Compositions for reducing intracellular protein glycosylation and methods of making the same

Abstract

The application belongs to the technical field of functional food, and particularly relates to a composition for reducing intracellular protein glycosylation and a preparation method thereof. The composition is composed of mulberry leaf extract and olive powder, and broccoli seed water extract and phyllanthus emblica powder can be added to form an enhanced composition. The mass ratio of the mulberry leaf extract, the broccoli seed water extract, the olive powder and the phyllanthus emblica powder in the enhanced composition is 1:5:1:1. When the composition is prepared by using the mass ratio, the glycosylation inhibition effect of the composition is better than that of aminoguanidine.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of functional food, and particularly relates to a composition for reducing intracellular protein glycosylation and a preparation method thereof. BACKGROUND

[0002] The non-enzymatic glycosylation reaction was first discovered by French chemist Louis-Camille Maillard in 1912 as the browning reaction of proteins and carbohydrates, which was later named the Maillard reaction. Forty years later, Hodge first proposed the basic chemical pathway of the Maillard reaction in 1953 and it was confirmed in the cooking of food. In the following 30 years, scientists have been studying the possible effects of the Maillard reaction in the life system.

[0003] Until 1981, Monnier and Cerami discovered the Maillard reaction in the body and its potential effects on biological aging. This reaction is generated by proteins and glucose in the body without the participation of enzymes through a series of reactions to form advanced glycosylation end products (AGEs). This reaction is called "non-enzymatic glycosylation" reaction to distinguish from glycosylation reaction of glycoprotein formed under the participation of enzymes, also known as glycation reaction. More broadly, non-enzymatic glycosylation refers to the reaction of reducing sugars with proteins, lipids or nucleic acids to form end products without enzyme catalysis.

[0004] It is currently believed that the classical non-enzymatic glycosylation process is divided into three stages. The first stage is the formation of Schiff base and Amadori product, the initial products of non-enzymatic glycosylation. Schiff base is formed by condensation of free amino group of protein and carbonyl group of glucose, and then Amadori rearrangement forms Amadori product, that is, the carbon-nitrogen double bond in Schiff base is protonated to form 1,2- enol form of glucose part, and then the enol form is interchanged, and the 2 position becomes ketone. The formation of Schiff base and Amadori product in this stage is reversible, and the formation of Schiff base is short, while the rearrangement of Schiff base to form Amadori product needs a long time.

[0005] Studies have shown that the formation of Schiff base and Amadori product does not change the color of the protein. The second stage of glycosylation is the formation of glycosylation carbonyl intermediates, which have much higher reactivity than glucose and directly act on the free amino group of the protein, have high physiological damage hazards, and correspond to the increase of oxygen free radicals in the body, which is called oxidative stress, also known as carbonyl stress, and these dicarbonyl compounds are called RCS.

[0006] These dicarbonyl compounds are mainly Methylglyoxal (MGO), Glyoxal (GO), and 3-Deoxyglucosone (3-DG).

[0007] They can be formed in several ways. They can be formed from the rearrangement of the first-stage Amadori product, or they can be formed from the Wolff pathway, i.e., the autoxidation of glucose to form the simplest dicarbonyl compound, GO. In addition to GO, glucose can form 2,3- enol structure through dehydration, and then form 3-DG and MGO. However, the reaction degree of glucose to form 3-DG and MGO is very low.

[0008] In addition, in vivo, carbonyl compounds such as glyceraldehyde and MGO can also be formed through physiological metabolic pathways. The unstable Schiff base loses erythrose or glyceraldehyde through the Namiki pathway, i.e., through reverse aldol condensation, oxidation or dehydration to form a carbonyl group, and then loses a primary amine to decompose to form GO, MGO and 3-DG.

[0009] In vivo, 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 intermediate substances formed by glucose through the polyol pathway. The polyol pathway and lipid peroxidation are part of the expanded non-enzymatic glycation process. The final product of glycation formed through lipid peroxidation is also called ALEs.

[0010] The third stage of glycation is the formation of Advanced Glycation End Products (AGEs), one of which is formed directly from the Amadori product. The Amadori product can be oxidized to form CML (Nε- (carboxymethyl) lysine) by losing one molecule of water, or it can be oxidized to form pentosidine through rearrangement, or it can be non-oxidatively rearranged to form glucose alkane.

[0011] CML, pentosidine and glucose alkane are all AGEs. Another way to form AGEs is that dicarbonyl compounds directly react with lysine residues and arginine residues on proteins. Some AGEs are produced during the cooking process of daily food. These AGEs are absorbed into the blood after being digested by the human body, and are transported to various tissues and organs, thereby causing cell damage and diseases. These AGEs, like those ingested into the human body in food or tobacco, are called exogenous AGEs, while those produced in the human body are called endogenous AGEs.

