CN115645431B

CN115645431B - Composition and application thereof

Info

Publication number: CN115645431B
Application number: CN202211588214.6A
Authority: CN
Inventors: 岳中宝; 吴新星; 汪玉芳; 贺瑞坤; 张旭光
Original assignee: BY Health Co Ltd
Current assignee: BY Health Co Ltd
Priority date: 2022-12-12
Filing date: 2022-12-12
Publication date: 2023-03-21
Anticipated expiration: 2042-12-12
Also published as: CN115645431A

Abstract

The present application provides a composition and uses thereof. The composition comprises malvidin-3-O-glucoside and myricetin, wherein the weight ratio of the malvidin-3-O-glucoside to the myricetin is (5-7): 1. After malvidin-3-O-glucoside and myricetin are combined, especially when the weight ratio of the malvidin-3-O-glucoside to the myricetin is (5 to 7): 1, all the components are mutually cooperated, thereby having obvious inhibition effect on AGEs and having obvious protection effect on HUVEC cell injury induced by high sugar and AGEs.

Description

Composition and application thereof

Technical Field

The application relates to the field of pharmaceutical preparations, in particular to a composition and application thereof.

Background

Advanced glycation end products (advanced glycation end products AGEs) refer to stable covalent adducts formed by macromolecules such as proteins, lipids or nucleic acids which spontaneously react with glucose or other reducing monosaccharides without the participation of enzymes. It is the end product of the non-enzymatic glycosylation reaction (Maillard reaction), and is the product of the binding of excess sugar to the protein. There are dozens of identified AGEs, such as carboxymethyl lysine (CML), carboxyethyl lysine (CEL), pyrrol, etc.

AGEs are derived from two sources in the body, one is that excess sugar and protein synthesize AGEs in the body, and the other is that AGEs present in food are taken into the body by eating. AGEs are able to combine with and destroy the tissue cells of the body. Normally, AGEs in the body can be cleared by the kidneys. However, AGEs accumulate in vivo with age or under certain pathological conditions, which may cause significant adverse effects such as damage to the body, interfere with normal physiological and biochemical processes in vivo, affect normal metabolism of the body, and cause the occurrence and development of diseases. In pathological conditions, AGEs may be present in the corresponding pathological tissues.

There are two main pathogenic mechanisms of AGEs. First, AGEs accumulate both intracellularly and extracellularly in all tissues and fluids, altering the structure and function of cellular proteins through glycation and cross-linking. Glycosylation of proteins affects their normal function by disrupting molecular conformation, interfering with receptor function, or altering enzymatic activity. In addition, AGEs are also cross-linked with other intracellular and extracellular molecules (such as lipids and nucleic acids) resulting in structural and functional changes that affect their normal function. Secondly, AGEs can interact with specific cell surface receptors, thereby altering intracellular signaling, gene expression, promoting reactive oxygen species production and activation of inflammatory pathways.

The current research proves that AGEs can accelerate the aging of human bodies and cause a plurality of chronic degenerative diseases, such as cardiovascular and cerebrovascular diseases, neurodegenerative diseases, osteoarticular diseases, kidney diseases and the like. Studies have shown that elevated levels of AGEs are a risk factor for the development of diabetes and complications. AGEs are independent risk factors for insulin resistance, and multiple regression analysis results show that AGEs levels are independently related to insulin resistance in healthy subjects. In another study, over 300 non-diabetic patients were examined to confirm that serum AGEs levels were independently correlated with HOMA-IR. The results of a 1 year clinical trial with 138 obese subjects with metabolic syndrome showed that a low AGEs diet improved insulin resistance and potentially reduced risk of type 2 diabetes in the test population compared to the high AGEs diet group without a substantial weight loss. In addition, the hyperglycemia of the diabetic patients can further promote the glycosylation reaction process, promote the increase of AGEs level and further cause the occurrence of related complications. The content of AGEs in liver cells and renal tubules of the diabetic patients is obviously increased through staining. Overexpression of β -growth factor and increased concentration of vascular endothelial growth factor are found in the glomeruli and tubules. AGEs binding to specific receptors results in the overexpression of cytokines that may play an important role in diabetic vascular complications, such as Diabetic Retinopathy (DR), diabetic Nephropathy (DN), atherosclerosis.

Increased AGEs levels are closely associated with decreased cognitive abilities in the elderly. Studies have shown that AGEs are significantly increased in the brain and central nervous system of patients with neurodegenerative diseases such as alzheimer's disease, and that increased AGEs levels are mainly concentrated in tissue regions with significant pathological features (e.g. hippocampus of alzheimer's disease). Furthermore, it was found that dietary AGEs or their precursors may lead to a loss of selective permeability of the blood brain barrier. Results of alzheimer mouse experiments show that exogenously ingested AGE can reduce SIRT1 expression, thereby increasing β -amyloid and plaque production through the depolymerin and metalloprotease pathways. A cross-sectional study with the elderly showed that high levels of AGEs diet were associated with a more rapid memory decline.

