WO2013040784A1

WO2013040784A1 - Lyophilizing insulin liposome powder, oral insulin complex preparation, preparation method therefor, and use thereof

Info

Publication number: WO2013040784A1
Application number: PCT/CN2011/080069
Authority: WO
Inventors: 范圣刚; 王志萍
Original assignee: Fan Shenggang; Wang Zhiping
Priority date: 2011-09-23
Filing date: 2011-09-23
Publication date: 2013-03-28

Abstract

A lyophilizing insulin liposome powder and oral insulin complex preparation for treatment of type II diabetes, and preparation method therefor. The lyophilizing insulin liposome powder according to the present invention is prepared in the following steps: to an aqueous HCl solution, adding insulin, and then adding a phosphate buffer solution and sodium cholate solution to give an aqueous mixture; co-dissolving cholesterol and lecithin in absolute ethyl alcohol and adding aqueous mannitol solution and sodium cholate solution, and homogenizing for 10-30 minutes, to give an oily mixture; uniformly mixing the aqueous mixture with the oily mixture and placing in a water bath at 2-6°C, subjecting to distillation under reduced pressure, to give a concentrate; and lyophilizing the concentrate.

Description

Insulin liposome freeze-dried powder, oral insulin composite preparation, preparation method and application thereof

The present invention relates to an insulin liposome lyophilized powder for treating type II diabetes, an oral insulin composite preparation, and a preparation method and application thereof.

Background technique

As a biochemical drug for lowering blood sugar, insulin has been used for the treatment of diabetes for nearly 100 years. It is still the drug of choice for patients with type 1 diabetes and is an indispensable drug for more and more patients with type 2 diabetes. At present, clinical treatment is more inclined to use insulin as early as possible for patients with type 2 diabetes, which can repair insulin cells and better reduce the incidence of diabetes. However, since insulin is a peptide drug, it is degraded by the proteolytic enzymes of the gastrointestinal tract without being absorbed, so the treatment of the oral route is almost ineffective. For this reason, insulin injections are commonly used in clinical practice. At the same time, in order to avoid the inconvenience and adverse effects of peripheral administration, and the simple onset of hypoglycemic agents, various non-injected insulin preparations have been designed at home and abroad, especially in various ways. Insulin-based, but the existing insulin produced by embedding still has low embedding rate, poor stability, and it is difficult to achieve industrial production and other weaknesses.

Invention disclosure

The technical problem to be solved by the present invention is to provide an insulin liposome lyophilized powder with high embedding rate and an oral insulin compound preparation for treating type II diabetes containing the insulin liposome freeze-dried powder, the oral insulin compound The preparation has good stability and is convenient for industrial production.

An insulin liposome lyophilized powder prepared according to the following steps:

1) Add insulin to the 0.01-0.02M HCl aqueous solution and dissolve it sufficiently to make the insulin concentration 2.6-3.4g/l, and then add the phosphate buffer with pH 6.5-7.5 and 0.1-0.2M to mix evenly. The concentration of insulin reaches 0.34-0.45 g / l, and the concentration of 0.01-0.02 g / ml sodium cholate solution is added to make the concentration of insulin reach 0.33-0.43 g / l to obtain an aqueous phase mixture;

2) Mix a mixture of cholesterol and lecithin with a mass ratio of 1: (1-3) in absolute ethanol, dissolve well and mix well, so that the concentration of the mixture is 0.018-0.025 g/ml, plus the concentration is 0.01-0.02g/ml aqueous solution of mannitol, the concentration of the mixture of cholesterol and lecithin is 0.011-0.013g/ml, and then the sodium cholate solution is added at a concentration of 0.01-0.02g/ml to make a mixture of cholesterol and lecithin. The concentration reaches 0.011-0.012g/ml, and the obtained liquid is placed in a high pressure homogenizer. 10-30 min, the oil phase mixture is obtained;

3) Mix the mixture of the aqueous phase and the oil phase in a volume ratio of (1.3-2.9): 1 in a container and place it at 2-6 °. After 2-4 hours in a water bath, anhydrous ethanol was removed under reduced pressure to obtain a concentrated liquid;

4) The concentrate is freeze-dried to obtain an insulin liposome lyophilized powder.