[0012] Because of the complexity of the formation process of AGEs, the generated AGEs are numerous, which can be classified according to their structure and properties. According to whether the structure of AGEs belongs to cross-linking type, it can be divided into non-cross-linking AGEs and cross-linking AGEs. Non-cross-linking AGEs only connect one amino acid residue of a protein, while cross-linking AGEs connect two amino acid residues of a protein, which can come from the same protein molecule or different protein molecules, forming intramolecular cross-linking and intermolecular cross-linking. Non-cross-linking AGEs include CML and methylglyoxal-derived hydroimidazolone-1, etc.

[0013] Cross-linking AGEs can be divided into Lys-Lys cross-linking and Lys-Arg cross-linking according to the category of cross-linked amino acids. Lys-Lys cross-linking AGEs include MOLD (methylglyoxal lysine dimer) and the like, and Lys-Arg cross-linking AGEs include glucosone and pentosidine. Some AGEs have the characteristics of spontaneous fluorescence, which can be divided into fluorescent cross-linking AGEs (such as pentosidine and cross-linking) and non-fluorescent cross-linking AGEs (such as glyoxal lysine dimer, alkyl formyl pyrrole glucoside).

[0014] It should be noted that not all the structures of AGEs have been determined. Some AGEs (such as carboxymethyl lysine) remain stable under acidic hydrolysis conditions and are relatively easy to detect; while other AGEs are easily decomposed under acid hydrolysis conditions, but can be preserved in the process of enzymatic hydrolysis, which makes the detection of such AGEs more difficult. Although the development of current mild hydrolysis technology of proteins reduces the destruction of acid-labile modified amino acids, the academic community still believes that only a small amount of AGEs can be detected in tissue proteins.

[0015] CML is the first AGE isolated and identified from in vivo glycosylated proteins, which can be generated by the oxidative degradation of Amadori products or the reaction of GO with lysine residues of proteins. This glycosylated lysine formed by multiple pathways has been detected in multiple tissues, such as blood vessels, bladder, cornea, kidney, brain, heart, etc. Because CML is widely distributed in the human body, it has become one of the main biomarkers of glycosylation reaction in the human body.

[0016] Pentosidine is the first cross-linking AGE discovered in vivo. It cross-links lysine and arginine residues in proteins, resulting in intra- or inter-molecular cross-linking. In addition, it is an AGE with autofluorescence, and other AGEs with aromatic chemical structures have been found to have similar fluorescence properties. Pentosidine can be derived from the Amadori product by oxidative rearrangement. Pentosidine is formed at a low rate in collagen, and another important cross-linking AGE, called glucosepane, is formed without the need for an oxidative step. It is approximately 20 times more abundant than pentosidine in the extracellular matrix and is currently the most abundant cross-linking AGE in the extracellular matrix. It is also a lysine-arginine cross-link.

[0017] Since Monnier proposed in 1981 that non-enzymatic browning of proteins in vivo might be related to aging and related pathologies, the "non-enzymatic glycosylation hypothesis of aging" revealed that non-enzymatic glycosylation products are one of the biomarkers of aging. Most AGEs accumulate in tissues with low turnover rates, including lens proteins and collagen, as individuals age. Dyer's comparative study of the glycosylation of human lens proteins and skin collagen showed that human skin collagen glycosylation increased by 33% from the age of 20 to 80. Verzijl analyzed the contents of three AGEs, CML, CEL (carboxyethyl lysine), and pentosidine, in the cartilage of 20 normal individuals aged 3-81 and the skin collagen of 26 normal individuals aged 19-91 and found that AGEs accumulated linearly with actual age in cartilage and skin collagen. The production and accumulation of these AGEs, on the one hand, cause damage to protein function by changing molecular recognition, and 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 reduction of viscoelasticity. These changes can produce a series of adverse pathological changes, including arteriosclerosis, atherosclerosis, kidney disease, retinopathy, and neuropathy.

[0018] In addition, AGEs can interact with their receptor RAGE, trigger oxidative stress and activate cell signaling pathways, leading to metabolic disorders of cells, tissues, and organs, and have pathogenic effects on a variety of chronic diseases related to aging, such as diabetes and atherosclerosis.

[0019] Our skin will continue to age as we age, and gradually show signs of aging. Skin aging can make the skin drier and thinner, and cause age spots. The skin will become less elastic and more rigid, with fine lines and wrinkles, and changes in skin color, which are visible signs of skin aging. The process of skin aging can be divided into two categories— intrinsic aging and extrinsic aging. Extrinsic aging is mainly due to exposure of the skin to harsh environments, which may be ultraviolet radiation or environmental pollution.

