Cornus officinalis flower extract with effects of resisting oxidation and reducing blood glucose and blood lipid as well as preparation method and application of Cornus officinalis flower extract

Abstract

The invention discloses a dogwood flower extract with anti-oxidation and blood sugar and lipid reducing effects as well as a preparation method and application thereof, and belongs to the technical field of traditional Chinese medicines. The invention aims to solve the technical problems of regulating blood sugar, insulin, blood fat and antioxidant level by activating a PI3K / AKT / mTOR pathway in vivo, improving liver, kidney and pancreas injuries and exerting anti-diabetic activity by reducing inflammatory factor expression. According to the key point of the technical scheme, the invention discloses a dogwood flower extract with the effects of resisting oxidation and reducing blood sugar and blood lipid. The active ingredients of the dogwood flower extract comprise morroniside, swertioside, loganin, ursolic acid, oleanolic acid, dogwood neoglycoside, gallic acid, protocatechuic acid, p-hydroxybenzoic acid, catechin, chlorogenic acid, caffeic acid, epicatechin, orientin, rutin, ferulic acid, benzoic acid, resveratrol, quercetin and cinnamic acid.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine technology, specifically to a Cornus officinalis flower extract with antioxidant, hypoglycemic and lipid-lowering effects, its preparation method and application. Background Technology

[0002] Cornus officinalis, a traditional Chinese tonic, is primarily used as a raw material in the production of various traditional Chinese medicines such as Liuwei Dihuang Wan and Huanshao Dan. In recent years, with in-depth research on Cornus officinalis, its role in dietary therapy and health maintenance has gained attention, leading to the emergence of more related health products. Furthermore, its dried fruit has a long history of use as a cosmetic ingredient. The diverse compounds and nutrients in Cornus officinalis have made it a focus of attention for scholars both domestically and internationally. However, we have found that existing research mainly concentrates on the fruit, pericarp, and kernel of Cornus officinalis, with the main components being iridoid glycosides, tannins, and flavonoids. Organic acids, polysaccharides, alkaloids, and polysaccharide compounds have also been reported. Research reports on Cornus officinalis flowers mostly focus on the morphology of tissues and organs; there are currently no reports on the chemical composition, content, and bioactivity evaluation of Cornus officinalis flower extracts.

[0003] Studies have found that the aqueous extract of Cornus officinalis can reduce the expression of P16 and P21 in D-galactose-induced senescent MRC-5 cells, and this effect may be related to its ability to slow down telomere shortening in MRC-5 cells. Related studies have shown that the complex of Cornus officinalis saponins and Dioscorea opposita saponins has a protective effect on the myocardium of diabetic rats. The complex was found to inhibit apoptosis, reduce ROS levels, significantly reduce caspase-3 protein expression, and upregulate Bax and Bcl-2 protein expression. Cornus officinalis polysaccharides and total glycosides have a promoting effect on the expression of NFR-1 mRNA, PGC-1α and gene in the myocardium of rats with acute myocardial infarction, thus playing a cardioprotective role.

[0004] Relevant literature retrieved: This document, published in China (CN102061175A) on January 30, 2013, discloses a method for extracting antioxidants from the kernels of Cornus officinalis. This invention relates to a method for preparing antioxidants from the kernels of Cornus officinalis. The main steps of this invention are as follows: defatting the kernels of Cornus officinalis, percolating with acetone, recovering the solvent, dispersing in water, extracting with ethyl acetate, separating the ethyl acetate extract, recovering the solvent, and thus obtaining the antioxidants from the kernels of Cornus officinalis.

[0005] This document, published in China (CN101974048A) on February 16, 2011, discloses a method for extracting mononosides from Cornus officinalis. The process steps include ultrasonic extraction of the raw material with ethanol, ethanol recovery from the extract, adsorption through a polar macroporous resin, purification and concentration via a water-to-ethanol gradient elution over a biomembrane, extraction with water-saturated n-butanol, crystallization with a mixed solution of ethyl acetate and dichloromethane, and drying under reduced pressure to obtain the product.

[0006] The journal *Food Science*, with the article titled "Research Progress on Effective Components and Pharmacological Effects of Cornus officinalis and Its Application in the Food Industry," Volume 24, published in 2003, discloses that Cornus officinalis contains various substances with medicinal value. The main components isolated from the pulp can be classified as: organic acids and esters (gallic acid, malic acid, tartaric acid), pentacyclic triterpenes and their esters (ursolic acid, oleanolic acid), iridoids (monoside, 7-O-methylmonoside, loganin, strychnine), amino acids (aspartic acid, glutamic acid, serine, threonine), and minerals (Ca, Mg, Fe, Cu, Zn, Se, Na, P, etc., 21 mineral elements). The pulp of Cornus officinalis is rich in effective components, with loganin, ursolic acid, and monoside being the main effective components with important pharmacological and physiological functions. Cornus officinalis flowers and fruits are products of different growth stages of Cornus officinalis, and their active ingredients will obviously differ.

[0007] The prior art represented by the aforementioned documents has at least the following unresolved technical problems or defects: (1) The purification process is complex and time-consuming. Relevant evidence is provided by CN101974048A, which describes an extraction method that employs various purification techniques, including adsorption with polar macroporous resin, biofilm purification and concentration, water-saturated n-butanol extraction, and crystallization from a mixed solution of ethyl acetate and dichloromethane. These steps are cumbersome and complex, which may result in a long extraction time and low production efficiency.

[0008] (2) There is a problem with the crystallization solvent. The relevant evidence is: CN101974048A, which uses a mixed solution of ethyl acetate and dichloromethane for crystallization. Dichloromethane is a toxic organic solvent that is harmful to the environment and human health. Special protective measures need to be taken during production and use, which increases the safety cost of production and environmental pressure.

[0009] (3) Insufficient consideration of raw material characteristics. The relevant evidence is: Publication No. CN101974048A. This invention document does not mention an effective treatment method for impurities such as polysaccharides in Cornus officinalis raw materials. This will not only increase the difficulty of extraction and may affect the purity and extraction efficiency of the product, but also increase the cost due to the high cost of the equipment required for the purification process. Summary of the Invention

[0010] The purpose of this invention is to provide: a Cornus officinalis flower extract with antioxidant, hypoglycemic and lipid-lowering effects, and related technologies, to solve technical problems such as antioxidant, hypoglycemic, and lipid-lowering effects, or combinations thereof.

[0011] In a first aspect, the present invention provides: a Cornus officinalis flower extract with antioxidant, hypoglycemic and lipid-lowering effects, wherein the active ingredients of the Cornus officinalis flower extract include monosodium glutamate, strychnos nuciferoside, loganin, ursolic acid, oleanolic acid, cornus officinalis glycoside, gallic acid, protocatechuic acid, p-hydroxybenzoic acid, catechin, chlorogenic acid, caffeic acid, epicatechin, strychnos nuciferoside, rutin, ferulic acid, benzoic acid, resveratrol, quercetin and cinnamic acid.

[0012] Preferably, the Cornus officinalis flower extract contains 2.96-6.63 mg / g of mononoside, 1.25-1.84 mg / g of thymol, 1.20-1.82 mg / g of loganin, 0.43-1.17 mg / g of ursolic acid, 17.31-24.55 mg / g of oleanolic acid, 2.83-5.67 mg / g of neo-cornuate, 12.73-76.49 mg / g of gallic acid, 1.12-1.69 mg / g of protocatechuic acid, 0.07-0.12 mg / g of p-hydroxybenzoic acid, and 1.4 mg / g of catechin. The total content of chlorogenic acid is 0.33-0.56 mg / g, caffeic acid is 0.49-8.31 mg / g, epicatechin is 2.75-16.63 mg / g, sennatin is 0.82-1.95 mg / g, rutin is 0.46-7.03 mg / g, ferulic acid is 1.27-14.72 mg / g, benzoic acid is 2.47-11.29 mg / g, resveratrol is 0.31-0.58 mg / g, quercetin is 3.13-18.24 mg / g, and cinnamic acid is 1.02-4.28 mg / g.

[0013] Secondly, the present invention provides a method for preparing the Cornus officinalis flower extract, comprising the steps of dissolving Cornus officinalis flowers in a solvent and extracting the Cornus officinalis flower extract.

[0014] This includes technical features: solvent and extraction.

[0015] The technical feature solvent is selected from at least one of water, methanol, ethanol, acetone, ethyl acetate, chloroform, and hexane; preferably at least one of water, methanol, and ethyl acetate; further preferably water; and even more preferably distilled water.

[0016] Preferably, the ratio of the powder to the solvent is 1:5-15; more preferably, it is 1:10.

[0017] Preferably, the process further includes a step of pulverizing the Cornus officinalis flowers before dissolving them in the solvent.

[0018] More preferably, the pulverizing method is selected from at least one of a high-speed electric grinder, a traditional Chinese medicine crusher, a high-speed universal pulverizer, a hammer mill, a vibratory mill, and an air jet mill; even more preferably, it is pulverized using a traditional Chinese medicine crusher.

[0019] More preferably, a drying step is included before the pulverizing operation.

[0020] More preferably, the drying process includes, but is not limited to, freeze drying, oven drying, air drying, blow drying, natural drying, vacuum drying, etc.; even more preferably, air drying; and most preferably, air drying in a dry, ventilated, and dark place.

[0021] The extraction method is selected from at least one of reflux extraction, ultrasound-assisted extraction, percolation extraction, cold soaking extraction, supercritical fluid extraction, microwave-assisted extraction, and accelerated solvent extraction. Preferably, the extraction method is a combination of reflux extraction and ultrasound-assisted extraction; More preferably, the ultrasound duration is selected from 15-60 min; even more preferably, it is 35 min.

[0022] More preferably, the reflux temperature is 20-60°C, and even more preferably 45°C.

[0023] More preferably, the reflux duration is 1-4 hours; even more preferably, it is 3 hours.

[0024] More preferably, the number of refluxes is 1-4 times; even more preferably, it is 3 times.

[0025] Preferably, the extraction process further includes a purification process.

[0026] More preferably, the purification process includes concentration, filtration, and drying.

[0027] More preferably, the concentration method is selected from at least one of rotary evaporator concentration, vacuum drying oven concentration, water bath heating concentration, oil bath / sand bath heating concentration, freeze drying concentration, and membrane concentration; more preferably, rotary evaporator concentration; and even more preferably, the rotary evaporator concentration temperature is 45°C.

[0028] More preferably, the filtration method is selected from at least one of atmospheric pressure filtration, reduced pressure filtration, centrifugal filtration, thermal filtration, and membrane filtration; more preferably, centrifugal filtration; even more preferably, the centrifugal filtration speed is 3000-15000 r / min; most preferably, 12500 r / min; even more preferably, the centrifugal filtration time is 1-10 min; most preferably, 3 min.

[0029] More preferably, the drying method includes, but is not limited to, freeze drying, oven drying, air drying, blow drying, natural drying, vacuum drying, etc.; even more preferably, vacuum drying; most preferably, the vacuum drying temperature is 45°C.

[0030] Preferably, the preparation method of the Cornus officinalis flower extract includes the following steps: (1) Pulverization: Place the Cornus officinalis flower sample in a dry, ventilated and dark place to dry, and then pulverize it into powder to obtain Cornus officinalis flower powder; (2) Extraction: Dissolve the Cornus officinalis flower powder obtained in step (1) in distilled water. The ratio of powder to distilled water is 1:10. Sonicate for 35 min and reflux at 45℃ for 3 h each time to obtain Cornus officinalis flower supernatant. (3) Purification: The supernatant obtained in step (2) was concentrated by rotary evaporation at 45°C, centrifuged at 12500 r / min for 3 min, the supernatant was collected, and vacuum dried at 45°C to obtain the Cornus officinalis flower extract.

[0031] Thirdly, the present invention provides the application of the Cornus officinalis flower extract in terms of antioxidant properties.

[0032] This includes technical features: applications.

[0033] Fourthly, the present invention provides the application of the Cornus officinalis flower extract in lowering blood sugar and lipids.

[0034] This includes technical features: applications.

[0035] Fifthly, the present invention provides a pharmaceutical composition having antioxidant, hypoglycemic and lipid-lowering effects, wherein the active ingredients include the above-mentioned Cornus officinalis flower extract and one or more pharmaceutically acceptable carriers, diluents and excipients.

