Plant extract composition with synergistic hypoglycemic activity and use thereof

Abstract

The present application relates to the technical field of plant extract, and provides a plant extract composition with synergistic hypoglycemic activity and application thereof, wherein the plant extract composition comprises mulberry leaf extract, white kidney bean extract and balsam pear powder with a mass ratio of 8:(1-24):(1-64). The present application optimizes the mass ratio of the mulberry leaf extract, the white kidney bean extract and the balsam pear powder, so that the plant extract with the mulberry leaf extract, the white kidney bean extract and the balsam pear powder as main raw materials has a significantly better effect on reducing blood sugar than the plant extract in the prior art. Meanwhile, the plant extract composition also has the effects of synergistically reducing the number of peripheral nerve neutrophils, improving peripheral nerve inflammation, synergistically enhancing the fluorescence intensity of peripheral nerves, improving peripheral nerve injury, and inhibiting the enzyme activity of bacteria-derived dipeptidyl peptidase-4.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of plant extracts, in particular to a plant extract composition with synergistic hypoglycemic activity and application thereof. BACKGROUND

[0002] Metabolic diseases are a class of diseases caused by disorders of metabolic processes, involving disorders of synthesis, decomposition, transformation or transport of substances in the body. Among them, high blood sugar related metabolic diseases are of great concern. At present, the methods for preventing and treating high blood sugar related metabolic diseases include adopting low-sugar, low-fat and high-fiber dietary habits, and taking hypoglycemic drugs, etc.

[0003] However, relying only on traditional dietary adjustment and chemical hypoglycemic drugs often has poor compliance, long-term side effects and significant individual differences. In recent years, more and more studies have shown that active extracts derived from natural plants (such as polyphenols, flavonoids, saponins, terpenes and alkaloids, etc.) play a multi-target, low-toxicity advantage in regulating glucose metabolism, improving insulin sensitivity, inhibiting gluconeogenesis, and reducing oxidative stress and chronic inflammation. For example, plant active substances such as puerarin and bitter gourd saponins have been widely used in functional foods and traditional Chinese medicine preparations, and their hypoglycemic effect and safety have been verified in clinical trials. These findings show that plant extracts, as an important part of food, health products and even drugs, are becoming one of the key strategies for preventing and intervening high blood sugar related metabolic diseases, and showing unique application value in the prevention, adjuvant therapy and even disease management of high blood sugar related metabolic diseases. SUMMARY

[0004] The present application provides a plant extract composition with synergistic hypoglycemic activity and application thereof, which uses mulberry leaf extract, white kidney bean extract and bitter gourd powder as raw materials to provide effective active ingredients for reducing blood sugar. In a specific ratio, the effect of reducing blood sugar can be significantly improved without toxic side effects.

[0005] Specifically, by reasonably compounding with other plant extracts with hypoglycemic or related regulating effects, it is found that when the mass ratio of mulberry leaf extract, white kidney bean extract and bitter gourd powder is 8:(1~24):(1~64), the hypoglycemic effect of the obtained composition is significantly better than that of other compounding ratios, which has very important technical significance for preparing hypoglycemic foods or drugs with better efficacy, more comprehensive and stable effects, and better compliance.

[0006] In a first aspect, the present application provides a plant extract composition comprising mulberry leaf extract, white kidney bean extract and bitter gourd powder in a mass ratio of 8:(1~24):(1~64).

[0007] According to the plant extract composition provided by the application, preferably, the mass ratio of mulberry leaf extract, white kidney bean extract and bitter gourd powder is 8: (1-8) : (1-22).

[0008] According to the plant extract composition provided by the application, preferably, the mass ratio of mulberry leaf extract, white kidney bean extract and bitter gourd powder is 8: (1-3) : (1-18).

[0009] According to the plant extract composition provided by the application, preferably, the mass ratio of mulberry leaf extract, white kidney bean extract and bitter gourd powder is 8: (1-3) : (1-8).

[0010] According to the plant extract composition provided by the application, preferably, the mass ratio of mulberry leaf extract, white kidney bean extract and bitter gourd powder is 8:3: (1-8).

[0011] According to the plant extract composition provided by the application, preferably, the mass ratio of mulberry leaf extract, white kidney bean extract and bitter gourd powder is 8: (1-3) : (3-4).

[0012] According to the plant extract composition provided by the application, preferably, the mass ratio of mulberry leaf extract, white kidney bean extract and bitter gourd powder is 8:3: (3-8).

[0013] 1-Deoxynojirimycin (1-DNJ) is a polyhydroxy piperidine alkaloid existing in mulberry trees, has a structure similar to that of alpha-1, 4-glucose, is an alpha-glucosidase inhibitor, has hypoglycemic function, antitumor and antiviral activity, and is an index component in mulberry leaf extract.

[0014] Phaseolin in white kidney bean extract is a natural alpha-amylase inhibitor, which is a glycoprotein in chemical nature, can effectively inhibit the decomposition of starch, and make it discharged outside the gastrointestinal tract. In addition, due to the large molecular weight, phaseolin does not enter the blood circulation system and does not act on the central nervous system, so that the appetite is not inhibited while losing weight, and there is no side effect even when the dosage is very high, and it has good safety. The alpha-amylase inhibitor activity is used as the index component of the white kidney bean extract in the application.

[0015] Saponins in bitter gourd are a kind of glycosides with triterpenes or steroids as aglycone, which are composed of sugar and aglycone. They can not only lower the blood glucose level of the body by regulating the uptake of glucose by body cells, but also relieve inflammation through various pathways. The bioactive components in bitter gourd include saponins, proteins, polysaccharides, polyphenols, sterols, etc. The total saponins are used as the index component of the bitter gourd powder in the application.