[0020] There are different theories on the origin of intrinsic aging. One theory is the Hayflick phenomenon of fibroblasts during culture, i.e., cellular senescence. Another intrinsic mechanism that leads to skin aging is the damage caused by free radicals that people accumulate as they age (free radical theory). Today, the theory of intracellular protein glycosylation caused by free radicals is widely accepted as a more general intrinsic aging mechanism in the skin. AGEs can cause changes in the biomechanical properties and biochemical changes of the skin, including the synthesis of activating molecules (such as extracellular matrix macromolecules, cytokines) and the activation of matrix metalloproteinases or matrix-degrading enzymes, and cause dysfunction of skin fibroblasts.

[0021] Collagen is a key, lifelong and most prevalent protein, which is the main component of the extracellular matrix in human skin tissue. Collagen polypeptides are assembled into a triple helix structure by three peptide chains, which can form microfibers, and in turn form larger fibrils or fibers in tissues. After fiber formation, collagen chains can be cross-linked and connected to each other under the action of enzymes, which enhances the structural and biochemical stability of the tissue.

[0022] The main role 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 change that occurs due to aging. After glycosylation of collagen, its structure changes, which adversely affects the biophysical and biomechanical properties of the tissue.

[0023] On the one hand, chemical reactions with free amino groups on the side chain of collagen molecules will directly affect the triple helix properties of collagen, and in turn affect the function of tissues and organs; on the other hand, the process of forming cross-linked AGEs leads to cross-linking within and between collagen molecules, which destroys the arrangement and spatial structure of collagen fibers during cross-linking, and causes the collagen in the dermis to become rigid and lose elasticity, making the skin thin and wrinkled, and ultimately leading to skin aging.

[0024] The accumulation of AGEs in skin tissue is a long process, and the rate of accumulation is related to the life and turnover rate of proteins. The half-life of collagen in skin tissue is 15 years, which makes it a potential target for glycosylation. In human skin collagen, there are various AGEs, including pentosidine, CML, glucose amine and arginine pyridine. Verzijl's analysis data of human skin samples show that AGEs in skin collagen accumulate linearly with age, and the content of CML, CEL or pentosidine increases by 3 to 4 times between the ages of 20 to 80. Monnier's research data show that glucose amine is the most abundant AGE in human skin collagen. In addition to the appearance of AGEs in the dermis layer of the skin, the presence of AGEs is also detected in the epidermis layer. In the epidermis, glycosylated keratin can cause the skin color to turn yellow and the water content of the stratum corneum to decrease, resulting in dry skin. In addition, the irradiation of sunlight also accelerates the formation of AGEs.

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

[0026] Aminoguanidine (AG) is the earliest synthetic AGEs inhibitor found in clinical trials, which can capture highly active carbonyl compounds, including methylglyoxal (MGO), deoxyglucose furanone (3-DG), etc., thereby blocking the conversion of carbonyl compounds to AGEs and the formation and development of AGEs. Pyridoxamine is an amine in vitamin B6, and its mechanism of action as an AGEs inhibitor is mainly to chelate metal ions and reduce the Maillard reaction. Carnosine is a dipeptide that inhibits AGEs through three mechanisms: intercepting ROS, reacting with carbonyl groups on proteins, and preventing cross-linking of glycosylated proteins. Although synthetic inhibitors have good effects in inhibiting the formation and development of glycosylation, most synthetic inhibitors have safety problems and side effects, such as liver damage, gastrointestinal diseases, headaches, etc., which limit their application range.

[0027] Active ingredients present in natural plants include polyphenolic compounds, polysaccharides and terpenoids, etc., which play an important role in inhibiting the development of AGEs, reducing protein glycosylation and antioxidant. Among them, polyphenolic compounds are the most effective antioxidants found, and there are literature reports that polyphenolic compounds have in vitro inhibitory activity of AGEs.

[0028] Polyphenol inhibitors can be divided into flavonoids, stilbenes, lignans and phenolic acids. Flavonoids are a class of polyphenols containing C6-C3-C6 structure, distributed in plants, vegetables, fruits and Chinese herbal medicines. They have been widely concerned due to their multiple benefits to human health, such as anti-glycation, antioxidant, anti-inflammatory, anti-viral and so on. Flavonoids can be divided into flavones, flavonols, flavanones, flavanols, chalcones and isoflavones according to their chemical structures.

[0029] Rutin, as a common flavonoid compound of food and medicine, can prevent the development of AGEs and has certain efficacy in preventing and treating hyperglycemia. Studies on the bovine serum albumin (BSA)-glucose (Glu) model have shown that the presence of rutin can effectively scavenge free radicals and more effectively inhibit the development of AGEs at each stage than AG. Quercetin, a flavonol widely present in Chinese herbal medicines, vegetables and fruits, has been proven by many researchers to have a preventive effect on protein glycosylation and AGEs formation. Quercetin can significantly inhibit the formation and development of carbonyl compounds during protein glycosylation. In addition, quercetin inhibits protein glycosylation in a dose-dependent manner and reduces the microenvironment changes in protein conformation caused by glycosylation. Compared with synthetic glycosylation inhibitors, natural glycosylation inhibitors have greater application potential in the prevention and treatment of diabetes and its related complications, and their ability to inhibit AGEs and glycosylation is more significant.