[0036] In pharmaceutical compositions, the term "composition" includes products comprising an active ingredient and an inert component (pharmaceutical-acceptable excipient) constituting a carrier, as well as any product obtained directly or indirectly from a combination, complexation, or aggregation of two or more components, or from the decomposition of one or more components, or from other types of reactions or interactions of one or more components. Therefore, the pharmaceutical compositions of the present invention include any composition prepared by mixing the Cornus officinalis flower extract, other active ingredients, and pharmaceutically acceptable excipients.

[0037] The pharmaceutical compositions of the present invention comprise the above-mentioned Cornus officinalis flower extract as an active ingredient, a pharmaceutically acceptable carrier, and optional other therapeutic ingredients or adjuvants. The pharmaceutical compositions include those suitable for oral, rectal, topical, and parenteral (including subcutaneous, intramuscular, and intravenous) administration, although the most suitable route in any particular case depends on the specific subject, the nature and severity of the disease to which the active ingredient is administered, and can be prepared by any method known in the pharmaceutical field.

[0038] Among them, the above-mentioned Cornus officinalis flower extract can interact with other active ingredients with antioxidant, hypoglycemic and lipid-lowering effects, or enhance the efficacy of diseases such as diabetes. Other active ingredients include SGLT-2 inhibitors, GLP-1 receptor agonists, α-glucosidase inhibitors and other natural active ingredients.

[0039] Representative SGLT-2 inhibitors include dapagliflozin and empagliflozin, whose core mechanism of action is to inhibit renal glucose reabsorption (promoting urinary glucose excretion); mildly reduce weight and blood pressure; protect the cardiovascular system (anti-oxidative stress), achieving both blood sugar and mild lipid reduction. While they lack direct antioxidant activity, they can improve oxidative stress. Representative GLP-1 receptor agonists include liraglutide and smegglutide, whose core mechanism of action is to promote insulin secretion and inhibit glucagon; delay gastric emptying (controlling postprandial blood glucose); reduce weight and blood lipids; and possess anti-inflammatory and antioxidant properties, showing significant blood sugar and lipid-lowering effects, making them suitable for obese diabetes. Representative α-glucosidase inhibitors include acarbose and voglibose, whose core mechanism of action is to inhibit intestinal α-glucosidase (delaying the breakdown of carbohydrates into glucose); mildly reduce postprandial blood glucose and triglycerides, achieving both blood sugar and mild lipid-lowering effects, but lacking direct antioxidant activity. Other naturally derived active ingredients, such as curcumin (derived from turmeric), puerarin (derived from kudzu root), berberine (Coptis chinensis, Phellodendron chinense), and astragalus polysaccharide (Astragalus membranaceus), can activate AMPK, enhance insulin sensitivity, inhibit the NF-κB inflammatory pathway, and reduce insulin resistance.

[0040] The active ingredient can be administered orally in solid or liquid dosage forms, such as capsules, tablets, lozenges, sugar lozenges, granules, and powders, and in liquid forms such as elixirs, syrups, emulsions, dispersions, and suspensions. The active ingredient can also be administered parenterally in sterile liquid dosage forms such as dispersions, suspensions, or solutions. Other dosage forms that can be used to administer the active ingredient include ointments, creams, drops, transdermal patches, or powders for topical administration; ophthalmic solutions or suspensions for ocular administration, i.e., eye drops; sprays or powder compositions for inhalation or intranasal administration; or creams, ointments, sprays, or suppositories for rectal or vaginal administration. Gelatin capsules contain the active ingredient and a powdered carrier, such as lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, etc. Similar diluents can be used to prepare compressed tablets. Both tablets and capsules can be formulated as sustained-release products to provide sustained release of the drug over several hours. Compressed tablets can be sugar-coated or film-coated to mask any unpleasant taste and protect the tablet from air, or they can be enteric-coated for selective disintegration in the gastrointestinal tract. Liquid dosage forms for oral administration may contain colorants and flavoring agents to increase patient acceptability. Generally, water, suitable oils, saline solutions, aqueous solutions of dextran (glucose), and related sugar solutions, and glycols such as propylene glycol or polyethylene glycol are suitable carriers for parenteral solutions. Solutions for parenteral administration preferably contain water-soluble salts of the active ingredient, suitable stabilizers, and buffering agents as needed. Antioxidants such as sodium bisulfite, sodium sulfite, or ascorbic acid, alone or in combination, are suitable stabilizers. Citric acid and its salts, and sodium EDTA may also be used. Furthermore, parenteral solutions may contain preservatives such as benzalkonium chloride, methylparaben, or propylparaben, and chlorobutanol. For inhalation administration, the compounds of the present invention can be conveniently delivered in aerosol form from pressurized packaging or a nebulizer. The Cornus officinalis flower extract can also be delivered in a formulated powder form, which can be inhaled with the aid of a powder inhaler device. A preferred delivery system for inhalation is a metered-dose inhaler (MDI) aerosol, which can be formulated as a suspension or solution of the Cornus officinalis flower extract of the present invention in a suitable propellant, such as a fluorocarbon or hydrocarbon. For ophthalmic administration, ophthalmic formulations can be formulated with a solution or suspension of the Cornus officinalis flower extract in a suitable ophthalmic carrier at a suitable weight percentage, thereby maintaining sufficient contact time between the compound and the ocular surface to allow the compound to penetrate into the cornea and internal regions of the eye.

[0041] Useful pharmaceutical dosage forms for administering the compounds of the present invention include, but are not limited to, hard and soft gelatin capsules, tablets, parenteral injections, and oral suspensions. When the Cornus officinalis flower extract of the present invention is administered stepwise or in combination with other therapeutic agents, the same dosage forms as described above can be used. When the drugs are administered in physical combinations, the dosage form and route of administration should be selected based on the compatibility of the combined drugs. The Cornus officinalis flower extract of the present invention can be administered as the sole active ingredient or in combination with a second active ingredient, the second active ingredient including those known to have antioxidant, hypoglycemic, and lipid-lowering effects.

[0042] The beneficial effects of this invention are as follows: The present invention has at least the following beneficial effects: 1. Extracts of Cornus officinalis flowers in different solvents are rich in polyphenols, flavonoids, polysaccharides, and proanthocyanidins, as well as a wide variety of polyphenols and iridoid glycosides. Analysis of the compound composition of water, methanol, and ethyl acetate extracts of Cornus officinalis flowers revealed that most of the compounds in the three extracts are consistent with those in the extract of Cornus officinalis fruit.

[0043] 2. Different extracts of Cornus officinalis flowers exhibited good antioxidant and hypoglycemic and lipid-lowering abilities, with the aqueous extract of Cornus officinalis flowers showing better effects than the extract of Cornus officinalis fruit. The study also found that the aqueous extract of Cornus officinalis flowers may achieve its hypoglycemic and lipid-lowering effects by binding with compounds such as Tetrahydroalstonine, ursolic acid, and monosodium glutamate and undergoing hydrogen bonding.

[0044] 3. This study is the first to investigate the preventive and therapeutic effects of Cornus officinalis flower water extract on type 2 diabetes mellitus (T2DM) mice via the PI3K / AKT / mTOR pathway. The Cornus officinalis water extract significantly improved abnormal blood glucose levels, liver and kidney damage, lipid metabolism abnormalities, oxidative stress, and inflammatory responses in diabetic mice. Simultaneously, it upregulated the mRNA and protein expression of PI3K, AKT, and mTOR, suggesting that the therapeutic effect of Cornus officinalis flower water extract on diabetes may be related to the activation of the PI3K / AKT / mTOR pathway.

[0045] 4. Compared with the prior art, the present invention provides a technical solution with a different technical concept, and its technical effect is equivalent to or slightly improved with the prior art. The difference between the technical concept of the present invention and the prior art includes, but is not limited to, the use of a combination of reflux extraction and ultrasound-assisted extraction methods, which greatly preserves the effective components in Cornus officinalis flowers and avoids the loss of effective components of the medicinal material.

[0046] Furthermore, based on the present invention: 1. Based on the comparison of Examples 1-3 and Comparative Examples 4-5, this invention employs a combination of techniques such as hot reflux extraction and ultrasound-assisted extraction, achieving new technical effects that further improve the utilization rate of Cornus officinalis and maximize the retention of effective components in Cornus officinalis flowers. The combined technical effect is superior to the sum of the effects of each individual technique.

[0047] 2. Based on the comparison of Examples 1-3 and Comparative Examples 1-3, the present invention, based on the existing Cornus officinalis fruit extract, discovered that the Cornus officinalis flower extract also contains the effective components of the Cornus officinalis fruit extract, and produced unexpected technical effects. The total polyphenol and total flavonoid content in the Cornus officinalis flower extract is higher than that in the Cornus officinalis fruit extract. Attached Figure Description

[0048] Figure 1 TIC results for extracts of Cornus officinalis flowers in different solvents.

[0049] Figure 2 Standard curves for six marker monomers of Cornus officinalis.

[0050] Figure 3 This is a standard curve diagram of 14 monomeric phenol reference standards.

[0051] Figure 4 The DPPH• scavenging rate of different extracts from Cornus officinalis flowers and fruits.

[0052] Figure 5 The scavenging rate of •OH by different extracts of Cornus officinalis flower and fruit.

[0053] Figure 6 Different extracts from Cornus officinalis flowers and fruits on ABTS + • Clearance rate.

[0054] Figure 7 The inhibitory effects of different extracts from Cornus officinalis flowers and fruits on α-glucosidase were investigated.

[0055] Figure 8 The inhibitory effects of different extracts from Cornus officinalis flowers and fruits on α-amylase were investigated.

[0056] Figure 9 The inhibitory effects of different extracts from Cornus officinalis flowers and fruits on pancreatic lipase.

[0057] Figure 10 This is an image of organs from mice with acute toxicity.

[0058] Figure 11 The images show the appearance of mice in each group.

[0059] Figure 12 Images of mouse liver, kidney, and pancreas.

[0060] Figure 13 The graphs show the blood glucose curves and AUC values ​​for OGTT and ITT in mice (A: OGTT blood glucose curves for each group of mice; B: ITT blood glucose curves for each group of mice; C: OGTT blood glucose AUC values ​​for each group of mice; D: ITT curve AUC values ​​for each group of mice; ##: Extremely significant difference compared to the control group). P<0.01 **: Significant difference compared to the model group () P<0.01 )).

[0061] Figure 14 A graph showing the levels of INS and ADPN in mouse serum (compared to the control group, #: p<0.05 ;##: P<0.01 Compared with the model group, *: p<0.05 ;**: P<0.01 ).

[0062] Figure 15 A graph showing the level of kidney function in mice (compared to the control group, #:). p<0.05 ;##: P<0.01 Compared with the model group, *: p<0.05 ;**: P<0.01 ).

[0063] Figure 16 A graph showing blood lipid levels in mice (compared to the control group, #:). p<0.05 ;##: P<0.01 Compared with the model group, *: p <0.05 ;**: P<0.01 ).

[0064] Figure 17 A graph showing serum ALT and AST levels in mice (compared to the control group, #:). p<0.05 ;##: P<0.01 Compared with the model group, *: p<0.05 ;**: P<0.01 ).

[0065] Figure 18 A graph showing the levels of inflammatory factors in mouse serum (compared to the control group, #:). p<0.05 ;##: P<0.01 Compared with the model group, *: p<0.05 ;**: P<0.01 ).

[0066] Figure 19 Antioxidant levels in mice (compared to the control group, #:) p<0.05 ;##: P<0.01 Compared with the model group, *: p<0.05 ;**: P<0.01 ).

[0067] Figure 20 Image of mouse liver stained with HE (×200).

[0068] Figure 21 HE staining images of mouse kidneys (×400) (Blank group (A); Model group (B); Low-dose group (C); Medium-dose group (D); High-dose group (E); Positive control group (F)).

[0069] Figure 22 HE staining images of mouse pancreas (×400) (A: blank group; B: model group; C: low-dose group; D: medium-dose group; E: high-dose group; F: positive control group)).

[0070] Figure 23 The expression levels of mouse kidney pathway-related factors mRNA (compared to the control group, #: p<0.05 ;##: P<0.01 Compared with the model group, *: p<0.05 ;**: P<0.01 ).

