Application of composition containing lycium barbarum polysaccharide

Abstract

The invention discloses application of a composition containing lycium barbarum polysaccharide. The lycium barbarum polysaccharide is used for achieving the effects of resisting aging and removing lipofuscin, particularly, the specific lycium barbarum polysaccharide LBP1C can more remarkably reduce the level of transcription factors GATA4, activate key transcription regulatory factors of autophagy, reduce aging-related secretion phenotypes and inhibit accumulation of lipofuscin, and the lycium barbarum polysaccharide LBP1C has the effects of delaying aging, whitening skin and promoting healthy aging.

Description

Uses of compositions containing wolfberry polysaccharides

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of Chinese patent application No. 202310929842.4 filed on July 26, 2023, the contents of which are incorporated herein by reference. Technical Field

[0003] The present invention relates to the field of plant polysaccharides, and in particular to the use of a composition containing wolfberry polysaccharides. Background Art

[0004] Wolfberry (wolfberry) is a traditional and precious Chinese medicinal herb, known as the "longevity fruit" in Europe, America, and many Asian countries. The "Shennong Bencao Jing" (Shennong's Classic of Materia Medica) lists it as a top-grade herb, and it has been mentioned in herbal compendiums throughout history. As a typical Chinese herbal medicine with both medicinal and medicinal properties, wolfberry has been included in the National Health Commission's list of substances that are both food and medicinal. The "Compendium of Materia Medica" (Compendium of Materia Medica) states that wolfberry has the benefits of "strengthening bones and muscles, maintaining a light and youthful appearance, resisting cold and heat, and improving complexion and whitening." While preliminary research has scientifically elucidated and investigated the mechanisms of wolfberry's "strengthening bones and muscles," the scientific implications and mechanisms of its anti-aging and whitening properties are still not fully understood. Numerous bioactive components have been isolated from wolfberry, including polysaccharides, carotenoids, flavonoids, and phenols. Studies have shown that wolfberry polysaccharides exhibit antioxidant, anti-inflammatory, anti-apoptotic, and cytoprotective properties. Studies have also shown that crude wolfberry polysaccharides can extend the lifespan of fruit flies by enhancing their antioxidant capacity. Lycium barbarum (Lycium barbarum) crude extract can extend the lifespan of nematodes and improve their tolerance to paraquat, UV-induced oxidative stress, and heat stress. Lycium barbarum crude polysaccharides can also improve cognition in D-galactose-induced aging mice, reduce brain lipid peroxidation and lipofuscin levels, increase skin superoxide dismutase (SOD) activity, and reduce skin malondialdehyde levels. In terms of skin whitening and protection, Lycium barbarum root extract can effectively inhibit intracellular tyrosinase activity and reduce melanin content. Prolonged exposure to UV radiation can lead to premature aging of the epidermis and dermis. UV radiation induces the mitogen-activated protein kinase (MAPK) signaling pathway, leading to collagen degradation. Lycium barbarum glycoconjugates (LbGp) have anti-apoptotic and antioxidant effects. One of the extracts, LbGp5, can promote the viability of human fibroblasts and increase type I collagen content. Lycium barbarum polysaccharide fraction (LBPF) can protect mouse skin from UV-induced collagen fiber breakage. Most research on goji berries' anti-aging and whitening mechanisms focuses on their antioxidant properties, such as their ability to increase superoxide dismutase (SOD) and catalase (CAT) activity and reduce malondialdehyde (MDA) levels. The signaling pathways involved in goji berries' anti-aging effects primarily involve activation of the mitogen-activated protein kinase (MAPK) signaling pathway, with no other studies reported.

[0005] Summary of the Invention

[0006] The purpose of the present invention is to overcome the problems existing in the prior art and provide the use of a composition containing wolfberry polysaccharides.

[0007] In order to achieve the above objectives, the first aspect of the present invention provides a use of a composition.

[0008] A second aspect of the present invention provides a method of using the composition.

[0009] The present invention uses natural substances present in wolfberry as active ingredients, which can be used safely within the experimental dosage range, and achieves the effects of anti-aging and clearing lipofuscin. In particular, the use of a specific wolfberry polysaccharide LBP1C can more significantly reduce the level of transcription factor GATA4, activate key transcriptional regulators of autophagy, reduce aging-related secretory phenotypes, and inhibit the accumulation of lipofuscin, with the effects of delaying aging, whitening and promoting healthy aging. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 shows the results of LBP1C delaying fibroblast senescence. (a) Isolation and purification of LBP1C. (b) Replicative senescence cell model. SA-β-gal staining of young and aged cells (Scale bar = 20 μm). (c), (d), (e) RT-qPCR analysis of P21, P16, and LAMINB1 mRNA levels in HDF cells treated with LBP1C for 6 days (control group treated with LBP1C-free cell culture medium) (two passages). (f) SA-β-gal staining of HDF cells treated with LBP1C for 6 days. Scale bar = 20 μm. (g) RT-qPCR analysis of P21 mRNA levels in HDF cells treated with LBP1A and LBP1B for 6 days. Data are expressed as mean ± SEM (n = 3). *p < 0.05, **p < 0.01 based on unpaired t-test.

[0011] Figure 2 shows the results of LBP1C-induced reduction of GATA4 levels and inhibition of the SASP. (a), (b), (c), and (e) RT-qPCR analysis of GATA4, IL-1β, and iNOS mRNA levels in HDF cells treated with LBP1C for 6 days (control group treated with LBP1C-free cell culture medium). (d) Western blot analysis of iNOS in HDF cells treated with LBP1C-containing and LBP1C-free culture medium for 6 days. Actin was used as a loading control. Data are presented as mean ± SEM (n = 3). *p < 0.05, **p < 0.01 based on unpaired t-test.

[0012] Figure 3 shows the results of LBP1C-induced increased nuclear translocation of TFEB and activation of autophagy. (a) Immunofluorescence analysis of HDF cells treated with medium containing or without LBP1C for 6 days, n = 100 cells. Green: TFEB, blue: Hoechst (nucleus). Scale bar = 30 μm. (b) Lysosome tracker staining of HDF cells treated with or without LBP1C (control group treated with medium without LBP1C). Scale bar = 50 μm. (c) RT-qPCR analysis of TFEB-targeted genes in HDF cells treated with medium containing or without LBP1C for 6 days. Data are presented as mean ± SEM (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001 based on unpaired t-test. (d) Western blot analysis of LC3II / LC3I in HDF cells treated with medium containing or without LBP1C for 6 days. Actin was used as a loading control. Data are representative of two independent experiments.