The increase of AGEs level is closely related to the occurrence of osteoarticular diseases. The glycosylation pathway is considered to be one of the important factors leading to osteoporosis. Elevated serum AGEs levels, such as pentoside and CML, are found in patients with osteoporosis. Studies have shown that non-enzymatic glycosylation is a new factor affecting bone remodeling. AGEs accumulated in the bone matrix affect osteoblast differentiation and proliferation by binding to its receptor. In these cells, binding of AGEs to RAGE activates NF-. Kappa.B, resulting in increased expression of cytokines, growth factors, and cell adhesion molecules. This triggers an inflammatory process and induces oxidative stress, leading to osteoblast dysfunction and bone remodeling disorders. One of the major age-related changes in articular cartilage is an increase in AGEs levels. From the age of 20 years ago, AGEs accumulate in the collagen and proteoglycans of articular cartilage. The accumulation of AGEs in articular cartilage also leads to an increase in the prevalence of osteoarthritis as it AGEs. Recent studies have found that elevated levels of AGEs can negatively affect articular cartilage by increasing its stiffness, increasing chondrocyte-mediated proteoglycan degradation, decreasing proteoglycan synthesis, and inducing degradation of the chondrocyte extracellular matrix (ECM).

In addition, numerous empirical studies have shown an association between elevated levels of AGEs and various diseases such as skin aging, polycystic ovary syndrome, wound healing, periodontitis, erectile dysfunction, anemia in elderly women, slow walking speed in elderly, peripheral neuropathy, peripheral arterial disease, obstructive sleep apnea, cancer, schizophrenia, alzheimer's disease, higher all-cause mortality, severity of coronary atherosclerosis, cardiovascular disease mortality, and metabolic syndrome in adults and children, among others.

AGEs play an important role in the development or progression of a variety of diseases, and lowering their levels is positive for health benefits. Methods for reducing circulating AGEs include inhibiting the formation of AGEs, accelerating the catabolism of existing AGEs or inhibiting the crosslinking of AGEs, and blocking the biological response of AGEs. Inhibition of AGEs formation may occur by several mechanisms, including aldose reductase, antioxidant activity, reactive dicarbonyl trapping, sugar autoxidation inhibition, and amino binding. Research shows that inhibiting AGEs generation is favorable to delaying senility.

Anthocyanins are compounds formed by combining anthocyanidins and sugars, are widely present in cell sap of flowers, fruits, stems, leaves and root organs of plants, are different in color from red, purple red to blue and are water-soluble pigments with bright colors. The anthocyanin belongs to flavonoid compounds, and the skeleton structure of the anthocyanin is 2-phenyl benzopyran cation. According to preliminary statistics, tens of thousands of plants of 27 families, 73 genera, have been found to contain anthocyanins. More than 500 anthocyanins are currently isolated from plants. Various anthocyanins can exhibit different shades of yellow, red, violet, black, etc., depending on their ability to form resonance structures, substituents on the C6-C3-C6 nucleus and environmental factors.

At present, more than 20 anthocyanins are known, and the most common anthocyanins are 6 anthocyanins, namely cyanidin (Cy), pelargonidin (Pg), delphinidin (Dp), petuniain (Pt), peoniflorin (Pn) and malvidin (Mv), and the anthocyanins are mainly different from one another in a group R ₁ And R ₂ A change in (c).

Free anthocyanidins are very unstable and are generally present in nature in the form of glycoside conjugates. Its glycoside form is more stable than aglycone and therefore exists mainly as glycoside (glycoside), i.e., anthocyanin, in plants. Typically these glycosides include mono-glucosides, di-glucosides and acyl derivatives. In the known anthocyanins or anthocyanidins, the majority are present in glycosidated form, the glycoside-forming sugars being mainly glucose, galactose, rhamnose, arabinose, xylose and the disaccharides and trisaccharides composed of these monosaccharides, the common diglycosides being sophorose, rutinose and sambucobiose, etc. The sugar and anthocyanin are linked by O-bonds, mainly at the 3-, 5-and 7-carbon positions, and a small proportion is linked at the 3' -carbon position, and almost all anthocyanins are glycosidated at the 3-position.

Further, anthocyanins in plants are also present as acylated anthocyanins, which are formed by ester bond bonding of organic acids, and the most common acids involved in glycosyl acylation are various hydroxycinnamic acid derivatives such as ferulic acid, caffeic acid, sinapic acid, etc., fatty acids such as malic acid, acetic acid, succinic acid, malonic acid, oxalic acid, etc., and p-hydroxybenzoic acid, etc. The number of hydroxyl groups in anthocyanin molecules, the methylation degree of hydroxyl groups, the type, quantity and position of sugars attached to anthocyanin molecules, the type and number of fatty acids or aromatic acids attached to sugar molecules, the different actions of anthocyanin molecules with other substances and the like cause the existence of various anthocyanins in nature.

