Application of exopolysaccharide TDG3 in preparation of product for promoting synthesis of skin collagen

Abstract

The invention discloses an application of an exopolysaccharide TDG3 in preparation of a product for promoting synthesis of skin collagen. The structural formula of the exopolysaccharide TDG3 is # imgabs0, and the exopolysaccharide TDG3 can remarkably promote mouse skin collagen synthesis and wound healing, has the advantages of high stability, easiness in obtaining, high safety, low production cost and the like, is suitable for being prepared into a composition or a product for promoting skin collagen synthesis and protecting and repairing a skin barrier, and has a wide application prospect.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial extracellular polysaccharides and relates to the application of extracellular polysaccharide TDG3 in preparing a product for promoting skin collagen synthesis. Background Art

[0002] Collagen represents a family of proteins found in all vertebrates and invertebrates, varying greatly in size, function, and tissue distribution. Members of the collagen family have diverse functions, are present in all organs, and provide rigidity and integrity to bones and skin. The diversity of these polypeptides demonstrates the critical role collagen plays in the structure and integrity of organs and tissues. Collagen fibrils are assembled from glycoproteins, and all members of the collagen family share a characteristic triple-helical structure composed of three α-chains. Of the 28 different types of collagen described to date, type I is the most abundant and most studied in humans. Type I collagen accounts for 90% of bone mass and is the primary component of human skin (80%), with type III collagen making up the remainder of skin collagen (15%). Collagen is the primary structural protein of the skin, comprising approximately 70% of the dry weight of the dermis and playing a key role in maintaining skin elasticity, strength, and hydration. However, with aging, the amount of collagen in the skin decreases, a process regulated not only by intrinsic aging factors but also by external environmental factors. Epidemiological studies have shown that collagen loss is one of the main drivers of skin aging, manifesting as sagging, wrinkling, and moisture loss. Therefore, the development of new, safer, and more effective natural medicines is crucial for the long-term management and regulation of skin aging.

[0003] Decreased skin collagen is a core biological hallmark of skin aging. Its pathogenesis involves a complex molecular signaling network and the combined effects of internal and external factors. In recent years, with the advancement of anti-aging research, interventions targeting reduced skin collagen have gained increasing attention. Oral collagen peptide supplementation, topical vitamin C application, and photodynamic therapy have been shown to improve skin collagen levels. However, the long-term effects of these approaches require further verification. Oral collagen supplements have limited absorption capacity, and their effectiveness varies from person to person. Some people may be allergic to their ingredients. Laser and radiofrequency therapy can cause redness, pain, and even burns. Furthermore, their effects are not long-lasting, requiring regular re-treatment to maintain them. Overall, reduced skin collagen is the result of a combination of internal and external factors, and its epidemiological characteristics reflect the complex interaction between individual aging processes and environmental exposure patterns. Future research is needed to further clarify the specific mechanisms in different populations to inform prevention and intervention strategies.

[0004] Tremella polysaccharide is a common and well-studied polysaccharide. Studies have shown that Tremella polysaccharide can activate related signaling pathways in skin fibroblasts, upregulate the expression of collagen genes, and thus promote collagen synthesis. After using skin care products added with Tremella polysaccharide for a period of time, the user's skin elasticity was significantly improved and wrinkles were reduced (MA X, YANGM, HE Y, et al. A review on the production, structure, bioactivities and applications of Tremella polysaccharides [J]. International Journal of Immunopathology and Pharmacology, 2021, 35: 20587384211000541.). In the treatment of burns, some hospitals use dressings containing chitosan polysaccharides. When a patient's wound comes into contact with this dressing, it can accelerate collagen synthesis in the wound, promote wound healing, and reduce scar formation (ZHANG M, FAN Z, ZHANG J, et al. Multifunctional chitosan / alginate hydrogel incorporated with bioactive glass nanocomposites enabling photothermal and nitric oxide release activities for bacteria-infected wound healing [J]. International Journal of Biological Macromolecules, 2023, 232: 123445.). However, many polysaccharides have poor stability under different environmental conditions, such as changes in temperature and pH. For example, the structure of certain marine polysaccharides is easily destroyed under high temperatures or extreme pH conditions, resulting in a decrease or even loss of their activity in promoting collagen synthesis. The process of extracting polysaccharides from natural raw materials is often complex and requires a lot of manpower, material resources, and financial resources. For example, extracting highly active polysaccharides from specific rare plants or microorganisms may require special extraction techniques and equipment, and the difficulty in obtaining raw materials leads to high production costs.

