Plant extract composition with synergistic hypoglycemic activity and application thereof

Abstract

The invention relates to the technical field of plant extracts, and provides a plant extract composition with synergistic hypoglycemic activity and application thereof, and the plant extract composition comprises a mulberry leaf extract, a white kidney bean extract and bitter gourd powder in a mass ratio of 8: (1-24): (1-64). By optimizing the mass ratio of the mulberry leaf extract, the white kidney bean extract and the bitter gourd powder, the blood sugar reducing effect of the plant extract taking the mulberry leaf extract, the white kidney bean extract and the bitter gourd powder as main raw materials is remarkably superior to that of a plant extract in the prior art. Meanwhile, the plant extract composition also has the effects of synergistically reducing the number of neutrophils around nerves, improving peripheral neuroinflammation, synergistically enhancing the fluorescence intensity of the peripheral nerves, improving peripheral nerve injury and inhibiting the enzymatic activity of a bacterium source dipeptidyl peptidase-4.

Description

Technical Field

[0001] The present invention relates to the technical field of plant extracts, and particularly relates to a plant extract composition having synergistic hypoglycemic activity and its application. Background Art

[0002] Metabolic diseases are a group of diseases caused by disorders in the metabolic process, involving obstacles in the synthesis, decomposition, transformation or transportation of substances in the body. Among them, hyperglycemia-related metabolic diseases have attracted much attention. Currently, methods for the prevention and treatment of hyperglycemia-related metabolic diseases include adopting a low-sugar, low-fat and high-fiber diet, as well as taking hypoglycemic drugs, etc.

[0003] However, relying solely on traditional dietary adjustments and chemical hypoglycemic drugs often has limitations such as poor compliance, side effects of long-term use and significant individual differences. In recent years, more and more studies have shown that bioactive extracts derived from natural plants (such as polyphenols, flavonoids, saponins, terpenoids and alkaloids, etc.) have played the advantages of multi-target and low toxicity 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 momordica saponins have been widely used in functional foods and traditional Chinese medicine preparations, and their hypoglycemic effects and safety have been verified in clinical trials. These findings indicate that plant extracts, as an important part of foods, health products and even drugs, are becoming one of the key strategies for preventing and intervening in hyperglycemia-related metabolic diseases, and demonstrating unique application value in the prevention, adjuvant treatment and even disease course management of hyperglycemia-related metabolic diseases. Summary of the Invention

[0004] The present invention provides a plant extract composition having synergistic hypoglycemic activity and its application, which provides effective active ingredients for hypoglycemia with mulberry leaf extract, white kidney bean extract and bitter gourd powder as raw materials, and can significantly improve the hypoglycemic effect when used in a specific ratio, and has no toxic side effects.

[0005] Specifically, by reasonably compounding with other plant extracts having hypoglycemic or related regulatory effects, the present invention finds 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 other compounding ratios, which has very important technical significance for preparing hypoglycemic foods or drugs with better curative effects, more comprehensive actions, more stability and better compliance.

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

[0007] According to the plant extract composition provided by the present invention, 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 present invention, 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 present invention, 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 present invention, 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 present invention, 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 present invention, 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 polyhydroxypiperidine alkaloid present in mulberry trees. It has a structure similar to that of α-1,4-glucose and is an α-glucosidase inhibitor, having hypoglycemic function, anti-tumor and antiviral activities. It is also an index component in mulberry leaf extract.

[0014] Phaseolin in white kidney bean extract (also known as Phaseolin) is a natural α-amylase inhibitor. Its chemical nature is a glycoprotein, which can effectively inhibit the decomposition of starch and make it excreted from the gastrointestinal tract. In addition, due to its large molecular weight, phaseolin does not enter the blood circulation system and does not act on the central nervous system. It does not suppress appetite while losing weight and has no side effects even at high doses, showing good safety. In the present invention, the α-amylase inhibitor activity is used as the index component of white kidney bean extract.

[0015] The saponins in bitter gourd are glycoside compounds with triterpenoid or steroid compounds as aglycones, composed of sugars and aglycones. It can not only reduce the body's blood sugar level by regulating the uptake of glucose by body cells, but also play a role in relieving inflammation through various ways. The bioactive components in bitter gourd include saponins, proteins, polysaccharides, polyphenols, sterols, etc. In the present invention, the total saponins are used as the index component of bitter gourd powder.

[0016] According to the plant extract composition provided by the present invention, the content of 1-deoxynojirimycin in the mulberry leaf extract is greater than or equal to 1.0 g / 100 g; Or, the activity of α-amylase inhibitor in the white kidney bean extract is greater than or equal to 1×10 5 U / g; Or, the total saponin content in the balsam pear powder is greater than or equal to 1.5% w / w.

[0017] According to the plant extract composition provided by the present invention, the content of 1-deoxynojirimycin in the mulberry leaf extract is greater than or equal to 1.0 g / 100 g, the activity of α-amylase inhibitor in the white kidney bean extract is greater than or equal to 1×10 5 U / g, and the total saponin content in the balsam pear powder is greater than or equal to 1.5% w / w.

[0018] The present invention discovers through a large number of experiments that using 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 α-amylase inhibitor activity greater than or equal to 1×10 5 U / g, and a balsam pear powder with a total saponin content greater than or equal to 1.5% w / w plays a key role in ensuring the hypoglycemic effect, improving peripheral nerve inflammation, and peripheral nerve injury of the above plant extract composition.

[0019] According to the plant extract composition provided by the present invention, the mulberry leaf extract is prepared by a water extraction method; the white kidney bean extract is prepared by a water extraction method; the balsam pear powder is prepared by a water extraction method.