[0013] Figure 4 shows that LBP1C reduces lipofuscin in HDF cells and Caenorhabditis elegans (C. elegans) and enhances motility in C. elegans. (a) Lipofuscin staining of HDF cells treated with culture medium containing and without LBP1C for 6 days (from P25 to P26). Scale bar = 20 μm. (b) Confocal imaging (lipofuscin) of C. elegans fed with bacteria containing and without LBP1C for 8 days. Scale bar = 50 μm. Green (488 nm). (c) Statistical analysis of the frequency of body bends per 30 seconds (15 worms, n = 3) in N2 C. elegans fed with bacteria containing and without LBP1C for 10 days. (d) Pharyngeal twitching in N2 C. elegans fed with bacteria containing or without LBP1C for 10 days (15 nematodes, n = 3). Data are presented as mean ± SEM. **p < 0.01, ***p < 0.001 based on unpaired t-test. (e) This figure shows that LBP1C delays aging by activating TFEB, which subsequently reduces the SASP and lipofuscin. DETAILED DESCRIPTION

[0014] The endpoints of the ranges and any values ​​disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoints of each range, the endpoints of each range and individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered to be specifically disclosed herein.

[0015] The present invention provides a use of a composition, characterized in that the composition contains Lycium barbarum polysaccharides. According to one embodiment of the present invention, the use is to delay aging (anti-aging), and can also be used in the preparation of a medicine, health product or skin care product for delaying aging.

[0016] According to one embodiment of the present invention, the use is whitening, and can also be used in the preparation of skin care products for whitening. "Whitening" refers to inhibiting the deposition of pigments to improve skin dullness and make the skin tone look even and bright.

[0017] According to another embodiment of the present invention, the use is to activate the key transcriptional regulatory factor of autophagy (nuclear translocation of Transcription Factor EB, TFEB), especially to increase the nuclear translocation ratio of TFEB. It can also be used in the preparation of drugs, health products or skin care products for activating key transcriptional regulatory factors of autophagy (or increasing the nuclear translocation ratio of TFEB).

[0018] According to another embodiment of the present invention, the use is to reduce the expression level of P16, P21, senescence-associated-β-galactosidase (SA-β-gal), transcription factor GATA4 or senescence-associated secretory phenotype (SASP), and can also be used in the preparation of a medicine or skin care product for reducing the expression level of P16, P21, SA-β-gal, transcription factor GATA4 or SASP.

[0019] According to another embodiment of the present invention, the use is to increase the expression level of LAMIN B1 or increase the level of cellular autophagy, and can also be used in the preparation of medicines, health products or skin care products for increasing the expression level of LAMIN B1 or increasing the level of cellular autophagy.

[0020] According to another embodiment of the present invention, the use is to inhibit the accumulation of lipofuscin, and can also be used in the preparation of medicines, health products or skin care products for inhibiting the accumulation of lipofuscin.

[0021] According to another embodiment of the present invention, the use is to promote healthy aging, and can also be used in the preparation of medicines, health products or skin care products for promoting healthy aging.

[0022] The present invention also provides a method for using the composition, characterized in that the method comprises: administering the composition containing Lycium barbarum polysaccharides to a subject or animal cells. The method may also comprise first preparing the composition according to the method described below, and then administering the composition to the subject or animal cells.

[0023] In one embodiment of the present invention, the composition can be used for delaying aging and / or whitening, that is, the method of using the composition of the present invention is a method for delaying aging and / or whitening.

[0024] In one embodiment of the present invention, the composition can be used to activate TFEB, that is, the method of using the composition of the present invention is a method of activating TFEB.

[0025] In one embodiment of the present invention, the composition can be used to reduce the expression level of P16, P21, SA-β-gal, transcription factor GATA4 or SASP, that is, the method of using the composition of the present invention is a method for reducing the expression level of P16, P21, SA-β-gal, transcription factor GATA4 or SASP.

[0026] In one embodiment of the present invention, the composition can be used to increase the expression level of LAMIN B1 or increase the level of cellular autophagy. That is, the method of using the composition of the present invention is a method for increasing the expression level of LAMIN B1 or a method for increasing the level of cellular autophagy.

[0027] In one embodiment of the present invention, the composition can be used to inhibit the accumulation of lipofuscin to achieve a whitening effect. That is, the method of using the composition of the present invention is a method for inhibiting the accumulation of lipofuscin.

[0028] In one embodiment of the present invention, the composition can be used to promote healthy aging, that is, the method of using the composition of the present invention is a method of promoting healthy aging.

[0029] In the present invention, the content of Lycium barbarum polysaccharide in the composition is preferably greater than or equal to 40wt% (such as 40wt%, 41wt%, 42wt%, 43wt%, 44wt%, 45wt%, 45.4wt%, 45.5wt%, 45.6wt%, 46wt%, 47wt%, 48wt%, 49wt%, 50wt%, 60wt% or any value or range therebetween), more preferably 40-50wt%. Lycium barbarum polysaccharide is also a polysaccharide derived from wolfberry, and its content can be measured by the phenol-sulfuric acid method.

[0030] In the present invention, the monosaccharide composition of the wolfberry polysaccharide includes mannose, rhamnose, gluconic acid, galacturonic acid, glucose, galactose, arabinose and xylose. The molar ratio of mannose, rhamnose, gluconic acid, galacturonic acid, glucose, galactose, arabinose and xylose in the monosaccharide composition of the wolfberry polysaccharide is 5-6:3-4:0.8-1.2:5-6:2-3:8-9:14-15:1, more preferably 13.1:8.3:2.5:13.8:5.6:20.0:34.2:2.4. Lycium barbarum polysaccharides whose monosaccharide composition falls within the above range will show better anti-aging and / or lipofuscin removal effects. The term "monosaccharide composition" used in the present invention refers to the composition of the basic units (monosaccharides) formed by glycosidic bonds (dehydration) to form polysaccharides.

[0031] In the present invention, the composition also contains at least one of protein, pigment and inevitable impurities. Preferably, The content of protein in the composition is 10-15wt% (as 10wt%, 11wt%, 11.2wt%, 11.4wt%, 11.5wt%, 11.6wt%, 11.8wt%, 12wt%, 13wt%, 14wt%, 15wt% or any value or arbitrary range between the above numerical values), more preferably 11-12wt%. The remaining ingredients of the composition of the present invention are pigment and inevitable impurities. Protein content can be measured by BSA colorimetry.

[0032] In the present invention, the weight average molecular weight distribution range of the macromolecular substance in the composition can be 20-700 kDa (e.g., 22 kDa, 22.1 kDa, 22.2 kDa, 22.5 kDa, 23 kDa, 25 kDa, 40 kDa, 60 kDa, 80 kDa, 100 kDa, 171 kDa, 172 kDa, 200 kDa, 300 kDa, 400 kDa, 500 kDa, 600 kDa, 664 kDa, 665 kDa, 700 kDa, or any value or range therebetween). The molecular weight can be measured by high performance gel permeation chromatography (HPGPC).