[0030] Mulberry leaf extract refers to the effective components extracted from mulberry leaves, which has various health benefits and application values. Common extraction processes include water extraction, ethanol extraction, ultrasonic extraction, etc. Taking water extraction as an example, the process flow is: mulberry leaf powder → sieving → extraction (hot water extraction) → concentration → spray drying → mulberry instant powder. The process of preparing mulberry leaf extract by hot water extraction can effectively extract active ingredients from mulberry leaves, while maintaining the original nutritional components and biological activity of mulberry leaves.

[0031] In recent years, a large number of studies have shown that mulberry leaf powder and mulberry leaf extract are rich in crude protein, amino acids, polyphenols, mineral elements and vitamins, and other nutrients, which have multiple biological activities and functions, such as immune regulation, antioxidant, anticancer, etc., and play an important role in human health. Therefore, mulberry leaf powder and mulberry leaf extract have broad application prospects in the fields of food, medicine and other fields.

[0032] Mulberry leaf powder contains a variety of vitamins, such as vitamin C, vitamin E, vitamin B1, vitamin B2, etc. These vitamins play an important role in human growth and development, immune system and nervous system, etc. Vitamin C can promote the synthesis of collagen, enhance skin elasticity; vitamin E can resist oxidation, delay aging; vitamin B1 can promote metabolism, maintain nerve function; vitamin B2 can promote protein synthesis and metabolism, maintain skin health, etc.

[0033] Mulberry leaf polysaccharide is a natural high molecular polysaccharide, its main component is a variety of macromolecular carbohydrates. Mulberry leaf polysaccharide can be divided into different components, including mannans, galacturonic acid and chitin, etc. Mulberry leaf polysaccharide has significant anti-inflammatory effect, can inhibit the occurrence of inflammatory reaction, reduce the harm of inflammatory reaction to the body. In addition, mulberry leaf polysaccharide can also promote the secretion of insulin, increase the utilization of glucose by the body, thereby reducing blood glucose level, and has the potential to assist in the treatment of diabetes.

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

[0035] Human immune function is the key to maintaining the stability of the internal environment of the body, including defense, clearance and monitoring of pathogens, etc. If the immune regulation function is abnormal, it may lead to the occurrence of autoimmune diseases. Mulberry leaf powder and mulberry leaf extract contain a variety of polysaccharide substances, such as mulberry leaf polysaccharide, β-glucan, etc., which have immune regulation effect, can enhance the body's immunity, prevent diseases such as cold. Therefore, appropriate consumption of mulberry leaf powder and mulberry leaf extract is helpful to regulate the human immune system.

[0036] Canarium album, also known as green fruit, is a fruit of the tree plant of the Rutales Rutales Oleaceae Oleaceae genus in China. It is a famous subtropical characteristic fruit in China. In addition to containing rich nutrients such as protein, fat, and carbohydrates, it is also rich in vitamins C, carotene, retinol, vitamin B1, vitamin B2, dietary fiber, and niacin, as well as elements such as calcium, iron, chromium, manganese, and aluminum. Olive is a traditional fruit of medicine and food, and modern research has found that it is rich in flavonoids and polyphenol natural active substances, with good antibacterial and antioxidant functions.

[0037] Flavonoids are contained in olives, mainly distributed in leaves and fruits. There are three kinds of flavonoids in olive fruits, namely kaempferol-3-O-β-D-glucoside, hyperoside and amentoflavone. However, the use of different materials and extraction methods will lead to differences in the types or contents of flavonoids extracted from olive fruits. Polyphenols are also contained in olive fruits, and researchers have extracted a variety of phenolic compounds with hepatoprotective function from olive leaves and fruits, including brevifolin, hyperoside, ellagic acid and 3,3'-methoxy ellagic acid. He Zhiyong et al. studied Fujian sandalwood olives and found that phenolic compounds in olives were mainly distributed in olive pulp, with abundant ellagic acid and tannic acid. The study of tannin components in olive bark showed that condensed tannin substances belong to proanthocyanidins and prodelphinidins, and in the structural units of proanthocyanidins and prodelphinidins, catechin and epigallocatechin, gallate was found.

[0038] Phyllanthus emblica L. is a perennial shrub or tree of Euphorbiaceae Phyllanthus. It is also known as Amla and Indian Gooseberry. Phyllanthus emblica is a kind of economic plant resources of food and medicine, and is one of the three health plants promoted by the World Health Organization around the world.