[0016] The 1-deoxynojirimycin content of the mulberry leaf extract is greater than or equal to 1.0 g / 100 g;

[0017] Alternatively, the alpha-amylase inhibitor activity of the white kidney bean extract is greater than or equal to 1 x 10 5 U / g;

[0018] Alternatively, the total saponin content of the bitter gourd powder is greater than or equal to 1.5% w / w.

[0019] The plant extract composition provided by the present application has a mulberry leaf extract with a 1-deoxynojirimycin content greater than or equal to 1.0 g / 100 g, a white kidney bean extract with an alpha-amylase inhibitor activity greater than or equal to 1 x 10 5 U / g, and a bitter gourd powder with a total saponin content greater than or equal to 1.5% w / w.

[0020] The present application has found, through a large number of experiments, that the mulberry leaf extract with a 1-deoxynojirimycin content greater than or equal to 1.0 g / 100 g, the white kidney bean extract with an alpha-amylase inhibitor activity greater than or equal to 1 x 10 5 U / g, and the bitter gourd powder with a total saponin content greater than or equal to 1.5% w / w, play a key role in ensuring the hypoglycemic effect and the improvement of peripheral nerve inflammation and peripheral nerve damage of the above-mentioned plant extract composition.

[0021] The plant extract composition provided by the present application has a mulberry leaf extract prepared by water extraction, a white kidney bean extract prepared by water extraction, and a bitter gourd powder prepared by water extraction.

[0022] In a second aspect, the present application also provides a preparation method of the plant extract composition as described above, which can be prepared by any suitable physical mixing method, which includes the step of mixing the mulberry leaf extract, the white kidney bean extract, and the bitter gourd powder. The order of mixing between the mulberry leaf extract, the white kidney bean extract, and the bitter gourd powder is not particularly specified, as long as they can be uniformly mixed. The mass ratio of the mulberry leaf extract, the white kidney bean extract, and the bitter gourd powder in the final mixture is 8:(1-24):(1-64).

[0023] In a third aspect, the present application also provides a plant extract composition for lowering blood sugar, which includes the plant extract composition as described above, or the plant extract composition prepared by the preparation method as described above, and the active ingredient for lowering blood sugar is derived from the mulberry leaf extract, the white kidney bean extract, and the bitter gourd powder.

[0024] In a fourth aspect, the present application also provides use of the plant extract composition as described above in maintaining healthy blood glucose level, improving diabetic complications, delaying progression of diabetes, or treating or preventing high blood glucose related metabolic diseases in a subject.

[0025] The use comprises administering the plant extract composition to the subject in an effective amount. The subject can be a human. The effective amount can range from 500 to 12000 mg per day, such as 500 mg per day, 550 mg per day, 600 mg per day, 650 mg per day, 700 mg per day, 750 mg per day, 800 mg per day, 850 mg per day, 900 mg per day, 950 mg per day, 1 g per day, 1.5 g per day, 2 g per day, 2.5 g per day, 3 g per day, 3.5 g per day, 4 g per day, 4.5 g per day, 5 g per day, 5.5 g per day, 6 g per day, 6.5 g per day, 7 g per day, 7.5 g per day, 8 g per day, 8.5 g per day, 9 g per day, 9.5 g per day, 10 g per day, 10.5 g per day, 11 g per day, 11.5 g per day, or 12 g per day.

[0026] In a fifth aspect, the present application also provides use of the plant extract composition for lowering blood glucose as described above in the preparation of a food product for maintaining healthy blood glucose level, improving diabetic complications, or delaying progression of diabetes.

[0027] In a sixth aspect, the present application also provides a food product composition comprising the plant extract composition for lowering blood glucose as described above; the food product composition is for maintaining healthy blood glucose level, improving diabetic complications, or delaying progression of diabetes.

[0028] According to the present application, the food product composition can be an oral liquid, a tea drink, a lozenge, a capsule, a drink, or an effervescent tablet.

[0029] The food product composition in the present application can also be a functional health product suitable for a subject.

[0030] The subject can be a mammal, preferably a human. The administration can be performed orally, enterally, by injection, by spraying, by physical or chemical mediation.

[0031] In a seventh aspect, the present application also provides use of the plant extract composition for lowering blood glucose as described above in the preparation of a medicament for treating and / or preventing high blood glucose related metabolic diseases.

[0032] According to the application, the high blood sugar related metabolic disease is selected from one or more of diabetes, diabetic neuropathy, diabetic nephropathy, diabetic retinopathy, diabetic cardiomyopathy, diabetic gangrene, diabetic cachexia, diabetic dyslipidemia, diabetic coronary heart disease, diabetic cerebrovascular disease, and diabetic peripheral vascular disease.

[0033] In an eighth aspect, the present application provides a pharmaceutical composition comprising the plant extract composition for lowering blood sugar as described above; the pharmaceutical composition is used for treating and / or preventing a high blood sugar related metabolic disease.

[0034] According to the pharmaceutical composition of the present application, the pharmaceutical composition is an injection, a tablet, a powder, a granule, a pill, a capsule, an oral liquid, a paste, a cream, or a spray.

[0035] According to the pharmaceutical composition of the present application, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

[0036] According to the pharmaceutical composition of the present application, the excipient is selected from one or a combination of two or more of a pharmaceutically acceptable diluent, an excipient, a filler, a binder, a humectant, a disintegrant, an absorption enhancer, a surfactant, an adsorption carrier, a lubricant, and a sustained release agent.