[0020] In a second aspect, the present invention also provides a preparation method of the plant extract composition as described above. The plant extract composition 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 balsam pear powder. There is no particular regulation on the mixing order among the mulberry leaf extract, the white kidney bean extract, and the balsam pear powder, as long as the three can be mixed evenly. The mass ratio of the mulberry leaf extract, the white kidney bean extract, and the balsam pear powder in the finally obtained mixture is 8:(1 - 24):(1 - 64).

[0021] In a third aspect, the present invention also provides a plant extract composition for hypoglycemia, including the plant extract composition as described above, or the plant extract composition prepared by the preparation method as described above. The hypoglycemic active ingredient is derived from the mulberry leaf extract, the white kidney bean extract, and the balsam pear powder.

[0022] In a fourth aspect, the present invention also provides the application of the plant extract composition as described above in maintaining a healthy blood glucose level, improving diabetic complications, delaying the progression of diabetes, treating or preventing hyperglycemia-related metabolic diseases in a subject.

[0023] The application includes administering the plant extract composition to a subject in an effective amount. The subject can be a human. The range of the effective amount can be 500 - 12000 mg / day, such as 500 mg / day, 550 mg / day, 600 mg / day, 650 mg / day, 700 mg / day, 750 mg / day, 800 mg / day, 850 mg / day, 900 mg / day, 950 mg / day, 1 g / day, 1.5 g / day, 2 g / day, 2.5 g / day, 3 g / day, 3.5 g / day, 4 g / day, 4.5 g / day, 5 g / day, 5.5 g / day, 6 g / day, 6.5 g / day, 7 g / day, 7.5 g / day, 8 g / day, 8.5 g / day, 9 g / day, 9.5 g / day, 10 g / day, 10.5 g / day, 11 g / day, 11.5 g / day or 12 g / day.

[0024] In a fifth aspect, the present invention also provides an application of the plant extract composition for reducing blood sugar as described above in the preparation of a food for maintaining a healthy blood sugar level, improving diabetic complications or delaying the progression of diabetes.

[0025] In a sixth aspect, the present invention also provides a food composition, comprising the plant extract composition for reducing blood sugar as described above; the food composition is used for maintaining a healthy blood sugar level, improving diabetic complications or delaying the progression of diabetes.

[0026] According to the food composition provided by the present invention, the food composition is an oral liquid, a tea drink, a lozenge, a capsule, a beverage or an effervescent tablet.

[0027] The food composition in the present invention can also be a functional health product suitable for the subject.

[0028] The subject can be a mammal, preferably a human. Administration can be carried out by oral, enterogastric, injection, spraying, physical or chemical mediated means.

[0029] In a seventh aspect, the present invention also provides an application of the plant extract composition for reducing blood sugar as described above in the preparation of a drug for treating and / or preventing hyperglycemia-related metabolic diseases.

[0030] According to the application provided by the present invention, the hyperglycemia-related metabolic diseases are 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.

[0031] In an eighth aspect, the present invention further provides a pharmaceutical composition, comprising the plant extract composition for lowering blood glucose as described above; the pharmaceutical composition is used for treating and / or preventing hyperglycemia-related metabolic diseases.

[0032] According to the pharmaceutical composition provided by the present invention, the pharmaceutical composition is an injection, tablet, powder, granule, pill, capsule, oral liquid, ointment, cream or spray.

[0033] According to the pharmaceutical composition provided by the present invention, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

[0034] According to the pharmaceutical composition provided by the present invention, the excipient is selected from one or a combination of two or more of a pharmaceutically acceptable diluent, excipient, filler, binder, wetting agent, disintegrant, absorption promoter, surfactant, adsorption carrier, lubricant and sustained release agent.

[0035] Dipeptidyl peptidase-4 is a serine protease that can rapidly cleave and inactivate incretins such as GLP-1 and GIP. Application of DPP-4 inhibitors can inhibit the degradation of GLP-1 and GIP, enhance the activity of incretins and neuropeptides, reduce fasting and postprandial glucose concentrations and glycated hemoglobin levels, and improve insulin sensitivity and β-cell function. Bacterial dipeptidyl peptidase-4, as an important intestinal bacterial host isoenzyme, 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 invention discovers that the above plant extract has certain technical effects in inhibiting the enzyme activity of bacterial dipeptidyl peptidase-4 and can be used as an inhibitor of bacterial dipeptidyl peptidase-4.

[0036] In a ninth aspect, the present invention further provides an in vitro method for inhibiting the enzyme activity of bacterial dipeptidyl peptidase-4, which comprises the step of contacting bacterial dipeptidyl peptidase-4 or cells containing bacterial dipeptidyl peptidase-4 with the plant extract composition as described above.

[0037] In a tenth aspect, the present invention further provides a use of a plant extract composition as a non-therapeutic purpose bacterial dipeptidyl peptidase-4 inhibitor in vitro.

[0038] The plant extract composition with synergistic hypoglycemic activity provided by the present invention and its application, by optimizing the mass ratio of mulberry leaf extract, white kidney bean extract and bitter gourd powder to 8:(1-24):(1-64), make the plant extract mainly composed of mulberry leaf extract, white kidney bean extract and bitter gourd powder significantly superior to the plant extracts in the prior art and the plant extract compositions under other compounding ratios in terms of blood glucose lowering effect.

