阅读说明：本技术 一种药物组合物及用于制备改善胰岛素抵抗、降血糖的药物的用途 (Pharmaceutical composition and application thereof in preparing medicines for improving insulin resistance and reducing blood sugar ) 是由 杨华 李萍 郑祖国 余玲玲 高雯 王可可 于 2020-12-31 设计创作，主要内容包括：本发明公开了一种药物组合物及用于制备改善胰岛素抵抗、降血糖的药物的用途。本发明发现,质量比为3:1的盐酸小檗碱和异甘草素具有协同降血糖、协同改善胰岛素抵抗的活性,实现1+1＞2的效果,二者形成的组合物可以用于制备降血糖、改善胰岛素抵抗的药物。(The invention discloses a pharmaceutical composition and application thereof in preparing a medicament for improving insulin resistance and reducing blood sugar. The invention discovers that berberine hydrochloride and isoliquiritigenin with the mass ratio of 3:1 have the activity of synergistically reducing blood sugar and synergistically improving insulin resistance, and the effect that 1+1 is more than 2 is realized, and the composition formed by the berberine hydrochloride and the isoliquiritigenin can be used for preparing the medicines for reducing blood sugar and improving insulin resistance.)

1. A pharmaceutical composition characterized by: consists of berberine hydrochloride and isoliquiritigenin.

2. The pharmaceutical composition of claim 1, wherein: the mass ratio of the two is 3: 1.

3. Use of the pharmaceutical composition of claim 1 or 2 for the preparation of a medicament for improving insulin resistance.

4. Use of the pharmaceutical composition of claim 1 or 2 for the preparation of a hypoglycemic medicament.

Technical Field

The invention belongs to the field of medicines, relates to a pharmaceutical composition and medical application thereof, and particularly relates to a pharmaceutical composition and application thereof in preparing medicines for improving insulin resistance and reducing blood sugar.

Background

Insulin Resistance (IR) refers to the condition where the biological effect of a target tissue is lower than that of a healthy person under the action of a normal dose of insulin, and is also called "decreased insulin sensitivity". IR is one of the main pathophysiological features of type 2 diabetes (T2DM), and is widely present in metabolic diseases such as obesity, non-alcoholic fatty liver disease (NAFLD), hypertension, hyperlipidemia, polycystic ovary syndrome (PCOS), and the like. Insulin mainly acts on peripheral tissues (muscle and fat) and liver, and can promote the uptake of skeletal muscle and fat tissues and utilize glucose at the periphery; it can promote glycogen synthesis in liver and inhibit hepatic gluconeogenesis. Epidemiological studies have shown that more than 80% of patients with T2DM have IR (Xiaojuan et al, progress in the study of insulin resistance and insulin signaling pathway abnormalities, Vol. 36, No. 21, 11/2020 in modern medicine and health).

Berberine and isoliquiritigenin are two very common natural products, and both have various biological activities. Research such as congratulation and the like finds that the berberine can improve insulin resistance (the research progress of action mechanism of the berberine for treating type 2 diabetes, the research of traditional Chinese medicine, 12 th year 2020). Leeyoungmi et al found that Isoliquiritigenin also had the effect of improving Insulin Resistance (Diareal Isoliquiritigenin at a Low Dose Insulin Resistance and NAFLD in Diet-Induced Obesity in C57BL/6JMice. International Journal of molecular sciences.2018).

However, at present, no report that berberine and isoliquiritigenin have synergistic effect on improving insulin resistance exists.

Disclosure of Invention

The invention aims to provide a pharmaceutical composition and application thereof in preparing medicines for improving insulin resistance and reducing blood sugar.

The above purpose of the invention is realized by the following technical scheme:

a pharmaceutical composition comprises berberine hydrochloride and isoliquiritigenin.

Preferably, the mass ratio of the berberine hydrochloride to the isoliquiritigenin is 3: 1.

The application of the pharmaceutical composition in preparing the medicine for improving insulin resistance.

The application of the pharmaceutical composition in preparing the hypoglycemic drug.

