Composition for reducing intracellular protein glycosylation and preparation method thereof

Abstract

The invention belongs to the technical field of functional food, and particularly relates to a composition for reducing protein glycosylation in cells and a preparation method thereof. The composition consists of mulberry leaf extract and olive powder, and broccoli seed water extract and phyllanthus emblica powder can be added to form the enhanced composition. The mass ratio of the mulberry leaf extract to the broccoli seed water extract to the olive powder to the phyllanthus emblica powder in the enhanced composition is 1: 5: 1: 1. When the composition is prepared according to the mass ratio, the glycosylation inhibition effect of the composition is superior to that of aminoguanidine.

Description

Technical Field

[0001] This application belongs to the field of functional food technology, specifically relating to a composition for reducing intracellular protein glycosylation and its preparation method. Background Technology

[0002] Non-enzymatic glycosylation was first discovered in 1912 by French chemist Louis-Camille Maillard, who observed the browning reaction of proteins with carbohydrates, and this reaction was later named the Maillard reaction. Forty years later, in 1953, Hodge first proposed the basic chemical pathway of the Maillard reaction, which was then confirmed in food cooking. For the next 30 years, scientists have been studying the potential impact of the Maillard reaction on living systems.

[0003] It wasn't until 1981 that Monnier and Cerami discovered the Maillard reaction in vivo and its potential role in biological aging. This reaction involves proteins and glucose in the body undergoing a series of reactions without the involvement of enzymes to produce advanced glycation end products (AGEs). This reaction is called "non-enzymatic glycosylation" to distinguish it from glycosylation, which involves the formation of glycoproteins with the participation of enzymes and is also known as glycaction. More broadly, non-enzymatic glycosylation refers to the complex reactions that can occur between reducing sugars and not only proteins, but also lipids or nucleic acids, without enzymatic catalysis to produce end products.

[0004] Currently, the classic non-enzymatic glycosylation process is considered to consist of three stages. The first stage involves the formation of the Schiff base and Amadori product, the initial products of non-enzymatic glycosylation. The Schiff base is formed by the condensation of a free amino group of protein with the carbonyl group of glucose, followed by Amadori rearrangement to form the Amadori product. Specifically, the carbon-nitrogen double bond in the Schiff base is protonated, causing the glucose portion to form a 1,2-enol form, followed by enol tautomerism, with the 2-position becoming a ketone. The formation of both the Schiff base and the Amadori product in this stage is reversible; the formation of the Schiff base is quick, while the rearrangement of the Schiff base to form the Amadori product requires a longer time.

[0005] Studies have shown that the formation of Schiff bases and Amadori products does not change the color of proteins. The second stage of glycosylation involves the formation of glycosylated carbonyl intermediates. These intermediates are far more reactive than glucose, acting directly on free amino groups in proteins and causing significant physiological damage. Corresponding to the oxidative stress caused by increased oxygen free radicals in the body, this is also known as carbonyl stress. These dicarbonyl compounds are called RCS.

[0006] These dicarbonyl compounds mainly include acetone aldehyde (Methylglyoxal, MGO), glyoxal (Glyoxal, GO), and 3-deoxyglucosone (3-DG).

[0007] These compounds can be formed through various pathways. They can be produced via rearrangement and degradation of the first-stage Amadori product, or via the Wolff pathway, where glucose undergoes auto-oxidation to form the simplest dicarbonyl compound, GO. Besides forming GO, glucose can also dehydrate to form a 2,3-enol structure, subsequently leading to 3-DG and MGO. However, the extent of this reaction from glucose to 3-DG and MGO is very low.

[0008] In addition, carbonyl compounds such as glyceraldehyde and MgO can also be generated in organisms through physiological metabolic pathways. Unstable Schiff bases undergo the Namiki pathway, which involves the loss of erythrose or glyceraldehyde through reverse aldol condensation, followed by oxidation or dehydration to form a carbonyl group, then the loss of a primary amine, and decomposition to generate GO, MgO, and 3-DG.

[0009] In living organisms, GO can also be formed by lipid peroxidation, MGO can be formed by the degradation of triose phosphate, and 3-DG can be formed by the degradation of fructose-3-phosphate. Triose phosphate and fructose-3-phosphate are intermediates formed from glucose via the polyol pathway. The polyol pathway and lipid peroxidation are part of an expanded non-enzymatic glycosylation process. The late-stage glycosylation products ultimately formed via lipid peroxidation are called ALEs.

[0010] The third stage of glycosylation involves the formation of non-enzymatic advanced glycation end products (AGEs), one of which is the direct formation via Amadori products. Amadori products, by removing one molecule of water, can undergo oxidative degradation to directly form CML (Nε-(carboxymethyl)lysine), or oxidative rearrangement to form pentasaccharides, or non-oxidative rearrangement to form glucoseane.

[0011] CML, pentasaccharides, and glucosane are all types of AGEs. Another way AGEs are formed is through the direct reaction of dicarbonyl compounds with lysine and arginine residues on proteins. Some AGEs are generated during the cooking process of everyday food. After being digested and absorbed by the body, these AGEs enter the bloodstream and are transported to various tissues and organs, leading to cell damage and disease. AGEs ingested through food or tobacco are called exogenous AGEs, while AGEs produced within the body are called endogenous AGEs.

[0012] Due to the complex formation process of AGEs and the wide variety of AGEs produced, they can be classified according to their structure and properties. Based on whether their structure is cross-linked, AGEs can be divided into non-cross-linked AGEs and cross-linked AGEs. Non-cross-linked AGEs connect to only one amino acid residue of a protein, while cross-linked AGEs connect to two amino acid residues of a protein. These two amino acids can come from the same protein molecule or different protein molecules, forming intramolecular and intermolecular cross-links. Non-cross-linked AGEs include CML and methylglyoxal-derived hydroimidazole-1, etc.