Researches find that some anthocyanins extracted from plants have a good effect of inhibiting AGEs formation, for example, the inhibition effect of anthocyanin compounds in the pulp of Lonicera caerulea on AGEs is stronger than that of other non-anthocyanin polyphenol compounds in the pulp, and the inhibition rate of the non-anthocyanin polyphenol compounds is 1.7 times that of the non-anthocyanin polyphenol compounds in the pulp. The prior art also finds that mulberry anthocyanin can obviously inhibit the generation of AGEs, and bird cherry anthocyanin and coreopsis tinctoria anthocyanin have good inhibition effect on the generation of AGEs.

However, anthocyanins extracted from plants are a mixture, and the components and content thereof are unstable, and the components and content of anthocyanins vary greatly depending on factors such as plant species, production area, year, season, and the like. A doctor thesis (research on correlation between polyphenol components of dry red wine at east foot of Helan mountain and antioxidant and anticancer activities thereof) (research on correlation between polyphenol components of dry red wine at east foot of Helan mountain and antioxidant and anticancer activities thereof [ D ] Yanling, university of northwest agriculture and forestry, 2019) researches the contents and biological activities of anthocyanin in grapes of different varieties and different years, and results show that the anthocyanin content in the grapes of different varieties and different years is greatly different, for example, the anthocyanin content in Cabernet Sauvignon grapes is 19.17 mg/L in 2011 and the anthocyanin content in 2009 is only 6.17 mg/L; secondly, 7 of 8 anthocyanin monomers detected in 2011 can not be detected in 2009 cabernet sauvignon; again, in 2013, the content of anthocyanin in grapes with Sira is more than 6 times that of grapes with cabernet sauvignon. Based on that the components and the content of anthocyanin are key factors influencing the biological activity of the anthocyanin, the anthocyanin components and the content of plant sources in different batches have great difference due to factors such as different plant varieties, production areas, years, seasons and the like, and further the biological activity of the anthocyanin has great difference. Therefore, it is difficult to form a product which effectively and stably inhibits the production of AGEs by using plant-extracted anthocyanins as raw materials.

Myricetin (myricetin) mainly exists in plants such as waxberry and camellia, and the alcohol extract is rich in a large amount of myricetin glycoside and other substances, has physiological activities such as oxidation resistance and aging resistance, can pass through a blood brain barrier, and has a certain effect of preventing neurodegenerative diseases and the like.

Disclosure of Invention

The application provides a composition and application thereof, and aims to provide a product which can efficiently and stably inhibit AGEs to achieve the purpose of reducing AGEs.

In a first aspect, the present application provides a composition, wherein the composition comprises malvidin-3-O-glucoside and myricetin, and the weight ratio of the malvidin-3-O-glucoside to the myricetin is (5 to 7): 1.

Further, the weight ratio of malvidin-3-O-glucoside to myricetin is 5.

Furthermore, the composition consists of malvidin-3-O-glucoside and myricetin.

In a second aspect, the present application provides a dietary supplement comprising an effective ingredient and an adjuvant, wherein the effective ingredient is a composition of any one of the above.

A third aspect of the present application provides a medicament comprising an active ingredient and a carrier, the active ingredient being a composition of any of the above, the medicament being a medicament for inhibiting AGEs, a medicament for treating AGEs-related injuries, diseases or symptoms, or a medicament for alleviating AGEs-related injuries, diseases or symptoms.

Further, the inhibition of AGEs is inhibition of the production of AGEs or promotion of the decomposition of AGEs; and/or AGEs-related damage is AGEs-mediated apoptosis increase, active oxygen content increase, inflammatory factor TNF-alpha overexpression, inflammatory factor ICAM-1 overexpression, inflammatory factor VCAM-1 overexpression, and/or mitochondrial ATP content decrease; and/or AGEs-related diseases are any one or more of cardiovascular and cerebrovascular diseases, neurodegenerative diseases and osteoarticular diseases; and/or AGEs-related symptoms are aging.

Further, the cardiovascular and cerebrovascular diseases are selected from diabetes and its complications, atherosclerosis; and/or the neurodegenerative disease is selected from alzheimer's disease, parkinson's disease, amyotrophic lateral sclerosis; and/or the osteoarticular disease is osteoporosis, osteoarthritis. A fourth aspect of the present application provides a use of a composition of any one of the above in the manufacture of a medicament or dietary supplement for inhibiting AGEs, treating AGEs-related injuries, diseases or symptoms, or alleviating AGEs-related injuries, diseases or symptoms.

Further, the cardiovascular and cerebrovascular diseases are selected from diabetes and its complications, atherosclerosis; and/or the neurodegenerative disease is selected from alzheimer's disease, parkinson's disease, amyotrophic lateral sclerosis; and/or the osteoarticular disease is osteoporosis, osteoarthritis.

After malvidin-3-O-glucoside and myricetin are combined, especially when the weight ratio of malvidin-3-O-glucoside to myricetin is (5-7): 1, the components are mutually synergistic, so that the composition has a remarkable inhibition effect on AGEs, and has an obvious protection effect on HUVEC cell damage induced by high-sugar and AGEs.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings needed to be used in the embodiments of the present application will be briefly described below, and it is obvious that the drawings described below are only some embodiments of the present application, and it is obvious for a person skilled in the art to obtain other drawings based on the drawings without any creative effort.