[0071] Figure 24 The expression levels of inflammatory cytokines mRNA in mouse kidneys (compared to the control group, #: p<0.05 ;##: P<0.01 Compared with the model group, *: p<0.05 ;**: P<0.01 ).

[0072] Figure 25 Immunohistochemical staining of mouse kidneys (×200) and positive area map (compared with the blank group, #: p< 0.05 ;##: P<0.01 Compared with the model group, * ：p<0.05 ;**: P<0.01 ). Detailed Implementation

[0073] The following non-limiting embodiments are intended to enable those skilled in the art to gain a more comprehensive understanding of the present invention, but do not limit the invention in any way. The following content is merely an exemplary description of the scope of protection claimed by the present invention, and those skilled in the art can make various changes and modifications to the present invention based on the disclosed content, and such changes should also fall within the scope of protection claimed by the present invention.

[0074] The present invention will be further described below by way of specific embodiments. Unless otherwise specified, all instruments, devices, equipment, reagents, products, etc., used in the embodiments of the present invention are obtained through conventional commercial means.

[0075] The information on raw materials used and experimental instruments and equipment is shown in Table 1 below: Table 1. Raw Material and Equipment Information

[0076] Example 1: Preparation of Cornus officinalis flower water extract (denoted as CFAE) Weigh 100g of Cornus officinalis flower powder, add 1000mL of distilled water, sonicate for 35min, and reflux at 45℃ three times for 3h each time. Centrifuge and combine the supernatants. Concentrate the extract by rotary evaporation at 45℃, centrifuge at 12500r / min for 3min, collect the supernatant, vacuum dry at 45℃, and store at 4℃ for later use.

[0077] Example 2: Preparation of Cornus officinalis flower methanol extract (denoted as CFME) The difference from Example 1 is that 1000 mL of distilled water was replaced with 500 mL of methanol, otherwise the same.

[0078] Example 3: Cornus officinalis flower ethyl acetate extract (denoted as CFEAE) The difference from Example 1 is that 1000 mL of distilled water was replaced with 500 mL of ethyl acetate, otherwise the same.

[0079] Comparative Example 1: Cornus officinalis fruit aqueous extract (denoted as CBAE) The difference from Example 1 is that the cornelian cherry flowers are replaced with cornelian cherry fruits, otherwise they are the same.

[0080] Comparative Example 2: Cornus officinalis fruit methanol extract (denoted as CBME) The difference from Example 2 is that the cornelian cherry flowers are replaced with cornelian cherry fruits, otherwise they are the same.

[0081] Comparative Example 3: Cornus officinalis fruit ethyl acetate extract (denoted as CBEAE) The difference from Example 3 is that the Cornus officinalis flowers are replaced with Cornus officinalis fruits, otherwise they are the same.

[0082] Comparative Example 4 Unlike Example 1, the extraction conditions were changed from "ultrasonic extraction for 35 min, reflux extraction at 45℃" to "hot reflux extraction at 100℃," while all other conditions remained the same. The contents of total polyphenols, total flavonoids, total polysaccharides, and proanthocyanidins in the obtained extract were 208.22 mg / g, 18.01 mg / g, 199.46 mg / g, and 0.29 mg / g, respectively. The contents of the six marker monomers—monoside, strychnine, loganin, ursolic acid, oleanolic acid, and cornus glycoside—were 3.21 mg / g, 1.37 mg / g, 1.19 mg / g, 4.85 mg / g, 20.28 mg / g, and 4.56 mg / g, respectively. The contents of the 14 monomers—phenolic gallic acid, protocatechuic acid, ... The contents of p-hydroxybenzoic acid, catechin, chlorogenic acid, caffeic acid, epicatechin, scutellarin, rutin, ferulic acid, benzoic acid, resveratrol, quercetin, and cinnamic acid were 61.25 mg / g, 0.89 mg / g, 0.09 mg / g, 7.22 mg / g, 0.40 mg / g, 0.29 mg / g, 2.44 mg / g, 0.68 mg / g, 0.31 mg / g, 11.86 mg / g, 0.20 mg / g, 2.85 mg / g, and 3.09 mg / g, respectively.

[0083] Comparative Example 5 Unlike Example 1, the extraction conditions were changed from "ultrasonic extraction for 35 min, reflux extraction at 45°C" to "microwave extraction, with microwave power set at 600W, temperature at 45°C, and time at 30 min," while all other conditions remained the same. The extracted extract contained 221.22 mg / g of total polyphenols, 20.01 mg / g of total flavonoids, 205.46 mg / g of total polysaccharides, and 0.41 mg / g of proanthocyanidins, respectively. The contents of the six marker monomers—monoside, strychnine, loganin, ursolic acid, oleanolic acid, and cornus glycoside—were 3.78 mg / g, 1.65 mg / g, 1.41 mg / g, 4.92 mg / g, 21.34 mg / g, and 4.89 mg / g, respectively. The extract also contained 14 monomers—phenolic gallic acid, protocatechuic acid, and para-glucan… The contents of hydroxybenzoic acid, catechin, chlorogenic acid, caffeic acid, epicatechin, scutellarin, rutin, ferulic acid, benzoic acid, resveratrol, quercetin, and cinnamic acid were 65.66 mg / g, 1.23 mg / g, 0.11 mg / g, 7.43 mg / g, 0.49 mg / g, 0.36 mg / g, 2.85 mg / g, 0.77 mg / g, 0.40 mg / g, 12.35 mg / g, 0.29 mg / g, 2.99 mg / g, and 3.68 mg / g, respectively.

[0084] Example 1: Identification and Quantitative Analysis of Active Components in Different Extracts of Cornus officinalis Flowers 1. Sample preparation: Weigh 25 mg of each sample powder from Examples 1-3 and Comparative Examples 1-3, and dilute to 25 mL with water to prepare 1 mg / mL Cornus officinalis flower water extract (denoted as CFAE), Cornus officinalis fruit water extract (denoted as CBAE), Cornus officinalis flower methanol extract (denoted as CFME), Cornus officinalis fruit methanol extract (denoted as CBME), Cornus officinalis flower ethyl acetate extract (denoted as CFEAE), and Cornus officinalis fruit ethyl acetate extract (denoted as CBEAE).

[0085] 2. Testing Experiment (1) Determination of total polyphenol content Add 0.8 mL of water and 1 mL of Folin-Ciocalteu reagent to 0.2 mL of control solution (0–1.5 g / L), shake well, incubate in the dark for 5 min, then add 2 mL of 7% sodium carbonate and 1 mL of water. Incubate at 45 °C in the dark for 25 min, and measure the absorbance at 760 nm. Plot a standard curve with the concentration on the x-axis and the measured value on the y-axis. The linear regression equation is as follows: Y =0.0112 x +0.0001, R² =0.9995. The absorbance values ​​of CFAE, CBAE, CFME, CBME, CFEAE, and CBEAE were measured according to the above method. The values ​​were substituted into the standard curve to calculate the total polyphenol content of different extracts. The results are expressed as gallic acid equivalents (mgGAE / g).

[0086] (2) Determination of total flavonoid content Prepare a rutin reference solution (0–1 mg / mL) using DMSO. Take 400 μL of the reference solution, add 120 μL of NaNO₂ (5%) and react for 6 min, then add 120 μL of aluminum nitrate (10%) and react for 6 min. Add 1.6 mL of 1M sodium hydroxide, incubate in the dark for 10 min, and measure the absorbance at 510 nm. Plot a standard curve with the concentration on the x-axis and the absorbance on the y-axis. The linear regression equation is as follows: Y =0.0297 x +0.0034, R² =0.9993. The absorbance values ​​of CFAE, CBAE, CFME, CBME, CFEAE, and CBEAE were determined using the method described above. The values ​​were then substituted into the standard curve to calculate the total flavonoid content of different extracts. The results were expressed as rutin equivalents (mgRE / g).

[0087] (3) Determination of total polysaccharide content Add 1 mL of phenol (6%) and 5 mL of concentrated sulfuric acid to 1 mL of glucose (0–3 mg / mL) control solution, mix well, let stand for 5 min, incubate at 30 °C for 15 min, and measure the absorbance at 490 nm. Plot a standard curve with glucose concentration on the x-axis and absorbance on the y-axis. The linear regression equation is as follows:Y =0.3609 x -0.0004, R² =0.9998. The absorbance values ​​of different extracts were determined using the method described above. The values ​​were then substituted into the standard curve to calculate the total polysaccharide content of CFAE, CBAE, CFME, CBME, CFEAE, and CBEAE. The results are expressed as (mg / g).

[0088] (4) Determination of proanthocyanidin content Prepare 5% vanillin and 10% hydrochloric acid solutions and mix them. Take 100 μL of 1-5 mg / mL proanthocyanidin control solution and add it to 500 μL of the above mixture. Incubate at 30℃ for 10 min and measure the absorbance at 500 nm. Plot a standard curve with concentration on the x-axis and absorbance on the y-axis. The regression equation is as follows: Y =0.0203 x +0.0935, R² =0.9996. The absorbance values ​​of different extracts were determined using the method described above. Substituting these values ​​into the standard curve, the proanthocyanidin contents of CFAE, CBAE, CFME, CBME, CFEAE, and CBEAE were calculated. Results are expressed in mg / g. The contents of total polyphenols, total flavonoids, total polysaccharides, and proanthocyanidins are shown in the table below: Table 2. Content of phytochemicals in different extracts of Cornus officinalis flowers and fruits

[0089] Note: Different lowercase letters in the same column of the table indicate that the content of extracts from different Cornus officinalis flowers and fruits varies significantly. P< 0.01 ).

[0090] It can be seen that the content differences among the active ingredients CFAE, CBAE, CFME, CBME, CFEAE, and CBEAE are extremely significant. P< 0.01 The total polyphenol content in CFAE was higher than that in other extracts, at 235.79±0.17 mg GAE / g, followed by CBME at 226.32±0.51 mg GAE / g, while CBEAE had the lowest content at 206.04±0.20 mg GAE / g. Meanwhile, the total polyphenol content in CFAE and CFME was higher than that in the ethyl acetate extract, indicating that water and methanol are more suitable for the precipitation of polyphenols from Cornus officinalis. The total flavonoid content in different extracts of Cornus officinalis was ranked as follows: CFAE>CFEAE>CBEAE>CBAE>CBME>CFME, with CFAE reaching a total flavonoid content of 23.70±0.14 mg RE / g. Furthermore, the total flavonoid content in the water extract and ethyl acetate extract of Cornus officinalis flower and fruit was higher than that in the methanol extract of Cornus officinalis flower and fruit. Regarding the total polysaccharide content, the detected content in different extracts of Cornus officinalis flower was significantly lower than that in the corresponding solvent extract of Cornus officinalis fruit.P<0.01 Among the different extracts of Cornus officinalis flower, CBAE had the highest content, reaching 340.13±0.89 mg / g; CFME had the lowest total polysaccharide content, at 42.26±0.78 mg / g. CFAE had the highest content among the different extracts of Cornus officinalis flower, at 228.47±0.68 mg / g, significantly higher than CFME and CFEAE. P<0.01 The total anthocyanin content in different extracts of Cornus officinalis, from highest to lowest, was CFEAE>CFME>CBME>CFAE>CBEAE>CBAE. Furthermore, the anthocyanin content in different extracts of Cornus officinalis flowers was higher than that in the corresponding solvent fruit extracts, indicating that anthocyanins are more abundant in Cornus officinalis flowers.

[0091] (5) Identification of chemical components in extracts of Cornus officinalis flowers and fruits S1. Chromatographic conditions: Column: UPLC-BEH-C18 column (100×2.1mm, 1.7μm), mobile phase: acetonitrile (A); 0.3% phosphoric acid (B), flow rate: 0.3mL / min, detection temperature: 30℃, injection volume: 1μL, detection wavelength: 200-460nm, elution program: 0-2min, 5%A; 2-5min, 10-20%A; 5-10min, 20-30%A; 10-20min, 30-45%A; 20-25min, 45-65%A; 25-50min, 65-80%A; 50-55min, 80-30%A; 55-60min, 30-10%A.