[0039] Meanwhile, the above-mentioned plant extract composition also has the effects of synergistically reducing the number of neutrophils around the nerves and improving peripheral nerve inflammation, synergistically enhancing the fluorescence intensity of peripheral nerves and improving peripheral nerve injury, and inhibiting the enzymatic activity of dipeptidyl peptidase-4 from bacteria in vitro. Brief Description of the Drawings

[0040] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0041] Figure 1 It is a test result diagram of Test Example 3 provided by the present invention.

[0042] Figure 2 It is a test result diagram of Test Example 4 provided by the present invention. Detailed Embodiments

[0043] To make the objectives, technical solutions, and advantages of the present invention clearer, the following will clearly and completely describe the technical solutions in the present invention with reference to the drawings in the present invention. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts fall within the scope of protection of the present invention.

[0044] In some specific embodiments of the present invention, a plant extract composition is first provided, which includes mulberry leaf extract, white kidney bean extract, and bitter gourd powder with a mass ratio of 8:(1 - 24):(1 - 64).

[0045] Taking the mass ratio of mulberry leaf extract, white kidney bean extract, and bitter gourd powder in the present invention as 8:(1 - 24):(1 - 64) as an example, this expression can be understood as: the mass ratio of mulberry leaf extract to white kidney bean extract satisfies 8:(1 - 24), and the mass ratio of mulberry leaf extract to bitter gourd powder satisfies 8:(1 - 64).

[0046] In some specific embodiments of the present invention, by weight, when the mulberry leaf extract is 8 parts, the number of parts of the white kidney bean extract can be any value or a range composed of any value among 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, 18 parts, 19 parts, 20 parts, 21 parts, 22 parts, 23 parts, and 24 parts.

[0047] In some specific embodiments of the present invention, by weight parts, when the mulberry leaf extract is 8 parts, the parts of bitter gourd powder can be any value or a range formed by any value among 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, 18 parts, 19 parts, 20 parts, 21 parts, 22 parts, 23 parts, 24 parts, 25 parts, 26 parts, 27 parts, 28 parts, 29 parts, 30 parts, 31 parts, 32 parts, 33 parts, 34 parts, 35 parts, 36 parts, 37 parts, 38 parts, 39 parts, 40 parts, 41 parts, 42 parts, 43 parts, 44 parts, 45 parts, 46 parts, 47 parts, 48 parts, 49 parts, 50 parts, 51 parts, 52 parts, 53 parts, 54 parts, 55 parts, 56 parts, 57 parts, 58 parts, 59 parts, 60 parts, 61 parts, 62 parts, 63 parts and 64 parts.

[0048] In some specific embodiments of the present invention, the mass ratio of the mulberry leaf extract, white kidney bean extract and bitter gourd powder is 8:(1 - 8):(1 - 22).

[0049] In some specific embodiments of the present invention, the mass ratio of the mulberry leaf extract, white kidney bean extract and bitter gourd powder is 8:(1 - 3):(1 - 18).

[0050] In some specific embodiments of the present invention, the mass ratio of the mulberry leaf extract, white kidney bean extract and bitter gourd powder is 8:(1 - 3):(1 - 8).

[0051] According to the plant extract composition provided by the present invention, preferably, the mass ratio of the mulberry leaf extract, white kidney bean extract and bitter gourd powder is 8:3:(1 - 8).

[0052] In some specific embodiments of the present invention, the mass ratio of the mulberry leaf extract, white kidney bean extract and bitter gourd powder is 8:(1 - 3):(3 - 4).

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

[0054] In some specific embodiments of the present invention, by weight parts, the plant extract composition includes 1 - 9 parts of white kidney bean extract, 1 - 18 parts of mulberry leaf extract and 1 - 18 parts of bitter gourd powder.

[0055] Specifically, in some specific embodiments of the present invention, the dosage of the white kidney bean extract can be any value or a range composed of any values among 1 part, 2 parts, 3 parts, 4 parts, 5 parts, 6 parts, 7 parts, 8 parts, and 9 parts. The dosage of the mulberry leaf extract can be any value or a range composed of any values among 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 dosage of the balsam pear powder can be any value or a range composed of any values among 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.

[0056] In some specific embodiments of the present invention, the content of 1-deoxynojirimycin in the mulberry leaf extract is greater than or equal to 1.0 g / 100 g; Or, the activity of α-amylase inhibitor in the white kidney bean extract is greater than or equal to 1×10 5 U / g; Or, the total saponin content in the balsam pear powder is greater than or equal to 1.5% w / w.

[0057] In some specific embodiments of the present invention, the content of 1-deoxynojirimycin in the mulberry leaf extract is greater than or equal to 1.0 g / 100 g, the activity of α-amylase inhibitor in the white kidney bean extract is greater than or equal to 1×10 5 U / g, and the total saponin content in the balsam pear powder is greater than or equal to 1.5% w / w.

[0058] In some specific embodiments of the present invention, the mulberry leaf extract is prepared by a water extraction method; the white kidney bean extract is prepared by a water extraction method; the balsam pear powder is prepared by a water extraction method.

[0059] In some specific embodiments of the present invention, there is also provided a preparation method of the above-mentioned plant extract composition, including the step of mixing the mulberry leaf extract, the white kidney bean extract, and the balsam pear powder. The mass ratio of the mulberry leaf extract, the white kidney bean extract, and the balsam pear powder in the obtained mixture is 8:(1~24):(1~64).

[0060] In some specific embodiments of the present invention, there is also provided a plant extract composition for reducing blood sugar, including the above-mentioned plant extract composition, or the plant extract composition prepared by the above-mentioned preparation method, and the active ingredient for reducing blood sugar is derived from the mulberry leaf extract, the white kidney bean extract, and the balsam pear powder.