[0039] Phyllanthus emblica is used in the treatment of cold, cough, sore throat, diarrhea, indigestion, heat and inflammation, skin eczema, and water and fire burns in the national folk medicine of China (Tibetan, Yi, Dai, Miao, Bai, Naxi, Lahu, Pumi, Wa, Achang, Jinuo, Buyei, Yao, Zhuang, as well as Mongolian, Uygur, etc. about 16).

[0040] Modern medicine has proved that the roots, stems, leaves, fruits and seeds of Phyllanthus emblica are rich in various bioactive substances, including organic acids, terpenes, flavonoids, polysaccharides, tannins, alkaloids, phytosterols, amino acids and vitamins; It has the characteristics of antioxidant, anticancer, anti-aging, cholesterol-lowering, anti-diabetic, immune regulation, anti-virus, anti-lipid abnormality, anti-apoptosis, antipyretic, analgesic, anti-inflammatory, liver protection, heart protection, anti-mutagenic, antibacterial and anti-diarrhea.

[0041] The Emblica fruit contains a rich source of free radical scavengers, such as resveratrol, gallic acid, tannic acid, lignan, quercetin, genistein, anthocyanin, hesperetin, kaempferol, superoxide dismutase, vitamin C, polysaccharides, tannins and organic acids, and has strong antioxidant capacity. Emblica extract can significantly increase the activity of superoxide dismutase in red blood cells of middle-aged and elderly people, reduce the content of lipid peroxidation, and scavenge hydroxyl radicals and superoxide radicals produced during metabolism.

[0042] In addition, Emblica polysaccharides also have the effect of scavenging superoxide radicals. The polyphenols, tannins and organic acids in Emblica can significantly reduce the content of malondialdehyde in the blood of mice. The methanol extract of Emblica dry fruit contains active ingredients such as tannin and old crane grass, which can effectively scavenge nitric oxide radicals (NO•). Emblica is rich in flavonoids, which can enhance the activity of endogenous antioxidants (such as catalase, superoxide dismutase, glutathione peroxidase and glutathione reductase) in the blood of mice, while reducing the level of lipid peroxidation, and protect cells against oxidative stress.

[0043] Emblica can enhance immune regulation by increasing the levels of induced T cells, suppressor T cells, immunoglobulin M, immunoglobulin G in serum, and the levels of serum albumin and globulin. In addition, the leaves, roots and bark of Emblica have been shown to have strong antioxidant capacity. Vitamin C in Emblica fruit has high stability, and its superoxide dismutase analogs have the characteristics of storage resistance, heat resistance and small molecule transdermal, and are rich in various free radical scavengers and endogenous antioxidants, which can effectively enhance the antioxidant function of the body, inhibit the production of free radicals and scavenge excess free radicals, and have important application value in the development of drugs, functional foods and cosmetics.

[0044] Thioglycosides are secondary metabolites present in cruciferous plants. These compounds are naturally stable, and when plant tissue is damaged, endogenous myrosinase is released, which hydrolyzes thioglycosides to produce a variety of active substances. Sulforaphane (1-isothiocyanato-4-methylsulfonylbutane, Sulfone) is one of the most important substances, which is rich in broccoli, especially in its seeds. Studies have shown that sulforaphane is one of the natural products with the best anticancer and anticancer effects and the strongest activity in vegetables so far, which has the ability to inhibit cancer cell proliferation, induce detoxification enzymes, block cell cycle and induce apoptosis, and has good prevention and inhibition effect on cancers including liver cancer, pancreatic cancer, lung cancer, rectal cancer, etc. In addition to the anticancer and anticancer activity, sulforaphane also has good effects in reducing blood pressure, protecting the heart, relieving diabetes, inhibiting bacteria, and improving schizophrenia and Alzheimer's disease, etc. At the same time, studies have shown that sulforaphane can also activate the Nrf2 pathway to regulate oxidative stress, thereby indirectly improving the antioxidant activity and immune response of the body. SUMMARY

[0045] Applicant has made in-depth research on protein glycosylation in cells, and found that mulberry leaf extract and olive powder combined in a certain proportion have the ability to significantly reduce protein glycosylation.

[0046] The present application first discloses a composition for reducing protein glycosylation, which is composed 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, dry, and crush.

[0049] Step 2: Take the mulberry leaf powder, add citric acid-sodium hydrogen phosphate buffer solution, add a complex enzyme preparation, and ultrasonically extract in a water bath.

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

[0051] Step 4: The supernatant is filtered under reduced pressure to obtain a clear extract.

[0052] Step 5: The clear extract is spray dried and crushed to obtain the finished product.

[0053] The complex enzyme preparation is cellulase, pectinase and hemicellulase.

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

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

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

[0057] To enhance the anti-glycosylation effect of the above composition, water extract of broccoli seeds and phyllanthus emblica powder can be added to the composition to form an enhanced composition.

[0058] The phyllanthus emblica powder is obtained by freeze-drying and then crushing the phyllanthus emblica.