[0013] Cross-linked AGEs can be classified into Lys-Lys cross-linked and Lys-Arg cross-linked types based on the type of amino acid involved in the cross-linking. Lys-Lys cross-linked AGEs include MOLD (methylglyoxal lysine dimer), while Lys-Arg cross-linked AGEs include glucosane and pentasaccharide. Some AGEs exhibit autofluorescence properties, which allows them to be further classified into fluorescent cross-linked AGEs (such as pentasaccharide and cross-linking agents) and non-fluorescent cross-linked AGEs (such as glyoxal lysine dimer and alkylformylpyrrole glucoside).

[0014] It is important to note that the structures of not all AGEs have been determined. Some AGEs (such as carboxymethyl lysine) remain stable under acidic hydrolysis conditions and are relatively easy to detect; while others are easily decomposed under acidic hydrolysis conditions but can survive during enzymatic hydrolysis, making these AGEs more difficult to detect. Although current advancements in mild protein hydrolysis techniques have reduced the destruction of acid-labile modified amino acids, the scientific community still believes that only a small number of AGEs can be detected in tissue proteins.

[0015] CML is the first AGE (glycated protein) isolated and identified from in vivo. It can be generated by the oxidative degradation of Amadori products or by the reaction of GO (glycated lysine) with lysine residues in proteins. This type of glycosylated lysine, formed through multiple pathways, has been detected in various tissues, such as blood vessels, bladder, cornea, kidneys, brain, and heart. Due to its widespread distribution in the human body, CML has become one of the main biomarkers of glycosylation reactions in humans.

[0016] Pentosaccharide was the first cross-linked AGE (Advanced Glyceryl Acetate) discovered in living organisms. It cross-links proteins by linking lysine and arginine residues together, resulting in intramolecular or intermolecular cross-linking. Furthermore, it is an AGE with autofluorescence properties, similar to other AGEs with aromatic chemical structures. Pentosaccharide can be obtained from Amadori products through oxidative rearrangement. Pentosaccharide has a low formation rate in collagen. Another important cross-linked AGE is glucosane, whose formation does not require an oxidation step. Its concentration in the extracellular matrix is ​​approximately 20 times that of pentosaccharide, making it the most important cross-linked AGE in the extracellular matrix to date. It is also a lysine-arginine cross-link.

[0017] Since Monnier proposed in 1981 that non-enzymatic browning of proteins in the body may be related to aging and related pathologies, this "non-enzymatic glycosylation aging hypothesis" has revealed that non-enzymatic glycosylation products are one of the biomarkers of aging. Most AGEs accumulate in tissue proteins with low turnover rates as an individual ages, including lens proteins and collagen. Dyer's comparative study on the degree of glycosylation of human lens proteins and skin collagen showed that human skin collagen glycosylation increased by 33% between the ages of 20 and 80. Verzijl analyzed the content of three AGEs—CML, CEL (carboxyethyl lysine), and pentasaccharide—in cartilage of 20 normal individuals aged 3-81 years and skin collagen of 26 normal individuals aged 19-91 years, and found that AGEs in both cartilage and skin collagen accumulated linearly with actual age. The production and accumulation of these AGEs, on the one hand, lead to protein functional damage by altering molecular recognition; on the other hand, AGE cross-linking can change the mechanical properties of structural proteins such as collagen, leading to hardening of blood vessels and tissues and decreased viscoelasticity. These changes can lead to a range of adverse pathological changes, including arteriosclerosis, atherosclerosis, nephropathy, retinopathy, and neuropathy.

[0018] In addition, AGEs interact with their receptor RAGE to trigger oxidative stress and activate cell signaling pathways, leading to metabolic disorders in cells, tissues and organs, and playing a pathogenic role in a variety of age-related chronic diseases (diabetes, atherosclerosis, etc.).

[0019] Our skin ages as we get older, gradually showing signs of aging. Skin aging makes it drier, thinner, and develops age spots. It becomes less elastic, stiffer, and develops fine lines and wrinkles. Skin tone also changes – these are all visible characteristics of skin aging. The skin aging process can be divided into two categories – internal aging and external aging. External aging is mainly due to the skin's exposure to harsh environments, such as ultraviolet radiation or environmental pollution.

[0020] There are different theories about the origin of intrinsic aging. One theory is the Hayflick phenomenon, or cellular senescence, that occurs during the culture of fibroblasts. Another intrinsic mechanism leading to skin aging is the damage caused by free radicals that accumulate with age (the free radical theory). Currently, the theory of intracellular protein glycosylation caused by free radicals is widely considered the more common intrinsic mechanism of skin aging. AGEs can cause changes in the biomechanical properties and biochemical changes of the skin, including activating the synthesis of molecules (such as macromolecules and cytokines in the extracellular matrix) and activating matrix metalloproteinases or matrix-degrading enzymes, as well as causing dysfunction of skin fibroblasts.

[0021] Collagen is a crucial, lifelong, and most ubiquitous protein, a major component of the extracellular matrix of human skin tissue. Collagen peptides consist of three peptide chains assembled into a triple helix structure. This structure can form microfibrils, which in turn form larger fibrils or fibers within the tissue. After fiber formation, the collagen chains can cross-link and interconnect under enzymatic action, enhancing the tissue's structural and biochemical stability.