[0061] In some specific embodiments of the present invention, there is also provided the use of the plant extract composition as described above in maintaining a healthy blood glucose level, improving diabetic complications, delaying the progression of diabetes, treating or preventing hyperglycemia-related metabolic diseases in a subject.

[0062] The use includes 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 / day, such as 500 mg / day, 550 mg / day, 600 mg / day, 650 mg / day, 700 mg / day, 750 mg / day, 800 mg / day, 850 mg / day, 900 mg / day, 950 mg / day, 1 g / day, 1.5 g / day, 2 g / day, 2.5 g / day, 3 g / day, 3.5 g / day, 4 g / day, 4.5 g / day, 5 g / day, 5.5 g / day, 6 g / day, 6.5 g / day, 7 g / day, 7.5 g / day, 8 g / day, 8.5 g / day, 9 g / day, 9.5 g / day, 10 g / day, 10.5 g / day, 11 g / day, 11.5 g / day or 12 g / day.

[0063] In some specific embodiments of the present invention, there is also provided the use of the plant extract composition for lowering blood glucose as described above in preparing a food for maintaining a healthy blood glucose level, improving diabetic complications or delaying the progression of diabetes.

[0064] In some specific embodiments of the present invention, there is also provided a food composition comprising the plant extract composition for lowering blood glucose as described above; the food composition is for maintaining a healthy blood glucose level, improving diabetic complications or delaying the progression of diabetes.

[0065] In some specific embodiments of the present invention, the food composition is an oral liquid, a tea drink, a lozenge, a capsule, a beverage or an effervescent tablet.

[0066] The food composition in the present invention can also be a functional health product suitable for the subject.

[0067] The subject can be a mammal, preferably a human. Administration can be carried out by oral, enterogastric, injection, spraying, physical or chemical-mediated means.

[0068] In some specific embodiments of the present invention, there is also provided the use of the plant extract composition for lowering blood glucose as described above in preparing a drug for treating and / or preventing hyperglycemia-related metabolic diseases.

[0069] In some specific embodiments of the present invention, the hyperglycemic-related metabolic diseases are 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.

[0070] In some specific embodiments of the present invention, 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 hyperglycemic-related metabolic diseases.

[0071] In some specific embodiments of the present invention, the pharmaceutical composition is an injection, tablet, powder, granule, pill, capsule, oral liquid, ointment, cream, or spray.

[0072] In some specific embodiments of the present invention, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

[0073] In some specific embodiments of the present invention, the excipient is selected from one or a combination of two or more of a pharmaceutically acceptable diluent, excipient, filler, binder, wetting agent, disintegrant, absorption enhancer, surfactant, adsorption carrier, lubricant, and sustained-release agent.

[0074] In some specific embodiments of the present invention, there is also provided an in vitro method for inhibiting the enzymatic activity of microbial dipeptidyl peptidase-4, which comprises the step of contacting microbial dipeptidyl peptidase-4 or a cell containing microbial dipeptidyl peptidase-4 with the plant extract composition as described above. The cell can be a bacterial cell expressing microbial dipeptidyl peptidase-4. Microbial dipeptidyl peptidase-4 may comprise the amino acid sequence of SEQ ID NO: 1.

[0075] In some specific embodiments of the present invention, there is also provided a non-therapeutic use of the plant extract composition as a microbial dipeptidyl peptidase-4 inhibitor in vitro.

[0076] The following combines some specific implementation cases, as well as Figure 1 - Figure 2 Describe the plant extract composition with synergistic hypoglycemic activity of the present invention and its applications.

[0077] For those not specifying specific techniques or conditions in the following examples, they shall be in accordance with the techniques or conditions described in the literature in the field or in accordance with the product specifications. For reagents or instruments not indicating the manufacturer, they are all conventional products that can be obtained through regular channels. Among them, the source information of some raw materials is as follows: Sample, solvent standard dilution water.

[0078] Metformin Hydrochloride Tablets (hereinafter referred to as metformin): white tablets, batch number ACR8818, Sino-American Shanghai Squibb Pharmaceutical Co., Ltd., with ultrapure water as the solvent.

[0079] Sitagliptin Phosphate Tablets (hereinafter referred to as sitagliptin): yellow tablets, batch number Y008396, Merck Sharp & Dohme (China) Investment Co., Ltd., with ultrapure water as the solvent.

[0080] Cyclocarya paliurus extract: prepared by water extraction method, with total flavonoids of 2.01 g / kg and total saponins of 1.0 g / 100 g.

[0081] Wheat bran extract, batch number XT20250118, Xi'an Boao Xintian Plant Development Co., Ltd.

[0082] Pancreatic oligopeptide: batch number 20241108820, Dezhou Lanli Biotechnology Co., Ltd.

[0083] Egg yolk powder: batch number 20230203, Zhejiang Aige Biotechnology Co., Ltd.

[0084] D-(+)-Glucose: batch number A2425167, Shanghai Aladdin Biochemical Technology Co., Ltd.

[0085] Absolute ethanol: batch number 20240123, Sinopharm Chemical Reagent Co., Ltd.

[0086] Methyl cellulose: batch number J16IS217502, Shanghai Yuanye Bio-Technology Co., Ltd.

[0087] Preparation Example 1 White kidney bean extract This preparation example provides a preparation method of white kidney bean extract, and the process is as follows: Using white kidney beans as raw materials, through raw material screening, pulverization, water extraction, sterilization, and spray drying to obtain a powdery or particulate substance, and the α-amylase inhibitor activity of the obtained product is greater than or equal to 5×10 4 U / g.