The insulin liposome lyophilized powder of the present invention is prepared according to the following steps:

1) Add insulin to 0.01M HCl solution and dissolve it sufficiently to make the concentration of insulin 2.9g/l. Then add phosphate buffer with pH of 7.0 and 0.2M to mix evenly, so that the concentration of insulin reaches 0.42g/l. , adding a concentration of 0.01 g / ml sodium cholate solution, so that the concentration of insulin reaches 0.40 g / l, to obtain a mixture of aqueous phase;

2) Dissolve a mixture of cholesterol and lecithin at a mass ratio of 1:3 in absolute ethanol, dissolve well and mix well, so that the concentration of the mixture is 0.020 g/ml, plus mannitol at a concentration of 0.02 g/ml. The aqueous solution is adjusted to a concentration of 0.0125 g/ml of cholesterol and lecithin, and then a sodium cholate solution having a concentration of 0.01 g/ml is added to bring the mixture concentration of cholesterol and lecithin to 0.011 g/ml, and the resulting liquid is placed at a high pressure. The homogenizer is homogenized for 10-30 min to obtain an oil phase mixture;

3) Mix the aqueous phase mixture and the oil phase in a volume ratio of 2.7:1 in a container and place it in a water bath at 2-6 ° C for 2-4 hours, then distill off the vacuum to remove anhydrous ethanol. Obtaining a concentrate;

An oral insulin combination preparation for treating type II diabetes, comprising the following components by weight:

The above insulin liposome lyophilized powder: 1 part;

Pueraria: 4-13 servings;

Trichosanthin: 9-29 servings;

Rehmannia: 5-15 ^;

Ginseng: 1-3 servings;

Astragalus: 3-9 servings;

Yam: 3-10 servings.

Oral insulin composite preparation for treating type II diabetes of the present invention,

The insulin liposome lyophilized powder according to claim 1 or 2: 1 part;

Pueraria: 8 servings;

Trichosanthin: 16 servings;

Rehmannia: 10 servings;

Ginseng: 2 servings;

Huang Wei: 6 servings; Yam: 6 servings.

The invention relates to a method for preparing an oral insulin compound preparation for treating type II diabetes, comprising the following steps: weighing and mixing the components in the oral insulin compound preparation in proportion, pulverizing through a 80 mesh sieve, wet granulation, and wet granules 60 70 V drying, dry granules are sifted with a 20 mesh sieve and filled into capsules.

The above-mentioned insulin liposome lyophilized powder is used for preparing a medicament for treating type II diabetes.

The above oral insulin complex preparation is used for preparing a medicament for treating type II diabetes.

The invention adds pueraria, trichosanthin, rehmannia, ginseng, astragalus and yam Chinese herbal medicine as auxiliary materials, wherein: pueraria: sweet, spicy, cool; spleen and stomach. Function and Indications: Decompression of muscles, fluid, rash, sun and diarrhea. "Compendium of Materia Medica" contains: Pueraria, cool, calm, sweet, with heat, fire, detoxification.

Trichosanthin: Shengjin, thirst quenching, lowering fire, moistening dryness, draining pus, swelling. For fever, polydipsia, lung heat dry cough, internal heat and thirst, sore swollen poison. "Materia Medica": Reduces fire and moisturizes, slips and quenches thirst, and ancients use diabetes to treat diabetes (ie diabetes). Passing through the water, stomach heat yellow, dry lips, swollen poison back, breast acne scars. "Medical Lin Shu Yao": lungs, gas, fire, Ning Xin, and diarrhea liver stagnation, slow liver urgency, clear bladder heat, short urinary urination, except Yang Ming and damp heat.

Rehmannia: nourishing yin, nourishing blood. Treatment of yin deficiency fever, thirst, vomiting blood, blood stasis, blood collapse, irregular menstruation, fetal movement, constipation. 1 "The Classic": The main folds and stagnation, wounds, bloody sputum, bone marrow, long muscles, soup for cold and heat accumulation, in addition to sputum. The living is especially good. 2 "Do not record": The main man has five labors and seven injuries, the woman is injured, the blood leaks from the blood, breaks the blood, sputum blood, and the large and small intestines, goes to the stomach to eat, fills the five internal organs, lacks internal injuries, passes blood, and benefits , Li and Ear. 3 "Pharmacology": Tonic deficiency, warm and qi, blood circulation, postpartum abdominal pain, the main vomiting blood. 4 "Japanese Huazi Materia Medica": Treatment of convulsions, heart and lung damage, vomiting blood, nose sputum, women collapsed in blood halo, help bones. 5 Wang Haogu: The main heart disease, the pain in the palm, the suffocation, the slumber, the hot and painful underfoot. 6 "Materia Medica is new": Treating blood deficiency and fever, often feeling hunger, tiredness and lying.