[0033] In the present invention, at least a portion (such as 10wt%, 11wt%, 12wt%, 13wt%, 14wt%, 15wt%, 16wt%, 17wt%, 18wt%, 19wt%, 20wt%, 25wt%, 30wt%, 40wt%, 50wt% or any value or any range between the above values) of the wolfberry polysaccharide in the composition is wolfberry polysaccharide LBP1C-2. The content of Lycium barbarum polysaccharide LBP1C-2 in the composition can be greater than or equal to 10wt% (such as 10wt%, 11wt%, 12wt%, 13wt%, 14wt%, 15wt%, 16wt%, 17wt%, 17.2wt%, 17.5wt%, 17.7wt%, 18wt%, 19wt%, 20wt%, 25wt%, 30wt%, 40wt%, 50wt% or any value or range therebetween), preferably 10-30wt%.

[0034] The molecular weight of LBP1C-2 is in the range of about 10 kDa to about 150 kDa, for example, about 10 kDa to about 110 kDa, about 10 kDa to about 80 kDa, about 10 kDa to about 60 kDa, about 50 kDa to about 120 kDa, or any other suitable range. The molar ratio of structural units provided by arabinose, galactose, galacturonic acid, and rhamnose in LBP1C-2 can be 30-70:20-60:0.1-10:0.1-10, preferably 49-50:33-34:8-9:8. For example, in certain embodiments, the molar ratio of structural units provided by arabinose, galactose, galacturonic acid, and rhamnose in LBP1C-2 is 49.9:33.6:8.5:8. The molecular weight can be a specific value or within a narrow range of about 10 kDa to about 150 kDa. Such molecular weight values ​​may be weight-average molecular weight (Mw) or number-average molecular weight (Mn). The values ​​of Mw and Mn may be close to each other because the Lycium barbarum polysaccharide LBP1C-2 is homogeneous and has a very narrow molecular weight distribution. The polydispersity (PD) index (i.e., the ratio of Mw to Mn) of Lycium barbarum polysaccharide LBP1C-2 may be in the range of about 1 to about 1.3, about 1 to about 1.2, or about 1 to about 1.1. In certain embodiments, the PD index is close to 1. Since the molecular weight of the polysaccharide in the raw wolfberry may change due to factors such as the growth environment and the harvest season, the molecular weight of the polysaccharide obtained from different batches may also be different.

[0035] High-performance gel permeation chromatography (HPGPC) analysis of LBP1C-2 revealed a single symmetrical peak, indicating a homogeneous polysaccharide. In certain embodiments, the structure of LBP1C-2 includes a backbone consisting of alternating 1,2-linked α-Rhap and 1,4-linked α-GalpA, as well as branches consisting of terminal (T)-, 1,3-, 1,6-, and 1,3,6-linked β-Galp, T-, 1,5-, and 1,3,5-linked α-Araf, and T-linked β-Rhap substituted at C-4 of the 1,2,4-linked α-Rhap. The structure of LBP1C-2 can be shown in Formula 1, Formula 2, or Formula 3, with Formula 1, Formula 2, and Formula 3 each representing the same structure in three different forms.

[0036] As shown in Formula 1-3, LBP1C-2 is composed of Ara, Gal, GalA, and Rha in a molar ratio of 49.9:33.6:8.5:8. Structural analysis reveals that LBP1C-2 is primarily composed of 1,2-α-Rha and 1,4-α-GalA as the backbone, with branches including T-α-Ara, 1,5-α-Ara, T-β-Rha, T-β-Gal, 1,3-β-Gal, 1,6-β-Gal, and 1,3,6-β-Gal attached to the C-4 position of the 1,2,4-α-Rha backbone sugar residue. The structural unit of LBP1C-2 has the structure shown in Formula 3, comprising a backbone (composed of 1,2-α-Rha, 1,2,4-α-Rha, and 1,4-α-GalA) and three branches (R1, R2, and R3).

[0037] In formula 1-3, n is 2 to 20. The molecular weight of the polysaccharide is proportional to the value of n. For example, when the molecular weight of the polysaccharide is 13.2 kDa, n is approximately 2.

[0038] In the present invention, the composition is derived from the water extraction and alcohol precipitation product of wolfberry. The composition can be obtained by water extraction and alcohol precipitation. Preferably, the composition is obtained by the following method:

[0039] (1) mixing wolfberry, enzyme and water for enzyme treatment to obtain an enzymatic hydrolyzate;

[0040] (2) subjecting the enzymatic hydrolysate to alcohol precipitation to obtain crude wolfberry polysaccharide;

[0041] (3) The crude wolfberry polysaccharide was loaded onto an anion exchange chromatography column and gradient eluted with deionized water and salt solutions of different ionic strengths.

[0042] In step (1), the amount of the enzyme used can be 1×10 7 -1.5×107 U(such as 10 7 U, 1.1×10 7 U, 1.2×10 7 U, 1.3×10 7 U, 1.4×10 7 U, 1.5×10 7 U or any value or any range between the above numerical values). The enzyme used may include cellulase, protease and amylase. The ratio between the enzyme activity units of cellulase, protease and amylase is 1:20-50:100-200. The ratio between the enzyme activity units of the protease and the cellulase can be 20, 25, 30, 33, 34, 35, 40, 45, 50 or any value or any range between the above numerical values. The ratio between the enzyme activity units of the amylase and the cellulase can be 100, 110, 120, 130, 140, 150, 160, 165, 167, 170, 180, 190, 200 or any value or any range between the above numerical values. In some embodiments of the invention, the weight ratio of cellulase, protease and amylase can be 100:10-25:20-40.

[0043] In the present invention, for the definition of enzyme activity unit, if the enzyme manufacturer has indicated it, the definition given by the manufacturer shall prevail; if the manufacturer does not indicate it, the definition in GB1886.174-2016 shall prevail.

[0044] The cellulase is a general term for a group of enzymes that can synergistically act on the β-1,4 glucosidic bonds of cellulose to degrade cellulose into short fibers, cellobiose, and glucose. It includes endo-β-glucanase (EG) that can arbitrarily cut the β-1,4 glucosidic bonds in cellulose molecules; exo-β-glucanase (CBH) that cuts the β-1,4 glucosidic bonds into cellobiose from the non-reducing end; and β-glucosidase (BG) that breaks down cellobiose into glucose. The enzymatic activity of cellulase is defined as follows: 1g of solid enzyme hydrolyzes the sodium carboxymethyl cellulose substrate at 50°C and pH 4.8 for 1h to produce reducing sugars equivalent to 1mg of glucose, which is one enzyme activity unit (U). In the present invention, the cellulase is preferably an acid cellulase.