[0037] Dipeptidyl peptidase-4 is a serine protease that rapidly inactivates GLP-1 and GIP and other intestinal insulinotropins. The use of DPP-4 inhibitors can inhibit the degradation of GLP-1 and GIP, enhance the activity of intestinal insulinotropins and neuropeptides, reduce fasting and postprandial glucose concentrations and glycosylated hemoglobin levels, and improve insulin sensitivity and beta cell function. Bacterial DPP-4, as an important intestinal bacterial host isozyme, can hydrolyze GLP-1 and significantly affect the occurrence and development of metabolic related diseases, and is a new target for anti-metabolic diseases. The present application finds that the above plant extract has certain technical effects in inhibiting the enzyme activity of bacterial DPP-4, and can be used as an inhibitor of bacterial DPP-4.

[0038] In a ninth aspect, the present application further provides an in vitro method for inhibiting the enzyme activity of bacterial DPP-4, comprising the step of contacting bacterial DPP-4 or a cell containing bacterial DPP-4 with the plant extract composition as described above.

[0039] In a tenth aspect, the present application further provides a non-therapeutic use of a plant extract composition as an inhibitor of bacterial DPP-4 in vitro.

[0040] The plant extract composition with synergistic hypoglycemic activity and application thereof are provided, and the mass ratio of mulberry leaf extract, white kidney bean extract and balsam pear powder is 8:(1-24):(1-64), so that the plant extract with mulberry leaf extract, white kidney bean extract and balsam pear powder as main raw materials has a significantly better effect on reducing blood sugar than the plant extract in the prior art and the plant extract composition with other compounding ratios.

[0041] Meanwhile, the plant extract composition has the effects of synergistically reducing the number of peripheral nerve neutrophils, improving peripheral nerve inflammation, synergistically enhancing the fluorescence intensity of peripheral nerves, improving peripheral nerve damage, and inhibiting the enzyme activity of bacteria-derived dipeptidyl peptidase-4 in vitro. BRIEF DESCRIPTION OF DRAWINGS

[0042] In order to more clearly illustrate the technical solutions of the present application or the prior art, the drawings needed in the following embodiments or prior art description will be briefly introduced. Obviously, the drawings in the following description are some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor.

[0043] Figure 1 is the test result graph of test example 3 provided by the present application.

[0044] Figure 2 is the test result graph of test example 4 provided by the present application. DETAILED DESCRIPTION

[0045] In order to make the purpose, technical solutions and advantages of the present application more clear, the technical solutions in the present application will be described clearly and completely in the following with reference to the drawings in the present application. Obviously, the described embodiments are some embodiments of the present application, but not all embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor belong to the scope of protection of the present application.

[0046] In some specific embodiments of the present application, a plant extract composition is first provided, which includes mulberry leaf extract, white kidney bean extract and balsam pear powder with a mass ratio of 8:(1-24):(1-64).

[0047] Taking the mass ratio of mulberry leaf extract, white kidney bean extract and balsam pear powder in the present application as an example, the expression can be understood as: the mass ratio of mulberry leaf extract and white kidney bean extract satisfies 8:(1-24), and the mass ratio of mulberry leaf extract and balsam pear powder satisfies 8:(1-64).

[0048] In some embodiments of the present application, when the mulberry leaf extract is 8 parts by weight, the fraction of white kidney bean extract can be any value or a range of values formed by any values selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 parts by weight.

[0049] In some embodiments of the present application, when the mulberry leaf extract is 8 parts by weight, the fraction of bitter gourd powder can be any value or a range of values formed by any values selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63 and 64 parts by weight.

[0050] In some embodiments of the present application, the mass ratio of the mulberry leaf extract, the white kidney bean extract and the bitter gourd powder is 8:(1-8):(1-22).

[0051] In some embodiments of the present application, the mass ratio of the mulberry leaf extract, the white kidney bean extract and the bitter gourd powder is 8:(1-3):(1-18).

[0052] In some embodiments of the present application, the mass ratio of the mulberry leaf extract, the white kidney bean extract and the bitter gourd powder is 8:(1-3):(1-8).

[0053] According to the plant extract composition provided by the present application, preferably, the mass ratio of the mulberry leaf extract, the white kidney bean extract and the bitter gourd powder is 8:3:(1-8).

[0054] In some embodiments of the present application, the mass ratio of the mulberry leaf extract, the white kidney bean extract and the bitter gourd powder is 8:(1-3):(3-4).

[0055] According to the plant extract composition provided by the present application, preferably, the mass ratio of the mulberry leaf extract, the white kidney bean extract and the bitter gourd powder is 8:3:(3-8).

[0056] In some embodiments of the present application, the plant extract composition comprises white kidney bean extract 1-9 parts by weight, mulberry leaf extract 1-18 parts by weight, and bitter gourd powder 1-18 parts by weight.

[0057] Specifically, in some embodiments of the present application, the white kidney bean extract can be in any value or range of values formed by any values selected from the group consisting of 1 part, 2 parts, 3 parts, 4 parts, 5 parts, 6 parts, 7 parts, 8 parts, and 9 parts. The mulberry leaf extract can be in any value or range of values formed by any values selected from the group consisting of 1 part, 2 parts, 3 parts, 4 parts, 5 parts, 6 parts, 7 parts, 8 parts, 9 parts, 10 parts, 11 parts, 12 parts, 13 parts, 14 parts, 15 parts, 16 parts, 17 parts, and 18 parts. The bitter gourd powder can be in any value or range of values formed by any values selected from the group consisting of 1 part, 2 parts, 3 parts, 4 parts, 5 parts, 6 parts, 7 parts, 8 parts, 9 parts, 10 parts, 11 parts, 12 parts, 13 parts, 14 parts, 15 parts, 16 parts, 17 parts, and 18 parts.

