'Hot-ground black jade' bitter gourd micro powder, extract and application thereof in preparation of hypoglycemic drugs

Abstract

The invention provides'hot-ground black jade 'bitter gourd micro powder, extract and application thereof in preparation of hypoglycemic drugs, and belongs to the technical field of biological medicines. The bitter gourd micro powder and extract prepared by the invention have efficient and lasting hypoglycemic effect, can keep good hypoglycemic effect after being orally taken, can reduce the level of total cholesterol, triglyceride and / or low-density lipoprotein in blood, and can relieve the lesion degree of liver, kidney and pancreas tissues of mice. The bitter gourd micro-powder and the bitter gourd extract do not change the body weight increase trend of type II diabetes db / db mice and have no obvious influence trend on the water intake and the food intake, and only the urine volume reduction can be observed from the appearance, so that the bitter gourd micro-powder and the bitter gourd extract prepared by the invention have small side effects, and the bitter gourd micro-powder and the bitter gourd extract can be used for treating diabetes mellitus type II diabetes mellitus type II diabetes mellitus type II diabetes mellitus type II diabetes mellitus type II mice. And the application has huge potential in preparation of novel diabetes medicines.

Description

Technical Field

[0001] The invention relates to the technical field of biomedicine, and in particular to "Reyan Black Jade" bitter melon micropowder, extract and application thereof in preparing hypoglycemic drugs. Background Art

[0002] Diabetes is a multifactorial chronic metabolic disease characterized by carbohydrate metabolism disorders. Its incidence rate is increasing year by year, causing serious impacts on individuals, social health and economic sectors. Diabetes is mainly divided into type I diabetes (insulin-dependent T1DM) and type II diabetes (non-insulin-dependent T2DM). T1DM is caused by genetic diseases or the inability of the pancreatic β cells to produce enough insulin. T2DM is caused by the combined effects of insulin receptor defects (insulin resistance) and insufficient insulin secretion (impaired β cell function), accounting for 90% of all diabetic patients. T2DM is closely related to obesity. The incidence and severity of diabetes are significantly increased in obese people, and may be further accompanied by macrovascular and microvascular complications. These complications may increase other diseases and cause varying degrees of damage to organs, such as acute complications (diabetic ketoacidosis, hypoglycemia, hyperosmolar hyperglycemic non-ketotic syndrome, lactic acidosis, etc.), microvascular (retinopathy, nephropathy, neuropathy) and macrovascular (cerebrovascular, cardiovascular, peripheral arterial disease) complications. T1DM is generally treated with insulin replacement therapy, while T2DM is treated with oral hypoglycemic drugs. The main drug treatments for T2DM include α-glucosidase inhibitors, sulfonylureas, glinides, biguanides, dipeptidyl peptidase-IV (DPP-IV) inhibitors, glucagon-like peptide-1 (GLP-1) receptor agonists, thiazolidinediones, and sodium-glucose co-transporter-2 (SGLT2) inhibitors. However, although general drugs for the treatment of diabetes have obvious therapeutic benefits, they are accompanied by significant side effects and even aggravate the condition. Therefore, it is particularly necessary to develop biological drugs for the treatment of diabetes.

[0003] Bitter melon is not only a vegetable, but also a medicine. It is deeply loved by people because it contains total bitter melon saponins, bitter melon plant protein powder, fatty acids, multiple vitamins, trace elements, etc. The use of bitter melon as a medicine was recorded as early as in the Compendium of Materia Medica, but the research on extracting effective ingredients for the treatment of diabetes began in the 1960s and 1970s. However, the hypoglycemic effect of bitter melon varies greatly due to different germplasms and preparation processes. Therefore, finding a bitter melon germplasm with good hypoglycemic effect and a new preparation process is of great significance for the development of new diabetes drugs. Summary of the invention

[0004] The purpose of the present invention is to provide "reyan black jade" bitter melon powder, extract and application thereof in the preparation of hypoglycemic drugs, so as to solve the problem of large side effects of chemical drugs in treating diabetes.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The invention provides a bitter melon micro powder, which is prepared by the following method: slicing the bitter melon and drying it, crushing it into powder, and passing it through a 100-120 mesh sieve to obtain the bitter melon micro powder.

[0007] The present invention also provides a bitter melon extract, which is prepared by the following method: S1. After drying bitter melon slices, crushing into powder, collecting bitter melon coarse powder with a sieve of >14 mesh;

[0008] S2. Mix the bitter melon coarse powder with water, perform ultrasonic extraction, collect the filtrate, and evaporate to dryness in a water bath to obtain the bitter melon extract.