[0088] According to the method of this preparation example, by changing the material-liquid ratio condition of water immersion extraction, white kidney bean extracts with the following different indexes are obtained: White kidney bean extract 1: α-amylase inhibitor activity is greater than or equal to 3×10 5 U / g.

[0089] White kidney bean extract 2: α-amylase inhibitor activity is greater than or equal to 1×10 5 U / g.

[0090] White kidney bean extract 3: α-amylase inhibitor activity is greater than or equal to 1×10 4 U / g.

[0091] Preparation Example 2 Mulberry Leaf Extract This preparation example provides a method for preparing mulberry leaf extract, and the process is as follows: Using mulberry leaves as raw materials, it is produced through processes such as water extraction, microfiltration, ultrafiltration, concentration, spray drying, and packaging. Among them, the mulberry alkaloid in the obtained mulberry leaf extract is ≥ 0.2 g / 100 g, and the content of 1-deoxynojirimycin is greater than or equal to 0.8 g / 100 g.

[0092] According to the method of this preparation example, mulberry leaf extracts with the following different indexes are obtained by changing the solid-liquid ratio conditions of water extraction: Mulberry Leaf Extract 1: 1-deoxynojirimycin (1-DNJ) 1.1 g / 100 g.

[0093] Mulberry Leaf Extract 2: 1-DNJ 1.0 g / 100 g.

[0094] Mulberry Leaf Extract 3: 1-DNJ 0.8 g / 100 g.

[0095] Preparation Example 3 Bitter Melon Powder This preparation example provides a method for preparing bitter melon powder, and the process is as follows: Using bitter melons as raw materials, it is processed into a solid through main processes such as water extraction, filtration, concentration, drying, pulverization, sieving, inspection, and packaging. Among them, the content of total saponins (calculated as ginsenoside Re) in the obtained solid is greater than or equal to 0.5% w / w.

[0096] According to the method of this preparation example, bitter melon powders with the following different indexes are obtained by changing the solid-liquid ratio conditions of water extraction: Bitter Melon Powder 1: The content of total saponins (calculated as ginsenoside Re) is greater than or equal to 1.5% w / w.

[0097] Bitter Melon Powder 2: The content of total saponins (calculated as ginsenoside Re) is greater than or equal to 0.5% w / w.

[0098] Experimental animals: Zebrafish are all raised in fish-raising water at 28 °C (water quality: 200 mg of instant sea salt is added to every 1 L of reverse osmosis water, the conductivity is 450 - 550 μS / cm; pH is 6.5 - 8.5; hardness is 50 - 100 mg / L CaCO 3 ), provided by the fish-raising center of Hangzhou Huante Company, and the experimental animal use license number is: SYXK (Zhe) 2022 - 0004. The feeding management meets the requirements of international AAALAC certification (certification number: 001458), and the IACUC ethical review number is: IACUC - 2025 - 11865 - 01.

[0099] Equipment and Consumable Information: Dissecting microscope: SZX7, OLYMPUS, Japan.

[0100] CCD camera: VertA1, Shanghai Tusen Vision Technology Co., Ltd.

[0101] Precision electronic balance: CP214, OHAUS, USA.

[0102] Ultrasonic cleaner: JP-010T, Shenzhen Jiemeng Cleaning Equipment Co., Ltd.

[0103] Full-automatic rapid sample grinder: JXFSTPRP-24L, Shanghai Jingxin Experimental Equipment Science and Technology Department.

[0104] High-speed refrigerated centrifuge: Heraeus Fresco17, ThermoFisher, Germany.

[0105] Bench-top large-capacity low-speed centrifuge: TD5A, Changsha Yingtai Instrument Co., Ltd.

[0106] Electrically focused continuously variable fluorescence microscope: AZ100, Nikon, Japan.

[0107] Blood glucose meter: ACCU-CHEK Performa, Roche Diagnostic Products (Shanghai) Co., Ltd.

[0108] Blood glucose test strips: batch number 478829, Roche Diagnostic Products (Shanghai) Co., Ltd.

[0109] Examples 1-8 and Comparative Examples 1-7 A plant extract composition is obtained by fully mixing mulberry leaf extract 1, white kidney bean extract 1, and bitter gourd powder 1 as raw materials. The weight parts of each component are shown in Table 1 below.

[0110] Table 1

[0111] Example 9 Basically the same as Example 2, except that: white kidney bean extract 1 is replaced with white kidney bean extract 2 in equal mass.

[0112] Example 10 Basically the same as Example 2, except that: mulberry leaf extract 1 is replaced with mulberry leaf extract 2 in equal mass.

[0113] Example 11 Basically the same as Example 2, except that: bitter gourd powder 1 is replaced with bitter gourd powder 2 in equal mass.

[0114] Comparative Example 8 Basically the same as Example 2, except that: the white kidney bean extract 1 is replaced with the white kidney bean extract 3 in equal mass.

[0115] Comparative Example 9 Basically the same as Example 2, except that: the mulberry leaf extract 1 is replaced with the mulberry leaf extract 3 in equal mass.

[0116] Comparative Example 10 A plant extract composition is obtained by fully mixing the following components in Table 2 as raw materials, and the weight parts of each component are as shown in Table 2 below.

[0117] Table 2

[0118] Comparative Example 11 A plant extract composition is obtained by fully mixing the following components in Table 3 as raw materials, and the weight parts of each component are as shown in Table 3 below.