[0058] In some embodiments of the present application, the mulberry leaf extract has a 1-deoxynojirimycin content of greater than or equal to 1.0 g / 100 g;

[0059] Alternatively, the white kidney bean extract has an alpha-amylase inhibitor activity of greater than or equal to 1 x 10 5 U / g;

[0060] Alternatively, the bitter gourd powder has a total saponin content of greater than or equal to 1.5% w / w.

[0061] In some embodiments of the present application, the mulberry leaf extract has a 1-deoxynojirimycin content of greater than or equal to 1.0 g / 100 g, the white kidney bean extract has an alpha-amylase inhibitor activity of greater than or equal to 1 x 10 5 U / g, and the bitter gourd powder has a total saponin content of greater than or equal to 1.5% w / w.

[0062] In some embodiments of the present application, the mulberry leaf extract is prepared by water extraction; the white kidney bean extract is prepared by water extraction; and the bitter gourd powder is prepared by water extraction.

[0063] In some embodiments of the present application, a method for preparing the plant extract composition as described above is also provided, comprising the step of mixing mulberry leaf extract, white kidney bean extract, and bitter gourd powder. The mass ratio of the mulberry leaf extract, the white kidney bean extract, and the bitter gourd powder in the resulting mixture is 8:(1-24):(1-64).

[0064] In some embodiments of the present application, there is also provided a plant extract composition for lowering blood sugar, comprising the plant extract composition as described above, or the plant extract composition prepared by the method as described above, wherein the active ingredients for lowering blood sugar are derived from mulberry leaf extract, white kidney bean extract and bitter gourd powder.

[0065] In some embodiments of the present application, there is also provided the use of the plant extract composition as described above for maintaining a healthy blood sugar level, improving diabetic complications, delaying the progression of diabetes, or treating or preventing high blood sugar related metabolic diseases in a subject.

[0066] The use comprises administering the plant extract composition to the subject in an effective amount. The subject can be a human. The effective amount can range from 500 to 12000 mg per day, such as 500 mg per day, 550 mg per day, 600 mg per day, 650 mg per day, 700 mg per day, 750 mg per day, 800 mg per day, 850 mg per day, 900 mg per day, 950 mg per day, 1 g per day, 1.5 g per day, 2 g per day, 2.5 g per day, 3 g per day, 3.5 g per day, 4 g per day, 4.5 g per day, 5 g per day, 5.5 g per day, 6 g per day, 6.5 g per day, 7 g per day, 7.5 g per day, 8 g per day, 8.5 g per day, 9 g per day, 9.5 g per day, 10 g per day, 10.5 g per day, 11 g per day, 11.5 g per day, or 12 g per day.

[0067] In some embodiments of the present application, there is also provided the use of the plant extract composition for lowering blood sugar as described above for the preparation of a food product for maintaining a healthy blood sugar level, improving diabetic complications, or delaying the progression of diabetes.

[0068] In some embodiments of the present application, there is also provided a food product composition comprising the plant extract composition for lowering blood sugar as described above; the food product composition is for maintaining a healthy blood sugar level, improving diabetic complications, or delaying the progression of diabetes.

[0069] In some embodiments of the present application, the food product composition is an oral liquid, a tea drink, a lozenge, a capsule, a drink, or an effervescent tablet.

[0070] The food product composition in the present application can also be a functional health product suitable for a subject.

[0071] The subject can be a mammal, preferably a human. The administration can be performed by oral, gastrointestinal, injection, spray, physical or chemical mediated means.

[0072] In some embodiments of the present application, there is also provided use of the plant extract composition for lowering blood sugar as described above in the manufacture of a medicament for treating and / or preventing a hyperglycemia-related metabolic disease.

[0073] In some embodiments of the present application, the hyperglycemia-related metabolic disease is selected from one or more of diabetes, diabetic neuropathy, diabetic nephropathy, diabetic retinopathy, diabetic cardiomyopathy, diabetic gangrene, diabetic cachexia, diabetic dyslipidemia, diabetic coronary heart disease, diabetic cerebrovascular disease, and diabetic peripheral vascular disease.

[0074] In some embodiments of the present application, there is also provided a pharmaceutical composition comprising the plant extract composition for lowering blood sugar as described above; the pharmaceutical composition is used for treating and / or preventing a hyperglycemia-related metabolic disease.

[0075] In some embodiments of the present application, the pharmaceutical composition is an injection, a tablet, a powder, a granule, a pill, a capsule, an oral liquid, a paste, a cream, or a spray.

[0076] In some embodiments of the present application, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

[0077] In some embodiments of the present application, the excipient is selected from one or a combination of two or more of a pharmaceutically acceptable diluent, an excipient, a filler, a binder, a humectant, a disintegrant, an absorption enhancer, a surfactant, an adsorption carrier, a lubricant, and a sustained release agent.

[0078] In some embodiments of the present application, there is also provided an in vitro method of inhibiting the enzymatic activity of a bacterial dipeptidyl peptidase-4, comprising the step of contacting the bacterial dipeptidyl peptidase-4 or a cell comprising the bacterial dipeptidyl peptidase-4 with the plant extract composition as described above. The cell can be a bacterial cell expressing the bacterial dipeptidyl peptidase-4. The bacterial dipeptidyl peptidase-4 can comprise the amino acid sequence of SEQ ID NO: 1.

