in item 5 above, weigh 10 grams of lecithin, 10 grams of polyoxyethylene hydrogenated castor oil (RH-40), and 8 grams of 95% ethanol, put them in a beaker, and dissolve them in a water bath at 37 °C. After magnetic stirring, set aside.

(2) According to the microemulsion prescription in Item 5 above, add 0.13 g of cholesterol to 3 g of isopropyl myristate, dissolve in a water bath at 37°C, and set aside.

(3) According to the microemulsion prescription in Item 5 above, add 10 grams of bile salts to 18 grams of water, dissolve in a 37°C water bath, and set aside.

(4) Slowly add the solution of step (2) to the solution prepared in step (1), stir evenly with a magnetic stirrer, and then add the solution prepared in step (3). Promptly obtain a kind of preferred microemulsion of the present invention.

Experimental example 1

The microemulsion physicochemical property of preparation example 2:

1. Particle size and shape:

The particle size and particle distribution coefficient PDI value were measured by a Malvern particle size analyzer, and the particle sizes were measured three times: 19.03nm, 19.23nm, and 19.20nm. The PDIs are: 0.165, 0.147, 0.147. The 40,000-fold electron microscope view shows that the shape of the microemulsion is a spherical body with a uniform size of nanoscale. The particle size is between 10 and 100nm, which meets the definition of microemulsion.

2. Stability:

(1) High-speed centrifugal acceleration test: Take an appropriate amount of the prepared microemulsion in a centrifuge tube, place it in a high-speed centrifuge, and centrifuge at 3500r min-1 at room temperature, centrifuge twice, 10min each time, and centrifuge After observation; the microemulsion still remains clear and transparent, and no oil-water layering is seen, indicating that the prepared microemulsion has good dynamic stability.

(2) Influence of 50°C water bath on the stability of microemulsion Take an appropriate amount of the prepared microemulsion in a vial, seal the bottle mouth, and place it in a 50°C water bath for observation. After 3 days of water bath, the microemulsion remains clear and transparent. No oil-water separation was observed, indicating that the prepared microemulsion was stable at high temperature.

(3) Investigation of the appearance stability of the microemulsion at room temperature The microemulsion was placed at room temperature for three months to observe the phenomenon. Results The microemulsion did not delaminate at room temperature, indicating that the microemulsion prepared according to the preferred microemulsion formulation was stable at room temperature. (4) Investigation on the appearance stability of the microemulsion in cold storage. The microemulsion was placed in the refrigerator (4°C) for three months. As a result, the cholesterol microemulsion did not separate at 4°C, and no borneol was precipitated, indicating that according to the preferred The microemulsion prepared by the microemulsion formulation is stable at low temperature.

Experimental example 2

The microemulsion biocompatibility of preparation example 2:

Sample preparation:

Culture medium: RPMI1640 culture medium containing 10% fetal bovine serum. Each liter of culture medium contains 10.4g of RPMI1640 medium powder, 100ml of fetal bovine serum, 100,000 units of penicillin, a small amount of phenol red, adjusted to pH 7.0-7.4 with aqueous sodium bicarbonate solution, and prepared by adding double distilled water.

Preparation of sample solution: 2ml of the microemulsion prepared according to Preparation Example 2 was sterilized, added with 20ml of culture solution, shaken and extracted at 37°C for 24 hours, filtered through a 0.45um sterile microporous membrane, and distributed for use. The extract was diluted to different concentrations of 25%, 50%, 75%, and 100% with RPMI1640 culture medium containing 10% fetal bovine serum.

Negative control solution: complete culture solution placed at 37°C for 24 hours.

Positive control solution: culture solution containing 0.5% phenol.

Cell line: mouse fibroblast cell line L929.

Cell culture:

Culture conditions: 37°C, 5% CO2, saturated humidity.

Test method: MTT method. Take L929 cells in the logarithmic growth phase, dilute them with culture medium to make L929 single cell suspension with a concentration of 5×104/ml, inoculate in each well of a 96-well culture plate, 0.20ml per well, and cultivate for 24h. L929 cells adhere to the wall. Aspirate the solution, add 0.20ml of negative control solution, microemulsion sample solution of different concentrations and positive control solution to 6 wells in sequence. After culturing for 48, 96, and 144 hours, the solution was aspirated, 0.20ml of fresh culture solution and 20ul of 5mg/ml MTT solution were added, shaken gently, and incubated at 37°C for 4 hours. Remove the solution, wash twice with 0.20ml normal saline, add 0.20ml DMSO (dimethyl sulfoxide) to each well, shake gently for 10min, and measure the absorbance OD value at 550nm by enzyme-linked immunoassay.