[0045] Protease is a general term for a class of enzymes that hydrolyze protein peptide chains. Protease activity is defined as the amount of enzyme required to hydrolyze casein per minute, releasing trichloroacetic acid-soluble matter at a wavelength of 275 nm with an absorbance equivalent to that of 1 microgram of tyrosine under assay conditions (37±0.2°C, pH 7). In the present invention, the protease is preferably papain. Papain is composed of 212 amino acids, has a molecular weight of 21,000, and is a sulfhydryl (-SH)-containing endopeptidase.

[0046] Amylases are enzymes capable of hydrolyzing α-1,4-glucosidic bonds and are classified as α-amylases and β-amylases based on the isomeric types of their hydrolysis products. Amylase activity is defined as one unit (U) of activity: 1g of solid enzyme liquefies 1g of soluble starch at 60°C and pH 6 for 1 hour. In the present invention, α-amylase is preferred.

[0047] In step (1), the amount of water used can be 10-30 L per kilogram of wolfberry on a dry basis.

[0048] In step (1), the conditions for the enzyme treatment may include: a temperature of 50-60° C. and a time of 0.5-2 h. After the enzyme treatment, the steps of enzyme inactivation, solid-liquid separation to obtain a liquid phase, and dialysis to remove small molecules may also be included.

[0049] In step (2), ethanol can be used for alcohol precipitation, and the amount of ethanol used can make the concentration of ethanol in the alcohol precipitation system be 70-90 vol% (such as 70 vol%, 75 vol%, 78 vol%, 80 vol%, 82 vol%, 85 vol%, 90 vol% or any value or any range between the above values).

[0050] In step (3), the anion exchange chromatography column can be a conventional chromatography column for separating biomacromolecules such as proteins and nucleic acids, preferably DEAE Sepharose TM Fast flow column. The eluent can be water and salt solutions of different ionic strengths (especially NaCl solutions of different ionic strengths) in sequence. The difference in salt concentration between salt solutions of adjacent concentrations is preferably 0.03-0.08M. The salt concentration of the salt solution with the lowest concentration can be 0.03-0.07M. The salt solutions of different ionic strengths are preferably salt solutions with salt concentrations of 0.03-0.07M, 0.08-0.12M, 0.18-0.22M, 0.3-0.5M, 0.55-0.65M, 0.75-0.85M, 0.9-1.2M, and 1.8-2.2M, and the eluent of the salt solution with an intermediate ionic strength (preferably 0.18-0.22M) (after drying) is collected as Lycium barbarum polysaccharide (composition) for stand-by use. The unit M refers to "mol / L".

[0051] In the present invention, the composition activates TFEB and reduces SASP, thereby delaying aging and reducing lipofuscin levels. The composition can be used in animals or animal cells. That is, the subject can be an animal, including mammals (such as humans) and / or nematodes (such as nematodes, particularly Caenorhabditis elegans). The animal cells can include mammalian cells, such as fibroblasts, particularly human fibroblasts.

[0052] In the present invention, the composition may further contain a carrier to formulate a specific form of a medicine, health product, or skin care product. A carrier refers to an inactive substance that serves as a carrier or medium for a drug or other active substance. The carrier may include, but is not limited to, solvents, cosolvents, colorants, preservatives, antimicrobial agents, fillers, binders, disintegrants, lubricants, surfactants, emulsifiers, suspending agents, or any combination thereof.

[0053] In the present invention, the composition can be used in the form of a dry powder or a solution, and the solvent can be water. The composition can be applied in any suitable manner, such as orally or externally (applied in the form of a facial mask), and can also be prepared into a suitable dosage form (such as tablets, capsules, solutions, essences, etc.). The composition can be used as a skin care product, and the skin care product can be a skin care product in the form of a facial mask, essence, cream, gel, emulsion, toner, lotion, loose powder, ointment, spray, cleanser, shower gel, or concealer stick.

[0054] When the composition of the present invention is formulated as a cream or gel, the carrier may be animal fiber, plant fiber, wax, paraffin, starch, tragacanth, cellulose derivatives, polyethylene glycol, silica gel, bentonite, silicon dioxide, talc or zinc oxide.

[0055] When the composition of the present invention is formulated as loose powder or spray, the carrier may be lactose, talc, silicon dioxide, aluminum hydroxide, calcium silicate or polyamide powder. When formulated as a spray, it may further contain a propellant such as chlorofluorocarbons, propane / butane or dimethyl ether.

[0056] When the composition of the present invention is formulated as a solution, emulsion, facial cleanser or shower gel, the carrier may use a solvent, solvating agent or emulsifier, such as water, ethanol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol oil, fatty acid glyceride, polyethylene glycol or fatty acid ester of sorbitan.

[0057] In the present invention, in order to enhance the effect of improving skin condition, the composition may further contain a substance that promotes skin absorption.

[0058] In the present invention, the dosage of the composition can be determined according to the condition of the subject, and can also be taken daily as a medicine, health food, or as a skin care product. In some embodiments, the dosage of the composition can be in the range of 0.001-500 mg / kg body weight, such as 0.01, 0.1, 1, 5, 10, 20, 30, 40, 50, 100, 200, 300, 400, 500 mg / kg body weight, or any value or range therebetween. The composition can be applied once, twice, or more than twice a day.

[0059] The present invention will be described in detail below through examples.

[0060] Example 1

[0061] 1 Materials and Methods

[0062] 1.1 Preparation and Characterization of LBP1C

[0063] 5 kg of dried wolfberry fruit was pulverized in a grinder. 1.0 kg of wolfberry powder was added to 20 L of deionized water. 30 g of cellulase (2000 U / g, Shandong Longkote Enzyme Preparation Co., Ltd.), 5 g of papain (400,000 U / g, Guangxi Nanning Pangbo Bioengineering Co., Ltd.), and 10 g of amylase (1,000,000 U / g, Guangxi Nanning Pangbo Bioengineering Co., Ltd.) were added at 55°C, and the mixture was stirred for 1 hour. The solution was heated to 100°C to inactivate the enzymes, followed by centrifugation. The supernatant was heated and concentrated to a small volume and dialyzed against running water (molecular weight cut-off 3500 Da) for 2 days. The dialyzed solution was then heated and concentrated again and centrifuged. Five volumes of 95% industrial ethanol were added to the supernatant, stirring continuously, and the solution was allowed to stand overnight. The precipitate was collected by centrifugation, washed three times with anhydrous ethanol and acetone, and dried in a 50°C oven to obtain crude wolfberry polysaccharide (LBP1) extracted by enzyme-water co-extraction.