Ginseng: A big qi, a solid, free, and soothing. "Medical Enlightenment" Record: Ginseng treatment of spleen and stomach yang deficiency and lung shortness of breath, short gas, less gas, Buzhongzhongzhong, diarrhea, spleen and stomach in the fire evil. "Indications" Record: ginseng supplement vitality, diarrhea, Shengjin liquid. "Minnan Materia Medica" records: ginseng treatment of yin and yang deficiency, lung weakness. Invigorating the qi and solidifying the table, diuretic toxic, drainage, and sore muscles. For qi deficiency and fatigue, eating less stool, qi sag, prolonged diarrhea, rectal prolapse, blood in the stool, sweating, qi deficiency and edema

Astragalus: It is difficult to collapse, long-term ulceration, blood deficiency, yellowing, internal heat and thirst; chronic nephritis proteinuria, diabetes.

Yam: Replenishing the spleen and nourishing the stomach, Shengjin Yifei, and tonifying the kidney. For spleen deficiency food, long-term diarrhea, lung deficiency and asthma, kidney deficiency, bring down, frequent urination, virtual heat and thirst.

The insulin liposome lyophilized powder provided by the invention has a high embedding rate and an insulin embedding rate of more than 90%; the oral insulin compound preparation containing the insulin liposome lyophilized powder has good stability and is preserved for 24 months. After that, the insulin content in the oral insulin combination still remained above 99%, while the entrapment rate of insulin liposomes was only reduced by 2% compared with 0 months. Left and right, it indicates that the leakage of insulin embedded in the liposome is not significant; the oral insulin composite preparation is convenient for industrial production due to high embedding rate and good stability. In the process of encapsulation of insulin liposome, anhydrous ethanol was used as solvent in the process of coating. Compared with ether and chloroform as solvents, the toxic side effect was small, and the binding of capsule to insulin solution was strong.

The embodiments described above are only intended to describe the preferred embodiments of the present invention, and are not intended to limit the scope of the present invention, and various embodiments of the present invention may be made by those skilled in the art without departing from the spirit of the invention. Modifications and improvements are intended to fall within the scope of the invention as defined by the appended claims. The best way to implement the invention

Example 1 Preparation of insulin liposome lyophilized powder

1) Add 100 mg of insulin to 30 ml of 0.01 M aqueous HCl solution and dissolve well. Add 200 ml of phosphate buffer solution of pH 6.5 and 0.1 M, and mix well. Add 10 ml of O.Olg/ml sodium cholate solution. , obtaining a mixture of aqueous phases;

2) Co-dissolve 0.5g of cholesterol and 0.5g of lecithin in 40ml of absolute ethanol, dissolve well and mix well, add 40ml of mannitol solution with concentration of O.Olg/ml and 10ml of O.Olg/ml. Sodium cholate solution, the obtained liquid is placed in a high-pressure homogenizer for 10 min to obtain an oil phase mixture;

3) The above aqueous phase mixture and the oil phase mixture are poured into a container and uniformly mixed and placed in a water bath at 2 ° C for 2 hours, and then anhydrous ethanol is distilled off under reduced pressure to obtain a concentrated liquid;

4) The concentrate is freeze-dried to obtain an insulin liposome lyophilized powder. Example 2 Preparation of oral insulin composite preparation

The insulin liposome lyophilized powder obtained in Example 1 was used as 1 part by weight, and then 4 parts of pueraria root, 9 parts of trichosanthin, 5 parts of rehmannia, 1 part of ginseng, 3 parts of astragalus, 3 parts of yam were uniformly mixed and pulverized. After 80 mesh sieve, wet granulation, the wet granules were dried at 60 ° C, and the dry granules were granulated with a 20 mesh sieve and filled into capsules. Example 3 Preparation of insulin liposome lyophilized powder

1) 400mg of insulin was added to 150ml of 0.02M HCl solution and dissolved thoroughly. Then add 1000ml, pH 7.5 0.2M phosphate buffer and mix well. Add 60ml of 0.02g/ml sodium cholate solution and mix well. Aqueous liquid mixture;