Result: See Table-1

Table 1

Relative cell viability of L929 cultured in biocompatible microemulsion determined by MTT spectrophotometry

It is generally believed that biomaterials with a relative cell survival rate RGR>75% have no toxic effect on cells, and experiments have shown that the relative cell survival rates of samples in each group are higher than 75%, which proves that the microemulsion formula has good biocompatibility.

The microemulsion of the present invention can be used to extract active ingredients in traditional Chinese medicinal plants.

Embodiment 1: Microemulsion extracts Andrographis paniculata:

(1) Microemulsion impregnation: Weigh 20g of Andrographis paniculata, measure 200ml of the microemulsion prepared according to the above preparation example 2, put the two in a conical flask, oscillate, after full contact, soak and extract at room temperature for 4 days, and filter with suction Get the filtrate. That is to obtain a preferred extract of the present invention.

(2) Ultrasonic microemulsion: Weigh 20 g of Andrographis paniculata, measure 200 ml of microemulsion prepared in Preparation Example 2 above, place the two in a conical flask, oscillate, after full contact, ultrasonic for 30 min, and filter to obtain the filtrate.

Embodiment 2: Microemulsion extracts Andrographis paniculata:

Microemulsion impregnation: Weigh 20 g of Andrographis paniculata medicinal material, measure 200 ml of microemulsion prepared according to the above Preparation Example 1, place the two in an Erlenmeyer flask, stir, after full contact, soak and extract at room temperature for 4 days, and suction filter to obtain the filtrate. That is, the extract of the present invention is obtained.

Comparative Example 1: Ethanol extraction of Andrographis paniculata:

(1) Ethanol impregnation: Weigh 20g of Andrographis paniculata, measure 200ml of 60% ethanol, put the two in a Erlenmeyer flask, oscillate, after full contact, impregnate and extract at room temperature for 4 days, and filter to obtain the filtrate.

(2) Ethanol ultrasound: Weigh 20g of Andrographis paniculata and 200ml of 60% ethanol, put the two in a Erlenmeyer flask, oscillate, after full contact, ultrasound for 30min, and filter to obtain the filtrate.

Experimental example 3

Comparison of extraction efficiency of different extracts:

Accurately measure 2ml of each of the above-mentioned Andrographis paniculata extracts into 10ml centrifuge tubes, add 6ml of methanol precisely, vortex shaker for 1min, let stand, and centrifuge at 10000r for 10min, transfer the supernatant to a 25ml volumetric flask, and methanol to volume , filtered through a 0.45μm filter membrane, and determined by high performance liquid chromatography. The chromatographic conditions are as follows:

Chromatographic column: Dima C18 column (150mm, 5μm), mobile phase is acetonitrile (A)-water (B) gradient elution: 0-5min, 22%-25%A, 5-30min, 25%-30% A, 30-50min, 30%-40%A; flow rate: 1.0ml·min-1; detection wavelength is 225nm and 254nm; injection volume: 20μL; column temperature: 25℃.

The assay results of each extract are as follows:

From the above results, it can be seen that microemulsion is used as an extraction solvent, and the extraction efficiency of andrographolide and dehydroandrographolide is higher than that of ethanol, whether it is immersion method or ultrasonic method. Among them, the extraction of dehydroandrographolide has a more advantageous effect, and the advantages of the microemulsion of the present invention for multi-component extraction are brought into play. The more prominent advantage is that the extraction efficiency of microemulsion as a solvent is significantly higher than that of ultrasonic extraction, indicating that the extraction solvent can reduce energy consumption and is a high-quality and efficient extraction solvent.

Dehydroandrographolide has anti-inflammatory and antipyretic effects, can inhibit the increase of capillary permeability and the development of inflammatory edema during inflammation, and can reduce the amount of inflammatory exudation. It has a good clinical effect on respiratory and intestinal infectious diseases. The advantage of extracting dehydroandrographolide from the biocompatible microemulsion of the present invention is that the biocompatible microemulsion containing dehydroandrographolide of the present invention can be directly orally used for the treatment of intestinal infectious diseases, without the need to extract the active ingredients from the microemulsion. milk separation.