[0022] The primary function of the collagen network is to provide a supportive extracellular framework for cells, promoting cell attachment, growth, differentiation, migration, and tissue morphogenesis. Collagen glycosylation is a non-enzymatic alteration that occurs due to aging. After glycosylation, collagen's structure changes, adversely affecting the biophysical and biomechanical properties of tissues.

[0023] On the one hand, the chemical reaction with the free amino groups on the side chains of collagen molecules will directly affect the triple helix properties of collagen, thereby affecting the function of tissues and organs. On the other hand, the formation of cross-linked AGEs leads to cross-linking within and between collagen molecules. During the cross-linking process, the arrangement and spatial structure of collagen fibers are destroyed, and the collagen in the dermis becomes stiff and loses its elasticity, making the skin thinner and wrinkled, ultimately leading to skin aging.

[0024] The accumulation of AGEs in skin tissue is a protracted process, with the accumulation rate correlated with protein lifespan and turnover rate. Collagen in skin tissue has a half-life of 15 years, making it a potential target for glycation. Various AGEs are present in human skin collagen, including pentosaccharin, CML, glucosane, and arginine pyridine. Verzijl's analysis of human skin samples showed that AGEs in skin collagen accumulate linearly with age, and the content of CML, CEL, or pentosaccharin increased 3 to 4 times between the ages of 20 and 80. Monnier's research data indicated that glucosane is the most abundant AGE in human skin collagen. Besides the dermis, AGEs are also detected in the epidermis. In the epidermis, glycated keratin leads to yellowing of the skin and reduced stratum corneum moisture content, causing dry skin. Furthermore, sun exposure accelerates AGE formation.

[0025] Existing glycosylation inhibitors can be divided into two categories: chemically synthesized inhibitors and natural inhibitors.

[0026] Aminoguanidine (AG), the first synthetic AGEs inhibitor discovered in clinical trials, can capture highly reactive carbonyl compounds, including methylglyoxal (MGO) and deoxyglucuronide (3-DG), thereby blocking the conversion of carbonyl compounds into AGEs and the formation and development of AGEs. Pyridoxamine, an amine in vitamin B6, acts as an AGEs inhibitor primarily by chelating metal ions and mitigating the Maillard reaction. Carnosine, a dipeptide, inhibits AGEs through three mechanisms: intercepting ROS, interacting with carbonyl groups on proteins, and preventing cross-linking of glycated proteins. Although synthetic inhibitors are effective in inhibiting the formation and development of glycation, most have safety concerns and side effects, such as liver damage, gastrointestinal disorders, and headaches, limiting their application.

[0027] Active ingredients found in natural plants include polyphenols, polysaccharides, and terpenes, which play important roles in inhibiting the development of advanced aging processes (AGEs), reducing protein glycosylation, and providing antioxidant protection. Among these, polyphenols have been identified as the most effective antioxidants, and literature has reported their in vitro inhibitory activity against AGEs.

[0028] Polyphenol inhibitors can be classified into flavonoids, stilbenes, lignans, and phenolic acids. Flavonoids are a class of polyphenolic compounds containing a C6-C3-C6 structure, distributed in plants, vegetables, fruits, and traditional Chinese medicines. They have attracted widespread attention due to their various health benefits (such as anti-glycation, antioxidant, anti-inflammatory, and antiviral effects). Based on their chemical structure, flavonoids can be further classified into flavonoids, flavonols, flavanones, flavanols, chalcones, and isoflavones.

[0029] Rutin, a common flavonoid compound found in both food and medicine, can inhibit the development of advanced glycation end products (AGEs) and has certain efficacy in preventing and treating hyperglycemia. Studies using the bovine serum albumin (BSA)-glucose (Glu) model have shown that rutin effectively scavenge free radicals and inhibits various stages of AGE development more effectively than AG. Quercetin, a flavonol widely found in traditional Chinese medicine, vegetables, and fruits, has had its preventive effects on protein glycosylation and AGE formation proven by numerous researchers. Quercetin can significantly inhibit the formation and development of carbonyl compounds during protein glycosylation. Furthermore, quercetin inhibits protein glycosylation in a dose-dependent manner, mitigating the microenvironmental changes in protein conformation caused by glycosylation. Compared to synthetic glycosylation inhibitors, natural glycosylation inhibitors have greater application potential in the drug prevention and treatment of diabetes and its related complications, exhibiting a more significant ability to inhibit AGEs and the glycosylation process.

[0030] Mulberry leaf extract refers to substances from which active ingredients are extracted from mulberry leaves, possessing various health benefits and application value. Commonly used extraction processes include water extraction, ethanol extraction, and ultrasonic extraction. Taking water extraction as an example, the process flow is: mulberry leaf powder → sieving → extraction (hot water extraction) → concentration → spray drying → mulberry leaf instant powder. The hot water extraction method for preparing mulberry leaf extract can effectively extract the active ingredients from mulberry leaves while preserving the original nutrients and biological activity of the leaves.

[0031] In recent years, numerous studies have shown that mulberry leaf powder and extracts are rich in various nutrients, including crude protein, amino acids, polyphenols, minerals, and vitamins. These components possess diverse biological activities and functions, such as immune regulation, antioxidation, and anti-cancer effects, playing a vital role in human health. Therefore, mulberry leaf powder and extracts have broad application prospects in the food, pharmaceutical, and other fields.

[0032] Mulberry leaf powder contains various vitamins, such as vitamin C, vitamin E, vitamin B1, and vitamin B2. These vitamins play important roles in human growth and development, the immune system, and the nervous system. Vitamin C can promote collagen synthesis and enhance skin elasticity; vitamin E can act as an antioxidant and delay aging; vitamin B1 can promote metabolism and maintain nerve function; and vitamin B2 can promote protein synthesis and metabolism, maintaining healthy skin.