FIG. 1 shows the results of enzyme-linked immunosorbent assay for detecting AGEs content in HUVEC cells of each test group in test example 2.

FIG. 2 shows the results of the measurement of cell viability in each test group by the CCK8 method in test example 2.

FIG. 3 shows the results of the enzyme reader in test example 2 detecting the production of reactive oxygen species ROS in each test group.

FIG. 4 shows the detection of apoptosis rate of each test group by Propidium Iodide (PI) staining method in test example 3.

FIG. 5 shows the results of qPCR assay for inflammatory factor TNF-. Alpha.expression in each test group in test example 3.

FIG. 6 shows the results of qPCR detection of the expression of the inflammatory factor ICAM-1 in each test group in test example 3.

FIG. 7 shows the results of qPCR detection of the expression of the inflammatory factor VCAM-1 in each test group in test example 3.

Fig. 8 shows the results of the measurement of the mitochondrial-related ATP index in each test group in test example 3.

Detailed Description

Embodiments of the present application will be described in further detail below with reference to the drawings and examples. The following detailed description of the embodiments and the accompanying drawings are provided to illustrate the principles of the application and are not intended to limit the scope of the application, i.e., the application is not limited to the described embodiments.

As described in the background art, the composition of the plant-extracted anthocyanin mixture of the prior art is unstable due to plant raw materials, extraction processes, and the like, and the extracted anthocyanin mixture is difficult to form an effective and stable product for inhibiting the production of AGEs. In order to solve the problem, the application researches a plurality of anthocyanin monomers, and discovers that the anthocyanin monomers are malvidin anthocyanin and malvidin-3-O-glucoside have good inhibition effect on AGEs. In addition, myricetin alone is not ideal for inhibiting AGEs. However, when malvidin-3-O-glucoside and myricetin are combined at a specific ratio, better AGEs inhibitory action than malvidin-3-O-glucoside is achieved.

Based on the above studies, an exemplary embodiment of the present application provides a composition, wherein the composition comprises malvidin-3-O-glucoside and myricetin, and the weight ratio of malvidin-3-O-glucoside to myricetin is (5 to 7): 1.

Experiments prove that after malvidin-3-O-glucoside and myricetin are combined, the components are mutually cooperated, so that the composition has a remarkable inhibiting effect on AGEs, has an obvious protection effect on HUVEC cell injury induced by high-sugar and AGEs, and has certain difference in different proportions.

The effect is more prominent when the ratio of malvidin-3-O-glucoside to myricetin is 5, or 6, or 7.

The composition of the present application may also include other anthocyanins, and in some embodiments, the composition consists of malvidin-3-O-glucoside and myricetin, with a significant synergistic effect.

Another exemplary embodiment of the present application provides a dietary supplement comprising an effective ingredient and an adjuvant, wherein the effective ingredient is a composition of any one of the above. By utilizing the dietary supplement, the function of inhibiting AGEs is realized, and the aim of resisting saccharification is further realized.

Another exemplary embodiment of the present application provides a medicament comprising an active ingredient and a carrier, wherein the active ingredient is an anthocyanin composition of any one of the above, and the medicament is a medicament for inhibiting AGEs, treating AGEs-related injuries, diseases or symptoms, or alleviating AGEs-related injuries, diseases or symptoms.

In some embodiments, said inhibiting AGEs is inhibiting the production of AGEs, or promoting the breakdown of AGEs; and/or AGEs-related damage is AGEs-mediated apoptosis increase, active oxygen content increase, inflammatory factor TNF-alpha overexpression, inflammatory factor ICAM-1 overexpression, inflammatory factor VCAM-1 overexpression, and/or mitochondrial ATP content decrease; and/or AGEs-related diseases are any one or more of cardiovascular and cerebrovascular diseases, neurodegenerative diseases and osteoarticular diseases; and/or AGEs-related symptoms are aging.

In some embodiments, the cardiovascular and cerebrovascular diseases are selected from diabetes and its complications, atherosclerosis; and/or neurodegenerative diseases selected from alzheimer's disease, parkinson's disease, amyotrophic lateral sclerosis; and/or the osteoarticular disease is osteoporosis, osteoarthritis.

In another exemplary embodiment of the present application, there is provided a use of an anthocyanin composition of any one of the above in the preparation of a medicament or dietary supplement for inhibiting AGEs, treating AGEs-related injuries, diseases or symptoms, or alleviating AGEs-related injuries, diseases or symptoms.

In some embodiments, the cardiovascular and cerebrovascular diseases are selected from diabetes and its complications, atherosclerosis; and/or the neurodegenerative disease is selected from alzheimer's disease, parkinson's disease, amyotrophic lateral sclerosis; and/or the osteoarticular disease is osteoporosis, osteoarthritis.