Experimental example 4

Comparison of Oral Bioavailability of Microemulsion Extract and Ethanol Extract

Blood sample collection and processing

The anesthetic urethane solution (5ml/kg) was injected into the ear vein of the rabbit in an awake state. After anesthesia, it was tied to the rabbit platform, the common carotid artery was separated, and the tube was buried. About 5ml of blank blood samples were collected respectively, anticoagulated with heparin, and collected After the blank blood, each group was administered intragastrically respectively. Example 1 Microemulsion-impregnated Andrographis paniculata extract and Comparative Example 1 Ethanol-impregnated Andrographis paniculata extract were each 5ml, respectively at 5min, 15min, 30min, 45min, 1h, 1.5, 2h after intragastric administration. About 5ml of blood samples were collected at 3h, 4h, 5h, and 7h, and anticoagulated with heparin.

The blood sample collected above was placed in a low-temperature centrifuge at 4° C. and centrifuged at 3000 r/min for 20 minutes to separate the plasma and set aside. Accurately take 1ml of plasma at each time point after administration, put it into a 10ml centrifuge tube with a stopper, add 6ml of chloroform, mix in a vortex mixer for 5min, centrifuge at 3000r/min for 20min, accurately draw 4ml of chloroform layer, and put it at 37 Evaporate to dryness in a water bath at ℃, dissolve the residue with 1ml of methanol ultrasonically, pass through a 0.45μm filter membrane, and enter high performance liquid phase to determine the content.

Preparation of standard curve

Accurately weigh 5.mg of andrographolide reference substance, dissolve it in methanol to the mark in a 50ml volumetric flask, and prepare a stock solution for later use. Take 7 blank 10ml stoppered centrifuge tubes, add 1ml of blank plasma accurately to each tube, and then accurately add standard stock solution 8, 16, 24, 32, 40, 48, 56μl respectively, and process according to the above blood sample processing method, according to the area The normalization method was used to calculate the standard curve equation. Chromatographic conditions C18 column (150mm, 5μm, Dima Company); mobile phase: methanol: water (50:50); flow rate: 1.0ml/min, detection wavelength λ=225nm, column temperature is room temperature.

According to the preparation method of the standard blood sample, under the chromatographic conditions of this test, the linear range is (0.1-2.3μg/ml), the peak area of the sample is plotted against the concentration, and the standard curve equation is obtained by linear regression: A=1.8394C-0.3068 (R2=0.9924) The linear relationship is better, as shown in Figure 1

result

Blood concentration of andrographolide extracted by two kinds of extraction solutions

After intragastric administration of the microemulsion extract of Example 1 and the ethanol extract of Comparative Example 1, the concentration of andrographolide in the plasma of rabbits is shown in Table 1

Table 1 Blood drug concentration after drug administration

The bioavailability parameters of andrographolide extracted from the two solutions are shown in Table 2 and the blood drug concentration-time curve Figure 2.

Table 2 Bioavailability parameters

The blood drug concentration of andrographolide extracted from microemulsion rises rapidly after oral administration, and the blood drug concentration reaches the peak at 30 minutes, and the blood drug concentration is at a high level in the time period of 0.5h to 2h, and forms another absorption in the time period of 2h to 3h After 3 hours, the plasma concentration began to decrease, forming obvious double absorption peaks. After oral administration of ethanol-extracted andrographolide to rabbits, the plasma concentration increased slowly, reached the peak at 3 hours, and then decreased rapidly. Compared with the ethanol extract, the relative bioavailability of the microemulsion extract can reach 142%, indicating that the oral absorption and utilization of the microemulsion extract is significantly higher than that of the ethanol extract.