[0009] The present invention also provides the use of bitter melon, the bitter melon micropowder and / or the bitter melon extract in preparing hypoglycemic drugs.

[0010] The present invention also provides the use of bitter melon, the bitter melon micropowder and / or the bitter melon extract in preparing a medicine for reducing the level of glycosylated hemoglobin.

[0011] The present invention also provides the use of bitter melon, the bitter melon micropowder and / or the bitter melon extract in preparing a medicine for reducing the level of total cholesterol, triglyceride and / or low-density lipoprotein in blood.

[0012] Preferably, the variety of bitter melon is "Reyan Black Jade".

[0013] The invention also provides a method for reducing glycosylated hemoglobin in the blood of mice, which comprises feeding the mice with the bitter melon micropowder or the bitter melon extract.

[0014] Preferably, the feeding dose of the bitter melon powder is 1.4-1.8 g / kg, more preferably 1.5-1.7 g / kg, more preferably 1.6 g / kg; the feeding dose of the bitter melon extract is 0.3-0.5 g / kg, more preferably 0.35-0.45 g / kg, more preferably 0.4 g / kg.

[0015] By adopting the above technical scheme, the present invention has the following beneficial effects: the bitter melon powder and bitter melon extract prepared by the present invention have efficient and lasting hypoglycemic effect, can maintain good hypoglycemic effect after oral administration, can reduce the total cholesterol, triglyceride, and / or low-density lipoprotein levels in the blood, and can reduce the degree of liver, kidney, and pancreatic tissue lesions in mice. The bitter melon powder and bitter melon extract of the present invention do not change the weight growth trend of type II diabetic db / db mice, lack a significant trend in the effect on water intake and food intake, and only a decrease in urine volume can be observed from the appearance, indicating that the bitter melon powder and bitter melon extract prepared by the present invention have little side effects and have great potential in the preparation of new diabetes drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The effect of bitter melon powder and bitter melon extract on blood sugar in mice. The left side shows the effect of bitter melon powder on blood sugar in mice, and the right side shows the effect of bitter melon extract on blood sugar in mice (Note: compared with the normal group and the diabetic model group, ##: p<0.01; compared with the drug administration group and the diabetic model group, *: p<0.05,

[0017] **:p<0.01).

[0018] Figure 2 This is the effect of bitter melon powder and bitter melon extract on the glycated hemoglobin level in mice (Note: compared with the normal group and the diabetic model group, ##: p<0.01; compared with the drug-treated group and the diabetic model group, *: p<0.05, **: p<0.01).

[0019] Figure 3 The figure shows the effects of bitter melon powder and bitter melon extract on the levels of total cholesterol (CHO), triglycerides (TD), high-density lipoprotein (HDL) and low-density lipoprotein (LDL) in mice (Note: compared with the normal group and the diabetic model group, ##: p<0.01; compared with the drug-treated group and the diabetic model group, *: p<0.05, **: p<0.01).

[0020] Figure 4 The effect of bitter melon powder and bitter melon extract on the pathological changes of liver tissue in mice (observed under 40× eyepiece).

[0021] Figure 5 The effect of bitter melon powder and bitter melon extract on the pathological changes of mouse kidney tissue (observed under 40× eyepiece).

[0022] Figure 6 The effect of bitter melon powder and bitter melon extract on the pathological changes of pancreatic tissue in mice (observed under 40× eyepiece). DETAILED DESCRIPTION

[0023] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0024] Metformin hydrochloride tablets (Glucophage ): Shanghai Bristol-Myers Squibb Pharmaceuticals Co., Ltd., batch number: AAW6138;

[0025] Cholesterol assay kit: Biosino Biotechnology Co., Ltd., batch number: 191651;

[0026] Triglyceride assay kit: Biosino Biotech Co., Ltd., batch number: 191031;

[0027] Low-density lipoprotein (LDL-C) test kit: Nanjing Jiancheng Bioengineering Institute, batch number: 20190613;

[0028] High-density lipoprotein (HDL-C) test kit: Nanjing Jiancheng Bioengineering Institute, batch number: 20190613;

[0029] Glycogen Assay Kit: SIGMA-ALDRICH, Lot#5E08K06460;

[0030] Mouse Ins / insulin Elisa Kit: SIGMA-ALDRICH, Lot#0528F0534;

[0031] A1CNow+ Glycated Hemoglobin Detector: Beijing Anpu Biochemical Technology Co., Ltd.;

[0032] Blood glucose meter: Roche Accu-ChekActive, with test strips, batch number: 24695431;

[0033] SpectraMax 190 full-wavelength microplate reader: Meigu Molecular Instruments (Shanghai) Co., Ltd.;

[0034] SpectraMax Gemini XPS fluorescence microplate reader: Meigu Molecular Instruments (Shanghai) Co., Ltd.