The dietary supplement or the medicament can also comprise auxiliary materials, for example, the dietary supplement comprises dietetically acceptable lithium-rich, the medicament comprises pharmaceutically acceptable auxiliary materials, and the auxiliary materials can be selected from common corresponding auxiliary materials, and can be selected according to specific dosage forms of the medicament or the dietary supplement.

In some embodiments, the above medicament or dietary supplement is in the form of an oral formulation or an injectable formulation. Preferably, the oral preparation is a tablet, powder, capsule, granule, pill, powder, paste, solid beverage or oral liquid. Or preferably, the injection preparation is injection or powder injection for injection.

The advantageous effects of the present application will be further described below with reference to examples and comparative examples.

Table 1 standard information used for the experiments:

experimental procedures

Bovine Serum Albumin (BSA) and Methylglyoxal (MGO) interact to generate AGEs, a compound to be detected is incubated with the BSA and the MGO together, and the amount of the AGEs generated is judged by detecting the change of a fluorescence value in a system, so that whether the compound has an inhibiting effect on the generation of the AGEs is evaluated. Aminoguanidine (AG) was used as a positive control.

The study was carried out using sterile black non-transparent 96-well plates, in which 90. Mu.L of 10 mg/ml BSA solution and 10. Mu.L of 1.25M MGO solution were added to each well, as shown in the following table, and then 10. Mu.L of the test solution or 10. Mu.L of physiological saline (negative control) was added to each well, and the positive control group was added with the positive drug solution, as designed. After the reaction solution was mixed well, fluorescence values at the start of the experiment (excitation wavelength Ex =370 nm, emission wavelength Em =440 nm) were measured with an M5 microplate reader. After the detection is finished, the sterile sealing plate is sealed by a membrane, and the black non-transparent 96-well plate is placed at 37 ℃ and is protected from light for 24 hours, and then the fluorescence value is detected again under the same condition. And 6-8 multi-well detection is carried out on each test object group and each control group, the increase of the fluorescence value of each well of each group is calculated, and the AGEs generation inhibition rate of each group is converted.

Table 2: 96-pore plate reaction system for preliminary screening test

Under the same reaction system and conditions, the test substances are detected in different gradient concentrations (50, 10, 1, 0.1, 0.03 and 0.01 mg/mL), 5 double-well detection is carried out on each concentration group of the test substances and each control group, and the IC50 value of the test substances is obtained by fitting the detection results by Graphpad.

Test example 1

The test results of the inhibition effect of the test substances bilberry mixed anthocyanin, malvidin-3-O-glucoside, myricetin, aminoguanidine, sample 1, sample 2, sample 3, sample 4 and sample 5 on AGEs generation were experimentally verified according to the experimental process. Tables 3 and 4 show the inhibitory effect of each of the above anthocyanins on the production of AGEs.

Wherein, the weight ratio of the components is,

sample 1: malvidin 3-O-glucoside: myricetin = 4;

sample 2: malvidin 3-O-glucoside: myricetin = 5;

sample 3: malvidin 3-O-glucoside: myricetin = 6;

sample 4: malvidin 3-O-glucoside: myricetin = 7;

sample 5: malvidin 3-O-glucoside: myricetin = 8;

sample 6: mixing cowberry fruit with anthocyanin.

TABLE 3

Remarking: p is<0.05 *^: compared with malvidin-3-O-glucoside, cowberry mixed anthocyanin and a positive control group, the composition has significant difference.

TABLE 4

It can be seen that the ratio combination samples of samples 2-4 are significantly better than other samples in their AGEs-inhibiting effect. Therefore, when the weight ratio of the malvidin-3-O-glucoside to the myricetin is (5-7): 1, the synergistic effect is obvious.

Test example 2 inhibition of high sugar-induced AGEs production and protection of umbilical vein endothelial cells

The experimental principle is as follows: HUVEC cells cultured in vitro were randomly divided into a control group, a model group (40 mmol/L glucose) and malvidin-3-O-glucoside group, a myricetin group, a sample 1 group, a sample 3 group, a sample 5 group, and a sample 6 group. High sugar induction was carried out at 40mmol/L and endothelial cells were intervened with different anthocyanins at 100. Mu. Mol/L for 24h.

The HVUECs are subjected to primary culture and subculture by adopting a modified Jaffe method and the like, and 3-6 generations of well-grown HVUECs are taken for experiment.

Experimental grouping and conditioned culture well-grown 3-6 generations of HVUECs were taken to prepare cell suspensions at 4.0X 10 ⁵ cell/well cell density was plated on 24-well plates, DMEM medium containing 10% fetal bovine serum was added, and the mixture was incubated at 37 ℃ and 5% CO ₂ Culturing in an incubator, and changing a serum-free culture solution when the HUVECs grow to be in a sub-fusion state, and continuously culturing for 12-24 h. Then adding a sample with the concentration of 100umol/L for pretreatment for 8 hours according to the experimental requirements, and then adding glucose with the concentration of 40mmol/L for action for 24 hours. Grouping: (1) blank control group: adding DMEM culture solution with the same amount as the medicine; (2) model group glucose (40 mmol/L) was added; (3) and (3) arranging more than 3 multiple holes in the high-sugar + sample group, continuously culturing for 24 hours, and centrifugally collecting cells and culture solution.