Has the advantages that:

the invention constructs a disease model of high sugar and insulin resistance based on high fat diet commonly used in the field, and finds that the composition of berberine hydrochloride and isoliquiritigenin (the mass ratio is 3:1) can effectively reduce blood sugar and improve insulin resistance. As known to those skilled in the art, on the premise that the berberine hydrochloride or isoliquiritigenin monomer is consistent with the total administration dose of the composition, the drug effect of the berberine hydrochloride and isoliquiritigenin composition (the mass ratio is 3:1) is obviously superior to that of the berberine hydrochloride or isoliquiritigenin monomer, which indicates that the berberine hydrochloride and the isoliquiritigenin necessarily have synergistic effect; otherwise, the potency of the composition should be between the potency of the two monomers (additive), or greater than the potency of each of the two monomers (antagonistic). Therefore, the berberine hydrochloride and the isoliquiritigenin with the mass ratio of 3:1 have the activity of synergistically reducing the blood sugar and synergistically improving the insulin resistance, the effect that 1+1 is more than 2 is realized, and the composition formed by the berberine hydrochloride and the isoliquiritigenin can be used for preparing the medicine for reducing the blood sugar and improving the insulin resistance.

Drawings

Figure 1 shows the body weight and diet values of the mice in each group.

FIG. 2 is a graph of fasting blood glucose values for each group of mice; note: ns represents no significant difference, p <0.05, p <0.01, p <0.001, p < 0.0001.

FIG. 3 is a glucose tolerance experiment in mice; note: ns represents no significant difference, p <0.05, p <0.01, p <0.001, p < 0.0001.

FIG. 4 is a mouse insulin resistance experiment; note: ns represents no significant difference, p <0.05, p <0.01, p <0.001, p < 0.0001.

Detailed Description

The following detailed description of the present invention is provided in connection with the accompanying drawings and examples, but not intended to limit the scope of the invention.

First, experimental material

1. Laboratory animal

Male C57BL/6J mice, 7-8 weeks old, weigh approximately 18-22g, source: zhejiang vitamin Tonglihua laboratory animal technology Co., Ltd. (SCXK (Zhe) 2019-.

2. Experimental reagent

Berberine hydrochloride (Dowman Stent Biotech Co., Ltd., product No. A0151, purity not less than 98%, Lot # MUST-20073011); isoliquiritigenin (Dowman ster Biotech Co., Ltd., product No. A0463, purity not less than 98%, Lot # MUST-20031903); metformin (MCE corporation, cat # HY-17471A, Lot # 29902); D-Glucose (Amresco, USA, Cat. No. 0188-1Kg, Lot #3277C 100); bovine insulin powder (Biosharp, cat # BS901, Lot #69116301), sodium chloride injection (agrimony). High fat diet (us Research Diets, cat # D12492, fat content 60%).

3. Laboratory apparatus

Ohm dragon blood sugar meter (HGM-114) and matched test paper (AS1)

Analytical balance (sartorius)

Electronic scale (Long xi electronic)

Constant temperature water bath (Kun mountain He Chuang)

Vortex instrument (the forest bell instrument)

Second, Experimental methods

1. Solution preparation

(1) Preparing animal medicines:

weighing berberine hydrochloride 180mg, isoliquiritigenin 180mg, metformin 600mg, berberine hydrochloride 135mg and isoliquiritigenin 45mg respectively, placing into 4 50mL centrifuge tubes, adding sodium chloride injection to constant volume to 30mL, vortex mixing, performing water bath ultrasound at 25 deg.C for 2 hr to obtain 6 mg/mL mixture^-1The berberine hydrochloride suspension or the isoliquiritigenin suspension is 20 mg/mL^-1Metformin solution and 6 mg. multidot.mL^-1The berberine hydrochloride/isoliquiritigenin mixed solution is stored at 4 ℃ and prepared once every two weeks.

(2) Preparing a glucose solution:

weighing 4g of D-glucose, dissolving in 20mL of pure water, vortexing to fully dissolve, and preparingTo a volume of 0.2 g/mL^-1For use in a mouse glucose tolerance test.