[0079] In some embodiments of the present application, there is also provided a non-therapeutic use of the plant extract composition as an inhibitor of bacterial dipeptidyl peptidase-4 in vitro.

[0080] Some specific embodiments of the present application are described below, and Figures 1-2 A plant extract composition having synergistic hypoglycemic activity and its use are described.

[0081] Unless otherwise specified in the following examples, the techniques or conditions described in the literature or according to the product specifications were used. Unless otherwise specified, the reagents or instruments used were conventional products that can be purchased through regular channels. Among them, the source information of part of the raw materials is as follows:

[0082] Sample, solvent standard dilution water.

[0083] Metformin hydrochloride tablets (hereinafter referred to as metformin): white tablets, batch number ACR8818, Shanghai Schering-Plough Pharmaceuticals Co., Ltd., solvent is ultrapure water.

[0084] Phosphoric acid sitagliptin tablets (hereinafter referred to as sitagliptin): yellow tablets, batch number Y008396, Hangzhou Merck Pharmaceuticals Co., Ltd., solvent is ultrapure water.

[0085] Cyclocarya paliurus extract: prepared by water extraction method, total flavonoids 2.01 g / kg, total saponins 1.0 g / 100 g.

[0086] Wheat bran extract, batch number XT20250118, Xi'an Boaoxintian Plant Development Co., Ltd.

[0087] Pancreatic oligopeptide: batch number 20241108820, Dezhou Lanli Biotechnology Co., Ltd.

[0088] Chicken egg yolk powder: batch number 20230203, Zhejiang Aige Biological Technology Co., Ltd.

[0089] D-(+) -glucose: batch number A2425167, Shanghai Aladdin Biochem Technology Co., Ltd.

[0090] Anhydrous ethanol: batch number 20240123, National Pharmaceutical Group Chemical Reagent Co., Ltd.

[0091] Methyl cellulose: batch number J16IS217502, Shanghai Yuanye Biological Technology Co., Ltd.

[0092] Preparation Example 1 White kidney bean extract

[0093] The present preparation example provides a preparation method of white kidney bean extract, the process is as follows:

[0094] Using white kidney beans as raw materials, through raw material screening, crushing, water extraction, sterilization, spray drying, a powdery or particulate material is prepared, the obtained product has an alpha-amylase inhibitor activity greater than or equal to 5 x 10 4 U / g.

[0095] According to the method of the present preparation example, by changing the solid-liquid ratio of water extraction, white kidney bean extracts with different indicators are obtained as follows:

[0096] White kidney bean extract 1: alpha-amylase inhibitor activity greater than or equal to 3 x 10 5 U / g.

[0097] White kidney bean extract 2: alpha-amylase inhibitor activity greater than or equal to 1 x 10 5 U / g.

[0098] White kidney bean extract 3: alpha-amylase inhibitor activity greater than or equal to 1 x 10 4 U / g.

[0099] Preparation Example 2 Mulberry leaf extract

[0100] The present preparation example provides a method for preparing a mulberry leaf extract, the process of which is as follows:

[0101] The mulberry leaf extract is produced using mulberry leaves as raw material, through water extraction, microfiltration, ultrafiltration, concentration, spray drying, and packaging, etc. The mulberry leaf extract obtained has mulbarbark ≥ 0.2 g / 100 g and 1-deoxynojirimycin content greater than or equal to 0.8 g / 100 g.

[0102] According to the method of the present preparation example, by changing the solid-liquid ratio of water extraction, mulberry leaf extracts with different indexes are obtained as follows:

[0103] Mulberry leaf extract 1: 1-deoxynojirimycin (1-DNJ) 1.1 g / 100 g.

[0104] Mulberry leaf extract 2: 1-DNJ 1.0 g / 100 g.

[0105] Mulberry leaf extract 3: 1-DNJ 0.8 g / 100 g.

[0106] Preparation Example 3 Bitter gourd powder

[0107] The present preparation example provides a method for preparing a bitter gourd powder, the process of which is as follows:

[0108] The solid bitter gourd powder is produced using bitter gourd as raw material, through water extraction, filtration, concentration, drying, crushing, sieving, testing, and packaging, etc. The solid obtained has total saponins (calculated as ginsenoside Re) content greater than or equal to 0.5% w / w.

[0109] According to the method of the present preparation example, by changing the solid-liquid ratio of water extraction, bitter gourd powders with different indexes are obtained as follows:

[0110] Bitter gourd powder 1: total saponins (calculated as ginsenoside Re) content greater than or equal to 1.5% w / w.

[0111] Bitter gourd powder 2: total saponins (calculated as ginsenoside Re) content greater than or equal to 0.5% w / w.

[0112] Experimental animals: Zebrafish were bred and provided by the fish breeding center of Hangzhou Huan Te Co., Ltd. and were maintained in water at 28℃ (water quality: 200 mg of instant sea salt was added to each 1 L of reverse osmosis water, conductivity was 450-550 μS / cm, pH was 6.5-8.5, and hardness was 50-100 mg / L CaCO3). The experimental animal use license number was SYXK (Zhe) 2022-0004. The breeding and management met the requirements of international AAALAC certification (certification number: 001458), and the IACUC ethical review number was IACUC-2025-11865-01.

[0113] Equipment and consumable information:

[0114] Dissecting microscope: SZX7, OLYMPUS, Japan.

[0115] CCD camera: VertA1, Shanghai Tushen Vision Technology Co., Ltd.

[0116] Precision electronic balance: CP214, OHAUS, USA.