[0005] In summary, while a variety of skin collagen-enhancing drugs have demonstrated promising clinical results, they are all associated with varying degrees of adverse reactions. Furthermore, not all patients respond adequately to existing drugs. Therefore, there is an urgent need to develop novel, low-cost, readily available, safer and more effective natural ingredients to provide new insights into the development of novel skin collagen-enhancing products. Summary of the Invention

[0006] The present invention provides an application of extracellular polysaccharide TDG3 in preparing a product for promoting skin collagen synthesis.

[0007] The exopolysaccharide TDG3 of the present invention is produced by Duganella sp. G3 and is composed of glucose, mannose and galactose in a molar ratio of 3:2:1. The glycosidic bond connection mode is: 4)-β-D-Glcp-(1→4)[α-D-Manp-(1→2)]-β-D-Manp-(1→4)-β-D-Glcp-(1→3)-β-D-Galp-(1→4)-β-D-Glcp-(1→3), and its structural formula is: Where n=100~100000.

[0008] The Durum G3 described in the present invention was deposited in the China Center for Type Culture Collection on January 18, 2024, with a deposit number of CCTCC NO: M 2024137, and the deposit address is Wuhan University, Wuhan, China.

[0009] The exopolysaccharide TDG3 of the present invention is prepared by the following steps:

[0010] The fermentation liquid of Duchenne sp. G3 was diluted with water to remove protein, and then 2 to 3 times the volume of ethanol was added to the supernatant. The precipitate was collected by centrifugation and dried to obtain a pure product of extracellular polysaccharide TDG3.

[0011] The protein removal method of the present invention adopts conventional methods in the art, such as adding 0.05-0.2% NaOH, filtering with a 20nm-2μm filter membrane, and ultrafiltration with an ultrafiltration membrane with a molecular weight cutoff of 5kD-50kD.

[0012] In a specific embodiment of the present invention, the above-mentioned extracellular polysaccharide TDG3 is prepared by the following steps:

[0013] The fermentation broth of D. dulcis G3 was mixed with twice the volume of pure water, and NaOH was added to a final concentration of 0.1% to 0.2%. The mixture was boiled for 10 to 20 minutes, and then the precipitate was filtered through a 1 μm filter membrane. Finally, 0.1% to 0.3% sodium acetate and 3 times the volume of ethanol were added and stirred evenly. The precipitate was collected by centrifugation and dried to obtain pure extracellular polysaccharide TDG3.

[0014] The product for promoting skin collagen synthesis of the present invention is a composition or product.

[0015] The compositions of the present invention include but are not limited to skin care products, pharmaceutical compositions, etc.

[0016] The products described in the present invention include but are not limited to medical dressings, medical devices, biomaterials, tissue engineering products, etc.

[0017] The pharmaceutical composition of the present invention can be administered by topical application or subcutaneous injection.

[0018] The pharmaceutical composition of the present invention can be administered to any animal, including humans and non-human animals, such as pets or livestock.

[0019] The administration concentration and dosage of the pharmaceutical composition of the present invention depends on the age, health and weight of the recipient, the frequency of treatment, the route of administration, etc.

[0020] In a specific embodiment of the present invention, in the pharmaceutical composition, the administration concentration of the extracellular polysaccharide TDG3 is 0.5 mg / mL to 20 mg / mL, and the dosage is 0.5 mg / kg to 2 g / kg.