Experimental example 5

The anti-inflammatory effect of the extract was characterized by the mouse ear swelling test induced by xylene; the antipyretic effect of the extract was characterized by the fever test of the rat induced by bacterial lipopolysaccharide

1. Effect of xylene-induced ear swelling in mice

The experimental mice were randomly divided into a model group, a prednisone acetate group, a microemulsion group in Example 1 and an ethanol group in Comparative Example 1 according to body weight, with 7 mice in each group. The experimental animals were administered continuously for 4 days, the model group was given tap water, the prednisone acetate group was administered at 0.2g/kg, the microemulsion group and the alcohol extraction group were administered at 2g/kg (equivalent to crude drug), fasting before the experiment (cannot help water ) 5h. 30 minutes after the last administration, ether was lightly anesthetized, and 0.01 mL of xylene was accurately applied to the front and back sides of the right ear of the mouse with a quantitative sampler, and the animals were sacrificed 30 minutes later by dislocation of the cervical spine. The ears were removed from the symmetrical parts of the left and right ears with a puncher with a diameter of 5 mm, and weighed. The difference between the weights of the two ears represented the degree of swelling. The swelling degree and inhibition rate of xylene-induced mouse ear swelling were calculated respectively. The results are shown in Table 3.

Table-3. Effect of p-xylene-induced ear swelling in mice

Note: Compared with blank group, **P<0.01; compared with alcohol extraction group, ^△△ P<0.05

2. Effect on LPS-induced fever in rats

The experimental grouping was the same as the mouse ear swelling experiment. Measure the anal temperature of rats with an electronic thermometer once a day for 3 consecutive days; measure 3 times continuously on the 4th day with an interval of 0.5h each time. , and the fluctuation is within 0.3°C) for the test. All animals were subcutaneously injected with LPS physiological saline solution at 300 μg/kg, their body temperature was measured 3.5 hours after injection, and they were randomly divided into 4 groups according to body temperature stratification, aspirin 0.2g/kg group and model control group, microemulsion 2g/kg group, Alcohol extraction 2g/kg group. At 4 hours, the corresponding drugs were given by intragastric administration (normal water was given to the model group), the rectal temperature was measured at 4.5 hours and 5.5 hours, and the temperature change value compared with the initial body temperature was calculated. The results are shown in Table-4.

Table-4. Effects on LPS-induced fever in rats

Note: Compared with the blank group, **P<0.01; compared with the alcohol extraction group, ^△ P<0.01 ^△△ P<0.05

In summary, the microemulsion prepared by the present invention is used as an extraction solvent for extracting Andrographis paniculata, which has higher extraction efficiency and higher oral bioavailability of the extract. Microemulsion extraction of Andrographis paniculata has outstanding advantages such as low energy consumption, high extraction efficiency, high absorption and utilization of the extract, and strong anti-inflammatory ability.

What have been described above are only some embodiments of the present invention. For those skilled in the art, without departing from the inventive concept of the present invention, several modifications and improvements can be made, and these all belong to the protection scope of the present invention.

Claims (7)

1. a microemulsion extraction method of Andrographis paniculata, it is characterized in that, comprise Andrographis paniculata medicinal material and biocompatible microemulsion fully contact a period of time, then suction filtration, obtain filtrate; Wherein said biocompatible microemulsion comprises lecithin, Cholesterol, bile salts and water, said biocompatible microemulsion also includes polyoxyethylene hydrogenated castor oil, isopropyl myristate and 95% ethanol, wherein lecithin, cholesterol, bile salts, polyoxyethylene hydrogenated castor oil , isopropyl myristate, 95% ethanol, water weight ratio: 1:0.013:1:1:0.3:8:18. 2. a microemulsion extracting method described in claim 1, wherein said biocompatible microemulsion adopts and comprises the following step method preparation: (1) Take a certain amount of lecithin, polyoxyethylene hydrogenated castor oil and 95% ethanol by weighing the prescription ratio, dissolve; (2) mixing prescription amount cholesterol and isopropyl myristate, dissolving; (3) adding the prescribed amount of bile salt into water and dissolving; (4) Add the solution of step (2) to the solution prepared in step (1), stir evenly, and then add the solution of step (3). 3. according to the microemulsion extraction method of claim 2, wherein the described dissolving of step (1), (2) and (3) is carried out in 37 ℃ of water baths. 4. The microemulsion extraction method according to claim 1 or 2, wherein said sufficient contact comprises shaking or stirring. 5. A microemulsion extract of Andrographis paniculata, characterized in that it is extracted according to any one of the microemulsion extraction methods of claims 1-4. 6. A medicine, health food, nutraceutical or cosmetic composition, which contains the microemulsion extract described in claim 5. 7. The pharmaceutical, health food, nutraceutical or cosmetic composition according to claim 6, wherein said composition is an oral preparation, a spray or an external application.