1) The content change of AGEs is detected by enzyme-linked immunosorbent assay, and the result is shown in figure 1, the ratio P of # to a control group is less than 0.05; * P <0.05 to model group; p <0.05 for the other sample groups.

2) The cell viability was measured by the CCK8 method using a detection kit (CCK-8 cell proliferation and cytotoxicity detection kit, japan Co., ltd.) according to the instructions, and the results are shown in FIG. 2, wherein the ratio of # to the control group P is less than 0.05 in FIG. 2; * P <0.05 to model group;

3) Detecting the generation of Reactive Oxygen Species (ROS) by using an active oxygen detection kit (Biyun, product number: S0033S) and a microplate reader according to an operation instruction, wherein the result is shown in figure 3, and the ratio P of the # to a control group is less than 0.05 in figure 3; * P <0.05 to model group; the ratio of P to other sample groups is <0.05.

As can be seen from FIGS. 1 to 3, sample 3 had the least AGEs, the highest cell viability and the least ROS, and was closest to the control group.

Test example 3 protective action of inhibiting AGEs on umbilical vein endothelial cell injury

The experimental principle is as follows: HUVEC cells cultured in vitro were randomly divided into a control group, a model group (200. Mu.g/mL AGEs), a malvidin-3-O-glucoside group, a myricetin group, a sample 1 group, a sample 3 group, a sample 5 group, and a sample 6 group. The obtained mixture is pretreated by 100 mu mol/L of each group of anthocyanin for 8 hours, and then 200 mu g/mL of AGEs are added to act on endothelial cells for 24 hours.

1) Propidium Iodide (PI) is a nuclear staining reagent capable of staining DNA, and can release red fluorescence after being embedded into double-stranded DNA. PI cannot pass through a living cell membrane, but only through a damaged cell membrane to stain nuclei. Therefore, PI is often used in conjunction with nuclear fluorescent probes such as DAPI to stain both live and dead cells. When PI and DAPI and other nuclear dyes are co-dyed, DAPI can be taken up by living cells and combined with DNA to be blue fluorescence under ultraviolet light; while PI stains dead cells to produce red fluorescence. The test results are shown in figure 4, where the # to control ratio P <0.05; * P <0.05 to model group; p <0.05 for the other sample groups.

2) Detecting the expression level of inflammatory factors by qPCR (quantitative polymerase chain reaction) by using a detection Kit (Beyofast ™ SYBR Green One-Step qRT-PCR Kit) according to the instruction, wherein the test result is shown in figures 5 to 7, and the ratio P of the # to a control group is less than 0.05 in figures 5-7; * P <0.05 to model group; p <0.05 for the other sample groups.

3) And performing mitochondrial related ATP index detection by using an ATP Assay Kit (ATP Assay Kit) (Biyunyan, product number S0026) according to the operation instruction, wherein the test result is shown in FIG. 8, and in FIG. 8, # is compared with the control groupP<0.05(ii) a * In comparison with model groupP<0.05. 4) Detecting the enzyme activity data of endothelial cell function NO (Biyuntian, product number S0021) and eNOS (eNOS enzyme-linked immunosorbent assay kit, nanjing institute of bioengineering) by using a detection kit according to the instruction, and testingThe results are shown in Table 5.

TABLE 5

The ratio of P to control group is less than 0.05; * P <0.05 to model group; p <0.05 for the other sample groups.

As can be seen in FIG. 4, the least significant apoptosis rate after sample 3 was affected was closest to the control group; as can be seen from fig. 5 to 7, sample 3 had minimal expression of each inflammatory factor after the effect, closest to the control group; as can be seen in fig. 8, sample 3 had the highest mitochondrial content after the effect, closest to the control group.

Test example 4 animal experiments (sample 3 with blood AGEs level)

The experimental method comprises the following steps:

1. animal grouping: mice of 2 months of age were divided into groups according to body weight, and randomly divided into 1 model group, malvidin-3-O-glucoside group, sample 1 group, sample 3 group, sample 5 group, sample 6 group, and 1 blank control group.

2. Preparing a feed: common feed: SPF grade maintenance feed; high AGEs feed: will be provided with ⁶⁰ The SPF-grade maintenance feed sterilized by Co irradiation is baked at 160 ℃ for 40 minutes to prepare a high advanced glycation end product feed (high AGEs feed), and the concentration of carboxymethyl lysine and carboxyethyl lysine in the high AGEs feed is determined to be more than 2 times of that of a control feed.