[0021] The pharmaceutical carrier used in the pharmaceutical composition of the present invention can be a pharmaceutical carrier conventionally used in the art, such as isotonic saline solution, isotonic glucose solution, or isotonic solution containing a buffer system, such as PBS buffer solution.

[0022] In a specific embodiment of the present invention, the pharmaceutical carrier used is 0.9% physiological saline.

[0023] Compared with the prior art, the present invention has the following significant advantages:

[0024] (1) The exopolysaccharide TDG3 of the present invention is a microbial exopolysaccharide. Compared with other chemically synthesized polysaccharides, it has the advantages of higher stability, easy availability, high safety, and low production cost.

[0025] (2) The extracellular polysaccharide TDG3 of the present invention can be used to protect the skin barrier and can effectively improve the skin collagen synthesis of mice. The extracellular polysaccharide TDG3 is safe and non-toxic, has strong pharmacological effects, and has good application prospects.

[0026] (3) The extracellular polysaccharide TDG3 of the present invention can regulate collagen synthesis on the skin surface and hair follicle growth, thereby enhancing the protection of the skin barrier.

[0027] (4) The extracellular polysaccharide TDG3 of the present invention can accelerate the wound healing rate in mice by promoting collagen production.

[0028] (5) The extracellular polysaccharide TDG3 of the present invention can specifically bind to receptors on the keratinocyte membrane and secrete cytokines to regulate the proliferation of fibroblasts and collagen synthesis. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is the infrared absorption spectrum of the exopolysaccharide TDG3 produced by Durumila sp. G3.

[0030] Figure 2 This is a liquid chromatogram of component determination after derivatization of the extracellular polysaccharide TDG3, where the markers are: Man (mannose), Glc (glucose), and Gal (galactose).

[0031] Figure 3 This is the gas chromatogram of the extracellular polysaccharide TDG3 after methylation and acetylation treatment.

[0032] Figure 4 (Left) heteronuclear single quantum coherence NMR spectrum and (right) heteronuclear multiple quantum correlation spectrum of the extracellular polysaccharide TDG3.

[0033] Figure 5 Histological evaluation of the dorsal skin of C57BL / 6 mice following application of the exopolysaccharide TDG3. Figure A shows hematoxylin-eosin (H&E) staining of the skin of mice in the Ctrl, HG, and LG groups; Figure B shows Masson staining of the skin of mice in the Ctrl, HG, and LG groups.

[0034] Figure 6 Histological evaluation of the dorsal skin of C57BL / 6 mice following subcutaneous injection of the exopolysaccharide TDG3. Figure A shows hematoxylin and eosin (H&E) staining of the skin of mice in the Ctrl, HG3, and LG3 groups; Figure B shows Masson staining of the skin of mice in the Ctrl, HG3, and LG3 groups.

[0035] Figure 7 To study the effect of applying extracellular polysaccharide TDG3 on the hydroxyproline content in the back skin tissue of mice.

[0036] Figure 8Figure 3: Effects of applying the exopolysaccharide TDG3 on the expression of collagen genes (Col1α1, Col2α1, and Col3α1) in the dorsal skin tissue of mice. Figure A shows the effect of exopolysaccharide TDG3 on the expression of Col1α1 in the dorsal skin tissue of mice; Figure B shows the effect of exopolysaccharide TDG3 on the expression of Col2α1 in the dorsal skin tissue of mice; and Figure C shows the effect of exopolysaccharide TDG3 on the expression of Col3α1 in the dorsal skin tissue of mice.

[0037] Figure 9 Figure 3: The effect of applying the exopolysaccharide TDG3 on wound healing in mice. Figure A shows the wound healing process in mice, and Figure B shows the quantitative assessment of wound size (initial wound diameter: 1.5 cm).

[0038] Figure 10 Representative fluorescence microscopy images of HaCaT cells and NIH3T3 cells treated with FITC-labeled exopolysaccharide TDG3 (100 μg / mL). Scale bar: 100 μm.