[0092] S2.MS Analysis Conditions: A Hydrogel™ high-performance liquid chromatography system was used, equipped with a Synapt G2 mass spectrometer featuring an electrospray ionization (ESI) source. A PDA detector was employed, with a detection wavelength of 200–400 nm. Mass spectrometry detection was performed using electrospray ionization and positive and negative ion mode detection. The ion source temperature was 100 °C, and the capillary voltage was 3 kV (ESI). + ) and 2.5kV (ESI) ‐ The parameters were: cone voltage 40V, desolvation gas temperature 350℃, collision energy 6.0eV, desolvation gas flow rate 600L / h, and cone gas flow rate 50L / h. The scanning range was 50-1200 (…). m / z ).

[0093] (6) UPLC determination of pharmacologically active content in extracts Six main active monomer components from different extracts of Cornus officinalis flowers and fruits were determined under the following conditions: Chromatographic column: Inersil ODS-3C 18Column (150 mm × 4.6 mm, 4 μm), mobile phase A:B = acetonitrile: 0.3% phosphoric acid, flow rate 1.0 mL / min, column temperature: 30 °C, injection volume: 10 μL, detection wavelengths 210, 218, and 240 nm. Elution conditions: 0–5 min, 80–90% B; 5–10 min, 70–80% B; 10–20 min, 60–80% B; 20–35 min, 40–60% B; 35–55 min, 20–40% B; 55–60 min, 20% B. Each sample was repeated three times.

[0094] Column: Inertsil / WondaSilC 18 (250 mm × 4.6 mm, 4 μm) column, injection volume: 10 μL; mobile phase A:B = acetonitrile: 0.2% glacial acetic acid, flow rate: 0.8 mL / min, elution program: 0–15 min, 94–95% B; 15–30 min, 70–94% B; 30–35 min, 55–70% B; 35–45 min, 45–55% B, column temperature: 30 °C, peak area of ​​monomeric phenol was determined at 280 nm. Each sample was repeated 3 times.

[0095] 3. Data Analysis All experiments were repeated three times. The results of the content of the six main pharmacologically active components and 14 monomeric phenols in CFAE, CBAE, CFME, CBME, CFEAE, and CBEAE are shown in Table 3.

[0096] The contents of mononoside, strychnine, and loganin in different extracts of Cornus officinalis flowers were significantly lower than those in the corresponding solvent extracts of Cornus officinalis fruits. Furthermore, the contents of mononoside varied significantly among different solvent extracts of Cornus officinalis flowers and fruits. P<0.01The mononoside content in the three solvents was CFME > CFAE > CFEAE, indicating that mononosides are easily affected by the extraction solvent and are more readily bound to methanol and water than ethyl acetate. The mononoside contents of CBME, CBEAE, and CBAE were 13.64±0.23 mg / g, 12.36±0.28 mg / g, and 8.73±0.15 mg / g, respectively, which were higher than those of CFME, CFEAE, and CFAE. Among CFAE, CBAE, CFME, CBME, CFEAE, and CBEAE, the CBAE loganin content was 11.95±0.29 mg / g, significantly higher than the other extracts; CFEAE had the lowest content, at only 1.20±0.04 mg / g. The contents of ursolic acid and oleanolic acid in CFAE were 5.13±0.03 mg / g and 24.55±0.47 mg / g, respectively, which were significantly higher than those in CBAE, CFME, CBME, CFEAE, and CBEAE. Furthermore, the oleanolic acid content in CFAE and CFME was higher than that in the corresponding solvent extract of Cornus officinalis fruit. P<0.01 The contents of CFEAE and CBEAE in the extracts were significantly lower than those in other extracts, at 0.43±0.05 mg / g and 17.31±0.23 mg / g, respectively. The contents of new cornosides detected in different extracts of Cornus officinalis flowers and fruits, from highest to lowest, were CBME>CFAE>CFME>CFEAE>CBAE>CBEAE, and there were highly significant differences in the contents among the different extracts of Cornus officinalis flowers and fruits. P<0.01 ).

[0097] The results of monomeric phenol content analysis for CFAE, CBAE, CFME, CBME, CFEAE, and CBEAE showed that resveratrol was not detected in CBAE, while the other monomeric phenols were detectable in different extracts of Cornus officinalis flowers and fruits. The differences in monomeric phenol content among CFAE, CBAE, CFME, CBME, CFEAE, and CBEAE were highly significant. P<0.01 The gallic acid content of CFAE and CFME was significantly higher than that of other extracts, at 76.49±0.02 mg / g and 32.20±0.29 mg / g, respectively; and the gallic acid content of different solvent extracts of Cornus officinalis flowers was higher than that of the corresponding solvent extracts of Cornus officinalis fruits. P<0.01 The contents of p-hydroxybenzoic acid and resveratrol were low in CFAE, CBAE, CFME, CBME, CFEAE, and CBEAE. Resveratrol was not detected in CBAE, but its content in CBAE and CBEAE was significantly higher than that in Cornus officinalis flower extract. P<0.01Furthermore, the resveratrol content in CFAE was lower than that in CFME and CFEAE, indicating that resveratrol is more readily bound to organic solvents and precipitated than water. CBME had the highest protocatechuic acid content at 31.40±0.33 mg / g, followed by CBAE at 8.06±0.07 mg / g, both significantly higher than the contents of different extracts of Cornus officinalis flowers. P<0.01 Meanwhile, the chlorogenic acid content in different extracts of Cornus officinalis fruit was higher than that in the corresponding extracts of Cornus officinalis flower, indicating that the chlorogenic acid content in Cornus officinalis flower is slightly lower than that in Cornus officinalis fruit. Furthermore, the contents of catechin, ferulic acid, benzoic acid, and cinnamic acid in CFAE were significantly higher than those measured in CBAE, CFME, CBME, CFEAE, and CBEAE; the contents of epicatechin, rutin, and quercetin in CFME extract were significantly higher than those in CFAE and CFEAE; CFEAE had the highest caffeic acid content (8.31±0.06 mg / g), followed by CBEAE (5.47±0.08 mg / g), both significantly higher than the measured contents in CFAE, CBAE, CFME, and CBME, indicating that ethyl acetate extracts of Cornus officinalis flower and fruit are more readily precipitated for caffeic acid than water and methanol.

[0098] Table 3. Main pharmacological components and content of 14 monomeric phenols in different extracts of Cornus officinalis flower and fruit.

[0099] Note: "-" indicates that it was not detected; different lowercase letters in the same row indicate that the content of Cornus officinalis flower and fruit extracts differs significantly. P<0.01 ).

[0100] Example 2: In vitro activity evaluation and analysis of different extracts from Cornus officinalis flowers 1. In vitro antioxidant activity study (1) DPPH • Scavenging ability Take 80 μL of each of the gradient concentrations of CFAE, CBAE, CFME, CBME, CFEAE, and CBEAE, respectively, and mix with 120 μL of 0.15 μg / μL DPPH. Shake well, in the dark for 0.5 h, and measure the OD value at 517 nm. A i ). DPPH was performed using water replacement to determine the CFAE, CBAE, CFME, CBME, CFEAE, and CBEAEOD values. A i0 CFAE, CBAE, CFME, CBME, CFEAE, and CBEAE were measured with water as a substitute for the OD values ​​of the blank group. A j The formulas for the clearance rates of different extracts from Cornus officinalis flowers and fruits are as follows: DPPH clearance rate (%) =

[0101] (2) •OH scavenging ability 80 μL of different concentrations of CFAE, CBAE, CFME, CBME, CFEAE, and CBEAE were respectively mixed with 80 μL of FeSO4, 80 μL of salicylic acid-ethanol solution, and 60 μL of H2O2. The mixtures were incubated at 37℃ in the dark for 0.5 h, and the absorbance was measured at 510 nm. A i The OD values ​​of CFAE, CBAE, CFME, CBME, CFEAE, and CBEAE were determined using H2O2 instead of water. A i0 ); CFAE, CBAE, CFME, CBME, CFEAE, CBEAE were measured by replacing the blank OD value with water ( A j The formulas for the clearance rates of different extracts from Cornus officinalis flowers and fruits are as follows: •OH removal rate (%) =

[0102] (3) ABTS + • Clearance Take 7mMABTS + • Add 528 μL of 2.4 mM potassium persulfate to 30 mL of the solution and incubate in the dark for 12 h. Dilute with ethanol to a concentration of 0.70 ± 0.02 at 734 nm. Take 1500 μL of the above solution and 50 μL of different concentrations of CFAE, CBAE, CFME, CBME, CFEAE, and CBEAE, mix well, and incubate in a water bath at 25 °C for 35 min. Measure the OD value at 734 nm. A i The mixture was replaced with water to determine the OD values ​​of CFAE, CBAE, CFME, CBME, CFEAE, and CBEAE. A i0 ); Different extracts of Cornus officinalis flowers and fruits were used to determine the OD values ​​of the blank group using water instead of water. A j The formulas for calculating the clearance rates of different extracts from Cornus officinalis flowers and fruits are as follows: ABTS + Clearance rate (%) =

[0103] 2. In vitro enzyme inhibitory activity assay (1) α-glucosidase inhibitory activity Add 150 μL of PBS (pH 6.8) and 50 μL of 1 U / mL enzyme solution to 150 μL of a gradient concentration of CFAE, CBAE, CFME, CBME, CFEAE, and CBEAE, and incubate at 37°C for 10 min. Add 150 μL of 2.5 mg / mL pNPG and incubate at 37°C for 30 min. Stop the reaction with 200 μL of sodium carbonate (1 M), and measure the OD at 405 nm. A CFAE, CBAE, CFME, CBME, CFEAE, and CBEAE were measured by replacing the blank OD with water, respectively. A 0), enzyme solution was replaced with buffer solution for OD measurement ( A si The formula for calculating the inhibition rate of different extracts from Cornus officinalis flowers and fruits is as follows: α-glucosidase inhibition rate (%) =

[0104] (2) α-Amylase inhibitory activity Take 50 μL of each of the following: FAE, CBAE, CFME, CBME, CFEAE, and CBEAE. Mix each with 50 μL of α-amylase (1.50 μg / mL). Add 50 μL of substrate (derived from corn DQTM 0.2 mg / mL) to initiate the reaction. Detect at 37℃ for 30 min at an emission wavelength of 535 nm and an excitation wavelength of 485 nm, and record the absorbance. A s The absorbance of the extract was measured using PBS instead of CFAE, CBAE, CFME, CBME, CFEAE, and CBEAE, respectively. A 0), PBS was used to replace amylase solution for absorbance detection ( A si (Acarbose was used as a positive control. The formula for calculating the inhibition rate of different extracts from Cornus officinalis flowers and fruits is as follows:) α-Amylase inhibition rate (%) =

[0105] (3) Pancreatic lipase inhibitory activity Add 50 μL of CFAE, CBAE, CFME, CBME, CFEAE, and CBEAE to 50 μL of enzyme solution (1 U / mL), mix, add 100 μL of substrate (0.75 mg / mL), and incubate at 37°C for 15 min. Detect the fluorescence values ​​of CFAE, CBAE, CFME, CBME, CFEAE, and CBEAE at an excitation wavelength of 360 nm and a detection wavelength of 465 nm. A i The detection time was 30 minutes. The blank group was detected by replacing CFAE, CBAE, CFME, CBME, CFEAE, and CBEAE with Tris-HCl solution. A0); the background fluorescence values ​​for CFAE, CBAE, CFME, CBME, CFEAE, and CBEAE are ( A i0 Tris-HCl solution was used instead of enzyme solution for detection. Orlistat was used as a positive control. The inhibition rates of different extracts from Cornus officinalis flowers and fruits were calculated using the following formulas: Pancreatic lipase inhibition rate (%) =

[0106] 3. Data Analysis All experiments were repeated three times, and results are expressed as mean ± SD. AutoDock was used to investigate the docking mechanisms of α-glucosidase, α-amylase, lipase, and other target proteins of popular research pathways. Principal component analysis (PCA) was performed using SIMCA-P14.1 to analyze the content of major substances, antioxidant IC50, and enzyme inhibition IC50 values ​​of CFAE, CBAE, CFME, CBME, CFEAE, and CBEAE. The comprehensive evaluation of antioxidant capacity among CFAE, CBAE, CFME, CBME, CFEAE, and CBEAE was performed using the APC method, calculated as follows:

[0107]

[0108] (1) DPPH • Scavenging ability Depend on Figure 4 As shown, CFAE, CBAE, CFME, CBME, CFEAE, and CBEAE all exhibited DPPH• scavenging effects at certain concentrations, and the scavenging effect was positively correlated with the concentration of different extracts from Cornus officinalis flowers and fruits. Among them, CFAE showed better DPPH• scavenging ability than CFME, CFEAE, and the three Cornus officinalis fruit extracts, with an overall trend similar to that of VC. At 2 mg / mL, CFAE achieved a DPPH• scavenging rate of 55%. When the concentration increased to 4 mg / mL, the scavenging rate reached 87%, significantly higher than CBAE, CFME, CBME, CFEAE, and CBEAE, and comparable to that of VC. In contrast, CBEAE at concentrations of 1-10 mg / mL showed lower DPPH• scavenging rates than CFAE, CFME, and CFEAE. Furthermore, it was found that at the same concentration, the DPPH• scavenging effect of Cornus officinalis flower extracts was CFAE > CFME > CFEAE, indicating that the polarity of the Cornus officinalis flower extract reagent affects its inhibitory ability on DPPH•.