3. The experimental method comprises the following steps: the samples were dissolved in purified water to prepare 1mg/ml stock solutions and the solutions were gavaged at doses ranging from 0.9 to 3 mg/kg. Wherein the model group and the sample group were given high AGEs feed and the blank control group was given normal feed for 1 month. Meanwhile, the sample group is perfused with the tested sample, and the model group and the blank control group are dosed with the same volume of solvent. 0.4ml of tail tip blood is taken as a baseline sample before intervention starts, 0.4ml of blood sample is taken from an animal which is sacrificed at the end of the experiment, and the content of the combined carboxymethyl lysine (CML) and the combined carboxyethyl lysine (CEL) in the blood pressure sample is detected by adopting a UPLC-MS method.

The experimental results are as follows: CML and CEL in the blood of model mice were significantly elevated compared to the blank control group (P < 0.05); compared with the model group, the malvidin-3-O-glucoside, myricetin, sample 1, sample 3, sample 5 and sample 6 can obviously reduce the CML and CEL contents in blood (P < 0.05), and the effect of sample 3 is obviously better than that of other groups (P < 0.05).

And (4) experimental conclusion: sample 3 can significantly reduce AGEs in the blood of mice and is significantly superior to other sample groups.

Test example 5 animal experiment (sample 3 acute toxicity experiment)

The experimental standard is as follows: the experiment was carried out according to the method prescribed in GB 15193.3-2014 national Standard for food safety acute peroral toxicity test.

Animal grouping: 20 SPF-grade SD rats are half male and female, and the individual values of the same sex individual weight are within the range of average +/-20%.

The experimental method comprises the following steps: weighing a proper amount of sample, adding a proper amount of deionized water, fully and uniformly mixing, and preparing a test object solution with the concentration of 0.25 g/mL. Before the test, the animals were fasted overnight (about 16 h) with free access to water. In the formal test, the animals in the test group are subjected to intragastric administration of the test solution according to the weight of 20 mL/kg, the test solution is subjected to intragastric administration for 2 times within 24h, the two times of intragastric administration are separated by about 4h, a small amount of feed is administered in the period, and the animals continue to fast for about 3 h after being subjected to the first administration of the test substance. After administration of the test subjects, the time of appearance and disappearance of signs of intoxication and the time of death were observed and recorded for a period of 14 days. The dead animals should be roughly dissected and visually examined for further histopathological examination if the abnormal tissue or organs are found. Weighing was carried out on days 0, 1, 3, 7 and 14, respectively.

The experimental results are as follows: the animals have no abnormal symptoms during the period of sample administration and 14 d observation period, the body weight is normally increased, no animal death is seen, and the LD50 is more than 10 g/kg body weight. At the end of the test, all animals were roughly dissected and visually observed without abnormality.

And (4) experimental conclusion: the sample is detected according to GB 15193.3-2014 acute oral toxicity test of national food safety standard, and has acute oral toxicity LD50 of more than 10 g/kg body weight for SD rat, and is practically nontoxic.

Test example 6 animal experiments (sample 3 and type II diabetes)

Based on the fact that elevated levels of AGEs are a risk factor for the development of diabetes and complications, and AGEs are an independent risk factor for insulin resistance, the present application further studies the relationship between sample 3 and type ii diabetes.

Experimental animal models and groups: streptozotocin (STZ, product of Sigma, dissolved in citrate buffer at pH4.5 at a concentration of 0.1 mmol/L, ready for use) was injected into the lower left abdominal cavity of rats at a dose of 60 mg/kgBW. The normal control group was injected with an equal volume of citrate buffer. After 72 hours, the tail blood of the rat is taken for measuring the blood sugar, and the rat with the blood sugar concentration of more than 16.7mmol/L is taken as the diabetic rat. Diabetic rats were randomly divided into 1 model group, sample 3 groups, and 1 blank control group was additionally provided.

The administration method comprises the following steps: the samples were dissolved in purified water to prepare 1mg/mL stock solutions and administered by gavage at a dose of 0.9-3 mg/kg. Sample 3 groups were gavaged with test samples, model groups and blank control groups were given the same volume of solvent.

Detection indexes are as follows: the main measurement indexes include body weight, glucose tolerance, fasting Blood Glucose (FBG), insulin (INS) content in serum, etc

The experimental results are as follows: the sample 3 can obviously reduce the FBG level and the serum INS content of the type II diabetic rat, and improve the weight loss and the insulin resistance symptoms of the type II diabetic rat.

Test example 7 animal experiments (sample 3 and Alzheimer's disease)

Based on the close correlation between increased AGEs levels and decreased cognitive abilities in the elderly, the present application further investigated the relationship between sample 3 and alzheimer's disease.

Experimental animal models and groups: 3x Tg-AD mice (Psen 1tm1Mpm Tg (APPSwe, tauP 301L) triple-transgenic AD mice on C57BL6 background), 4-month-old AD mice were randomly divided into 1 model group, sample 3 group, and 1 blank control group.

The administration method comprises the following steps: the samples were mixed into the mouse feed at a dose of 0.9-3mg/kg and administered by free feeding for a period of 3 months. The group of sample 3 was given the feed supplemented with the test sample, and the model group and the blank control group were given the maintenance feed supplemented with the placebo.