[0033] Mulberry leaf polysaccharide is a natural high-molecular-weight polysaccharide, mainly composed of various large-molecule carbohydrates. It can be divided into different components, including mannan, galacturonic acid, and chitosan. Mulberry leaf polysaccharide has significant anti-inflammatory effects, inhibiting inflammatory responses and reducing their harmful effects on the body. Furthermore, it can promote insulin secretion and increase the body's utilization of glucose, thereby lowering blood sugar levels and showing potential as an adjunct treatment for diabetes.

[0034] Mulberry leaf powder and extract are rich in flavonoids and polyphenols, natural antioxidants that effectively inhibit the generation of free radicals and protect cells from oxidative damage, thus playing an antioxidant role. This is helpful in preventing aging and diseases such as cancer. Rutin, a flavonoid compound with high content, has good antioxidant and anti-inflammatory effects. Gamma-aminobutyric acid (GABA) in mulberry leaf powder and extract is a neurotransmitter that can promote brain tissue metabolism and restore brain cell function, while also improving cerebral blood circulation, enhancing the activity of angiotensin-converting enzyme I, and having a significant blood pressure-lowering function.

[0035] The human immune system is crucial for maintaining homeostasis, including defense against, clearance of, and surveillance of pathogens. Abnormal immune regulation can lead to autoimmune diseases. Mulberry leaf powder and extracts contain various polysaccharides, such as mulberry leaf polysaccharides and β-glucan, which have immunomodulatory effects, enhancing immunity and preventing illnesses such as colds. Therefore, appropriate consumption of mulberry leaf powder and extracts can help regulate the human immune system.

[0036] Green olives (Canarium album), also known as green fruit, are the fruit of a tree belonging to the genus *Olive* in the family Burseraceae, order Rutales, suborder Rutaceae. They are a famous subtropical fruit in my country. Besides being rich in protein, fat, and carbohydrates, they are also rich in vitamin C, carotene, retinol, vitamin B1, vitamin B2, dietary fiber, and niacin, as well as elements such as calcium, iron, chromium, manganese, and aluminum. Olives are a traditional food and medicine, and modern research has found that they are rich in flavonoids and polyphenols, possessing excellent antibacterial and antioxidant properties.

[0037] Flavonoids in olives are mainly distributed in the leaves and fruits. Three flavonoid compounds are found in olive fruits: kaempferol-3-O-β-D-glucoside, hyperoside, and cephalotaxine. However, different materials and extraction methods result in variations in the types and amounts of flavonoids extracted from olive fruits. Olives also contain polyphenols. Researchers have extracted several hepatoprotective phenolic compounds from olive leaves and fruits: brevifolin, hyperoside, ellagic acid, and 3,3'-methoxyellagic acid. He Zhiyong et al. studied *Olive santalinus* from Fujian and found that phenolic substances are mainly distributed in the olive pulp, with gallic acid and ellagic acid being particularly abundant. Studies on tannin components in olive stem bark show that the condensed tannins belong to the proanthocyanidin and prodelphin types, and gallic esters were found in the structural units of proanthocyanidins and prodelphin—catechin and epigallocatechin.

[0038] Phyllanthus emblica L., belonging to the genus Phyllanthus in the family Euphorbiaceae, is a perennial shrub or tree. It is also known as Amla (ancient name, medicinal name), Yugan (Fujian and Taiwan), Dianganlan (Yunnan), "Amla," and "Indian Gooseberry," among other names. Phyllanthus emblica is an economically valuable plant resource with both medicinal and edible uses, and is one of the three health-promoting plants encouraged for cultivation worldwide by the World Health Organization.

[0039] Phyllanthus emblica is widely used in traditional Chinese medicine among ethnic minorities (including Tibetans, Yi, Dai, Miao, Bai, Naxi, Lahu, Pumi, Wa, Achang, Jino, Buyi, Yao, Zhuang, Mongolians, and Uyghurs, among about 16 other ethnic groups) to treat colds, coughs, sore throats, diarrhea, indigestion, fever, inflammation, promote saliva production and moisten the lungs, treat skin eczema, and burns.

[0040] Modern medicine has proven that the roots, stems, leaves, fruits, and seeds of Phyllanthus emblica are rich in a variety of bioactive substances, including organic acids, terpenes, flavonoids, polysaccharides, tannins, alkaloids, phytosterols, amino acids, and vitamins. They possess antioxidant, anticancer, anti-aging, cholesterol-lowering, antidiabetic, immunomodulatory, antiviral, anti-dyslipidemia, anti-apoptotic, antipyretic, analgesic, anti-inflammatory, hepatoprotective, cardioprotective, antimutagenic, antibacterial, and antidiarrheal properties.

[0041] The fruit of the Phyllanthus emblica contains abundant free radical scavengers, such as resveratrol, gallic acid, tannic acid, lignans, quercetin, genistein, anthocyanins, hesperidin, kaempferol, superoxide dismutase, vitamin C, polysaccharides, tannins, and organic acids, exhibiting strong antioxidant capabilities. Phyllanthus emblica extract can significantly increase the activity of superoxide dismutase in erythrocytes of middle-aged and elderly individuals, reduce lipid peroxidation levels, and scavenge hydroxyl and superoxide free radicals generated during metabolism.