2) Co-dissolve 2.5 g of cholesterol and 7.5 g of lecithin in 450 ml of absolute ethanol, dissolve well and mix well, add 400 ml of mannitol solution with a concentration of 0.02 g/ml and 40 ml of cholic acid at a concentration of 0.02 g/ml. Sodium solution, put the liquid obtained It is placed in a high-pressure homogenizer for 30 minutes to obtain an oil phase mixture;

3) Pour the mixture of the aqueous phase and the oil phase obtained above into a container, mix well and place in a water bath at 6 ° C for 4 hours, then distill off the absolute ethanol under reduced pressure to obtain a concentrated liquid;

4) The concentrate is freeze-dried to obtain an insulin liposome lyophilized powder. Example 4 Preparation of oral insulin composite preparation

The insulin liposome lyophilized powder obtained in Example 3 was used as 1 part by weight, and then 13 parts of pueraria root, 29 parts of trichosanthin, 15 parts of rehmannia, 3 parts of ginseng, 9 parts of scutellaria, and 10 parts of yam were uniformly mixed and pulverized. After 80 mesh sieve, wet granulation, the wet granules were dried at 70 ° C, and the dry granules were granulated with a 20 mesh sieve and filled into capsules. Example 5 Preparation of insulin liposome lyophilized powder

1) Add 200mg of insulin to 70ml of 0.01M HCl solution and dissolve it thoroughly, then add 400ml, pH7.0 0.2M phosphate buffer to mix well, then add 20ml of O.Olg/ml sodium cholate solution and mix evenly. Obtaining a mixture of aqueous phases;

2) Co-dissolve 0.5g of cholesterol and 1.5g of lecithin in 100ml of absolute ethanol, dissolve well and mix well, add 60ml of mannitol solution with a concentration of 0.02g/ml and 20ml of ooze of O.Olg/ml. The sodium salt solution is placed in a high-pressure homogenizer for 20 minutes to obtain an oil phase mixture;

3) Pour the mixture of the aqueous phase and the oil phase obtained above into a container, mix well, and place in a water bath at 6 ° C for 3 hours, then distill off the absolute ethanol under reduced pressure to obtain a concentrated liquid;

4) The concentrate is freeze-dried to obtain an insulin liposome lyophilized powder. Example 6 Preparation of Oral Insulin Combination Preparation

The insulin liposome lyophilized powder obtained in Example 5 was used as 1 part by weight, and 8 parts of pueraria root, 16 parts of trichosanthin, 10 parts of rehmannia, 2 parts of ginseng, 6 parts of jaundice, and 6 parts of yam were uniformly mixed and pulverized. After 80 mesh sieve, wet granulation, the wet granules were dried at 65 °C, and the dry granules were sifted with 20 mesh sieves and filled into capsules. Example 7 Preparation of insulin liposome lyophilized powder

1) Add 270mg of insulin to 100ml of 0.01M HCl solution and dissolve it thoroughly, then add 500ml, pH7.0 0.1M phosphate buffer to mix well, then add 30ml of O.Olg/ml sodium cholate solution and mix well. Obtaining a mixture of aqueous phases; 2) 1.5 g of cholesterol and 1.5 g of lecithin are dissolved in 160 ml of absolute ethanol, fully dissolved and uniformly mixed, and 80 ml of a mannitol solution having a concentration of 0.01 g/ml and 30 ml of a cholesteric solution having a concentration of O.Olg/ml are added. The sodium salt solution is placed in a high-pressure homogenizer for 20 minutes to obtain an oil phase mixture;

4) The concentrate is freeze-dried to obtain an insulin liposome lyophilized powder. Example 8 Preparation of oral insulin composite preparation

The insulin liposome lyophilized powder obtained in Example 7 was used as 1 part by weight, and 8 parts of pueraria root, 16 parts of trichosanthin, 10 parts of rehmannia, 2 parts of ginseng, 6 parts of astragalus, and 6 parts of yam were uniformly mixed and pulverized. After 80 mesh sieve, wet granulation, the wet granules were dried at 65 °C, and the dry granules were sifted with 20 mesh sieves and filled into capsules. Example 9 Detection of insulin embedding rate of insulin liposome freeze-dried powder