[0109] (2) •OH scavenging ability The scavenging ability of Cornus officinalis flower and fruit extracts against •OH in different solvents is shown in the table below. Figure 5When the concentration of CFAE was 0.1-1 mg / mL and 4-8 mg / mL, its scavenging ability for •OH was superior to that of CBAE, CFME, CBME, CFEAE, and CBEAE. At 1-4 mg / mL, the scavenging effect of CFAE for •OH was comparable to that of CBAE. Furthermore, at 6 mg / mL, only CFAE achieved a •OH scavenging rate of 95%, while the scavenging rates of CBAE, CFME, CBME, CFEAE, and CBEAE were all below 90%. At concentrations of 4-8 mg / mL, the scavenging rates of CFME and CFEAE were higher than those of CBME and CBEAE at the same concentrations, but significantly lower than those of the two aqueous extracts at the same concentrations. This indicates that among extracts using different polar solvents, the aqueous extracts are more effective than those using methanol and ethyl acetate.

[0110] (3) ABTS + • Clearance The effects of different solvents on ABTS of Cornus officinalis flower and fruit extracts + •See clearance rate results Figure 6 . CFAE, CBAE, CFME, CBME, CFEAE, CBEAE vs. ABTS + • The scavenging ability of all components has a certain scavenging effect, and it is related to the concentrations of CFAE, CBAE, CFME, CBME, CFEAE, and CBEAE. The scavenging rates of CFAE at concentrations of 0.1-0.2 mg / mL and 0.6-1.0 mg / mL are greater than those of CBAE, CFME, CBME, CFEAE, and CBEAE. However, the scavenging rate of CBEAE at 0.5 mg / mL is greater than that of CFAE at this concentration. Meanwhile, 0.6 mg / mL CFAE and CBEAE also have a greater scavenging effect on ABTS. + • Its scavenging effect is comparable to that of Vitamin C, and significantly higher than that of CBAE, CFME, and CFEAE. Furthermore, at the same concentration, the ethyl acetate extracts of Cornus officinalis flower and fruit showed better scavenging effect on ABTS. + • Its cleansing effect is superior to that of Cornus officinalis flower and fruit methanol extracts.

[0111] (4) In vitro antioxidant IC50 The antioxidant IC50 results of different extracts from Cornus officinalis flowers and fruits are shown in Table 4.

[0112] Table 4. Antioxidant IC50 values ​​of different extracts from Cornus officinalis flowers and fruits

[0113] Note: Different lowercase letters in the same column of the table indicate that the IC50 differences between extracts are extremely significant. P<0.01 ).

[0114] A smaller IC50 value indicates a better scavenging ability for the corresponding free radical. We found that the IC50 values ​​of CFAE, CBAE, CFME, CBME, CFEAE, and CBEAE showed highly significant differences in their scavenging effects on different free radicals. P<0.01 Studies have shown that, in terms of DPPH• and •OH scavenging ability, different extracts prepared from Cornus officinalis flowers have lower IC50 values ​​than the corresponding Cornus officinalis fruit extracts, indicating that Cornus officinalis flowers are superior to the corresponding Cornus officinalis fruit extracts in scavenging DPPH• and •OH. Furthermore, it was found that for DPPH• scavenging ability, the IC50 values ​​among extracts in different polar solvents were ranked as follows: water extract > alcohol extract > ethyl acetate extract, indicating that the aqueous phase is more suitable than the other two solvents for extracting DPPH• scavenging substances from Cornus officinalis flowers and fruits; for ABTS... + • Scavenging ability: The IC50 value of CFAE is less than that of CBAE, CFME, CBME, CFEAE, and CBEAE, indicating that CFAE is effective against ABTS. + • It has the best scavenging ability compared to other extracts, and the ethyl acetate extracts of Cornus officinalis flower and fruit have been found to have an effect on ABTS. + • Stronger scavenging ability than the methanol extracts of Cornus officinalis flowers and fruits.

[0115] (5) Evaluation of comprehensive antioxidant index The APC index results of extracts of Cornus officinalis flowers and fruits in different polar solvents are shown in Table 5.

[0116] Table 5. APC Index of Different Extracts from Cornus officinalis Flowers and Fruits

[0117] Note: In the same column of the table, a, b, c, and d indicate that there are highly significant differences among the extracts. P<0.01 ).

[0118] It can be seen that the comprehensive antioxidant capacity of extracts of Cornus officinalis flowers and fruits in different polar solvents, from strongest to weakest, is VC>CFAE>CFEAE>CBAE>CFME>CBME>CBEAE. Among them, the antioxidant capacity of CFAE is comparable to that of the positive control VC. At the same time, there are significant differences among CFAE, CBAE, CFME, CBME, CFEAE, CBEA, and PC indices. p<0.05 ).

[0119] (6) α-glucosidase inhibitory activity The inhibitory activities of extracts of Cornus officinalis flowers and fruits on α-glucosidase in different polar solvents are shown in the table below. Figure 7CFAE, CBAE, CFME, CFEAE, and CBEAE can all inhibit α-glucosidase, and their inhibitory effects are concentration-dependent. CFME shows greater inhibitory effects on α-glucosidase than CFAE, CBAE, CBME, CFEAE, and CBEAE at concentrations of 1-10 mg / mL. CFAE and CFME show higher inhibition rates than CBAE and CBME at a concentration of 10 mg / mL. CFAE at 20-25 mg / mL achieves an inhibition rate of 99% on α-glucosidase, significantly higher than the inhibition rates of other extracts of Cornus officinalis flower and fruit, and acarbose at the same concentration.

[0120] (7) α-Amylase inhibitory activity Depend on Figure 8 It was found that CFAE, CBAE, CFME, CBME, CFEAE, and CBEAE all exhibited certain inhibitory abilities against α-amylase, and these abilities were concentration-dependent. Furthermore, it was observed that among different extracts of Cornus officinalis flowers and fruits, CFAE and CBAE showed stronger inhibitory activities against α-amylase than CFME, CFEAE, CBME, and CBEAE. At different solvent extract concentrations of Cornus officinalis flowers and fruits ranging from 1 to 16 mg / mL, there were highly significant differences in the inhibition rates of CFAE, CBAE, CFME, CBME, CFEAE, and CBEAE. P<0.01 Meanwhile, when the concentration of CFAE reached 1 mg / mL, its inhibitory effect was significantly greater than that of CBAE, CFME, CBME, CFEAE, and CBEAE, and similar to that of acarbose, indicating that CFAE has a good inhibitory effect on α-amylase.

[0121] (8) Pancreatic lipase inhibitory activity Depend on Figure 9 It was found that CFAE, CBAE, CFME, CBME, CFEAE, and CBEAE all inhibited α-glucosidase, and their inhibitory effects were positively correlated with the extract concentration. The inhibition rate of lipase by 0.1-1 mg / mL CFAE was greater than that of CBAE, CFME, CBME, CFEAE, and CBEAE at the same concentration; the inhibitory effect of 10 mg / mL CFAE on lipase was comparable to that of orlistat. Meanwhile, the ethyl acetate extract of Cornus officinalis flower and fruit at 5 mg / mL showed a greater inhibition rate of lipase than other extracts, while the methanol extract of Cornus officinalis flower and fruit had the lowest inhibition rate at this concentration compared to other extracts. Furthermore, we found that the inhibition rates of 10 mg / mL CFAE, CBAE, CFME, CBME, CFEAE, and CBEAE were close to those of orlistat, indicating that CFAE, CBAE, CFME, CBME, CFEAE, and CBEAE all have strong inhibitory effects on lipase.

[0122] (9) Enzyme inhibitory activity IC50 The IC50 results of the extracts of Cornus officinalis flowers and fruits in different polar solvents on the inhibition of three enzymes are shown in Table 6.

[0123] Table 6. Enzyme inhibition ability (IC50 values) of different extracts from Cornus officinalis flowers and fruits.

[0124] Note: Different lowercase letters in the same column of the table indicate that the differences between CBAE, CFME, CBME, CFEAE, and CBEAE are extremely significant. P< 0.01 ).

[0125] As shown in the table, the IC50 values ​​of CFAE for inhibiting α-glucosidase and α-amylase are lower than those of CBAE, CFME, CBME, CFEAE, and CBEAE, indicating that CFAE's inhibitory ability against α-glucosidase and α-amylase is superior to other solvent extracts of Cornus officinalis flowers and fruits. Meanwhile, the IC50 value of CFEAE for inhibiting α-glucosidase is higher than that of CFME but lower than that of CBEAE. Furthermore, there are highly significant differences in the IC50 values ​​of CFAE, CBAE, CFME, CBME, CFEAE, and CBEAE for inhibiting amylase. P<0.01 The IC50 values ​​of CFAE and CBAE for lipase inhibition were significantly greater than those of CFME, CBME, CFEAE, and CBEAE, indicating that the aqueous extracts of Cornus officinalis flowers and fruits exhibited superior inhibitory ability compared to the alcoholic extracts and ethyl acetate extracts of Cornus officinalis flowers and fruits.

[0126] Example 3: Aqueous extract of Cornus officinalis flower inhibited inflammatory response in diabetic nephropathy mice via the PI3K / AKT / mTOR pathway. 1. Acute toxicity test Four Kunming mice (half male and half female) were randomly divided into a control group and a treatment group. After 16 hours of fasting with free access to water, the treatment group was administered CFAE by gavage at the maximum therapeutic concentration (400 mg / mL) and maximum administration volume (40 mL / kg / dose), while the control group received an equal dose of water. The treatment was repeated 12 hours later, with fasting but free access to water throughout the treatment period. After 7 days of routine feeding and observation, no mice died. Therefore, the MTD method was used to determine the acute oral toxicity of the extract.

[0127] Twenty mice (half male and half female) were randomly divided into a control group and a treatment group. Both groups were administered the drug according to the above-described administration conditions. Mice were observed for 14 consecutive days, during which time changes in body weight, food and water intake, general condition, and mortality were recorded for each group.

[0128] 2. Establishment of animal models Ninety Kunming mice were housed in an environment of 22℃ and 50% humidity. After 7 days of acclimatization, the control group was fed a normal diet, while the rest were fed a high-fat, high-sugar diet. Four weeks later, the high-fat, high-sugar mice were intraperitoneally injected (once daily for 3 days) with 50 mg / kg STZ citrate buffer (pH 4.5) to establish a type 2 diabetes mellitus (T2DM) model, while the remaining mice were injected with an equal volume of physiological saline. After modeling, mice with a random blood glucose level ≥16.7 mmol / L were randomly divided into a model group, a low-dose Cornus officinalis flower water extract group, a medium-dose Cornus officinalis flower water extract group, a high-dose Cornus officinalis flower water extract group, and a positive control group.

[0129] Gavage dosage: The dosage of Cornus officinalis flower water extract in each treatment group was 500 mg / kg CFAE, 1000 mg / kg CFAE, and 2000 mg / kg CFAE, respectively; the positive control group received 200 mg / kg metformin hydrochloride; the blank control and model groups received 200 mg / kg physiological saline by gavage. Gavage was administered once daily for 4 weeks. During the gavage period, the blank control group continued to be fed a normal diet, while the Cornus officinalis flower water extract gavage groups and the positive control group continued to be fed a high-fat, high-sugar diet. Blood glucose, body weight, water intake, and food intake were measured in the mice during the period.