[0042] In addition, amla polysaccharides also have the effect of scavenging superoxide free radicals. Polyphenols, tannins, and organic acids in amla can significantly reduce malondialdehyde levels in mouse blood. The methanol extract of dried amla fruit contains active ingredients such as tannins and geranyl oleoresin, which can effectively scavenge nitric oxide (NO•) free radicals. Amla is rich in flavonoids, which can enhance the activity of endogenous antioxidants (such as catalase, superoxide dismutase, glutathione peroxidase, and glutathione reductase) in mouse blood, while reducing lipid peroxidation levels and protecting cells against oxidative stress.

[0043] Phyllanthus emblica can enhance immunomodulatory function by increasing serum levels of induced T cells, suppressor T cells, immunoglobulin M, immunoglobulin G, as well as serum albumin and globulin levels. Furthermore, the leaves, roots, and bark of Phyllanthus emblica have been shown to possess strong antioxidant capabilities. The vitamin C in Phyllanthus emblica fruit is highly stable, and its superoxide dismutase analogues exhibit storage resistance, heat resistance, and small-molecule transdermal properties. It is also rich in various free radical scavengers and endogenous antioxidants, effectively enhancing the body's antioxidant function, inhibiting free radical production, and scavenging excess free radicals. Therefore, it has significant application value in the development of pharmaceuticals, functional foods, and cosmetics.

[0044] Glucosinosides are secondary metabolites found in cruciferous plants. These compounds are naturally stable; when plant tissues are damaged, endogenous myrosinase is released, and the glucosinosides are hydrolyzed to produce various active substances. Sulforaphane (1-isothiocyanate-4-methanesulfonylbutane) is one of the most abundant substances, particularly found in broccoli seeds. Studies have shown that sulforaphane is one of the most potent and effective natural products found in vegetables to date, exhibiting the ability to inhibit cancer cell proliferation, induce detoxification enzymes, arrest the cell cycle, and induce apoptosis. It has shown good preventive and inhibitory effects against cancers including liver cancer, pancreatic cancer, lung cancer, and rectal cancer. In addition to its anti-cancer activity, sulforaphane also shows good efficacy in lowering blood pressure, protecting the heart, alleviating diabetes, inhibiting bacteria, and improving schizophrenia and Alzheimer's disease. Meanwhile, studies have shown that sulforaphane can also activate the Nrf2 pathway to regulate oxidative stress responses, thereby indirectly improving the body's antioxidant activity and immune response. Summary of the Invention

[0045] The applicant has conducted in-depth research on intracellular protein glycosylation and found that a combination of mulberry leaf extract and olive powder in a certain proportion has the ability to significantly reduce protein glycosylation.

[0046] This application first discloses a composition for reducing protein glycosylation, consisting of mulberry leaf extract and olive powder.

[0047] The extraction method of the mulberry leaf extract is as follows:

[0048] Step 1: Take mulberry leaves, rinse them, dry them, and then crush them.

[0049] Step 2: Take mulberry leaf powder, add citrate-disodium hydrogen phosphate buffer, add compound enzyme preparation, and extract by ultrasonication in a water bath.

[0050] Step 3: Heat the extract to inactivate enzyme activity, centrifuge, and collect the supernatant.

[0051] Step 4: Filter the supernatant under reduced pressure to obtain a clear extract.

[0052] Step 5: Clarify the extract, spray dry, and pulverize to obtain the finished product.

[0053] The compound enzyme preparation consists of cellulase, pectinase, and hemicellulase.

[0054] The dosage of the compound enzyme preparation, based on the mass of mulberry leaf powder, is 1.5% cellulase, 1.0% pectinase, and 0.5% hemicellulase.

[0055] The olive powder is obtained by freeze-drying and then pulverizing green olives.

[0056] The mass ratio of mulberry leaf extract to olive powder in the composition is 1:1 to 10, preferably 1:2 to 5, and more preferably 1:5.

[0057] To enhance the anti-glycation effect of the above composition, broccoli seed water extract and amla powder can be added to the composition to form an enhanced composition.

[0058] The amla powder is obtained by freeze-drying amla and then pulverizing it.

[0059] The enhanced composition contains mulberry leaf extract, olive powder, broccoli seed water extract, and amla powder in a mass ratio of 1:5:1:1.

[0060] This application further discloses the use of the enhanced composition in the preparation of functional foods that reduce intracellular protein glycosylation.

[0061] The beneficial effects of this application are as follows: This application first discloses a composition for reducing intracellular protein glycosylation, wherein the composition consists of mulberry leaf extract and olive powder, and broccoli seed water extract and amla powder can be added to further form an enhanced composition. The mass ratio of mulberry leaf extract, broccoli seed water extract, olive powder, and amla powder in the enhanced composition is 1:5:1:1. After preparation, the glycosylation inhibition effect of this composition is superior to that of aminoguanidine.

[0062] Explanation of abbreviations:

[0063] AGEs: Advanced Glycation End Products

[0064] RAGE: Receptor for Advanced Glycosylation End Products

[0065] MGO: Methylglyoxal

[0066] GO: Glyoxal

[0067] 3-DG: 3-Deoxyglucosone

[0068] CML: Nε-(carboxymethyl)lysine (carboxymethyl lysine)

[0069] CEL: Nε-Carboxyethyl Lysine (Carboxyethyl Lysine)

[0070] MOLD: Methylglyoxal Lysine Dimer

[0071] ALES: Late-stage lipid peroxidation products

[0072] ROS: Reactive Oxygen Species

[0073] GABA: γ-aminobutyric acid (neurotransmitter)

[0074] ELISA: Enzyme-linked immunosorbent assay

[0075] PBS: Phosphate Buffer

[0076] BSA: Bovine serum albumin

[0077] DMSO: Dimethyl sulfoxide (organic solvent)