The contents of the capsules obtained in Examples 2, 4, 6 and P 8 were accurately weighed to 2.5 g each, and 10 ml of three distilled waters were separately added to uniformly disperse the contents and dissolve the lyophilized powder at 4 ° C in a low temperature centrifuge. The Chinese medicine powder was removed by centrifugation at 12000 rpm for 10 min, and the supernatant was taken for ultrafiltration with a filter membrane having a molecular weight cut off of 12,000. The free insulin solution was passed through a filter membrane to measure the volume of each free insulin solution, respectively, according to the Pharmacopoeia of the People's Republic of China 2010 edition 2 The insulin test method measures the free insulin concentration by HPLC, and the chromatographic conditions are as follows. Embedding rate = (1-free insulin concentration X free insulin solution volume / total insulin mass) X 100%, where total insulin mass refers to the embedding system when embedding in Examples 1, 3, 5, and 7 The total insulin quality. The results of the embedding rate test are shown in Table 1.

The chromatographic conditions: using octadecylsilane bonded silica as a filler; 0. 2mol / L sulfate buffer (take anhydrous sodium sulfate 28.4g, dissolved in water, add phosphoric acid 2. 7ml, water 800ml, with The ethanolamine was adjusted to a pH of 2.3, and water was added to 1000 ml) - acetonitrile (volume ratio 74: 26)) as a mobile phase at a flow rate of 1.0 ml/min; the column temperature was 40 ° C; and the detection wavelength was 214 nm.

Table 1 Insulin liposome lyophilized powder embedding rate test results

The results showed that: insulin liposome freeze-dried powder can embed more than 90% of insulin, and the embedding rate is high. Example 10 Detection of insulin stability in oral insulin combination preparation

The oral insulin complex preparations obtained in Examples 2, 4, 6 and 8 were stored at a temperature of 25 ± 2 ° C and a humidity of 60% ± 10% at 0, 3, 6, 9, 12, 18, 24 Sampled separately in months. The contents of the capsules obtained in Examples 2, 4, 6 and P 8 were accurately weighed to 2.5 g each, and 10 ml of an aqueous solution of 0.1 mol/L hydrochloric acid was added to uniformly disperse the contents and the lyophilized powder was dissolved, and allowed to stand overnight. The liposome was ruptured, and the Chinese medicinal powder was removed by centrifugation at 10 ° C and 12000 rpm for 10 min in a low temperature centrifuge. The supernatant was taken and the volume of each supernatant was measured. According to the Pharmacopoeia of the People's Republic of China, the 2010 edition of the second insulin test method was HPLC. The method measures the insulin concentration in the supernatant. Percentage of retained insulin = insulin concentration in supernatant X supernatant volume / total insulin mass X 100%, where total insulin mass refers to embedding when embedded in Examples 1, 3, 5, and 7 The total insulin quality in the system. The specific test results are shown in Table 2.

Table 2 Results of insulin stability test in oral insulin combination preparation

After 24 months of storage, the insulin in the oral insulin combination was still very stable, retaining more than 99% of insulin. Example 11

The oral insulin complex preparations obtained in Examples 2, 4, 6 and 8 were stored at a temperature of 25 ± 2 ° C and a humidity of 60% ± 10% at 0, 3, 6, 9, 12, 18, 24 Sampled separately in months. The insulin embedding rate test was carried out according to the method of Example 9, that is, the stability of the oral insulin complex preparation and the stability of the liposome in the oral insulin complex preparation were examined to see if there was liposome instability in the oral insulin complex preparation. , resulting in the leakage of the embedded insulin into free insulin, the results are shown in Table 3.

Table 3 insulin embedding rate stability test

Sample

0 months 3 months 6 months 9 months 12 months 18 months 24 months embedding rate (%) Example 2 91.7% 91.7% 91.0% 91.1% 90.9% 90.3% 90.1% Example 4 91.2% 91.2% 90.7% 90.5% 90.5% 90.4% 90.0% Example 6 92.0% 92.0% 91.0% 91.0% 90.8% 90.6% 90.2 % Example 8 91.5% 91.5% 91.0% 91.1% 90.9% 90.3% 90.1% The results showed that after 24 months of storage of the oral insulin complex, the insulin embedding rate was only about 2% lower than that at 0 months. It shows that the stability of liposome is good and the stability of oral insulin compound preparation is good. Example 12 Hypoglycemic experiment of oral insulin complex preparation on type 2 diabetic SD rat model

1. Test drug: Oral insulin composite preparation prepared in Example 8; Positive control: Jiangtangning capsule, specification: 0.4 g/granule.