[0064] DEAE Sepharose TM Rapid flow column separation of crude polysaccharides: about 6 g of enzyme-water co-extraction wolfberry crude polysaccharide (LBP1) was dissolved in 60 mL of deionized water, centrifuged to remove insoluble matter, and the supernatant was loaded on DEAE Sepharose TM The rapid flow column was gradient eluted with deionized water and NaCl solutions of different ionic strengths (0.05M, 0.1M, 0.2M, 0.4M, 0.6M, 0.8M, 1.0M, and 2.0M) in sequence. The samples were collected by an automatic collector at a flow rate of 12mL / 15min. The polysaccharide content was detected by the sulfuric acid-phenol method, and its OD was determined. 490 Elution profiles were plotted, and identical fractions were combined, concentrated under reduced pressure, and dialyzed (molecular weight cutoff: 3500 Da). The dialysis bag contents were collected and freeze-dried to yield 0.05 M elution fractions of LBP1A, 0.1 M elution fractions of LBP1B, and 0.2 M elution fractions of LBP1C. LBP1C was the primary polysaccharide used in the next experiments (oven-dried at 50°C).

[0065] 200 mg of the polysaccharide LBP1C was dissolved in 2 mL of 0.2 M NaCl and centrifuged at 4000 rpm for 10 minutes. The supernatant was separated by chromatography on a Sephacryl HR S-300 gel column and eluted with 0.2 M NaCl at a flow rate of 3-6 mL / 15 minutes. The elution curve was plotted at a wavelength of 490 nm using the sulfuric acid-phenol method. Five absorption peaks were detected from the elution curve. The second absorption peak was collected and combined for HPGPC analysis. The single, symmetrical absorption peak with a weight-average molecular weight of 12-720 kDa was the Lycium barbarum polysaccharide LBP1C-2. After concentration, dialysis, and freeze-drying, approximately 30 mg of LBP1C-2 was obtained. However, the same procedure was repeated with LBP1A and LBP1B, but no detectable amount of LBP1C-2 was obtained.

[0066] Determination of the Physicochemical Properties of LBP1C: The total sugar content of LBP1C was determined by the phenol-sulfuric acid method (Dubois, M., Gilles, K., Hamilton, J.K., Rebers, P.A., & Smith, F. (1951). A Colorimetric Method for the Determination of Sugars. Nature, 168(4265), 167–167). The protein content of LBP1C was determined by the BSA colorimetric method (see the instructions for the BCA kit). The results showed that the total sugar and protein contents of LBP1C were 45.52 wt% and 11.5 wt%, respectively, with the remainder consisting of pigments and unavoidable impurities.

[0067] Molecular weight determination: On a Shodex 804 (8.0 mm × 300 mm, exclusion limit 4 × 10 5 Da) and Shodex802 column (8.0 mm × 300 mm, exclusion limit 1 × 10 4Homogeneity and molecular weight were measured by high-performance gel permeation chromatography (HPGPC) on an Agilent 1260 HPLC system equipped with a flow rate of 0.5 mL / min and 0.1 M NaNO3 as the mobile phase. The sample was prepared into a 4 mg / mL solution using the mobile phase, and 10 μL of the solution was injected each time. The eluent was monitored by RI and UV detectors, and the column temperature was maintained at 25°C (Jin, C., Du, Z., Lin, L., Zhou, L., Li, S., Liu, Q., & Ding, K. (2017). Structural Characterization of Mannoglucan from Dendrobium nobile Lindl and the Neuritogenesis-Induced Effect of Its Acetylated Derivative on PC-12 Cells. Polymers, 9(12), 399.). The detection results of LBP1C showed four peaks. The weight-average molecular weights of Peaks 1, 2, and 3 are 664,115 Da, 171,337 Da, and 22,172 Da, respectively. Peak 4 is the peak of the mobile phase NaNO3. For LBP1C-2, its weight-average molecular weight (Mw), number-average molecular weight (Mn), and polydispersity (PD) index are approximately 13,181 Da, 10,750 Da, and 1.22, respectively.

[0068] Monosaccharide Composition Determination: Samples were hydrolyzed with 2 M trifluoroacetic acid (TFA) at 110°C for 4 hours. The released monosaccharides were derivatized with 1-phenyl-3-methyl-5-pyrazolone (PMP) and analyzed by HPLC on an XDB-C18 column (250 x 4.6 mm, 5 μm), eluting with acetonitrile / phosphate buffer (15.5:84.5 by volume, pH 6.7) at a flow rate of 1 mL / min. UV detection was set at 254 nm. The monosaccharide identity and content (as measured by peak area) were determined by comparing the retention time and peak area of ​​the test residues with those of monosaccharide standards (mannose (Man), rhamnose (Rha), gluconic acid (GlcA), galacturonic acid (GalA), arabinose (Ara), xylose (Xyl), galactose (Gal), and glucose (Glc).

[0069] The results showed that LBP1C contained Man, Rha, GlcA, GalA, Glc, Gal, Xyl, and Ara in molar ratios of 13.1:8.3:2.5:13.8:5.6:20.0:2.4:34.2, respectively, while the monosaccharide compositions (molar ratios) of LBP1A and LBP1B deviated from those of 5-6:3-4:0.8-1.2:5-6:2-3:8-9:14-15:1. LBP1C-2 was composed of Ara, Gal, GalA, and Rha in a molar ratio of 49.9:33.6:8.5:8.

[0070] NMR analysis: One-dimensional and two-dimensional NMR spectra were measured at 25°C on a Bruker AVANCE III 500M NMR spectrometer. The structure of LBP1C-2 was confirmed by reference to the NMR spectra. The results showed that the structure of LBP1C-2 is shown in Formula 1-3.

[0071] Cell treatment: Dry powder LBP1C was prepared in culture medium at a final concentration of 0.4 mg / mL and treated for approximately 20 generations of cells, with treatment lasting for 2 generations and approximately 6 days. Nematode treatment: 5 mg / mL of LBP1C was mixed into OP50 bacterial solution and fed to nematodes (natural feeding), starting from the first day of treatment and continuing for 10 days.

[0072] 1.2 Human fibroblast culture

[0073] Human dermal fibroblasts (HDF) (purchased from ScienCell Research Laboratories, catalog number: 2320) were used for experimental studies of cell senescence. The cells were cultured in DMEM medium containing 10% fetal bovine serum (FBS) (purchased from Gibco, catalog number: 10099-141), 100 units / ml penicillin, and 100 μg / ml streptomycin in a 5% CO2 incubator at 37°C and 95% humidity.