[0078] NBT: Nitroblue tetrazolium (Amadori product detection reagent)

[0079] OPD: o-phenylenediamine (dicarbonyl compound derivatizing reagent)

[0080] DMEM: Dulbecco Modified Eagle Medium

[0081] FBS: Fetal bovine serum

[0082] MTT: Thiazol Blue (cell viability assay reagent)

[0083] TMB: 3,3',5,5'-Tetramethylbenzidine

[0084] Proclin 300: Preservative. Detailed Implementation

[0085] The present application is further illustrated below with reference to specific embodiments, but the embodiments do not limit the present application in any way. Unless otherwise specified, the reagents, methods, and equipment used in this application are conventional reagents, methods, and equipment in this technical field. Unless otherwise specified, the reagents and materials used in the following embodiments are all commercially available.

[0086] Example 1: Preparation of Mulberry Leaf Extract

[0087] Step 1: Take fresh, impurity-free mulberry leaves, rinse them, dry them at 40~50℃ until the moisture content is ≤8%, crush them and pass them through an 80~100 mesh sieve, and seal them for later use.

[0088] Step 2: Take mulberry leaf powder, add citrate-disodium hydrogen phosphate buffer at pH 5.0 at a material-liquid ratio of 1:15, add compound enzyme preparation (cellulase 1.5%, pectinase 1.0%, hemicellulase 0.5%, based on the mass of mulberry leaf powder), and extract by ultrasonication in a 45℃ water bath (frequency 40kHz, power 300W) for 40 minutes.

[0089] Step 3: Rapidly heat the extract to 85℃ and maintain for 10 minutes to inactivate enzyme activity, then centrifuge at 4000~5000r / min for 15 minutes and collect the supernatant.

[0090] Step 4: The supernatant is filtered under reduced pressure through a 0.45μm microporous membrane to obtain a clear extract.

[0091] Step 5: Spray dry the clarified liquid (inlet air temperature 160℃, outlet air temperature 80℃), pulverize it through a 100-mesh sieve to obtain the finished product (moisture content ≤5%), and store it in a dark, sealed container.

[0092] Example 2: Preparation of anti-glycation composition

[0093] Table 1 Formulation design of anti-glycation compositions

[0094] .

[0095] Preparation method:

[0096] Step 1: Take mulberry leaf extract, olive powder, amla powder, and broccoli seed water extract, grind them, and pass them through a 100-mesh sieve for later use.

[0097] Step 2: Take the prescribed amount of mulberry leaf extract and olive powder, and mix them evenly;

[0098] Step 3: Add the prescribed amount of broccoli seed water extract to the mixture obtained in Step 2 and mix well;

[0099] Step 4: Add the prescribed amount of amla powder to the mixture obtained in Step 3, mix well, and package to obtain the final product.

[0100] Note: If a certain component is not present in the composition formulation, the steps of crushing and mixing that component are skipped during the preparation process.

[0101] Example 3: Determination of the effect of the composition of Example 2 on reducing protein glycosylation

[0102] 3.1 Establishment of an in vitro non-enzymatic protein glycosylation model

[0103] Fetal bovine serum albumin (30 mg / mL), glucose solution (300 mg / mL), and aminoguanidine solution (60 μg / mL) were prepared using 0.2 mol / L phosphate-buffered saline (PBS, pH 7.4, 0.2% proclin 300 as preservative). Mulberry leaf extract (60 μg / mL), broccoli seed aqueous extract (60 μg / mL), olive powder (60 μg / mL), amla powder (60 μg / mL), and the various formulations obtained in Example 2 (60 μg / mL, based on the mass of mulberry leaf extract) were dissolved in DMSO.

[0104] Table 2: Sample preparation methods for in vitro protein non-enzymatic glycosylation model experiments

[0105] .

[0106] The above samples were cultured in a 37 ℃ constant temperature incubator for 10 days. After 10 days, the culture medium was collected for the determination of Amadori products, dicarbonyl compounds, fluorescent late glycosylation products, and non-crosslinked late glycosylation products (CML). The determination methods are as follows:

[0107] Amadori product determination:

[0108] 0.2 mL of culture medium and 0.8 mL of NBT reagent (0.3 mmol / L) were added to 2 mL of carbonate buffer (100 mmol / L, pH=10.1). After reacting at 40 °C for 1 h, the OD value was measured at 530 nm using a microplate reader. The OD value represents the amount of Amadori product generated.

[0109] Determination of the content of dicarbonyl compounds:

[0110] Take 1 mL of culture medium into a 30 kD ultrafiltration tube and centrifuge at 4500 G for 12 min. Take 900 μL of the filtrate from the outer tube, add 20 μL of acetic acid and 30 μL of 100 mmol / L OPD solution to a sample vial, mix, purge with nitrogen, and derivatize at 40 ℃ for 80 min. Analyze the reaction solution and standards quinoxaline and 2-methylquinoxaline using ultra-high performance liquid chromatography-tandem quadrupole time-of-flight mass spectrometry. Determine GO, MGO, and 3-DG using pre-column derivatization high performance liquid chromatography. After the derivatization reaction of the culture medium, analyze using high performance liquid chromatography.

[0111] The high performance liquid chromatography (HPLC) conditions were as follows: column: WondaCract ODS-2 C18 column (150 mm × 4.6 mm × 5 μm); column temperature: 25 ℃; flow rate: 0.8 mL / min; injection volume: 10 μL; UV detector: 313 nm; mobile phase: phase A was 10 % (V / V) methanol aqueous solution (0.2% acetic acid), phase B was methanol (0.2% acetic acid); elution conditions: time min (phase A concentration), 0 (90%) - 12 (0%) - 15 (0%) - 16 (90%) - 20 (90%).