[0059] The mass ratio of the mulberry leaf extract, the olive powder, the water extract of broccoli seeds, and the phyllanthus emblica powder in the enhanced composition is 1:5:1:1.

[0060] The application further discloses a use of the enhanced composition for preparing a functional food for reducing protein glycosylation in cells.

[0061] The application has the following beneficial effects: the application first discloses a composition for reducing protein glycosylation in cells, which is composed of mulberry leaf extract and olive powder, and can be further composed of water extract of broccoli seeds and phyllanthus emblica powder to form an enhanced composition. The mass ratio of the mulberry leaf extract, the water extract of broccoli seeds, the olive powder, and the phyllanthus emblica powder in the enhanced composition is 1:5:1:1. After being prepared into the composition, the glycosylation inhibition effect of the composition is better than that of aminoguanidine.

[0062] Abbreviation explanation:

[0063] AGEs: Advanced Glycation End products

[0064] RAGE: Receptor for AGEs

[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: Lipid peroxidation advanced products

[0072] ROS: Reactive Oxygen Species

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

[0074] ELISA: Enzyme-linked immunosorbent assay

[0075] PBS: Phosphate buffered saline

[0076] BSA: Bovine serum albumin

[0077] DMSO: Dimethyl sulfoxide (organic solvent)

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

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

[0080] DMEM: Dulbecco's modified Eagle's medium

[0081] FBS: Fetal bovine serum

[0082] MTT: Thiazolyl blue (cell viability detection reagent)

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

[0084] proclin 300: Preservative. DETAILED DESCRIPTION

[0085] The present application will be further described in conjunction with specific examples, but the examples do not limit the present application in any form. Unless otherwise specified, the reagents, methods and equipment used in the present application are conventional reagents, methods and equipment in the technical field. Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0086] Example 1 Preparation of mulberry leaf extract

[0087] Step 1: Take fresh mulberry leaves without impurities, rinse, and dry at 40-50°C to a moisture content of ≤8%, crush through an 80-100 mesh sieve, and seal for use.

[0088] Step 2: Take the mulberry leaf powder, add pH 5.0 citric acid-disodium hydrogen phosphate buffer at a solid-liquid ratio of 1:15, add a complex enzyme preparation (cellulase 1.5%, pectinase 1.0%, hemicellulase 0.5%, based on the mass of the mulberry leaf powder), and extract by ultrasonic bath at 45°C (frequency 40 kHz, power 300 W) for 40 minutes.

[0089] Step 3: The extract was quickly heated to 85℃ for 10 minutes to deactivate the enzyme, and then centrifuged at 4000-5000 r / min for 15 minutes. The supernatant was collected.

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

[0091] Step 5: The clear extract was spray-dried (inlet temperature 160℃, outlet temperature 80℃), and then ground through a 100 mesh sieve to obtain the finished product (moisture content ≤5%) which was stored in a light-proof container.

[0092] Example 2: Preparation of an anti-glycosylation composition

[0093] Table 1: Formula design of the anti-glycosylation composition

[0094] .

[0095] Preparation method:

[0096] Step 1: The mulberry leaf extract, olive powder, emblic powder, and water extract of broccoli seeds were ground and passed through a 100 mesh sieve for use.

[0097] Step 2: The prescribed amount of mulberry leaf extract and olive powder were mixed evenly.

[0098] Step 3: The prescribed amount of water extract of broccoli seeds was added to the mixture obtained in Step 2 and mixed evenly.

[0099] Step 4: The prescribed amount of emblic powder was added to the mixture obtained in Step 3 and mixed evenly, and then packaged.

[0100] Note: If a certain component is not included in the formula of the composition, then the steps of grinding and mixing that component are skipped during preparation.

[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 model of non-enzymatic glycosylation of proteins

[0103] 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 buffer (PBS, pH=7.4, 0.2% proclin 300 as a preservative). The mulberry leaf extract (60 μg / mL), water extract of broccoli seeds (60 μg / mL), olive powder (60 μg / mL), emblic powder (60 μg / mL), and each formula composition of Example 2 (60 μg / mL, based on the mass of mulberry leaf extract) were dissolved in DMSO.

[0104] Table 2: Sample preparation method for in vitro protein non-enzymatic glycation model test

[0105] .

[0106] The above sample was incubated in a 37 °C constant temperature incubator for 10 days. After 10 days, the culture solution was taken to determine the Amadori product, dicarbonyl compound, fluorescent advanced glycation product, and non-crosslinked advanced glycation product CML. The determination method is as follows:

[0107] Amadori product determination:

[0108] 0.2 mL of the culture solution 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), and reacted at 40 °C for 1 h. The OD value was determined at 530 nm by an enzyme marker, and the amount of Amadori product was represented by the OD value.