2. Experimental animals: Male SPF Wistar rats, 150, 180-200g, provided by Shandong University Experimental Animal Center, certificate number: SCXK (Lu) 2003004, NO.0006088.

3. Experimental design and grouping:

1) Animal model: Rats were fed for 3 days after purchase, and 10 rats were randomly selected to measure normal blood glucose level. After fasting for 24 hours, intraperitoneal injection of streptozotocin 50 mg/kg (dissolved in a concentration of 0.01 g/ml) Aqueous citric acid). The fasting blood glucose was measured once at 72 h after injection, and the rats with unsuccessful modeling were excluded.

2) Experimental grouping: The qualified and screened qualified rats were randomly divided into model control group, Jiangtangning group and high, medium and low dose groups of test drugs according to blood glucose level and body weight balance, totaling 5 groups, each group 9 only.

3) Route of administration: Oral

4) Dosage: Jiangtangning Capsule, the clinical equivalent of the rat is 0.648g/kg, and it is configured to be 0.0648g/ml before administration, and it is administered at 10ml/kg. Oral insulin compound preparation: The oral insulin compound preparation prepared in Example 8 is 4 mg=1 IU, and the doses of the high, medium and low dose groups of the test drug are set to be 4, 2, 1 times the clinical equivalent amount of the oral insulin compound preparation, S卩: 28.8 mg/kg, 14.4 mg/kg, 7.2 mg/kg. Before administration, they were formulated into 2.88 mg/ml, 1.44 mg/ml, 0.72 mg/ml, and administered at 10 ml/kg.

5) Experimental method

5.1 Dosing and testing:

One dose: The day after grouping, 2 hours after fasting, the blood glucose levels were measured at 1 h, 2 h, 4 h, and 8 h after administration.

Administration for several consecutive days: After one administration, the administration was continued for 4 days, and after 2 hours of fasting per day, it was administered once a day. On the sixth day, the blood glucose level after fasting for 2 hours was measured.

5.2 Measurement index: The blood glucose level at different time points after one administration and the blood glucose level after continuous administration for several days. 5.3 Statistical Methods: All data were expressed as X±s, and t-tests were used between groups.

4. Experimental results:

1) Pre-grouping blood glucose values:

As shown in Table 4, the blood glucose level of the rats was significantly increased after modeling, which was significantly different from that of normal rats. After blood glucose was used as an indicator, there was no difference between the model group and the blood glucose group of each test drug. Blood glucose levels of each group after model selection (X±s, n=9)

Group blood glucose (mmol/L)

Normal group 7.12±1.11

Model control group 24.43 ± 4.26

Jiangtangning Group 24.39±4.23

High dose group 24.43 ±3.98

Medium dose group 24.71 ±3.65

Low dose group 24.6±3.76

2) Blood glucose changes after one dose

The tail-breaking method was used to measure the blood glucose levels of lh, 2h, 4h and 8h after one dose. The results showed that the high, medium and low dose groups significantly reduced the blood glucose level in rats at l-4h, compared with the model control group. There are significant differences. The hypoglycemic control group also had a significant effect on lowering blood glucose, and its intensity of action was similar to that of the low dose group. The results are shown in Table 5.

Table 5 - Changes in blood glucose levels of each group after sub-administration (X ± s, n = 9) Group dose (mg / kg) lh 2h 4h 8h Model control group 23.81 ± 2.26 21.67 + 2.81 17.98 ± 1.84 5.13 ± 2.18 Tangning Group 648 14.32±7.47 11.24±7.67 12.91 ±2.87 5.08±0.97 High dose group 28.8 5.61±2.65 4.64±0.98 9.09±3.29 4.1±1.06 Medium dose group 14.4 12.01±3.39 7.51 ±2.24 11.5±2.54 4.77±1.57 Low dose Group 7.2 16.78±6.40 14.8±6.63 13.31±5.29 4.98±0.99

3) Changes in blood glucose after continuous administration for several days

Five days after continuous administration, the blood glucose levels of the rats in each group were directly measured after fasting for 2 hours on the sixth day, as shown in Table 6.