[0035] Homozygous BKS-DB mice and wild-type BKS-DB mice were provided by Jiangsu Jicui Yaokang Biotechnology Co., Ltd. (Experimental Animal Certificate No.: 201902730, License No.: SCXK(Su)2018-0008).

[0036] The seeds of 21 bitter melon germplasms were analyzed for their in vitro hypoglycemic activity. The inhibitory activity of different samples on glucosidase was tested to preliminarily determine the hypoglycemic level. The bitter melon material '9-18' (Reyan Black Jade) with excellent comprehensive activity was selected for further in vivo hypoglycemic activity test.

[0037] Example 1. Preparation of "hot-milled black jade" bitter melon powder

[0038] The bitter melon slices are dried, crushed into powder, and passed through a 100-120 mesh pharmacopoeia sieve to obtain bitter melon fine powder.

[0039] Example 2. Preparation of "Reyan Heiyu" bitter melon extract

[0040] After drying the bitter melon slices, crush them into powder, and collect the coarse powder with a pharmacopoeia sieve of >14 mesh. Weigh a certain amount of bitter melon coarse powder, add 20 times the volume of hot water (50℃) and ultrasonically extract for 1h, filter and collect the filtrate, repeat the extraction 3 times, combine the filtrates of 3 times, evaporate to dryness in a water bath, and obtain the bitter melon water extract.

[0041] Example 3. Analysis of hypoglycemic activity of "Reyan Heiyu" bitter melon in vivo

[0042] 83 6-week-old male homozygous BKS-DB mice (hereinafter referred to as db / db mice) and 11 6-week-old wild-type BKS-DB mice were adaptively raised for 7 days. After fasting for 8 hours, the tail vein terminal blood of the mice was collected and the fasting blood glucose was measured with a blood glucose meter. The db / db mice with fasting blood glucose values ​​>10.0mmol / L were considered to have formed a diabetic hyperglycemia model. The diabetic model mice were randomly divided into 8 groups with 10 mice in each group: model group, 3 bitter melon micropowder dose groups, 3 bitter melon extract dose groups, and positive control drug group. Each group of mice was placed in the same breeding cage. When the mice were 8 weeks old, the corresponding drugs were given to each group of mice by gavage:

[0043] Bitter melon powder 0.4 (low dose), 0.8 (medium dose), 1.6 g / kg (high dose);

[0044] Bitter melon extract 0.1 (low dose), 0.2 (medium dose), 0.4 g / kg (high dose);

[0045] The positive control drug was metformin 0.2 g / kg.

[0046] Ten wild-type mice were selected as the normal control group and given drinking water in equal proportion by gavage as in the model group for 9 consecutive weeks.

[0047] The glucose and glycosylated hemoglobin levels in the blood of each group of mice were measured weekly. The results are shown in Table 1 and Figure 1 shown.

[0048] Table 1 Effects of the tested samples on blood glucose in mice

[0049]

[0050] The results showed that the significant hypoglycemic effect of bitter melon extract and powder can last from the second week of intervention to the ninth week. In the ninth week of treatment, the blood glucose level of mice in the model group was 35.0mmol / L, the blood glucose level of mice in the high-dose group of bitter melon powder was 25.8mmol / L, the blood glucose level of mice in the high-dose group of bitter melon extract was 30.7mmol / L, and the blood glucose level of mice in the positive drug group was 23.1. Compared with the model group, the blood glucose reduction levels reached 26.3%, 12.3% and 34% respectively. The blood glucose reduction activity of bitter melon powder was significantly higher than that of the extract.

[0051] Glycated hemoglobin (HbA1c) is formed when hemoglobin in red blood cells is wrapped by blood glucose. The amount of glucose bound to hemoglobin is proportional to the total glucose content in the blood system, so the higher the blood glucose level, the higher the HbA1c level. HbA1c can reflect the average blood glucose level over a period of about 2 to 3 months. Figure 2 The glycated hemoglobin (HbA1c) levels of mice in each group after 9 weeks of drug intervention were measured by Figure 2 It can be seen that the level of glycated hemoglobin (HbA1c) in diabetic model mice was significantly increased, and both metformin and bitter melon test samples could significantly reduce the level of glycated hemoglobin (HbA1c). The amount of glycated hemoglobin is proportional to the total glucose content in the blood. The high-dose group of bitter melon micropowder, the high-dose group of bitter melon extract, and the positive drug group can significantly reduce the level of glycated hemoglobin in the blood of model mice, and compared with the model group, the reduction levels reached 25.8%, 24.6%, and 27.0%, respectively.