[0119] Table 3

[0120] Comparative Example 12 A plant extract composition is obtained by fully mixing the following components in Table 4 as raw materials, and the weight parts of each component are as shown in Table 4 below.

[0121] Table 4

[0122] Test Example 1 Test on the hypoglycemic effect of single components of plant extracts on zebrafish Test method: Randomly select 5dpf wild-type AB strain zebrafish in a beaker, and 30 zebrafish are treated in each beaker (experimental group). The test samples to be measured are respectively given (obtained by dissolving the plant extract in water, and the concentration is shown in Table 1). The drug used for the positive control is metformin, and its concentration is 400 μg / mL. At the same time, a normal control group and a model control group are set up, and the volume of each beaker is 25 mL. Except for the normal control group, the other experimental groups are given 1.5 mg / mL egg yolk powder in water during the day and 30 mg / mL glucose in water at night to establish a high-sugar and high-fat model of zebrafish. After treatment at 28 °C for 2 days, the zebrafish are washed 3 times with standard dilution water, and the data are collected with a blood glucose meter to analyze and statistically analyze the glucose level of zebrafish. The auxiliary hypoglycemic efficacy of the test samples is evaluated based on the statistical analysis results of this index. The statistical processing results are expressed as mean±SE. Statistical analysis is performed using SPSS software, and p<0.05 indicates that the difference is statistically significant.

[0123] Table 5

[0124] In the table, compared with the model control group, indicates p < 0.05, indicates p < 0.01, indicates p < 0.001.

[0125] Test Example 2: Test on the hypoglycemic effect of the plant extract composition on zebrafish Test method: Basically the same as Test Example 1, the differences are as follows: In addition to the test samples in Test Example 1, the test samples to be measured also include the plant extract compositions of Examples 1 to 11 and Comparative Examples 1 to 12, and in the test samples containing plant extracts, the total concentration of the plant extracts 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.

[0126] Table 6

[0127] It can be seen from the data in the above table that the positive control group, Control Groups 1 to 3, Examples 1 to 11, and Comparative Examples 1 to 12 all have the effect of assisting hypoglycemia. Among them, at a concentration of 125 μg / mL, the effect of assisting hypoglycemia of Examples 1 to 11 is better than that of Control Groups 1 to 3 and is significant. However, the effect of assisting hypoglycemia of Comparative Examples 1 to 12 and Control Groups 1 to 3 does not all show significant differences. This indicates that under the same raw material composition and the same experimental concentration conditions, the compositions of Examples 1 to 11 not only have a significantly better hypoglycemic effect than the single-component plant extracts but also are significantly higher than the compositions under other ratios.

[0128] Test Example 3: Effect of the plant extract composition on the number of neutrophils around the nerves of zebrafish Test method: Randomly select 5-dpf (days post-fertilization) transgenic neutrophil green fluorescent zebrafish (MPX (myeloperoxidase)) in a beaker, with 30 zebrafish in each beaker (experimental group). The test samples were administered in water solution (at a concentration of 125 μg / mL, and the experimental groups are shown in Table 7). The drug in the positive control was sitagliptin, with a concentration of 350 μg / mL. At the same time, a normal control group and a model control group were set up, and the volume of each beaker was 25 mL. Except for the normal control group, the other experimental groups were given 1.5 mg / mL egg yolk powder in water solution during the day and 30 mg / mL glucose in water solution at night to establish a high-sugar and high-fat zebrafish model. After treatment at 28 °C for 2 days, 10 zebrafish were randomly selected from each experimental group and placed under a fluorescence microscope for photographing. The NIS-Elements D 3.20 advanced image processing software was used to analyze and collect data, and the number of neutrophils around the nerve was analyzed. The hypoglycemic effect of the sample was evaluated based on the statistical analysis results of this index. The statistical treatment results were expressed as mean ± SE. Statistical analysis was performed using SPSS software, and p < 0.05 indicated that the difference was statistically significant. Among them, the plant extract in control group 4 was bitter gourd powder 1, the plant extract in control group 4 was mulberry leaf extract 1, and the plant extract in control group 6 was white kidney bean extract 1. The test results are as Figure 1 shown in Table 7 below, Figure 1 wherein, 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 compositions 1-8 correspond to Examples 1-8 in sequence.

[0129] Table 7

[0130] It can be seen from the data in the above table that compared with the model control group, control groups 4-6, Examples 1-8, and Comparative Examples 1-7 all have the effect of assisting in hypoglycemia, specifically manifested as reducing the number of neutrophils around the nerve and improving peripheral nerve inflammation. Among them, at a concentration of 125 μg / mL, the intervention groups of Examples 1-7 were significantly better than control groups 4-6 and Comparative Examples 1-7 in assisting in reducing peripheral nerve inflammation. This indicates that under the same raw material composition and the same experimental concentration conditions, appropriately proportioned white kidney bean extract, mulberry leaf extract, and bitter gourd powder have a synergistic effect in reducing the number of neutrophils around the nerve and improving peripheral nerve inflammation.