[0109] Dicarbonyl compound content determination:

[0110] 1 mL of the culture solution was taken in a 30 kD ultrafiltration tube, centrifuged at 4500 G for 12 min. 900 μL of the outer tube filtrate was mixed with 20 μL of acetic acid and 30 μL of 100 mmol / L OPD liquid in a sample bottle, and nitrogen was filled. The reaction was carried out at 40 °C for 80 min. The reaction liquid and standard quinoxaline, 2-methyl quinoxaline were analyzed by using ultra-high performance liquid chromatography tandem quadrupole time-of-flight mass spectrometry. Pre-column derivatization high performance liquid chromatography was used to determine GO, MGO and 3-DG. After the culture solution was derivatized, it was analyzed by high performance liquid chromatography.

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

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

[0113] Fluorescent advanced glycation product determination:

[0114] The fluorescence values in the culture solution were measured using a fluorescence spectrophotometer at excitation / emission wavelengths (ex / em) of 370 / 440 nm and 335 / 385 nm according to the method of Pageon, and the inhibition rate of the composition of Example 2 or aminoguanidine on fluorescent late-stage 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 to which the composition of Example 2 or AG is added; and B0 is the fluorescence value of the corresponding blank control of the group to which the composition of Example 2 or AG is added.

[0117] The inhibition of each group of samples on fluorescent late-stage glycosylation products is expressed as an inhibition rate (%).

[0118] Non-cross-linked late-stage glycosylation product CML determination:

[0119] The content of CML in the culture solution was determined using enzyme-linked immunoassay (ELISA). The sample was added to a micro-well coated with CML antibody, and then combined with 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 determined at a wavelength of 450 nm using an enzyme-labeled instrument.

[0120] The amount of non-cross-linked late-stage glycosylation product CML is expressed as an OD value.

[0121] The data obtained are shown in the following table:

[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, which are formed from the imine intermediate (Schiff base) between the free amino group or N-terminal amino group on the protein and the reducing sugar through Amadori rearrangement.

[0125] The second stage of the glycosylation reaction is the formation of carbonyl intermediates, which mainly include GO, MGO and 3-DG, and can directly act on the free amino group in the protein, causing high physiological damage. Therefore, reducing the content of dicarbonyl compounds can effectively inhibit the non-enzymatic glycosylation of proteins and reduce the production of AGEs.

[0126] In the later stage of glycation reaction, there are several pathways to form complex AGEs. One of them is that Amadori product directly undergoes oxidation or non-oxidation, rearrangement, degradation, and the other is that dicarbonyl compounds directly react with lysine residues and arginine residues on proteins. Both of these two pathways eventually form fluorescent AGEs, such as pentosidine formed by direct oxidative degradation of Amadori product, MOLD formed by reaction of MGO with two lysines, etc. Therefore, different excitation wavelengths can be used to detect various fluorescent AGEs.

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

[0128] As can be seen from the above table data, the positive control group (sample 12) uses aminoguanidine as a glycosylation inhibitor, which significantly inhibits the formation of Amadori product, dicarbonyl compound, AGEs and CML product compared with sample 13 without adding any glycosylation inhibitor. Samples 1-4 use mulberry leaf extract, water extract of broccoli seeds, olive powder and phyllanthus niruri powder as potential glycosylation inhibitors, and there is no obvious difference in numerical change compared with sample 13. These four components only show weak glycosylation inhibition, among which mulberry leaf extract is the best.

[0129] Samples 5-8 use formulations 1-4 of Example 2 as glycosylation inhibitors, respectively. Formulations 1-4 all contain four components, only the addition amount of olive powder is different, and the corresponding glycosylation inhibition effect increases with the increase of the addition amount of olive powder. When the addition amount of olive powder is 50 g, the corresponding sample 7, the glycosylation inhibition effect of this group of samples even exceeds the positive control group aminoguanidine. However, when the addition amount of olive powder exceeds 50 g, reaches 100 g, the glycosylation inhibition effect of this group of samples (sample 8) is actually reduced, so the composition of Example 2 formulation 3 is the best proportion of 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, and formulations 6 and 7 both contain three components. It can be seen that the generation amount of glycosylation products of these three groups of samples is higher than that of samples 5-8 (i.e. the composition of formulations 1-4 treatment group), which indicates that the glycosylation inhibitor effect of the composition of four components is better than that of two or three components, and the glycosylation inhibitor effect of the composition of three components is better than that of two components.

[0131] In summary, the composition comprising mulberry leaf extract, water extract of broccoli seeds, olive powder and phyllanthus emblica powder has a significant protein glycosylation inhibition effect. When the mass ratio of the four components is 1:5:1:1, the glycosylation inhibition effect is best. The second is 1:2:1:1, 1:10:1:1 and 1:1:1:1.