[0130] 3. Animal handling Mice were fasted for 8 hours before the end of the experiment, but allowed free access to water, and their oral glucose tolerance test (OGTT) was measured. One day later, the intraperitoneal time (ITT) results were measured. After fasting overnight, mice were anesthetized with ether, their whiskers were removed, blood was collected from their sinuses, and they were euthanized by cervical dislocation. Liver, kidney, and pancreas samples were collected from each group, and relevant organ indices were measured.

[0131] Blood samples: After standing at room temperature for 30 minutes, centrifuge at 3000 r / min for 10 minutes at 4℃, collect serum, and store at -20℃ for later use.

[0132] Tissue samples: A portion of tissue from each animal was fixed with 4% paraformaldehyde solution, and the remaining tissue was stored at -80°C for later use.

[0133] 4. Measurement of serum-related factors The levels of INS, ADPN, BUN, Scr, TC, TG, HDL-C, LDL-C, ALT, and AST in mice in the blank group, model group, low-dose CFAE group, medium-dose CFAE group, high-dose CFAE group, and positive group were detected using a kit.

[0134] The levels of TNF-α, MCP-1, IL-6 and IL-1β were determined using an ELISA kit.

[0135] 5. Determination of renal tissue homogenate and biochemical indicators Kidney tissue was collected, added to ice-cold physiological saline (1:9), homogenized, centrifuged at 3000 r / min for 10 min, and the supernatant was collected and stored at -20℃. The contents of SOD, GSH-Px, CAT, and MDA in tissue homogenates of blank group, model group, low-dose CFAE group, medium-dose CFAE group, high-dose CFAE group and positive group were detected.

[0136] 6. Histopathological observation (1) Tissue embedding: The tissue was dehydrated 6 times with gradient ethanol (75%-100%) for 60 min; xylene: ethanol (1:1) clearing for 10 min; xylene clearing for 10 min, 2 times; xylene: wax (1:1) immersion for 30 min, immersion for 1 h again, repeated 3 times; after immersion for 1 h, let stand overnight; trim the wax block and cut 5 μm sections with a pathological microtome.

[0137] (2) HE staining: (1) Dewax twice with xylene, 10 min each time; (2) Soak in 100% ethanol for 5 min, repeat once; soak in 95% and 80% ethanol for 5 min each, repeat once; (3) Place in H2O for 5 min; (4) Stain with hematoxylin for 0.5-2 min, rinse with running water for 5 min to return to blue; (5) Stain with eosin for 10 min; (6) Dehydrate with 80% ethanol for 2 min, dehydrate twice with 95% and 100% ethanol, 10 min each time; (7) Clear with xylene for 10 min, repeat twice; (8) Mount with neutral resin and store.

[0138] 7. Immunohistochemistry The protein expression levels of PI3K, AKT, mTORC1, and SREBP-1 in the liver were detected by immunohistochemistry.

[0139] The methods and steps are as follows: Sections were dewaxed with xylene for 30 min, 10 min per wash; dehydrated with an ethanol gradient (100%–50%) for 5 min per wash; washed three times with PBS-T (0.01 M, pH 7.4) for 5 min per wash; infiltrated with 1% Triton-100 for 30 min; boiled in CBS (0.01 M, pH 6.0) for 20 min, cooled, and then soaked in 3% H2O2 for 30 min; washed with PBS for 5 min, repeated three times; circled around the tissue with an immunohistochemical pen; blocked with 5% BSA at room temperature for 20 min. Wash with PBS-T for 5 min, repeat 3 times; add primary antibody, incubate at 37℃ for 2 h, wash with PBS-T for 2, 3, 5, and 8 min respectively; add secondary antibody and incubate at 37℃ for 1.5 h; wash again with PBS-T for 2, 3, 5, and 8 min each time; add DAB chromogenic solution, develop for 2.5 min, then wash with PBS-T for 2, 3, 5, and 8 min; counterstain with hematoxylin for 1 min; dehydrate with gradient ethanol (75-100%) for 5 min, clear with xylene for 5 min, repeat once; mount with neutral resin, bake and store.

[0140] 8. Determination of short-chain fatty acids Before euthanizing the mice, feces from each group were collected using sterile centrifuge tubes and stored at -80℃. 500 mg of fecal samples from the blank group, model group, low-dose CFAE group, medium-dose CFAE group, high-dose CFAE group, and positive group were added to 2 mL of extraction solvent (composed of 0.1 mol / L oxalic acid, 40 mmol / L sodium azide, and 0.1 mmol / L hexanoic acid), ground in liquid nitrogen, shaken for 60 min, centrifuged at 14000g for 10 min, and filtered through a 0.45 μm organic filter membrane to obtain the supernatant.

[0141] Gas chromatography analysis was performed using a Shimadzu GC-2014C system equipped with a flame ionization detector (FID), an air generator, and a hydrogen generator (Shanghai Quanpu Scientific Instruments Co., Ltd.). Chromatographic separation was achieved on a DB-5 column (30m × 0.25mm × 0.25μm) with hydrogen and air flow rates of 40 and 300 mL / min, respectively. The carrier gas flow rate was 20 mL / min, the injection volume was 1 μL, and the split ratio was 30:1. The temperature program was as follows: initial oven temperature: 50℃, 3 min; ramp to 120℃ at 35℃ / min and hold for 2 min; then ramp to 180℃ at 20℃ / min and hold for 3 min; FID and injection port temperatures were 250℃.

[0142] 9. Results and Analysis (1) Oral acute toxicity analysis The results of the acute oral toxicity test of Cornus officinalis flower water extract on mice are shown in Table 7. Figure 10 .

[0143] Table 7. Changes in mouse body weight, food and water intake

[0144] The results showed that no mice died after administration or during the feeding period. The mice in the Cornus officinalis flower water extract gavage group showed no difference in behavior and general condition compared to the control group, and no signs of poisoning were observed. At the same time, there were no significant changes in mouse weight, food and water intake during the experiment. Gross dissection revealed no obvious lesions in the organs of the two groups of mice, indicating that the maximum tolerable daily dose of Cornus officinalis flower water extract in mice is ≥32g / kg. This confirms that Cornus officinalis flower water extract is non-toxic to mice and can be used for further experimental research.

[0145] (2) Changes in mouse morphology, liver, kidneys and pancreas The morphological results of each group of mice are shown in the figure. Figure 11 It can be observed that the model group mice are thinner than the control group mice, with dirty and messy fur and sparser whiskers and hair; the low and medium dose Cornus officinalis flower water extract gavage groups showed increased body size compared to the model group, while the high dose Cornus officinalis flower water extract treatment group and the positive control group had body size comparable to the control group, and their fur was neat and complete.

[0146] from Figure 12 It was found that the liver, kidneys, and pancreas in the model group were lighter in color and smaller in shape compared with the blank group. The low and medium doses of Cornus officinalis flower water extract administered by gavage showed improvement compared with the model group. The high dose of Cornus officinalis flower water extract administered by gavage and the positive control group showed better improvement. This indicates that Cornus officinalis flower water extract can restore the liver, kidneys, and pancreas damage caused by STZ-induced T2DM, and the high dose extract is comparable to the positive control.

[0147] (3) Changes in mouse body weight Table 8 shows the results of T2DM model establishment and mouse body weight during gavage administration of Cornus officinalis flower water extract.

[0148] Table 8. Changes in mouse body weight (g) during gavage during model establishment

[0149] Note: Compared with the blank group, #: p<0.05 ;##: P<0.01 Compared with the model group, *: p<0.05 .

[0150] It can be seen that after feeding with a high-fat, high-sugar diet combined with STZ modeling, the body weight of mice in the treatment groups showed significant differences compared with the blank group. p<0.05The mice in the model group showed a significant decrease in body weight, and their weight decreased progressively throughout the entire modeling and treatment process. The mice in the low, medium, and high dose treatment groups of Cornus officinalis flower water extract showed a decreasing trend in body weight during weeks 1, 2, and 3, but their body weight increased to some extent in week 4 after administration. These results indicate that Cornus officinalis flower water extract can increase the body weight of T2DM mice, and this effect is related to the dose of Cornus officinalis flower water extract. Furthermore, the high-dose Cornus officinalis flower water extract gavage group showed an effect comparable to the positive control group.

[0151] (4) Changes in the amount of food and water consumed by mice Table 9 shows the results of food intake during the gavage treatment in mice to establish the mouse model. It can be seen that the food intake of the control group mice remained relatively stable throughout the entire gavage treatment process, and there was a highly significant difference in food intake between the control group and other treatment groups. P<0.01 The food intake of mice in the model group gradually increased during the gavage treatment for modeling, exhibiting polyphagia symptoms of type 2 diabetes mellitus (T2DM). Furthermore, significant differences were observed between the mice in the model group and the different dose groups of Cornus officinalis flower water extract and the positive control group at week 1 of gavage treatment. p<0.05 There were highly significant differences in weeks 3 and 4. P<0.01 Meanwhile, it was found that after treatment with Cornus officinalis flower water extract, the food intake of mice in different doses of Cornus officinalis flower water extract gavage group decreased significantly in week 4, which was similar to the trend of food intake in the positive control group, indicating that Cornus officinalis flower water extract has a certain alleviating effect on polyphagia symptoms in T2DM.

[0152] Table 9. Changes in food intake in mice during gavage during model establishment (g)

[0153] Note: Compared with the blank group, #: p<0.05 ;##: P<0.01 Compared with the model group, *: p<0.05 ;**: P<0.01 .

[0154] Table 10 shows the results of water intake during the gavage administration in mice during the modeling process. Water intake in the control group remained relatively stable during the gavage administration period, showing no significant increasing trend. Compared with the control group, water intake increased in both the model group and the group treated with Cornus officinalis flower water extract after modeling, and the differences were highly significant. P<0.01 The mice exhibited symptoms of type 2 diabetes mellitus (T2DM) with polydipsia. It was also found that the model group experienced a continuous increase in water intake throughout the process. In contrast, the water intake of the various dose groups of Cornus officinalis flower water extract and the positive control group increased during weeks 1, 2, and 3 after gavage, but decreased in week 4. Furthermore, the water intake of mice in the medium and high dose Cornus officinalis flower water extract gavage groups was lower than that of the positive control group in week 4, indicating that Cornus officinalis flower water extract can effectively alleviate polydipsia symptoms in T2DM.

[0155] Table 10. Changes in water intake (mL) in mice during gavage during model establishment

[0156] Note: Compared with the blank group, #: p<0.05 ;##: P<0.01 Compared with the model group, *: p<0.05 ;**: P<0.01 .

[0157] (5) Changes in blood glucose concentration in mice The results of random blood glucose concentrations in mice during the modeling process are shown in Table 11. After the modeling was completed, the blood glucose levels in mice in each dose group of Cornus officinalis flower water extract and the positive control group showed a significant increase compared to the control group, and the levels were significantly different. P< 0.01 Meanwhile, except for the control group, the random blood glucose levels of all mice were ≥16.7 mmol / L. Combined with the aforementioned polydipsia and polyphagia symptoms, this indicates that the T2DM model was successfully established. Throughout the process, the blood glucose levels in the model group continued to rise. In the low-dose Cornus officinalis flower water extract gavage group, random blood glucose levels continued to rise in weeks 1, 2, and 3, showing a significant decreasing trend in week 4. In contrast, the blood glucose levels in the medium- and high-dose Cornus officinalis flower water extract gavage groups and the positive control group increased in weeks 1 and 2, showing a decreasing trend in weeks 3 and 4. Furthermore, the blood glucose concentration in the high-dose Cornus officinalis flower water extract gavage group was lower than that in the positive control group in week 4, indicating that gavage administration of Cornus officinalis flower water extract can inhibit the rise in blood glucose in T2DM to a certain extent.

[0158] Table 11. Changes in blood glucose concentration in mice during gavage administration (mmol / L)

[0159] Note: Compared with the blank group, #: p<0.05 ;##: P<0.01 Compared with the model group, *: p<0.05 ;**: P<0.01 .