[0131] Test Example 4 Effect of Plant Extract Composition on the Number of Neutrophils around the Nerve in Zebrafish Test method: Randomly select 5 dpf transgenic zebrafish with green fluorescent peripheral nerves (Hb9) into a beaker, and 30 zebrafish are treated in each beaker (experimental group). The sample is dissolved in water and given (at a concentration of 125 μg / mL, and the experimental groups are shown in Table 8). The drug used for the positive control is sitagliptin, and its concentration is 350 μg / mL. At the same time, a normal control group and a model control group are set up, and the volume of each cup is 25 mL. Except for the normal control group, the other experimental groups are given 1.5 mg / mL egg yolk powder dissolved in water during the day and 30 mg / mL glucose dissolved in water at night to establish a high-sugar and high-fat zebrafish model. After treatment at 28 °C for 2 days, 10 zebrafish are randomly selected from each experimental group and placed under a fluorescence microscope for photographing. The NIS-Elements D 3.20 advanced image processing software is used to analyze and collect data, and the fluorescence intensity of the peripheral nerves is analyzed. The hypoglycemic effect of the sample is evaluated based on the statistical analysis results of this index. The statistical processing results are expressed as mean ± SE. Statistical analysis is 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 as Figure 2 shown in Table 8 below, Figure 2 in which, 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 in sequence.

[0132] Table 8

[0133] It can be seen from the data in the above table that compared with the model control group, control groups 7-9, Examples 1-8, and Comparative Examples 1-7 all have the effect of assisting hypoglycemia, specifically manifested as enhancing the fluorescence intensity of the peripheral nerves and improving peripheral nerve damage. Among them, at a concentration of 125 μg / mL, the intervention groups of Examples 1, 2, 3, 4, 6, and 7 have significantly better effects in assisting in enhancing peripheral nerve damage than control groups 7-9 and Comparative Examples 1-7. This indicates that under the conditions of the same raw material composition and the same experimental concentration, properly proportioned white kidney bean extract, mulberry leaf extract, and bitter gourd powder have a synergistic effect in enhancing the fluorescence intensity of the peripheral nerves and improving peripheral nerve damage.

[0134] Test Example 5 In vitro test for inhibiting the activity of bacterial dipeptidyl peptidase-4 Test method: (1) The DPP4 gene was cloned into the pET22b vector and a 6×His tag was added to the C-terminus. Then the resulting plasmid was transformed into Escherichia coli BL21(DE3). The bacteria were grown at 37 °C until the OD600 reached 1.0 - 1.2 and the expression of the recombinant protein was induced with 0.2 mmol / L isopropyl-β-D-thiogalactoside (IPTG) at 18 °C. After overnight induction, the bacteria were harvested and lysed by sonication, and the protein was purified by Ni2+ affinity resin (Ni-NTA, Qiagen) in a buffer containing 20 mmol / L Tris-HCl (pH 8.0) and 200 mmol / L NaCl. Among them, the amino acid sequence of the bacterial DPP4 protein (SEQ ID NO: 1) is as follows: MRKVSLALLLCLLCLAGMAQGQKALDLKDITSGRFRPENIQGVIPMPDGEHYTQMSADGTQIIKYSFRTGEKVEVIFDVNQARECDFKNFDSYQFSPDGDKLLIATRTTPIYRHSYTAVHYIYPLKRNDKGVTTNNIIERLSDGGPQQVPVFSPDGTMIAFVRDNNIFLVKLLYGNSESQVTEDGKQNSVLNGIPDWVYEEEFGFNRALEFSADNTMIAFIRFDESEVPSYSFPMFAGEAPQITPLKDYPGEYTYKYPKAGYPNSKVEVRTYDIKSHVTRTMKLPIDADGYIPRIRFTKDASKLAVMTLNRHQDRFDLYFADPRSTLCKLVLRDESPYYIKENVFDNIKFYPETFSLLSERDGFSHLYWYSMGGNLIKKVTNGKYEVKDFLGYDEADGSFYYTSNEESPLRKAVYKIDKKGKKLKLSQREGTNTPLFSQSMKYYMNKFSNLDTPMLVTLNDNTGKTLKTLINNDQLKQTLSGYAIPQKEFFTFQTTDGVTLNGWMMKPANFSTSKKYPVLMYQYSGPGSQQVLDTWGISWETYMASLGYIVVCVDGRGTGGRGEAFEKCTYLKIGVKEAKDQVETALYLGKQPYVDKDRIGIWGWSYGGYMTLMSMSEGTPVFKAGVAVAAPTDWRFYDTIYTERFMRTPKENAEGYKESSAFTRADKLHGNLLLVHGMADDNVHFQNCAEYAEHLVQLGKQFDMQVYTNRNHGIYGGNTRQHLYTRLTNFFLNNL. The bacterial DPP4-encoding gene can be obtained by expression and purification from Escherichia coli or prepared by commissioning a commercial gene synthesis company.

[0135] (2)Accurately weigh mulberry leaf extract 1, white kidney bean extract 1, bitter melon powder 1, and the aforementioned Examples 1-8, Comparative Examples 1-7, and Comparative Example 10. Prepare the above monomers and compositions into 0.1 mg / ml and 5 mg / mL solutions with DMSO for activity testing. Using the method in the DPP-4 inhibitor screening kit (Sigma, #MAK203), replace the human DPP4 protein with the bacterial DPP4 protein (obtained in step (1)) to test the above samples. The calculation method of dipeptidyl peptidase-4 inhibitory activity (%): Inhibition rate (%) = (activity of control well - activity of sample well) / activity of control well × 100%. Statistically analyze the experimental data, and calculate the inhibitory activities of each sample at this concentration as shown in Table 9. The results of statistical processing are expressed as mean ± SE. Use SPSS software for statistical analysis, and p < 0.05 indicates that the difference is statistically significant. Among them, the plant extract in Control Group 10 is bitter melon 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.

[0136] Table 9

[0137] As can be seen from the data in the above table, compared with the control group, Examples 1, 2, 4, 5, 6, and 7 showed synergistic effects at a concentration of 0.1 mg / ml. The inhibitory activity of the composition against bacterial DPP4 in vitro was higher than that of the monomers. Compared with Comparative Example 10, there was no significant difference in the inhibitory activity of bacterial DPP4 in vitro between Examples 2 and 4 and Comparative Example 10.