[0074] 1.3 Cultivation of Nematodes

[0075] N2 wild-type nematodes were cultured at a temperature of 20°C and a humidity of approximately 50%, and were fed with Escherichia coli strain OP50.

[0076] 1.4 Western blot

[0077] After preparing the electrophoresis sample with 5× SDS-PAGE loading buffer, the protein was separated using 8% SDS-PAGE gel and then transferred to NC membrane. The membrane was blocked with 5% skim milk powder (prepared with TBST, tris buffered saline + Tween 20) at room temperature for 1.5h (when the detection protein is biotinylated, milk is not blocked and TBST is used directly). Incubate with the corresponding primary antibody at 4°C overnight. Use HRP-conjugated secondary antibody to label the primary antibody and use enhanced chemiluminescence kit and ChemiDoc XRS + The color development was performed using a BioRad instrument, and the color development solution was ultrasensitive luminescent solution (Thermo Scientific, 89880). Antibodies: Anti-iNOS antibody (Santa Cruz, sc-7271), Anti-LC3B Antibody (Cell Signaling Technology, 2775S), Anti-P62 Antibody (MBL, PM045).

[0078] 1.5 Real-time fluorescence quantitative PCR

[0079] In the SYBR Green method, an excess of SYBR fluorescent dye is added to the PCR reaction system. The SYBR fluorescent dye specifically incorporates into double-stranded DNA, emitting a fluorescent signal. Unincorporated SYBR dye molecules do not emit any fluorescent signal, ensuring that the increase in fluorescent signal is completely synchronized with the increase in PCR product. After obtaining reverse-transcribed cDNA, the quantitative PCR detection system is shown in Table 1.

[0080] Table 1

[0081] Table 2

[0082] Quantitative PCR was performed on ABI7500, and the primers are shown in Table 2. For each sample to be tested, three independently prepared RNA samples were selected for reverse transcription into cDNA, and each cDNA sample was tested three times. The final C(t) values ​​were averaged, and the target gene was normalized with the internal reference gene, and the C(t) values ​​were averaged with 2 -△△Ct The relative changes in mRNA expression of the corresponding genes were calculated using the qPCR method. Quantitative PCR conditions included denaturation at 94°C for 10 minutes, followed by 40 cycles of denaturation at 94°C for 10 seconds, annealing at 60°C for 15 seconds, and extension at 72°C for 20 seconds, followed by extension at 72°C for 90 seconds. The melting curve of the products was monitored simultaneously.

[0083] 1.6SA-β-gal staining

[0084] Aspirate the cell culture medium, wash once with PBS or HBSS, add 1 ml of β-galactosidase (β-gal) staining fixative, and fix for 15 minutes at room temperature. Aspirate the cell fixative, wash the cells three times with PBS or HBSS for 3 minutes each, then aspirate the PBS or HBSS and add 1 ml of staining solution to each well. Incubate at 37°C for 12-16 hours and observe under a standard light microscope. Count the cells in the upper, lower, left, right, and center fields of view for each well and count >200 cells stained blue.

[0085] 1.7 Lipofuscin detection

[0086] Cells: Sudan Black staining: Remove cell culture medium, wash once with PBS, add 4% PFA, fix at room temperature for 15 minutes, and wash three times with PBS for 2 minutes each. Incubate in 70% ethanol for 2 minutes, then incubate with 0.7% Sudan Black (prepared freshly) diluted in 70% ethanol for 2-8 minutes. Observe the staining under a microscope to prevent precipitation. Remove Sudan Black and incubate in 50% ethanol for 2 minutes. Wash three times with distilled water for 2 minutes each, then add 40% glycerol. Observe and photograph under a standard microscope.

[0087] C. elegans: In vivo fluorescence spectroscopy is a classic method for detecting lipofuscin in C. elegans. Autofluorescence from the nematode intestine under 340 nm excitation and 430 nm emission is lipofuscin. Images were acquired using the Andor high-speed rotating disk live cell fluorescence imaging system, and statistical analysis of the images was performed using Imaris software.

[0088] 1.8 Immunofluorescence

[0089] Human fibroblasts were cultured in glass-bottomed dishes, the medium was removed, and the cells were washed three times with PBS. The cells were fixed with 4% paraformaldehyde for 15 minutes at room temperature and washed three times with PBS for 5 minutes each. The cells were permeabilized with 0.5% Triton X-100 (in PBS) for 10 minutes at room temperature and washed three times with PBS for 5 minutes each. Blocking was performed with 1% BSA (in PBS + 0.05% Tween) at room temperature for 1 hour. The cells were then incubated with the primary antibody (Anti-TFEB antibody (CST, D2O7D)) at 4°C overnight. After washing with PBST three times for 10 minutes each to remove nonspecific binding, the cells were incubated with a fluorescein-conjugated secondary antibody for 1 hour at room temperature. The cells were washed with PBST three times for 10 minutes each and observed using a laser confocal microscope at 63× magnification using an oil-immersed lens.

[0090] 1.9 C. elegans movement detection

[0091] A drop of M9 buffer was placed on a 3.5 cm uncoated NGM medium. A single nematode to be tested was placed in the M9 droplet and allowed to acclimate for 30 seconds. The number of body bends within 30 seconds was then counted under a stereomicroscope. The number of pumpings within 30 seconds was also counted under natural feeding conditions. Fifteen nematodes were analyzed for each experiment.

[0092] 1.10 Statistical Analysis

[0093] The experimental data were statistically analyzed using SPSS 22.0 statistical software. Measurement data were expressed as x ± s and all were normally distributed. Two independent sample t tests were used to compare the two groups.