[0160] (6) Changes in organ indices in mice As shown in Table 12, the liver and pancreas organ indices of the model group mice were significantly larger than those of the control group. P<0.01 The kidneys were also significantly enlarged. p<0.05 This indicates that type 2 diabetes mellitus (T2DM) causes organ swelling in mice, leading to abnormal organ indices in the liver, kidneys, and pancreas. Simultaneously, it was found that after 4 weeks of gavage administration of Cornus officinalis flower water extract, the organ indices in the low, medium, and high dose Cornus officinalis flower water extract gavage groups and the positive control group showed some signs of recovery. In particular, the liver and pancreas organ indices in the high-dose Cornus officinalis flower water extract gavage group were significantly lower than those in the model group. P<0.01The results indicate that the water extract of Cornus officinalis flower can reduce or improve liver, kidney and pancreas damage in patients with type 2 diabetes mellitus (T2DM) and relieve symptoms such as congestion and hyperplasia in the three organs.

[0161] Table 12. Mouse organ index / %

[0162] Note: Compared with the blank group, #: p<0.05 ;##: P<0.01 Compared with the model group, *: p<0.05 ;**: P<0.01 .

[0163] (7) Changes in ITT and OGTT The results of mouse OGTT, ITT, and area under the curve (AUC) are shown below. Figure 13 . Figure 13 In (A), mice in the low, medium, and high dose Cornus officinalis flower water extract gavage groups and the positive control group reached peak blood glucose levels 15 minutes after gavage with glucose. Blood glucose levels in the Cornus officinalis flower water extract-treated groups and the positive control group slowly recovered within 15-120 minutes. In the blank control and model groups, blood glucose levels reached peak levels after 30 minutes, rapidly decreased within 30-60 minutes, and then slowly decreased after 60-120 minutes. Based on... Figure 13 The results of (C) showed that the AUC of mice in groups other than the blank control group was significantly increased. P<0.01 ), while the AUC of each dose group of Cornus officinalis flower water extract was significantly smaller than that of the model group (), P<0.01 ).

[0164] according to Figure 13 The results showed that blood glucose levels decreased to peak levels within 0-60 minutes after insulin administration to each group of mice. Within 60-120 minutes, blood glucose levels significantly rebounded in the model group and the groups treated with different doses of Cornus officinalis flower water extract, while the blood glucose recovery rate in the control group was relatively slow. Figure 13 (D) It can be seen that the AUC of the model group and each group treated with Cornus officinalis flower water extract was significantly increased compared with the blank group. P<0.01 The AUC in each treatment group was significantly lower than that in the model group. P< 0.01 In summary, all doses of Cornus officinalis flower water extract significantly inhibited / restored glucose and insulin levels in diabetic mice. P<0.01 ).

[0165] (8) Effects on serum INS and ADPN in mice according to Figure 14The results showed that the INS and ADPN levels in the model group mice were significantly lower than those in the blank group. P<0.01 After treatment with Cornus officinalis flower water extract via gavage, the levels of INS and ADPN in mice in each dose group recovered compared to the model group. Furthermore, there was no significant difference in INS and ADPN levels between the medium- and high-dose Cornus officinalis flower water extract gavage groups and the control group, indicating that Cornus officinalis flower water extract has a certain inhibitory effect on the decrease in INS and ADPN in T2DM mice.

[0166] (9) Effects on renal function in mice Depend on Figure 15 It can be seen that the renal function indicators of the model group mice showed a significant increase compared with those of the blank group. P< 0.01 This indicates kidney damage in diabetic mice. Figure 15 In (A), the BUN levels in mice in the low-dose group and the positive group treated with Cornus officinalis flower water extract were significantly lower than those in the model group. p<0.05 ), while the medium and high doses of Cornus officinalis flower water extract administered by gavage showed a highly significant reduction compared to the model group ( P<0.01 Furthermore, the BUN levels in the medium- and high-dose Cornus officinalis flower water extract gavage groups were comparable to those in the control group. Figure 15 In (B), the Scr level results showed significant differences between the low- and medium-dose Cornus officinalis flower water extract gavage groups and the blank control group. p<0.05 The mice in the high-dose Cornus officinalis flower water extract gavage group showed no significant difference from the model group, while the mice in the model group showed a significant difference. p<0.05 The results showed no significant difference between the control group and the control group. These results indicate that diabetes causes kidney damage in mice, and the water extract of Cornus officinalis flower has a good inhibitory effect on this damage.

[0167] (10) Effects on blood lipid levels in mice Depend on Figure 16 It can be seen that the levels of TG, TC, and LDL-C in the model group mice were significantly increased compared with the control group. P<0.01 ), while HDL-C levels decreased significantly ( P<0.01 This indicates that T2DM caused abnormal blood lipid levels in mice.

[0168] like Figure 16 As shown in (A), after oral administration of Cornus officinalis flower water extract, the TG content in the low- and medium-dose Cornus officinalis flower water extract oral administration groups showed a significant decreasing trend. p<0.05 The high-dose group showed an extremely significant reduction effect. P<0.01 Furthermore, the high-dose Cornus officinalis flower water extract gavage group showed stronger therapeutic effects than the positive control group. Figure 16In (B), the low-dose Cornus officinalis flower water extract gavage group showed a lower TC than the model group, but the difference was not highly significant. p<0.05 The reduction effect was more significant in the medium and high doses of Cornus officinalis flower water extract group compared to the positive control group. P<0.01 ).

[0169] exist Figure 16 As shown in (C) and (D), the LDL-C content in all doses of Cornus officinalis flower water extract administered by gavage showed a highly significant decreasing trend compared to the model group. P<0.01 The effect of the decrease was dose-dependent with respect to the water extract of Cornus officinalis flowers. Furthermore, the high-dose Cornus officinalis flower water extract gavage group showed significantly better therapeutic effects than the positive control group. Additionally, the HDL-C levels in all Cornus officinalis flower water extract gavage groups were significantly higher than those in the model group. P<0.01 The levels of Cornus officinalis flower water extract in the high-dose group were not significantly different from those in the control group, indicating that Cornus officinalis flower water extract has a certain therapeutic effect on dyslipidemia in diabetic mice, and the effect is correlated with the dose of Cornus officinalis flower water extract.

[0170] (11) Effects on liver function in mice Figure 17 This indicates that the liver function indicators in the model group mice showed a clear increase compared to the control group. P<0.01 This indicates that type 2 diabetes mellitus (T2DM) causes liver damage in mice. The ALT and AST levels in the various doses of Cornus officinalis flower water extract administered by gavage were significantly improved compared to the model group. P<0.01 The levels of AST in the low, medium, and high dose Cornus officinalis flower water extract gavage groups decreased by 14%, 20%, and 30%, respectively. Meanwhile, the high dose Cornus officinalis flower water extract gavage group showed a significant difference in AST levels compared to the control group. p<0.05 The results were comparable to those of the positive control group. This indicates that the water extract of Cornus officinalis flowers can, to some extent, inhibit liver damage caused by diabetes.

[0171] (12) Effects on serum inflammatory factors in mice pass Figure 18 It can be seen that the levels of inflammatory factors in the model group were significantly higher than those in the control group. P<0.01 This indicates that diabetes can trigger an inflammatory response in the body. After treatment with Cornus officinalis flower water extract via gavage, all dosage groups showed a highly significant reduction in TNF-α, IL-6, and IL-1β compared to the model group. P<0.01 The low-dose Cornus officinalis flower water extract gavage group showed no significant difference in MCP-1 content compared to the model group, but the medium- and high-dose Cornus officinalis flower water extract treatment groups showed a significant reduction in MCP-1 compared to the model group. p<0.05The levels were similar to those in the positive control group. Meanwhile, the levels of TNF-α and MCP-1 in the low-dose Cornus officinalis flower water extract gavage group were significantly different from the control group. p<0.05 ), while the IL-6 content showed extremely significant differences ( P<0.01 In summary, the water extract of Cornus officinalis flower can alleviate the inflammatory response caused by diabetes, and the degree of inflammatory response shows a certain dose-dependent relationship with the water extract.

[0172] (13) Effects on antioxidant levels in mouse kidneys Depend on Figure 19 As shown in (A), (B), and (C), the levels of SOD, CAT, and GSH-Px in the model group showed a significantly lower effect compared to the blank group. P<0.01 The levels of antioxidant enzymes in all dose groups of Cornus officinalis flower water extract were higher than those in the model group, and the rate of increase was dose-dependent. Meanwhile, the antioxidant enzyme levels in the high-dose Cornus officinalis flower water extract gavage group were close to those in the positive control group, with a significant difference in SOD levels compared to the model group. p<0.05 The levels of CAT and GSH-Px were significantly different from those in the model group. P<0.01 This indicates that the high-dose group of Cornus officinalis flower water extract has a good protective effect against antioxidant enzymes.

[0173] like Figure 19 As shown in (D), the MDA level in the model group showed a significant increase compared to the control group. P<0.01 After gavage administration of Cornus officinalis flower water extract, the MDA levels in each treatment group showed a significant decrease compared to the model group. p<0.05 The levels of these substances decreased by 17%, 20%, and 24%, respectively. In summary, the aqueous extract of Cornus officinalis flowers can, to some extent, improve oxidative stress induced by diabetes in mice and enhance the body's antioxidant capacity.

[0174] (14) Pathological analysis Depend on Figure 20 It was found that the hepatocytes in the control group mice had normal morphology, normal arrangement, and intact tissue structure. In the model group mice, hepatocytes were swollen, with inflammatory cell infiltration, disordered arrangement, and irregular morphology; lipid droplets were deposited within the hepatocytes, and cell nuclei were damaged. While some hepatocytes in the low-dose Cornus officinalis flower water extract group still showed incomplete morphology compared to the model group, hepatocyte damage was somewhat improved. Compared to the model group, the medium- and high-dose Cornus officinalis flower water extract groups showed normal hepatocyte arrangement and reduced inflammatory cells, indicating that liver damage and fatty degeneration were improved.

[0175] Depend on Figure 21It was found that the glomeruli and tubules of the kidneys in the control group mice were clearly structured and intact, with no inflammatory cell infiltration or mesangial proliferation. In the model group, the mesangial area was dilated, and the tubular cells showed vacuolar degeneration, inflammatory cell infiltration was observed in the tubulointerstitium, and plasma proteins were leaking from Bowman's capsule. After gavage treatment, the vacuolar degeneration and mesangial proliferation of the renal tubules were alleviated in the low-dose Cornus officinalis flower water extract treatment group, while the inflammatory cell infiltration was significantly improved in the medium- and high-dose Cornus officinalis flower water extract treatment groups, with no significant plasma protein leakage and significant improvement in renal tubular epithelial cell edema.

[0176] Depend on Figure 22 It was observed that the islet cells in the control group were tightly arranged and clearly structured. In the model group, islet atrophy, inflammatory cell infiltration, and endothelial cell proliferation indicated islet destruction. Low-dose Cornus officinalis flower water extract could alleviate islet inflammatory cell infiltration to some extent. In the medium- and high-dose Cornus officinalis flower water extract treatment groups, islet cell arrangement was more regular, islet morphology was more regular, and the number of islet cells increased, while inflammatory cell infiltration was significantly improved.

[0177] (15) Analysis of renal mRNA expression levels To investigate the mechanism of action of Cornus officinalis flower water extract on type 2 diabetes mellitus (T2DM), RT-qPCR was used to detect genes related to the PI3K / AKT / mTOR pathway. The results are shown below. Figure 23 It was found that the expression levels of PI3K, AKT, and mTORC1 in the kidneys of model group mice were decreased compared with those in the blank group, while the relative expression level of SREBP-1 was significantly increased. P<0.01 After treatment with Cornus officinalis flower water extract via gavage, both the medium- and high-dose groups showed a significant increase in efficacy compared to the model group. P<0.01 In mice treated with low-dose Cornus officinalis flower water extract, the expression levels of AKT and mTORC1 in the kidneys showed a significant upward trend compared to the model group. P<0.01 While PI3K levels increased compared to the model group, the increase was not statistically significant. Meanwhile, the expression levels of SREBP-1 in mice at each dose of Cornus officinalis flower water extract after gavage showed a significant decreasing trend compared to the model group. P<0.01 The expression level in the high-dose Cornus officinalis flower water extract group was comparable to that in the positive control group.