[0138] At a concentration of 5 mg / ml, Examples 2, 4, and 5 showed synergistic effects. The inhibitory activity of the composition against bacterial DPP4 in vitro was higher than that of the monomers; compared with Comparative Example 10, Examples 2 and 4 had better effects.

[0139] Finally, it should be noted that the above examples are only used to illustrate the technical solutions of the present invention and are not intended to limit them; although the present invention 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 perform equivalent replacements for some of the technical features; and these modifications or replacements 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 invention.

Claims

1. A plant extract composition, characterized in that It includes mulberry leaf extract, white kidney bean extract and bitter melon powder in a mass ratio of 8:(1~24):(1~64).

2. The plant extract composition according to claim 1, characterized in that: The mass ratio of mulberry leaf extract, white kidney bean extract and bitter melon powder is 8:(1~8):(1~22).

3. The plant extract composition according to claim 2, characterized in that: The mass ratio of mulberry leaf extract, white kidney bean extract and bitter melon powder is 8:(1~3):(1~18).

4. The plant extract composition according to claim 3, characterized in that: The mass ratio of mulberry leaf extract, white kidney bean extract and bitter melon powder is 8:(1~3):(1~8).

5. The plant extract composition according to claim 4, characterized in that: The mass ratio of mulberry leaf extract, white kidney bean extract and bitter melon powder is 8:3:(1~8).

6. The plant extract composition according to claim 4, characterized in that: The mass ratio of mulberry leaf extract, white kidney bean extract and bitter melon powder is 8:(1~3):(3~4).

7. The plant extract composition according to claim 6, characterized in that: The mass ratio of mulberry leaf extract, white kidney bean extract and bitter melon powder is 8:3:(3~8).

8. The plant extract composition according to any one of claims 1 to 7, characterized in that The 1-deoxynojirimycin content of the mulberry leaf extract is greater than or equal to 1.0 g / 100 g; Alternatively, the α-amylase inhibitor activity of the white kidney bean extract is greater than or equal to 1×10 5 U / g; Alternatively, the total saponin content of the bitter melon powder is greater than or equal to 1.5% w / w.

9. The plant extract composition according to any one of claims 1 to 7, characterized in that The 1-deoxynojirimycin content of the mulberry leaf extract is greater than or equal to 1.0 g / 100 g, and the α-amylase inhibitor activity of the white kidney bean extract is greater than or equal to 1×10 5 U / g, and the total saponin content of the bitter melon powder is greater than or equal to 1.5%w / w.

10. The plant extract composition according to any one of claims 1 to 7, characterized in that: The mulberry leaf extract is prepared by water extraction; the white kidney bean extract is prepared by water extraction; and the bitter melon powder is prepared by water extraction.

11. The method for preparing the plant extract composition according to any one of claims 1 to 10, characterized in that: The main raw materials are mulberry leaf extract, white kidney bean extract and bitter melon powder in a mass ratio of 8:(1-24):(1-64).

12. A plant extract composition for lowering blood sugar, characterized in that: The invention comprises the plant extract composition according to any one of claims 1 to 10, or the plant extract composition prepared by the preparation method according to claim 11, wherein the active ingredient for lowering blood sugar is derived from the mulberry leaf extract, the white kidney bean extract and the bitter melon powder.

13. Use of the plant extract composition for lowering blood sugar according to claim 12 in preparing food for maintaining healthy blood sugar levels, improving diabetic complications or delaying the progression of diabetes.

14. A food composition, characterized in that It comprises the plant extract composition for lowering blood sugar as described in claim 10; the food composition is used to maintain healthy blood sugar level, improve diabetic complications or delay the progression of diabetes.

15. The food composition according to claim 14, characterized in that The food composition is oral liquid, tea, lozenge, capsule, drink or effervescent tablet.

16. Use of the plant extract composition for lowering blood sugar according to claim 12 in the preparation of a medicament for treating and / or preventing hyperglycemia-related metabolic diseases.

17. The use according to claim 16, characterized in that: 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.

18. A pharmaceutical composition, characterized in that The plant extract composition for lowering blood sugar as claimed in claim 12; the pharmaceutical composition is used for treating and / or preventing hyperglycemia-related metabolic diseases.

19. The pharmaceutical composition according to claim 18, characterized in that: The pharmaceutical composition is an injection, tablet, powder, granule, pill, capsule, oral solution, ointment, cream or spray.

20. The pharmaceutical composition according to claim 18 or 19, characterized in that: The pharmaceutical composition further comprises pharmaceutically acceptable excipients.

21. The pharmaceutical composition according to claim 20, characterized in that: The auxiliary material is selected from one or a combination of two or more of pharmaceutically acceptable diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption promoters, surfactants, adsorption carriers, lubricants and sustained-release agents.

22. An in vitro method for inhibiting the enzymatic activity of bacterial dipeptidyl peptidase-4, comprising the step of contacting bacterial dipeptidyl peptidase-4 or cells containing bacterial dipeptidyl peptidase-4 with the plant extract composition according to any one of claims 1 to 10 or the plant extract composition prepared by the preparation method according to claim 11.

23. Use of the plant extract composition according to any one of claims 1 to 10 or the plant extract composition prepared by the preparation method according to claim 11 as a bacterial dipeptidyl peptidase-4 inhibitor in vitro for non-therapeutic purposes.

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