(3) Preparing an insulin mother solution:

weighing 1mg bovine insulin powder, dissolving in 1mL pure water, vortex mixing, performing ultrasonic treatment in ice water bath for about 10min to obtain 1 mg/mL^-1The insulin mother liquor is subpackaged and stored at the temperature of minus 20 ℃ for standby.

(4) Preparing an insulin use solution:

before use, take 1 mg. mL^-1Adding 21.56mL of sodium chloride injection into 40 μ L of insulin mother liquor, and diluting 540 times to obtain 0.05 u/mL^-1And (4) insulin diluent for mouse insulin tolerance test.

2. Animal feeding

The experimental mice are raised in the experimental animal center of Chinese pharmaceutical university, the environmental conditions are controlled, the 12-hour illumination/12-hour dark cycle is maintained, the temperature is 23 +/-3 ℃, the relative humidity is 55 +/-15%, food and water are freely obtained, and drinking water and padding are replaced every three days.

3. Animal grouping, modeling and administration

After the mice are adapted to the environment for one week, the mice are divided into 6 groups according to the weight of the mice, and each group contains 8-14 mice. The six groups are respectively as follows: blank control (ND); a set of models (HFD); metformin 200 mg/kg^-1Administration group (Met); berberine hydrochloride 60 mg/kg^-1Administration group (BBR); isoliquiritigenin 60 mg/kg^-1Administration group (ISL); berberine hydrochloride/isoliquiritigenin 60 mg/kg^-1Combination group (B + I (3: 1)). The diet and dosing schedule for the six groups were as follows:

and ND group: feeding with common feed, and feeding with normal water, wherein the sodium chloride injection is administered every morning.

HFD group: feeding with high fat feed, drinking water normally, and infusing sodium chloride injection every morning.

BBR group: feeding high-fat feed, normally drinking water, and administering berberine hydrochloride suspension in a dose of 60mg kg in the morning^-1。

ISL group: feeding high-fat fodder, normally drinking water, and irrigating isoliquiritigenin suspension daily in morning at a dose of 60mg kg^-1。

Group B + I (3: 1): feeding high-fat fodder, normally drinking water, and feeding berberine hydrochloride/isoliquiritigenin mixed solution with dosage of 60mg kg in morning^-1。

Body weight, food intake of each group of mice was recorded weekly during dosing and used as a later data statistic.

4. Fasting Blood Glucose (FBG) assay in mice

Before the experiment, the mice are fasted for about 15 hours and normally drunk, 4-14 mice are randomly selected from each group for detection, before the detection, each mouse is marked by a marker, a tail tip of the mouse is cut by scissors for about 1-2mm, the mouse is gently stroked towards the tail tip from the proximal end by hands, a blood drop of a glucometer inserted with test paper in advance and close to the tail tip is made, the test paper automatically absorbs the blood drop, the reading is displayed, and the fasting blood glucose of each mouse is recorded. The detection process is kept quiet.

5. Mouse oral glucose tolerance (OGTT) assay

The mice are fasted for about 15 hours before the experiment, the mice are normally drunk, 6 mice are randomly selected from each group for detection, and each mouse is marked and weighed before the detection. Fasting blood glucose was first determined (as described above) and recorded for each mouse. Then use 0.2 g.mL^-1The glucose solution is used for intragastric administration, and the dosage is 2 g.kg^-1Timing is started after the first mouse is filled with the stomach, all mice are filled at a constant speed for about 20 s/mouse, and the blood glucose value of each mouse is measured by a blood glucose meter at time points of 15 min, 30 min, 60 min, 90 min and 120min after the timing is started, so that the accuracy of the time points is ensured, and the recording is carried out. The detection process is kept quiet.