[0178] like Figure 24 As shown, the expression levels of inflammatory factor mRNA in the model group mice were significantly higher than those in the control group. P<0.01 This indicates that diabetes can induce an inflammatory response in mice. After treatment with Cornus officinalis flower water extract via gavage, the expression levels of related factors mRNA in mice at all doses of Cornus officinalis flower water extract showed significantly lower levels. P<0.01Furthermore, the degree of decrease was dose-dependent with respect to the water extract of Cornus officinalis flowers. In the high-dose Cornus officinalis flower water extract group, the expression level of TNF-α in mice was comparable to that in the positive control group, while the expression level of MCP-1 was lower than that in the positive control group, indicating that the water extract of Cornus officinalis flowers can effectively improve the inflammatory response caused by diabetes.

[0179] (16) Analysis of renal AKT, PI3K, mTORC1 and SREBP-1 expression levels like Figure 25 As shown, the expression levels of PI3K, AKT, and mTOR proteins in the model group all showed a significant decrease compared to the control group. P<0.01 SREBP-1 expression levels showed a clear increase compared to blank levels. P<0.01 The aqueous extract of Cornus officinalis flower can alleviate the increase in SREBP-1 protein and improve the trend of significantly decreased expression of PI3K, AKT, and mTOR proteins, and this effect is dose-dependent. This indicates that the aqueous extract of Cornus officinalis flower can exert its therapeutic effect on diabetes by upregulating the expression levels of PI3K, AKT, and mTOR proteins.

[0180] (17) Short-chain fatty acids in mouse feces The main metabolic products of dietary carbohydrates are SCFAs. According to the results in the table below, the levels of acetic acid, propionic acid, isobutyric acid, and n-butyric acid in the model group showed a significant decrease compared to the control group. P<0.01 While isovaleric acid levels also showed a decreasing trend, the decrease was not significant. After gavage administration of Cornus officinalis flower water extract, mice in all dosage groups showed a significant recovery compared to the model group, and the degree of recovery was dose-dependent on the amount of Cornus officinalis flower water extract. Specifically, the high-dose Cornus officinalis flower water extract group had higher levels of acetic acid and isovaleric acid than the positive control group, at 1.33±0.04 mg / g and 0.17±0.00 mg / g, respectively. These findings demonstrate that Cornus officinalis flower water extract exhibits a significant regulatory effect on SCFA disorders induced by type 2 diabetes mellitus (T2DM), and this effect is dose-dependent.

[0181] Table 13. SCFA content in mouse feces (mg / g)

[0182] Note: Compared with the blank group, #: p<0.05 ;##: P<0.01 Compared with the model group, *: p<0.05 ;**: P<0.01 .

[0183] Verification of technical effectiveness and / or analysis of technical problem solving The contents of ursolic acid, oleanolic acid, gallic acid, catechin, ferulic acid, benzoic acid, and cinnamic acid in the aqueous extract of Cornus officinalis flowers, the contents of epicatechin, rutin, and quercetin in the methanol extract of Cornus officinalis flowers, and the content of caffeic acid in the ethyl acetate extract of Cornus officinalis flowers were all significantly higher than those in the extracts of Cornus officinalis fruit in different solvents. In summary, the extracts of Cornus officinalis flowers contain a rich variety of compounds and can be used as a material for further investigation of its in vitro and in vivo antioxidant, hypoglycemic, and lipid-lowering physiological activities.

[0184] Cornus officinalis flower water extract removes DPPH•, •OH, and ABTS. + The IC50 values ​​of the aqueous extract of Cornus officinalis were 1.01±0.03 mg / mL, 0.83±0.08 mg / mL, and 0.12±0.00 mg / mL, respectively, which were significantly lower than the IC50 values ​​of other polar solvents of Cornus officinalis flowers and the extract of Cornus officinalis fruit. Meanwhile, the APC index of the aqueous extract of Cornus officinalis flowers was second only to VC, reaching 99.05%. In vitro assays of the inhibitory activities of three enzymes showed that the IC50 values ​​of the aqueous extract of Cornus officinalis flowers against these three hypoglycemic and lipid-lowering enzymes were 1.26±0.17 mg / mL, 0.56±0.13 mg / mL, and 0.04±0.00 mg / mL, respectively, which were significantly different from the IC50 values ​​of the methanol extract, ethyl acetate extract, and different extracts of Cornus officinalis fruit. P<0.01 The results indicate that the aqueous extract of Cornus officinalis flowers has stronger antioxidant capacity and hypoglycemic and lipid-lowering effects than other extracts. PCA results show that there are significant differences and complete separation between different extracts of Cornus officinalis flowers and fruits, and the degree of separation is affected by the compounds and their activity.

[0185] The results showed that Cornus officinalis flower extract possessed good antioxidant, hypoglycemic, and lipid-lowering abilities, and the aqueous extract of Cornus officinalis flower was more effective than other extracts, making it a suitable material for evaluating the therapeutic mechanism of type 2 diabetes mellitus (T2DM). Furthermore, theoretical predictions of the therapeutic efficacy of Cornus officinalis flower aqueous extract for T2DM revealed extremely strong binding affinity between Tetrahydroalstonine, Telocinobufagin, ursolic acid, and PI3K, AKT, and mTOR. In conclusion, Cornus officinalis flower aqueous extract can serve as a material for exploring the in vivo therapeutic mechanism of T2DM.

[0186] OGTT / ITT results showed that mice in the Cornus officinalis flower aqueous extract gavage group exhibited improved glucose / insulin tolerance compared to the model group. Serum factor assays showed that, compared to the model group, the Cornus officinalis flower aqueous extract gavage group significantly upregulated INS, HDL-C, and ADPN levels. P<0.01 The levels of BUN, Scr, TG, TC, LDL-C, ALT, AST, TNF-α, MCP-1, IL-6, and IL-1β were significantly reduced. P<0.01Simultaneously, the detection of antioxidant enzyme activity and MDA content in kidney homogenate showed that the MDA content in the Cornus officinalis flower water extract gavage group was significantly lower than that in the model group. P<0.01 Antioxidant enzyme activity showed a significant increase. P<0.01 Pathological results showed that the aqueous extract of Cornus officinalis flowers could effectively alleviate the damage to liver, kidney, and pancreatic islet cells caused by diabetes. Furthermore, compared with the model group, mice in the gavage groups administered different doses of the aqueous extract of Cornus officinalis flowers showed significantly increased mRNA and protein expression levels of PI3K, AKT, and mTOR. P<0.01 ), while the expression levels of SREBP-1 and inflammatory factor mRNA showed a significant decrease. P<0.01 Meanwhile, the water extract of Cornus officinalis flower can significantly restore the reduced content of short-chain fatty acids, and the degree of restoration is dose-dependent.

[0187] In summary, different extracts of Cornus officinalis flowers are rich in various substances and have certain in vitro antioxidant and hypoglycemic and lipid-lowering functions. Among them, the water extract of Cornus officinalis flowers is more effective than other extracts. It may exert its anti-diabetic activity by activating the PI3K / AKT / mTOR pathway in vivo to regulate blood sugar, insulin, blood lipids and antioxidant levels, improve liver, kidney and pancreatic damage, and reduce the expression of inflammatory factors.

[0188] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A Cornus officinalis flower extract with antioxidant, hypoglycemic, and lipid-lowering effects, characterized in that, The active ingredients of the Cornus officinalis flower extract include mononoside, strychnoside, loganin, ursolic acid, oleanolic acid, cornus officinalis glycoside, gallic acid, protocatechuic acid, p-hydroxybenzoic acid, catechin, chlorogenic acid, caffeic acid, epicatechin, strychnosine, rutin, ferulic acid, benzoic acid, resveratrol, quercetin, and cinnamic acid.

2. The Cornus officinalis flower extract according to claim 1, characterized in that, The extract of Cornus officinalis flower contains mononoside at a mass content of 2.96-6.63 mg / g, daunoside at a mass content of 1.25-1.84 mg / g, loganin at a mass content of 1.20-1.82 mg / g, ursolic acid at a mass content of 0.43-1.17 mg / g, oleanolic acid at a mass content of 17.31-24.55 mg / g, neo-cornuate glycosides at a mass content of 2.83-5.67 mg / g, gallic acid at a mass content of 12.73-76.49 mg / g, protocatechuic acid at a mass content of 1.12-1.69 mg / g, p-hydroxybenzoic acid at a mass content of 0.07-0.12 mg / g, and catechins at a mass content of 1.43- The total content of chlorogenic acid is 0.33-0.56 mg / g, caffeic acid is 0.49-8.31 mg / g, epicatechin is 2.75-16.63 mg / g, scutellarin is 0.82-1.95 mg / g, rutin is 0.46-7.03 mg / g, ferulic acid is 1.27-14.72 mg / g, benzoic acid is 2.47-11.29 mg / g, resveratrol is 0.31-0.58 mg / g, quercetin is 3.13-18.24 mg / g, and cinnamic acid is 1.02-4.28 mg / g.

3. The method for preparing Cornus officinalis flower extract according to any one of claims 1-2, characterized in that, The preparation method includes the steps of: dissolving Cornus officinalis flowers in a solvent and extracting the Cornus officinalis flower extract.

4. The preparation method according to claim 3, characterized in that, The solvent is selected from at least one of water, methanol, ethanol, acetone, ethyl acetate, chloroform, and hexane; preferably at least one of water, methanol, and ethyl acetate; more preferably water; and even more preferably distilled water. Preferably, the ratio of the powder to the solvent is 1:5-15; more preferably, it is 1:

10.

5. The preparation method according to claim 3, characterized in that, The preparation method further includes a step of pulverizing the Cornus officinalis flowers before dissolving them in the solvent; Preferably, the pulverization method is selected from at least one of a high-speed electric grinder, a traditional Chinese medicine crusher, a high-speed universal pulverizer, a hammer mill, a vibratory mill, and an air jet mill; more preferably, it is a traditional Chinese medicine crusher. Preferably, the pulverization operation includes a drying step; more preferably, the drying includes, but is not limited to, freeze drying, oven drying, air drying, blow drying, natural drying, vacuum drying, etc.; even more preferably, air drying; most preferably, air drying in a dry, ventilated and light-protected place.

6. The preparation method according to claim 3, characterized in that, The extraction method is selected from at least one of reflux extraction, ultrasound-assisted extraction, percolation extraction, cold soaking extraction, supercritical fluid extraction, microwave-assisted extraction, and accelerated solvent extraction; preferably, the extraction method is a combination of reflux extraction and ultrasound-assisted extraction; more preferably, the ultrasound duration is selected from 15-60 min; even more preferably, it is 35 min; more preferably, the reflux temperature is 20-60℃, even more preferably, it is 45℃; more preferably, the reflux duration is 1-4 h; even more preferably, it is 3 h; more preferably, the number of refluxes is 1-4 times; even more preferably, it is 3 times.

7. The preparation method according to claim 3, characterized in that, The extraction process also includes a purification process. Preferably, the purification process includes concentration, filtration, and drying. Preferably, the concentration method is selected from at least one of rotary evaporator concentration, vacuum drying oven concentration, water bath heating concentration, oil bath / sand bath heating concentration, freeze drying concentration, and membrane concentration; preferably, rotary evaporator concentration is used; more preferably, the rotary evaporator concentration temperature is 45°C. Preferably, the filtration method is selected from at least one of atmospheric pressure filtration, reduced pressure filtration, centrifugal filtration, hot filtration, and membrane filtration; more preferably, centrifugal filtration; even more preferably, the centrifugal filtration speed is 3000-15000 r / min; most preferably, 12500 r / min; even more preferably, the centrifugal filtration time is 1-10 min; most preferably, 3 min. Preferably, the drying method includes, but is not limited to, freeze drying, oven drying, air drying, blow drying, natural drying, vacuum drying, etc.; more preferably, vacuum drying; and even more preferably, the vacuum drying temperature is 45°C.

8. The use of the Cornus officinalis flower extract according to any one of claims 1-7 in terms of antioxidant properties.

9. The use of the Cornus officinalis flower extract according to any one of claims 1-7 in lowering blood sugar and lipids.

10. A traditional Chinese medicine preparation with antioxidant, hypoglycemic, and lipid-lowering effects, characterized in that, The active ingredients of the traditional Chinese medicine preparation include the Cornus officinalis flower extract as described in any one of claims 1-7 and pharmaceutically acceptable excipients.

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