[0052] At the end of the drug intervention, mice in each group were weighed after fasting overnight, anesthetized with isoflurane, and blood was collected from the eye sockets with a capillary glass tube. Whole blood was taken to determine the content of glycosylated hemoglobin, and the remaining blood was centrifuged at 4000rpm to obtain serum and stored at -80°C for testing. The animals were then killed by dislocating the cervical vertebrae, and the liver, kidney, and pancreas tissues were fixed in 4% paraformaldehyde, and pathological sections were made and HE staining was used to observe pathological changes.

[0053] According to the instructions of the CHO, TG, HDL, LDL, glycogen and insulin kits, the levels of the above biochemical indicators in serum were determined.

[0054] The results are as follows Figure 3 , Figure 4 , Figure 5 and Figure 6As shown. The results showed that the high-dose group of bitter melon micropowder group, the high-dose group of bitter melon extract group, and the positive drug group could significantly reduce the level of triglyceride (TD) in the blood of model mice. Compared with the model group, the reduction levels were 37.6%, 36.7%, and 42.8%, respectively. The high-dose group of bitter melon micropowder group, the high-dose group of bitter melon extract group, and the positive drug group could significantly reduce the level of total cholesterol (CHO) in the blood of model mice. Compared with the model group, the reduction levels were 32.6%, 25.4%, and 23.8%, respectively. The high-dose group of bitter melon micropowder group, the high-dose group of bitter melon extract group, and the positive drug group could significantly reduce the level of low-density lipoprotein (LDL) in the blood of model mice. Compared with the model group, the reduction levels were 37.8%, 39.4%, and 43.6%, respectively, and had no significant effect on HDL-C. Figures 4 to 6 It can be seen that both bitter melon ultrafine powder and bitter melon extract can reduce the degree of liver, kidney and pancreatic tissue lesions in model mice in a dose-dependent manner. And from daily observations, bitter melon ultrafine powder and bitter melon extract cannot change the weight gain trend of db / db mice, and have no significant trend in the effect on water intake and food intake. Only the reduction in urine volume can be observed from the appearance.

[0055] As can be seen from the above examples, the present invention provides "reyan black jade" bitter melon powder, extract and its application in the preparation of hypoglycemic drugs. The bitter melon powder and extract of the present invention can effectively reduce blood sugar level, blood glycosylated hemoglobin content and blood total cholesterol (CHO), triglyceride (TD) and low-density lipoprotein (LDL) levels, and can reduce the degree of liver, kidney and pancreatic tissue lesions in mice.

[0056] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A bitter melon extract, characterized in that Prepared by the following method: S1. After the bitter melon slices were dried, they were crushed into powder, and the bitter melon coarse powder with a sieve of> 14 mesh was collected; S2. Mix the bitter melon coarse powder with water, perform ultrasonic extraction, collect the filtrate, and evaporate to dryness in a water bath to obtain the bitter melon extract.

2. The bitter melon extract according to claim 1, characterized in that The mass volume ratio of bitter melon coarse powder to water is 1g:18-22mL.

3. The bitter melon extract according to claim 2, characterized in that The temperature of the water is 48-52° C., and the extraction time is 50-70 minutes.

4. A bitter melon powder, characterized in that: The method is prepared by the following method: the bitter melon slices are dried, crushed into powder, and passed through a 100-120 mesh sieve to obtain bitter melon micro powder.

5. Use of bitter melon, the bitter melon extract according to claim 1 and / or the bitter melon powder according to claim 4 in the preparation of hypoglycemic drugs.

6. Use of bitter melon, the bitter melon extract according to claim 1 and / or the bitter melon powder according to claim 4 in the preparation of a medicament for reducing glycosylated hemoglobin levels.

7. Use of bitter melon, the bitter melon extract according to claim 1 and / or the bitter melon powder according to claim 4 in the preparation of a medicament for reducing the level of total cholesterol, triglycerides, and / or low-density lipoprotein in blood.

8. The use according to claim 5 or 6, characterized in that: The variety of bitter melon is "reyan black jade".

9. A method for reducing glycosylated hemoglobin in the blood of mice, characterized in that: Feeding mice with the bitter melon extract according to claim 1 or the bitter melon powder according to claim 4.

10. The method according to claim 9, characterized in that The feeding dosage of the bitter melon powder is 1.4-1.8 g / kg, and the feeding dosage of the bitter melon extract is 0.3-0.5 g / kg.

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