Detection indexes are as follows: the learning and memory level of each group of mice is detected by adopting behavioural experiments such as Morris water maze, diving platform and dark avoidance; and detecting related pathological indexes of Abeta and Tau in the brain of the mouse.

The experimental results are as follows: sample 3 can significantly improve learning and memory ability of AD mice, reduce expression levels of brain A beta and Tau-related pathological proteins, and improve Alzheimer's disease.

Test example 8 animal experiments (sample 3 and atherosclerosis)

Based on the increased AGEs levels and the close correlation between endothelial cell damage and atherosclerosis, the present application further investigated the relationship between sample 3 and atherosclerosis.

Experimental animal models and groups: c57BL/6J mice of males at 4 weeks of age were fed with a high-fat diet for 12 weeks to establish an atherosclerosis model. The atherosclerosis model mice were randomly divided into 1 model group, sample 3 groups, and 1 blank control group was additionally provided.

The administration method comprises the following steps: the samples were dissolved in purified water to prepare 1mg/mL stock solutions and administered by gavage at a dose of 0.9-3 mg/kg. Sample 3 groups were gavaged with test samples, model groups and blank control groups were given the same volume of solvent. Prophylactic administration was started from 6 weeks after the start of high-fat diet feeding for 6 weeks.

Detection indexes are as follows: detecting mouse blood lipid level, inflammation and active oxygen index (ROS, TNF alpha, ICAM, VCAM) and the like.

The experimental results are as follows: sample 3 was able to significantly improve the lipid metabolism including a decrease in TC, LDL-C levels in atherosclerotic mice; by reducing the levels of ROS and inflammatory factors (TNF alpha, ICAM and VCAM), the endothelial function of the mice is improved, thereby slowing the occurrence and the development of atherosclerosis.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent should be subject to the appended claims.

Claims

1. A composition for inhibiting AGEs, or treating AGEs-related injuries or AGEs-related diseases, or relieving AGEs-related injuries or AGEs-related diseases is disclosed, wherein the composition consists of malvidin-3-O-glucoside and myricetin, and the weight ratio of the malvidin-3-O-glucoside to the myricetin is (5 to 7): 1.

2. A composition as in claim 1, wherein the weight ratio of malvidin-3-O-glucoside to myricetin is 5.

3. A dietary supplement capable of inhibiting AGEs comprises effective components and auxiliary materials, wherein the effective components are a composition, the composition comprises malvidin-3-O-glucoside and myricetin, and the weight ratio of the malvidin-3-O-glucoside to the myricetin is (5 to 7): 1.

4. A dietary supplement according to claim 3, wherein the weight ratio of malvidin-3-O-glucoside to myricetin is 5.

5. The dietary supplement of claim 3 or 4, wherein said composition consists of said malvidin-3-O-glucoside and said myricetin.

6. A medicament comprising an active ingredient and a carrier, wherein the active ingredient is the composition of claim 1 or 2, and the medicament is a medicament for inhibiting AGEs, treating AGEs-related damage or AGEs-related diseases, or relieving AGEs-related damage or AGEs-related diseases.

7. The medicament according to claim 6, wherein the inhibition of AGEs is inhibition of AGEs production or promotion of AGEs degradation; and/or the AGEs-related damage is AGEs-mediated apoptosis increase, active oxygen content increase, inflammatory factor TNF-alpha overexpression, inflammatory factor ICAM-1 overexpression, inflammatory factor VCAM-1 overexpression, and/or mitochondrial ATP content decrease; and/or the AGEs-related diseases are any one or more of cardiovascular and cerebrovascular diseases and neurodegenerative diseases.

8. The medicament according to claim 7, wherein the cardiovascular and cerebrovascular diseases are selected from diabetes and its complications, atherosclerosis; and/or the neurodegenerative disease is selected from alzheimer's disease, parkinson's disease, amyotrophic lateral sclerosis.

9. Use of the composition of claim 1 or 2 for the preparation of a medicament, the composition being an active ingredient, the medicament being a medicament for treating, or relieving, AGEs-related diseases or AGEs-related injuries by inhibiting AGEs, the AGEs-related injuries being injuries of umbilical vein endothelial cells by AGEs, the AGEs-related diseases being any one or more of cardiovascular and cerebrovascular diseases and neurodegenerative diseases; the cardiovascular and cerebrovascular diseases are selected from diabetes and complications thereof, atherosclerosis; the neurodegenerative disease is selected from Alzheimer's disease, parkinson's disease, and amyotrophic lateral sclerosis.

10. The use according to claim 9, wherein the inhibition of AGEs is inhibition of the production of AGEs or promotion of degradation of AGEs; and/or the AGEs-related damage is manifested by AGEs-mediated apoptosis increase, active oxygen content increase, over-expression of inflammatory factor TNF-alpha, over-expression of inflammatory factor ICAM-1, over-expression of inflammatory factor VCAM-1, and/or reduction of mitochondrial ATP content.