[0094] 2. Results

[0095] 2.1LBP1C delays fibroblast senescence

[0096] Lycium barbarum (Goji berry) is a complex polysaccharide, each of which may have distinct functions. LBP1C is a polysaccharide extracted from the fruit. Dried, mature Lycium barbarum fruit is first extracted using a combined enzyme (cellulase, papain, and amylase)-water extraction method to obtain crude Lycium barbarum polysaccharide (LBP1). This crude Lycium barbarum polysaccharide LBP1 was isolated and purified using anion exchange (DEAE) and gel filtration chromatography (Sephacryl S-200HR and Sephacryl S-300HR) to obtain the polysaccharide LBP1C (Figure 1(a)). Its anti-aging and lipofuscin-clearing properties were subsequently investigated. Replicative senescence refers to the phenomenon in which normal cells cultured in vitro cease growth after a limited number of divisions, resulting in significant changes in cell morphology and physiological metabolic activity. In addition to most tumor cells and embryonic stem cells, cells of different ages and from different biological donors exhibit replicative senescence. The anti-aging effects of LBP1C were investigated using replicatively senescent human fibroblasts (HDFs) as a model. First, we characterized the young and senescent cells of the replicative senescent cell model. We used the SA-β-gal enzyme detection kit to detect HDF P10 and HDF P25 cells. The proportion of SA-β-gal-positive cells at P25 was significantly higher than that at P6 (Figure 1(b)). The results showed that P6 cells were young cells, defined as young cells (early passage, EP), and P25 cells were senescent cells (late passage, LP). HDF cells at passage P20 were treated with LBP1C, LBP1A, and LBP1B for two passages (6 days) and then assayed for senescence-related markers. The mRNA expression levels of the senescence markers P16 and P21 were significantly decreased in the LBP1C-treated group compared with the control group (Figures 1(c) and 1(d)), whereas the mRNA level of the senescence marker P16 remained unchanged in the LBP1A and LBP1B-treated groups (Figure 1(g)). The loss of LAMIN B1, a hallmark of cellular senescence, was significantly increased in the LBP1C-treated group compared with the control group (Figure 1(e)). The number of SA-β-gal-positive cells also decreased significantly in the LBP1C-treated group (Figure 1(f)). These results indicate that LBP1C delays senescence in human fibroblasts.

[0097] 2.2LBP1C reduces GATA4 levels and inhibits SASP levels

[0098] Next, the mechanism by which LBP1C delays cellular senescence was investigated. GATA4 plays a key role in activating the senescence process. GATA4 is inhibited by autophagy, but when cells age, autophagy levels decrease, GATA4 begins to accumulate, and its expression induces the expression of genes associated with the SASP. It was found that compared with the control group, the level of GATA4 in the LBP1C-treated group was significantly decreased (Figure 2(a)), the SASP factor interleukin-1β (IL-1β) was also significantly decreased in the LBP1C-treated group (Figure 2(b)), and the mRNA and protein levels of iNOS were also significantly reduced (Figures 2(c), 2(d), 2(e)). These results indicate that LBP1C reduces GATA4 levels and inhibits the SASP in senescent cells.

[0099] 2.3LBP1C increases TFEB nuclear entry and activates autophagy

[0100] LBP1C can reduce GATA4 levels, which are inhibited by autophagy. Therefore, we speculated that LBP1C activates autophagy, thereby reducing GATA4 levels. We first examined the nuclear importation of TFEB, a key transcriptional regulator of lysosomal biogenesis and autophagy. Immunofluorescence results showed that the LBP1C-treated group increased the proportion of TFEB nuclear import compared to the control group (Figure 3(a)). Lysosome counts were further analyzed using a lysosomal dye, revealing a significantly higher number of lysosomes in the LBP1C-treated group compared to the control group (Figure 3(b)). Analysis of TFEB downstream genes revealed that lysosomal hydrolases, lysosomal membrane proteins, and functionally related vesicular ATPases were significantly upregulated in the LBP1C-treated group (Figure 3(c)), further demonstrating that LBP1C promotes autophagy. Further analysis of autophagy levels revealed a significant increase in LC3II / I levels compared to the control group, indicating a significant increase in autophagy (Figure 3(d)). These results suggest that LBP1C activates autophagy by increasing TFEB nuclear import.

[0101] 2.4LBP1C reduces lipofuscin levels in senescent cells and nematodes

[0102] LBP1C increases autophagy, which clears cellular aggregates such as lipofuscin. Therefore, further investigation of its effects on lipofuscin levels at the cellular and individual levels was conducted. The lipofuscin content in the LBP1C-treated group was significantly lower than that in the control group (Figure 4(a)). In nematodes, 5 mg / mL of LBP1C was mixed with OP50 bacterial culture and treated and fed to the nematodes starting on day one. Lipofuscin levels and motility were then measured. Lipofuscin levels in nematodes treated with LBP1C for 8 days were significantly lower than in the control group (Figure 4(b)). Several indicators of nematode motility began to show differences from day 10, when the nematodes entered the senescent stage. LBP1C significantly increased the nematode's movement frequency and feeding rate (Figures 4(c) and 4(d)), indicating that LBP1C mitigates the decline in motility caused by aging and promotes healthy aging in nematodes.

[0103] In summary, the present study demonstrates that LBP1C increases lysosomal biogenesis and enhances autophagy by increasing TFEB nuclear import. This increased autophagy not only reduces the transcription factor GATA4, thereby suppressing the aging-related SASP phenotype, but also reduces lipofuscin production during aging, thus delaying aging (Figure 4(e)).

[0104] 3. Discussion

[0105] The present invention investigates the anti-aging and potential whitening effects and mechanisms of LBP1C, a polysaccharide component isolated from wolfberry, at the cellular and nematode system levels. The study found that LBPC1 reduces markers of naturally senescent cells and lowers the SASP, reducing lipofuscin levels in cells and nematodes, improving nematode motility, and promoting healthy aging. Mechanistically, LBPC1 was found to promote the nuclear import of TFEB, increasing lysosomal content and cellular autophagy, thereby reducing levels of GATA4, a regulator of the SASP and further reducing the SASP. Simultaneously, activation of the autophagy-lysosome system promotes the degradation of the intracellular substrate lipofuscin, thereby reducing lipofuscin levels. The present invention identifies LBP1C, the active ingredient in wolfberry that is responsible for its anti-aging and lipofuscin-inhibiting effects, and reveals its novel mechanism, providing a scientific basis for the widespread application of wolfberry.

[0106] 3.1 Using a natural aging model to study the anti-aging effects of Lycium barbarum LBP1C and discover its efficacy in promoting healthy aging

[0107] This study investigated the efficacy and mechanism of LBP1C using naturally aging human fibroblast models and naturally aging nematode models, revealing its ability to reduce SASP and lipofuscin. Compared to many previous studies conducted using accelerated aging mouse models or oxidative treatment or stimulation, this study better simulates the natural use of goji berries in daily life, providing users with practical reference information. The new anti-aging goal is "healthy aging," aiming to achieve "aging without aging," meaning maintaining a higher level of physical function than normal even in old age. Previous studies have revealed how exercise alleviates endoplasmic reticulum reduction stress, thereby promoting healthy aging. This study found that LBP1C can improve the motor ability of aging nematodes, demonstrating that goji berries promote healthy aging in nematodes. As a traditional Chinese medicine with both medicinal and edible properties, goji berries have important practical value in promoting healthy aging.