Table 6 Changes in blood glucose levels of each group after continuous administration for many days (X±s, n=9)

Dose (mg/kg) blood glucose (mmol/L) Model control group - 18.78 ± 3.43 hypoglycemic group 648 18.32 ± 3.27 high dose group 28.8 13.84 ± 4.37

Medium dose group 14.4 15.33 ± 5.78 Low dose group 7.2 17.53 ± 5.24

4) Conclusion of efficacy evaluation

Through one-time administration and multiple administration, the change of blood glucose level of the composite preparation in the type II diabetic rat model was observed. It can be considered that: oral administration of the oral insulin composite preparation has a clear reduction effect on blood sugar, and the effective time is presented. Significant dose-effect relationship; lower blood glucose levels compared to the model group after multiple doses. Industrial applicability

The insulin liposome lyophilized powder provided by the invention has a high embedding rate and an insulin embedding rate of more than 90%; the oral insulin compound preparation containing the insulin liposome lyophilized powder has good stability and is preserved for 24 months. After that, the insulin content in the oral insulin compound preparation remained above 99%, and the embedding rate of insulin liposome was only reduced by about 2% compared with 0 months, indicating that it was embedded in the liposome. The situation of insulin leakage is not significant; the oral insulin complex preparation is convenient for industrial production due to high embedding rate and good stability. In the process of encapsulation of insulin liposome, anhydrous ethanol was used as the solvent. Compared with diethyl ether and chloroform, the toxic side effect was small, and the binding of the envelope to the insulin solution was strong.

Claims

Rights request

1. An insulin liposome lyophilized powder characterized by the following steps:

2) Mix a mixture of cholesterol and lecithin with a mass ratio of 1: (1-3) in absolute ethanol, dissolve well and mix well, so that the concentration of the mixture is 0.018-0.025 g/ml, plus the concentration is 0.01-0.02g/ml aqueous solution of mannitol, the concentration of the mixture of cholesterol and lecithin is 0.011-0.013g/ml, and then the sodium cholate solution is added at a concentration of 0.01-0.02g/ml to make a mixture of cholesterol and lecithin. The concentration reaches 0.011-0.012g/ml, and the obtained liquid is placed in a high-pressure homogenizer for 10-30 minutes to obtain an oil phase mixture;

2. The insulin liposome lyophilized powder according to claim 1, which is prepared according to the following steps:

3) Mix the aqueous phase mixture and the oil phase in a volume ratio of 2.7:1 in a container and place it in a water bath at 2-6 ° C for 2-4 hours, then evaporate under reduced pressure to remove anhydrous ethanol. Obtaining a concentrate;

3. An oral insulin composite preparation for treating type II diabetes, comprising: the following components by weight: the insulin liposome lyophilized powder according to claim 1 or 2: 1 part;

Pueraria: 4-13 servings;

Trichosanthin: 9-29 servings;

Rehmannia: 5-15 servings; Ginseng: 1-3 servings;

Astragalus: 3-9 servings;

Yam: 3-10 servings.

The oral insulin complex preparation for treating type II diabetes according to claim 3, which comprises the following components in terms of weight:

The insulin liposome lyophilized powder according to claim 1 or 2: 1 part;

8 servings;

Trichosanthin: 16 servings;

Rehmannia: 10 servings;

Ginseng: 2 servings;

Huang Wei: 6 servings;

Yam: 6 servings.

A method for preparing an oral insulin compound preparation for treating type II diabetes according to claim 3, which comprises the steps of: weighing the components of the oral insulin compound preparation in proportion and mixing them uniformly, pulverizing 80 mesh sieve, wet granulation, dry granules were dried at 60 70 °C, dry granules were sifted with 20 mesh sieves, and filled into capsules.

6. A method for preparing an oral insulin complex preparation for treating type II diabetes according to claim 4, comprising the steps of: weighing the components of the oral insulin complex preparation in proportion and mixing them uniformly, pulverizing 80 mesh sieve, wet granulation, dry granules were dried at 60 70 °C, dry granules were sifted with 20 mesh sieves, and filled into capsules.

The use of the insulin liposome lyophilized powder according to claim 1 or 2 for the preparation of a medicament for treating type II diabetes.

The use of the oral insulin complex preparation according to claim 3 for the preparation of a medicament for treating type II diabetes.

9. The use of an oral insulin complex preparation according to claim 4 for the preparation of a medicament for treating type II diabetes