[0132] Example 4: Anti-protein glycosylation effect of the composition of Example 2 in the biomimetic dermis model

[0133] 4.1 Construction containing cell senescence dermis equivalent

[0134] Ribose (100 mmol / L) and Example 2 Formulation 1-7 compositions (100 μg / mL of mulberry leaf extract) were added to collagen (4 mg / mL), and Example 2 compositions were not added as a negative control, 100 μg / mL aminoguanidine as a positive control, and collagen without ribose as a blank control. Incubate in a 37°C constant temperature incubator for 24d to obtain pre-glycosylated collagen solution.

[0135] The pre-glycosylated collagen solution and 20 mmol / L acetic acid solution were added to the outer tube of the 50 mL 10 kD ultrafiltration tube (the ultrafiltration tube was compatible with 50% volume fraction acetic acid), centrifuged at 4500xg for 20 min, repeated 3 times to remove ribose, small molecules in Example 2 composition and aminoguanidine in pre-glycosylated collagen.

[0136] Mix the pre-glycosylated collagen with the untreated collagen in equal volume (to prevent the pre-glycosylated collagen from not forming a gel).

[0137] Mix 1 mL of 10-fold concentrated DMEM, 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 liquid (0.1 mol / L NaOH solution, adjust pH to 7.2-7.4), add 40 μL of FBS resuspended human skin fibroblasts (7.5x10 6 Place in a 37°C constant temperature incubator.

[0139] 4.2 Cell proliferation

[0140] Cell proliferation was detected after the cells were cultured in the dermis equivalent for 3 days, and the cell viability value was used to represent the inhibitory effect of each test sample on the glycosylation of extracellular matrix collagen. (When collagen is glycosylated, its promotion of cell proliferation decreases, resulting in a decrease in cell viability. And with the increase of collagen glycosylation, the cell viability becomes lower. When the glycosylation inhibitor is added, the collagen glycosylation is inhibited, the stronger the inhibitory effect, the lower the degree of glycosylation, and the higher the cell viability, that is, the cell viability and the effect of the glycosylation inhibitor are proportional).

[0141] MTT was dissolved in DMEM at a concentration of 5 mg / mL, sterile filtered. Add 900 μL DMEM and 100 μL prepared MTT solution to the 12-well plate, and add the gel to the cell culture well. Put the well into a 37℃, 5% CO2 incubator, and gently shake for 3h. Remove the MTT culture solution and wash with PBS twice. Add 1 mL DMSO to each well, and place in the incubator for 1h, gently shaking. Transfer the DMSO solution to a 96-well plate, and measure the absorbance at a wavelength of 490 nm using a microplate reader. Calculate the cell viability according to the following formula, and convert it to the relative cell viability relative to the control group.

[0142]

[0143] In the formula:

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

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

[0146] A0 is the absorbance of the blank group (blank DMEM containing only MTT, without cells).

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

[0148] Table 4: Viability detection of each group of cell samples (n=3)

[0149]

[0150] As can be seen from the above table data, similar to Example 3, the cell viability of Example 2 Formula 3 group is 88.2%, only less than the blank control group, better than the positive control group. Second is Example 2 Formula 2 and Formula 4, third is Example 2 Formula 1, Formula 6 and Formula 7, and the last is Example 2 Formula 5. Based on this data, the same conclusion as Example 3 can be drawn, that is, the composition composed of mulberry leaf extract, water extract of broccoli seeds, olive powder and phyllium powder has a significant protein glycosylation inhibition effect, and when the mass ratio of the four components is 1:5:1:1, the glycosylation inhibition effect is the best. Second is 1:2:1:1, 1:1:10:1 and 1:1:1:1.

[0151] The above examples are the preferred embodiments of the present application, but the embodiments of the present application are not limited by the above examples, and any other changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application should be equivalent replacement methods, which are all included in the protection scope of the present application.

Claims

1. A composition for reducing protein glycosylation, comprising, The composition is composed of mulberry leaf extract, olive powder, water extract of broccoli seed and phyllanthus emblica powder, and the mass ratio is 1:5:1:

1.

2. The composition of claim 1, wherein The extraction method of the mulberry leaf extract is as follows: Step 1: take mulberry leaves, rinse, dry and crush; Step 2: take the mulberry leaf powder, add citric acid-disodium hydrogen phosphate buffer solution and add a composite enzyme preparation, and then ultrasonically extract in a water bath; Step 3: heat the extract to inactivate the enzyme activity, centrifuge and take the supernatant; Step 4: reduce the pressure of the supernatant to filter, and obtain a clear extract; Step 5: spray dry the clear extract, crush and obtain the finished product; The composite enzyme preparation is composed of cellulase, pectinase and hemicellulase.

3. The composition of claim 2, wherein The olive powder is obtained by freeze-drying and crushing green olives.

4. The composition of claim 1, wherein The phyllanthus emblica powder is obtained by freeze-drying and crushing phyllanthus emblica.

5. Use of the composition according to any one of claims 1-4 for the preparation of a functional food for reducing protein glycosylation.

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