6. Mouse insulin tolerance assay

Before the experiment, the mice are fasted for about 4 hours, water is normally drunk, 6-9 mice are randomly selected from each group for detection, and each mouse is marked and weighed before the detection. Fasting blood glucose was first determined (as described above) and recorded for each mouse. Then use 0.05 u.mL^-1The insulin solution is injected into the abdominal cavity, and the dosage is 0.5 u.kg^-1Timing is started after the injection of the first mouse is finished, all mice are injected at a constant speed of about 20 s/mouse, and the blood glucose meter is used for measuring the time interval of the injection of each mouse at 15, 30, 60, 90 and 120min time points after the timing is startedThe blood sugar value ensures the accuracy of the time point and records. The detection process is kept quiet.

7. Data processing

All plots and statistics were performed by GraphPad prism8.0 and data are expressed as mean Standard Error (SE). Statistical analysis by t-test, differences of p <0.05 were considered to have statistical significance (.;, p < 0.05;. p, p < 0.01;. p, p < 0.001;. p, 0.0001; ns, no significance).

Third, experimental results

1. Body weight and food intake of mice

As shown in fig. 1, the amount of food intake after the mice were high-fat diet was significantly lower than that of the normal group, but the body weight of the mice after the high-fat diet was higher than that of the normal group; compared with the high-fat model group, the single administration and the combined administration have no obvious influence on the body weight and the food consumption of the mice.

2. Fasting blood sugar of mouse

As shown in table 1 and fig. 2, it can be seen from table 1 and fig. 2 that fasting blood glucose can be significantly increased after mice are taken for 4 weeks and 6 weeks after high fat diet, and metformin can significantly reduce fasting blood glucose, BBR and ISL have no significant efficacy at week 4, but fasting blood glucose can be significantly reduced at week 6, and the combination group can significantly reduce fasting blood glucose at week 4, and the effect of reducing fasting blood glucose at week 6 is more significant than that of the single-dose group.

TABLE 1

3. Oral glucose tolerance in mice

As shown in table 2 and fig. 3, it can be seen from table 2 and fig. 3 that after the mice had been taken for 6 weeks with high fat, the OGTT-AUC in the model group was significantly increased, i.e., the glucose tolerance was significantly decreased, metformin and the single-dose group and the combined-dose group were able to significantly improve the glucose tolerance, and the effect was the best in the 3:1 group, and the drug effect in the combined-dose group was significantly better than that in the single-dose group.

TABLE 2

4. Insulin tolerance in mice

As shown in table 3 and fig. 4, it can be seen from table 3 and fig. 4 that after the mice are on high fat diet for 6 weeks, the ITT-AUC in the model group is significantly increased, i.e. the insulin tolerance is significantly decreased, metformin and the single administration group and the combined administration group can significantly improve the glucose tolerance, and the effect is best in the 3:1 group, and the drug effect of the combined administration group is significantly better than that of the single administration group.

TABLE 3

In conclusion, the invention constructs a disease model of high glucose and insulin resistance based on high fat diet commonly used in the field, and finds that the composition of berberine hydrochloride and isoliquiritigenin (mass ratio is 3:1) can effectively reduce blood sugar and improve insulin resistance. As known to those skilled in the art, on the premise that the berberine hydrochloride or isoliquiritigenin monomer is consistent with the total administration dose of the composition, the drug effect of the berberine hydrochloride and isoliquiritigenin composition (the mass ratio is 3:1) is obviously superior to that of the berberine hydrochloride or isoliquiritigenin monomer, which indicates that the berberine hydrochloride and the isoliquiritigenin necessarily have synergistic effect; otherwise, the potency of the composition should be between the potency of the two monomers (additive), or greater than the potency of each of the two monomers (antagonistic). Therefore, the berberine hydrochloride and the isoliquiritigenin with the mass ratio of 3:1 have the activity of synergistically reducing the blood sugar and synergistically improving the insulin resistance, the effect that 1+1 is more than 2 is realized, and the composition formed by the berberine hydrochloride and the isoliquiritigenin can be used for preparing the medicine for reducing the blood sugar and improving the insulin resistance.

The above-described embodiments are intended to be illustrative of the nature of the invention, but those skilled in the art will recognize that the scope of the invention is not limited to the specific embodiments.