[0112] The content of GO, MGO and 3-DG is expressed by peak area.

[0113] Assay of fluorescent late-stage glycosylation products:

[0114] Using Pageon's method, fluorescence values ​​in the culture medium were measured at excitation / emission wavelengths (ex / em) of 370 / 440 nm and 335 / 385 nm using a fluorescence spectrophotometer, and the inhibition rate of the composition of Example 2 or aminoguanidine on fluorescent late glycosylation products was calculated according to the following formula.

[0115]

[0116] In the formula: A1 is the fluorescence value of the negative control; A0 is the fluorescence value of the group containing only BSA; B1 is the fluorescence value of the group containing the composition of Example 2 or AG; B0 is the fluorescence value of the corresponding blank control of the group containing the composition of Example 2 or AG.

[0117] The inhibition rate (%) represents the inhibition of fluorescent late glycosylation products by each group of samples.

[0118] Determination of CML in non-crosslinked late-stage glycosylation products:

[0119] The content of CML in the culture medium was determined using enzyme-linked immunosorbent assay (ELISA). Samples were added to microwells coated with CML antibody, which then bound to horseradish peroxidase-labeled CML antibody to form an antibody-antigen-enzyme-labeled antibody complex. After washing, TMB was added for color development, and the OD value was measured at 450 nm using an ELISA reader.

[0120] The amount of non-crosslinked late-stage glycosylation product CML generated is expressed as OD value.

[0121] The obtained data is shown in the table below:

[0122] Table 3: Effect of the composition of Example 2 on the content of Amadori products (n=3)

[0123] .

[0124] Amadori products are the products of the first stage of non-enzymatic glycosylation of proteins. They are generated by the Amadori rearrangement of imine intermediates (Schiff bases) formed between free amino or N-terminal amino groups of proteins and reducing sugars.

[0125] The second stage of glycosylation is the formation of carbonyl intermediates, mainly GO, MGO, and 3-DG. These intermediates can directly act on free amino groups in proteins and cause significant physiological damage. Therefore, reducing the content of dicarbonyl compounds can effectively inhibit non-enzymatic glycosylation reactions of proteins and reduce the production of AGEs.

[0126] In the later stages of glycosylation, complex AGEs can form through various pathways. One is through direct oxidation or non-oxidation, rearrangement, and degradation of the Amadori product; the other is through the direct reaction of dicarbonyl compounds with lysine and arginine residues on the protein. Both pathways ultimately lead to fluorescent AGEs. For example, the direct oxidative degradation of the Amadori product can form pentasaccharides, and MGO can react with two lysine residues to form MOLD, which has fluorescent crosslinking properties. Therefore, various fluorescent AGEs can be detected by using different excitation wavelengths.

[0127] CML was the first type of AGE isolated and identified from the human body. It is widely present in glycosylated proteins in various tissues. It is a non-crosslinked AGE and is mainly formed by two pathways: oxidative degradation of Amadori products and reaction of GO with lysine residues on proteins.

[0128] As can be seen from the data in the table above, the positive control group (sample 12) used aminoguanidine as a glycosylation inhibitor. Compared with sample 13, which did not add any glycosylation inhibitor, it significantly inhibited the formation of Amadori products, dicarbonyl compounds, AGEs, and CML products. Samples 1-4 used mulberry leaf extract, broccoli seed water extract, olive powder, and amla powder as potential glycosylation inhibitors, respectively. Compared with sample 13, there was no significant difference in the numerical changes. These four components only showed weak glycosylation inhibitory effects, among which mulberry leaf extract was the best.

[0129] Samples 5-8 were sequentially formulated using formulations 1-4 of Example 2 as glycosylation inhibitors. Formulations 1-4 each contained four components, differing only in the amount of olive powder added. The glycosylation inhibition effect increased with increasing olive powder content. When the olive powder content was 50g, corresponding to sample 7, the glycosylation inhibition effect of this group even exceeded that of the positive control group (aminoguanidine). However, when the olive powder content exceeded 50g, reaching 100g, the glycosylation inhibition effect of this group (sample 8) decreased. Therefore, the composition of formulation 3 of Example 2 was considered the optimal glycosylation inhibitor composition.

[0130] Samples 9-11 correspond to formulations 5-7 of Example 2, respectively. Formulation 5 contains only mulberry leaf extract and olive powder, while formulations 6 and 7 each contain three components. It can be seen that the amount of glycosylation products generated in these three groups of samples is higher than that in samples 5-8 (i.e., the combination treatment groups of formulations 1-4). This indicates that the glycosylation inhibitory effect of the four-component combination is better than that of the two- or three-component combination, and the glycosylation inhibitory effect of the three-component combination is better than that of the two-component combination.

[0131] In summary, the composition of mulberry leaf extract, broccoli seed water extract, olive powder, and amla powder exhibits a significant inhibitory effect on protein glycosylation. The optimal glycosylation inhibition effect is achieved when the mass ratio of the four components is 1:5:1:1, followed by 1:2:1:1, 1:10:1:1, and 1:1:1:1.

[0132] Example 4: Anti-glycation effect of the composition of Example 2 in a biomimetic dermal model

[0133] 4.1 Construction of dermal equivalent containing cellular senescence

[0134] Ribose (100 mmol / L) and the compositions of Examples 2, Formulations 1-7 (100 μg / mL based on mulberry leaf extract mass) were added to collagen (4 mg / mL). A negative control was used without the composition of Example 2, a positive control was used with 100 μg / mL aminoguanidine, and a blank control was used for collagen without added ribose. The mixture was incubated at 37 °C for 24 days to obtain a preglycosylated collagen solution.