[0108] 3.2 Discovery of a new mechanism by which Lycium barbarum LBP1C activates autophagy through TFEB to delay aging

[0109] This study reveals a novel mechanism by which LBP1C, an active ingredient in wolfberry, activates TFEB, promoting lysosome formation and autophagy, thereby exerting anti-aging and lipofuscin-clearing effects. Studies have shown that autophagy significantly declines during aging. Autophagy is a crucial component of protein quality control and cellular homeostasis. This decline in autophagy directly leads to the accumulation of GATA4, a key transcription factor in the SASP, which activates the SASP and contributes to aging. LBP1C significantly increases autophagy, potentially inhibiting aging. Furthermore, levels of IL-1β, which is associated with the SASP, and iNOS, which is associated with inflammation, are significantly reduced. The activities of corresponding aging markers, P16, P21, and β-gal, are also significantly decreased. The decline in nematode motility associated with aging is also reversed. Increased autophagy is also one of the factors that contributes to the reduction of lipofuscin. This study provides new scientific evidence for understanding the whitening effects of wolfberry. Previous studies have primarily focused on its antioxidant properties, scavenging free radicals and inhibiting lipid oxidation, thereby reducing lipofuscin. This study reveals that wolfberry reduces lipofuscin by activating autophagy to clear lipofuscin precursors. In summary, the present invention extracts new compounds from wolfberry and provides a new mechanism for its anti-aging and potential whitening effects.

[0110] Whitening actually encompasses two aspects: beauty (cell rejuvenation) and whiteness (reduced lipofuscin and other deposits). This study reveals the novel role and molecular mechanism of LBP1C, a potent ingredient in wolfberry, in delaying aging and removing lipofuscin deposits, explaining the scientific implications of wolfberry's benefits of slimming, anti-aging, complexion enhancement, and whitening, as documented in the Compendium of Materia Medica.

[0111] The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited thereto. Within the technical concept of the present invention, various simple variations of the technical solution of the present invention may be made, including combining the various technical features in any other appropriate manner. These simple variations and combinations should also be regarded as disclosed in the present invention and fall within the scope of protection of the present invention.

Claims

1. A use of a composition, characterized in that: At least part of the Lycium barbarum polysaccharide in the composition is Lycium barbarum polysaccharide LBP1C-2; the monosaccharide composition of the Lycium barbarum polysaccharide LBP1C-2 is arabinose, galactose, galacturonic acid and rhamnose; Wherein, the use is to delay aging and / or whiten the skin; And / or, the use is to activate the key transcriptional regulator of autophagy (TFEB); and / or, the use is to reduce the expression level of P16, P21, senescence-associated-β-galactosidase (SA-β-gal), transcription factor GATA4 or senescence-associated secretory phenotype (SASP); And / or, the use is to increase the expression level of LAMIN B1 or the level of cell autophagy; And / or, the use is to inhibit the accumulation of lipofuscin; And / or, the use is to promote healthy aging.

2. The use according to claim 1, wherein The content of Lycium barbarum polysaccharide in the composition is greater than or equal to 40wt%.

3. The use according to claim 2, wherein The content of Lycium barbarum polysaccharide in the composition is 40-50wt%.

4. The use according to any one of claims 1 to 3, wherein The monosaccharide composition of the wolfberry polysaccharide includes mannose, rhamnose, gluconic acid, galacturonic acid, glucose, galactose, arabinose and xylose, and the molar ratio of mannose, rhamnose, gluconic acid, galacturonic acid, glucose, galactose, arabinose and xylose in the monosaccharide composition of the wolfberry polysaccharide is 5-6:3-4:0.8-1.2:5-6:2-3:8-9:14-15:1; And / or, the composition further contains protein, pigment and inevitable impurities, the content of the protein is 10-15wt%; the balance is pigment and inevitable impurities; And / or, the weight average molecular weight distribution range of the macromolecular substances in the composition is 20-700 kDa.

5. The use according to any one of claims 1 to 4, wherein The composition also contains protein, pigment and inevitable impurities, wherein the content of the protein is 11-12 wt %; the balance is the pigment and inevitable impurities.

6. The use according to any one of claims 1 to 5, wherein The molar ratio of arabinose, galactose, galacturonic acid and rhamnose in the monosaccharide composition of wolfberry polysaccharide LBP1C-2 is 30-70:20-60:0.1-10:0.1-10.

7. The use according to claim 6, wherein The molar ratio of arabinose, galactose, galacturonic acid and rhamnose in the monosaccharide composition of wolfberry polysaccharide LBP1C-2 is 49-50:33-34:8-9:

8.

8. The use according to any one of claims 1 to 7, wherein The weight average molecular weight of Lycium barbarum polysaccharide LBP1C-2 is 10-150 kDa; And / or, the content of Lycium barbarum polysaccharide LBP1C-2 in the composition is greater than or equal to 10 wt %.

9. The use according to claim 8, wherein The weight average molecular weight of Lycium barbarum polysaccharide LBP1C-2 is 50-120 kDa; And / or, the content of Lycium barbarum polysaccharide LBP1C-2 in the composition is 10-30wt%.

10. The use according to any one of claims 1 to 9, wherein The structure of Lycium barbarum polysaccharide LBP1C-2 is shown in Formula 3: In formula 3, n=2-20.

11. The use according to any one of claims 1 to 10, wherein The preparation method of the composition comprises: (1) mixing wolfberry, enzyme and water for enzyme treatment to obtain an enzymatic solution; (2) subjecting the enzymatic hydrolyzate to alcohol precipitation to obtain crude wolfberry polysaccharide; (3) The crude wolfberry polysaccharide is loaded onto an anion exchange chromatography column and gradient eluted with deionized water and salt solutions of different ionic strengths.

12. The use according to claim 11, wherein The difference in salt concentration between adjacent salt solutions used in gradient elution is 0.03-0.08M; And / or, an eluate of a saline solution having a salt concentration of 0.18-0.22 M is taken as the source of the composition.

13. A method of using a composition, characterized in that: The method comprises: applying a composition containing Lycium barbarum polysaccharides to a subject or animal cells, wherein at least part of the Lycium barbarum polysaccharides in the composition are Lycium barbarum polysaccharides LBP1C-2; the monosaccharide composition of the Lycium barbarum polysaccharides LBP1C-2 is arabinose, galactose, galacturonic acid and rhamnose; Wherein, the composition is used for delaying aging and / or whitening; And / or, the composition is used to activate a key transcriptional regulator of autophagy (TFEB); and / or, the composition is used to reduce the expression level of P16, P21, senescence-associated-β-galactosidase (SA-β-gal), transcription factor GATA4, or senescence-associated secretory phenotype (SASP); And / or, the composition is used to increase the expression level of LAMIN B1 or the level of cellular autophagy; and / or, the composition is used to inhibit the accumulation of lipofuscin; And / or, the composition is for promoting healthy aging.

14. The method according to claim 13, wherein: The composition is the composition according to any one of claims 2 to 12.

Images