[0117] Ultrasonic cleaner: JP-010T, Shenzhen Jiemeng Cleaning Equipment Co., Ltd.

[0118] Full-automatic sample rapid grinding instrument: JXFSTPRP-24L, Shanghai Jingxin Experimental Equipment Technology Department.

[0119] High-speed refrigerated centrifuge: Heraeus Fresco17, ThermoFisher, Germany.

[0120] Tabletop high-capacity low-speed centrifuge: TD5A, Changsha Yingtai Instrument Co., Ltd.

[0121] Electric focusing continuous zoom fluorescence microscope: AZ100, Nikon, Japan.

[0122] Blood glucose meter: ACCU-CHEK Performa, Roche Diagnostics (Shanghai) Co., Ltd.

[0123] Blood glucose meter test paper: batch number 478829, Roche Diagnostics (Shanghai) Co., Ltd.

[0124] Examples 1-8 and Comparative Examples 1-7

[0125] A plant extract composition was obtained by fully mixing mulberry leaf extract 1, white kidney bean extract 1, and balsam pear powder 1 as raw materials. The weight fractions of each component are shown in Table 1 below.

[0126] Table 1

[0127]

[0128] Example 9

[0129] The same as Example 2 except that white kidney bean extract 1 is replaced with white kidney bean extract 2.

[0130] Example 10

[0131] The same as Example 2 except that mulberry leaf extract 1 is replaced with mulberry leaf extract 2.

[0132] Example 11

[0133] The same as Example 2 except that bitter gourd powder 1 is replaced with bitter gourd powder 2.

[0134] Comparative Example 8

[0135] The same as Example 2 except that white kidney bean extract 1 is replaced with white kidney bean extract 3.

[0136] Comparative Example 9

[0137] The same as Example 2 except that mulberry leaf extract 1 is replaced with mulberry leaf extract 3.

[0138] Comparative Example 10

[0139] A plant extract composition was obtained by thoroughly mixing each of the ingredients in Table 2 below, using each of the ingredients as a raw material, and the weight parts of each of the ingredients are shown in Table 2 below.

[0140] Table 2

[0141]

[0142] Comparative Example 11

[0143] A plant extract composition was obtained by thoroughly mixing each of the ingredients in Table 3 below, using each of the ingredients as a raw material, and the weight parts of each of the ingredients are shown in Table 3 below.

[0144] Table 3

[0145]

[0146] Comparative Example 12

[0147] A plant extract composition was obtained by thoroughly mixing each of the ingredients in Table 4 below, using each of the ingredients as a raw material, and the weight parts of each of the ingredients are shown in Table 4 below.

[0148] Table 4

[0149]

[0150] Test Example 1 Hypoglycemic effect test of single component of plant extract on zebrafish

[0151] Test method: 5dpf wild type AB strain zebrafish were randomly selected in beakers, and 30 zebrafish were treated in each beaker (experimental group). The test sample (dissolved in water, the concentration is shown in Table 1) was given, and the positive control drug was metformin with a concentration of 400 μg / mL. Normal control group and model control group were set up at the same time, and the volume of each beaker was 25 mL. Except for the normal control group, 1.5 mg / mL chicken egg yolk powder was given in the water during the day and 30 mg / mL glucose was given in the water at night to establish the high glucose and high fat model of zebrafish. After 2 days of treatment at 28℃, the zebrafish were washed 3 times with standard dilution water, and the data were collected with a blood glucose meter. The glucose level of zebrafish was analyzed and statistically analyzed to evaluate the auxiliary hypoglycemic effect of the test sample. The statistical processing result was expressed as mean ± SE. Statistical analysis was performed using SPSS software, and p<0.05 indicated that the difference was statistically significant.

[0152] Table 5

[0153]

[0154] In the table, compared with the model control group, p<0.05, p<0.01, p<0.001.

[0155] Test Example 2 Hypoglycemic effect test of plant extract composition on zebrafish

[0156] Test method: basically the same as Test Example 1, except that the test sample in Test Example 1 also includes plant extract compositions of Examples 1-11 and Comparative Examples 1-12, and the total concentration of plant extract in the test sample containing plant extract is 125 μg / mL. The test results are shown in Table 6. Among them, the drug in the positive control group is metformin, the plant extract in control group 1 is bitter gourd powder 1, the plant extract in control group 2 is mulberry leaf extract 1, and the plant extract in control group 3 is white kidney bean extract 1.

[0157] Table 6

[0158]

[0159] As can be seen from the data in the above table, the positive control group, control groups 1-3, examples 1-11, and comparative examples 1-12 all have auxiliary hypoglycemic efficacy. Among them, at a concentration of 125 μg / mL, the auxiliary hypoglycemic efficacy of examples 1-11 is better than that of control groups 1-3, and has significance. While the auxiliary hypoglycemic efficacy of comparative examples 1-12 is not significantly different from that of control groups 1-3. This shows that under the same raw material composition and the same experimental concentration conditions, the composition of examples 1-11 not only has a hypoglycemic effect significantly better than that of a single component of plant extract, but also significantly higher than that of the composition under other ratios.