[0135] The preglycosylated collagen solution and 20 mmol / L acetic acid solution were added to the inner tube of a 10 kD ultrafiltration tube (the ultrafiltration tube is compatible with 50% acetic acid by volume) in 50 mL of the outer tube. The mixture was centrifuged at 4500×g for 20 min, and repeated 3 times to remove ribose, small molecules and aminoguanidine from the preglycosylated collagen.

[0136] Mix preglycated collagen with untreated collagen in equal volumes (to prevent the preglycated collagen from failing to form a gel).

[0137] Mix 1 mL of DMEM at a concentration of 10x, 1.2 mL of 0.1 mol / L NaOH, and 2.1 mL of water.

[0138] Mix 200 μL of collagen solution with an appropriate amount of neutralizing solution (0.1 mol / L NaOH solution, adjusted to pH 7.2-7.4), and add 40 μL of human skin fibroblasts resuspended in FBS (7.5 × 10⁻⁶ cells). 6 Add the cells (per mL) to a polytetrafluoroethylene culture dish (15 mm, 5 mm) and place it in a 37 ℃ constant temperature incubator.

[0139] 4.2 Cell proliferation

[0140] Cell proliferation was assessed after 3 days of culture in dermal equivalents. Cell viability was used to represent the inhibitory effect of each experimental group on extracellular matrix collagen glycosylation. (When collagen undergoes glycosylation, its promoting effect on cell proliferation decreases, leading to reduced cell viability. Furthermore, cell viability decreases further with increasing collagen glycosylation. When glycosylation inhibitors are added, collagen glycosylation is inhibited. The stronger the inhibition and the lower the degree of glycosylation, the higher the cell viability; that is, cell viability is directly proportional to the effect of glycosylation inhibitors.)

[0141] Dissolve MTT in DMEM to a concentration of 5 mg / mL and filter aseptically. Add 900 μL of DMEM and 100 μL of the prepared MTT solution to a 12-well plate, and add the gel to the cell culture wells. Incubate the wells at 37 °C with 5% CO2 and gently agitate for 3 hours. Aspirate the MTT culture medium and carefully wash twice with PBS. Add 1 mL of DMSO to each well and incubate for 1 hour with gentle agitation. Transfer the DMSO solution to a 96-well plate and measure the absorbance at 490 nm using a microplate reader. Calculate cell viability using the following formula and convert it to relative cell viability compared to the control group.

[0142]

[0143] In the formula:

[0144] A1 is the absorbance of the sample group (DMEM containing samples, cells, and MTT);

[0145] A2 is the absorbance of the control group (containing cells and MTT, and blank DMEM without samples);

[0146] A0 is the absorbance value of the blank group (blank DMEM containing only MTT and no cells).

[0147] The data is shown in the table below:

[0148] Table 4: Cell viability assay for each group of cells (n=3)

[0149]

[0151] As shown in the table above, similar to Example 3, the cell viability of Formula 3 in Example 2 was 88.2%, second only to the blank control group and superior to the positive control group. Next were Formulas 2 and 4 in Example 2, followed by Formulas 1, 6, and 7 in Example 2, with Formula 5 being the least effective. Based on this data, the conclusion of Example 3 can also be drawn: the composition of mulberry leaf extract, broccoli seed water extract, olive powder, and amla powder has a significant inhibitory effect on protein glycosylation. The glycosylation inhibition effect is best when the mass ratio of the four components is 1:5:1:1. Next are 1:2:1:1, 1:1:10:1, and 1:1:1:1.

[0152] The above embodiments are preferred embodiments of this application, but the implementation of this application is not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of this application shall be considered equivalent substitutions and shall be included within the protection scope of this application.

Claims

1. A composition for reducing protein glycosylation, characterized in that, It is composed of mulberry leaf extract, olive powder, broccoli seed water extract and amla powder.

2. The composition according to claim 1, characterized in that, The extraction method of the mulberry leaf extract is as follows: Step 1: Take mulberry leaves, rinse them, dry them, and then crush them; Step 2: Take mulberry leaf powder, add citrate-disodium hydrogen phosphate buffer, add compound enzyme preparation, and extract by ultrasonication in a water bath; Step 3: Heat the extract to inactivate enzyme activity, centrifuge, and collect the supernatant; Step 4: Filter the supernatant under reduced pressure to obtain a clear extract; Step 5: Clarify the extract, spray dry, and pulverize to obtain the finished product; The compound enzyme preparation consists of cellulase, pectinase and hemicellulase.

3. The composition according to claim 2, characterized in that, The olive powder is obtained by freeze-drying and then pulverizing green olives.

4. The composition according to claim 2, characterized in that, The mass ratio of mulberry leaf extract to olive powder in the composition is 1:1-10.

5. The composition according to claim 4, characterized in that, The mass ratio of mulberry leaf extract to olive powder in the composition is 1:2-5.

6. The composition according to claim 4, characterized in that, The mass ratio of mulberry leaf extract to olive powder in the composition is 1:

5.

7. The composition according to claim 1, characterized in that, The amla powder is obtained by freeze-drying amla and then pulverizing it.

8. The composition according to claim 2, characterized in that, The mass ratio of mulberry leaf extract, olive powder, broccoli seed water extract, and amla powder in the composition is 1:5:1:

1.

9. Use of the composition according to any one of claims 1-8 for preparing functional foods that reduce protein glycosylation.

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