[0160] Test Example 3 Effect of plant extract composition on the number of perineural neutrophils of zebrafish

[0161] Test method: 5 dpf (days post fertilization) transgenic neutrophil green fluorescent zebrafish (MPX (myeloperoxidase)) were randomly selected in beakers, and 30 zebrafish were treated in each beaker (experimental group). The test sample (125 μg / mL concentration, see Table 7 for experimental groups) was given by water dissolution, and the drug in the positive control was sitagliptin, with a concentration of 350 μg / mL. A normal control group and a model control group were also set up, and each cup had a capacity of 25 mL. Except for the normal control group, the rest of the experimental groups were given 1.5 mg / mL chicken egg yolk powder by water dissolution during the day and 30 mg / mL glucose by water dissolution at night to establish a high-sugar high-fat zebrafish model. After 2 days of treatment at 28°C, 10 zebrafish were randomly selected from each experimental group and placed under a fluorescence microscope for photography. NIS-Elements D 3.20 advanced image processing software was used for analysis and data collection, and the number of perineural neutrophils was analyzed. The statistical analysis results of this indicator were used to evaluate the auxiliary hypoglycemic efficacy of the sample. The statistical processing results are expressed as mean ± SE. Statistical analysis was performed using SPSS software, and p<0.05 indicates that the difference is statistically significant. Among them, the plant extract in control group 4 is bitter gourd powder 1, the plant extract in control group 4 is mulberry leaf extract 1, and the plant extract in control group 6 is white kidney bean extract 1. The test results are shown in Figure 1 and Table 7 below, Figure 1 Among them, the normal group corresponds to the normal control group, the model group corresponds to the model control group, sitagliptin corresponds to the positive control group, monomer control group 1 corresponds to control group 4, monomer control group 2 corresponds to control group 5, monomer control group 3 corresponds to control group 6, and composition 1-8 corresponds to examples 1-8, respectively.

[0162] Table 7

[0163]

[0164] From the data in the above table, compared with the model control group, control groups 4-6, examples 1-8, and comparative examples 1-7 all have the auxiliary blood glucose lowering effect, which is specifically manifested in reducing the number of peripheral nerve neutrophils and improving peripheral nerve inflammation. Among them, at a concentration of 125 μg / mL, the auxiliary peripheral nerve inflammation lowering effect of the intervention groups of examples 1-7 is significantly better than that of control groups 4-6 and comparative examples 1-7. This shows that under the same raw material composition and the same experimental concentration conditions, the properly proportioned white kidney bean extract, mulberry leaf extract, and bitter gourd powder have the synergistic effect of reducing the number of peripheral nerve neutrophils and improving peripheral nerve inflammation.

[0165] Test Example 4: Effect of plant extract composition on the number of peripheral nerve neutrophils of zebrafish

[0166] Test method: 5 dpf transgenic peripheral nerve green fluorescent zebrafish (Hb9) were randomly selected in beakers, and 30 zebrafish were treated in each beaker (experimental group). The samples were given in water (concentration of 125 μg / mL, see Table 8 for experimental groups), and the positive control drug was sitagliptin with a concentration of 350 μg / mL. A normal control group and a model control group were also set up, and the capacity of each beaker was 25 mL. Except for the normal control group, the rest of the experimental groups were given 1.5 mg / mL chicken egg yolk powder in water during the day and 30 mg / mL glucose in water at night to establish a high-sugar high-fat zebrafish model. After 2 days of treatment at 28°C, 10 zebrafish were randomly selected from each experimental group and placed under a fluorescence microscope for photography. The peripheral nerve fluorescence intensity was analyzed and data were collected using NIS-Elements D 3.20 advanced image processing software, and the statistical analysis results of the index were used to evaluate the auxiliary blood glucose lowering effect of the samples. The statistical processing results are expressed as mean ± SE. Statistical analysis was performed using SPSS software, and p<0.05 indicates that the difference is statistically significant. Among them, the plant extract in control group 7 is bitter gourd powder 1, the plant extract in control group 8 is mulberry leaf extract 1, and the plant extract in control group 9 is white kidney bean extract 1. The test results are shown in Figure 2 and Table 8 below, Figure 2 Among them, sitagliptin corresponds to the positive control group, monomer control group 1 corresponds to control group 7, monomer control group 2 corresponds to control group 8, monomer control group 3 corresponds to control group 9, and compositions 1-8 correspond to examples 1-8, respectively.

[0167] Table 8

[0168]

[0169] From the data in the above table, compared with the model control group, the control groups 7-9, examples 1-8 and comparative examples 1-7 all have the auxiliary hypoglycemic effect, which is specifically manifested in enhancing the fluorescence intensity of the peripheral nerve around the nerve and improving the peripheral nerve injury. Among them, at the concentration of 125 μg / mL, the auxiliary effect of examples 1, 2, 3, 4, 6 and 7 on enhancing the peripheral nerve injury is significantly better than that of control groups 7-9 and comparative examples 1-7. This shows that under the same raw material composition and the same experimental concentration, the properly matched white kidney bean extract, mulberry leaf extract and bitter gourd powder have the synergistic effect of enhancing the fluorescence intensity of the peripheral nerve around the nerve and improving the peripheral nerve injury.

[0170] Test Example 5: In vitro inhibition of bacterial dipeptidyl peptidase-4 activity test

[0171] Test method:

[0172] (1) The DPP4 gene was cloned into the pET22b vector and a 6xHis tag was added at the C-terminus. The obtained plasmid was then transformed into E. coli BL21 (DE3). The bacteria were grown at 37°C to an OD600 of 1.0-1.2, and the expression of recombinant protein was induced at 18°C with 0.2 mmol / L of isopropyl-β-D-thiogalactoside (IPTG). After overnight induction, the bacteria were harvested and lysed by ultrasonication, and the protein was purified by Ni2+ affinity resin (Ni-NTA, Qiagen) in a buffer containing 20 mmol / L Tris-HCl (pH 8.0), 200 mmol / L NaCl. The amino acid sequence of the bacterial DPP4 protein (SEQ ID NO: 1) is as follows:

[0173] MRKVSLALLLCLLCLAGMAQGQKALDLKDITSGRFRPENIQGVIPMPDGEHYTQMSADGTQIIKYSFRTGEKVEVIFDVNQARECDFKNFDSYQFSPDGDKLLIATRTTPIYRHSYTAVHYIYPLKRNDKGVTTNNIIERLSDGGPQQVPVFSPDGTMIAFVRDNNIFLVKLLYGNSESQVTEDGKQNSVLNGIPDWVYEEEFGFNRALEFSADNTMIAFIRFDESEVPSYSFPMFAGEAPQITPLKDYPGEYTYKYPKAGYPNSKVEVRTYDIKSHVTRTMKLPIDADGYIPRIRFTKDASKLAVMTLNRHQDRFDLYFADPRSTLCKLVLRDESPYYIKENVFDNIKFYPETFSLLSERDGFSHLYWYSMGGNLIKKVTNGKYEVKDFLGYDEADGSFYYTSNEESPLRKAVYKIDKKGKKLKLSQREGTNTPLFSQSMKYYMNKFSNLDTPMLVTLNDNTGKTLKTLINNDQLKQTLSGYAIPQKEFFTFQTTDGVTLNGWMMKPANFSTSKKYPVLMYQYSGPGSQQVLDTWGISWETYMASLGYIVVCVDGRGTGGRGEAFEKCTYLKIGVKEAKDQVETALYLGKQPYVDKDRIGIWGWSYGGYMTLMSMSEGTPVFKAGVAVAAPTDWRFYDTIYTERFMRTPKENAEGYKESSAFTRADKLHGNLLLVHGMADDNVHFQNCAEYAEHLVQLGKQFDMQVYTNRNHGIYGGNTRQHLYTRLTNFFLNNL. The bacterial DPP4-encoding gene can be obtained from E. coli expression purification or commissioned commercial gene synthesis company to prepare.

[0174] (2) Accurately weigh mulberry leaf extract 1, white kidney bean extract 1, bitter gourd powder 1, and the aforementioned examples 1-8, comparative examples 1-7, and comparative example 10. Prepare 0.1 mg / ml and 5 mg / ml solutions of the above monomers and combinations in DMSO for activity testing. Test the above samples using the method in the DPP-4 inhibitor screening kit (Sigma, #MAK203) with bacterial DPP4 protein (obtained in step (1)) instead of human DPP4 protein. Calculate the dipeptidyl peptidase-4 inhibition activity (%) as follows: inhibition rate (%) = (control well activity - sample well activity) / control well activity x 100%. Statistically analyze the experimental data and calculate the inhibition activity of each sample at the concentration as shown in Table 9. The statistical processing results are expressed as mean ± SE. Perform statistical analysis using SPSS software, and p<0.05 indicates that the difference is statistically significant. Among them, the plant extract in control group 10 is bitter gourd powder 1, the plant extract in control group 11 is mulberry leaf extract 1, and the plant extract in control group 12 is white kidney bean extract 1.

[0175] Table 9

[0176]

[0177] From the above table data, it can be seen that, compared with the control group, examples 1, 2, 4, 5, 6, and 7 exhibit synergistic effects at a concentration of 0.1 mg / ml, and the in vitro bacterial DPP4 inhibition activity of the combination is higher than that of the monomer activity. Compared with comparative example 10, the in vitro bacterial DPP4 inhibition activity of examples 2 and 4 does not show significant difference from that of comparative example 10.

[0178] At a concentration of 5 mg / ml, examples 2, 4, and 5 exhibit synergistic effects, and the in vitro bacterial DPP4 inhibition activity of the combination is higher than that of the monomer activity; compared with comparative example 10, examples 2 and 4 are more effective.

[0179] Finally, it should be noted that the above examples are only used to illustrate the technical solutions of the present application, and not to limit them; although the present application has been described in detail with reference to the foregoing examples, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing examples, or make equivalent substitutions for part of the technical features; and these modifications or substitutions do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. A pharmaceutical composition, characterized in that, The pharmaceutical composition is used to treat and / or prevent hyperglycemia-related metabolic diseases; the pharmaceutical composition is composed of mulberry leaf extract, white kidney bean extract and bitter melon powder in a mass ratio of 8:3:(3~8); the treatment and / or prevention of hyperglycemia-related metabolic diseases is to reduce the number of neutrophils around nerves, improve peripheral nerve inflammation, and enhance peripheral nerve fluorescence intensity and improve peripheral nerve damage. The 1-deoxynojirimycin content of the mulberry leaf extract is greater than or equal to 1.0 g / 100 g; The α-amylase inhibitor activity of the white kidney bean extract is greater than or equal to 1×10⁻⁶. 5 U / g; The total saponin content of the bitter melon powder is greater than or equal to 1.5% w / w; The mulberry leaf extract was prepared by water extraction. The white kidney bean extract was prepared by water extraction. The bitter melon powder was obtained by water extraction.

2. The pharmaceutical composition according to claim 1, characterized in that, The pharmaceutical composition is an injection, tablet, powder, granule, pill, capsule, oral liquid, ointment, cream, or spray.

3. The pharmaceutical composition according to claim 1 or 2, characterized in that, The pharmaceutical composition also includes pharmaceutically acceptable excipients.

4. The pharmaceutical composition according to claim 3, characterized in that, The excipients are selected from one or more combinations of pharmaceutically acceptable fillers, binders, wetting agents, disintegrants, absorption enhancers, surfactants, adsorbents, lubricants, and sustained-release agents.

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