[0039] Figure 11 Figure 3 shows the effects of extracellular polysaccharide TDG3 on collagen-related genes in co-cultured NIH3T3 cells. Figure A shows the effect of extracellular polysaccharide TDG3 on the expression level of MMP-3 in NIH3T3 cells; Figure B shows the effect of extracellular polysaccharide TDG3 on the expression level of Col1α1 in NIH3T3 cells; Figure C shows the effect of extracellular polysaccharide TDG3 on the expression level of Col3α1 in NIH3T3 cells. DETAILED DESCRIPTION

[0040] Unless otherwise specified, all technical and scientific terms used in the present invention have the meanings commonly understood by those skilled in the art to which the present invention belongs. The specific embodiments and examples described below are for illustrative purposes only and are not intended to limit the present invention. Unless otherwise specified, the reagents or materials used in the following examples can be purchased commercially or synthesized with reference to existing methods. The present invention will be further described below in conjunction with the examples and accompanying drawings.

[0041] Example 1

[0042] Screening, 16S sequence identification and fermentation purification of exopolysaccharide from D. dulcis G3

[0043] 1. Screening of D. dulcis G3:

[0044] (1) The screening medium formula is: KH2PO4 1 g / L, CaCl2 0.1 g / L, MgCl2·6H2O 0.3 g / L, FeSO4 0.0125 g / L, KNO3 2 g / L, sucrose 20 g / L, pH 7.2. For solid medium, add 15 g / L agar. Sterilize the medium at 121°C for 20 min.

[0045] (2) Screening method: Take the soil from Nanjing Garden into a 2mL EP tube, add 1mL of physiological saline, shake evenly, centrifuge at 50g for 1min, remove the precipitated soil particles, take 500μL of supernatant and centrifuge at 12000g for 3min, discard the supernatant, then blow the precipitated bacteria with 1mL of physiological saline, rinse, centrifuge, and finally dissolve in 200μL of physiological saline to prepare soil leachate. Dilute the soil leachate and spread it on a solid plate culture medium, and culture it at 28℃ for 2-3 days. After the colonies grow, pick the single colonies with sticky polysaccharides on the surface according to the morphological characteristics of the colonies, dilute and spread them on a new solid culture medium, and culture it at 28℃. After the colonies grow, pick the single colonies again and dilute and spread them, repeat this process until the colonies grown on the solid culture medium have a uniform morphology. And test its fermentation potential with liquid screening culture medium.

[0046] 2. Molecular Identification: The 16S rDNA of the selected single colony strain (see SEQ ID NO. 1 in the sequence listing) was subjected to Blast comparison. The sequence similarity with Duganella sp. PAMC 2743 was as high as 99.02%, and the strain was identified as Duganella sp. and named Duganella G3.

[0047] 3. Production and purification method of exopolysaccharide TDG3 produced by Durum G3:

[0048] (1) The fermentation liquid medium used was formulated as follows: sucrose 20–50 g / L, KNO3 1–4 g / L, NaH2PO4 0.5–2 g / L, CaCl2 0.02–0.1 g / L, MgSO4·7H2O 0.1–0.5 g / L, FeSO4·7H2O 0.01–0.06 g / L, MnSO4·H2O 0.005–0.01 g / L, ZnCl2 0.01–0.02 g / L, pH 7. The solid medium was prepared by adding 15 g / L agar powder to the liquid medium.

[0049] (2) Fermentation process: First, single colonies were picked onto a solid plate and cultured at 28°C and 230 rpm for 2 days to obtain highly viable colonies. Then, the colonies were picked into 10 mL of liquid culture medium and cultured at 28°C and 230 rpm for 2 days as seed liquid. Then, the fermentation was expanded at a 2% inoculation ratio and cultured for 2 to 3 days to obtain a high-viscosity fermentation liquid.

[0050] (3) Purification: Purification was performed using NaOH and filtration. The specific process was as follows: the fermentation broth was mixed with twice the volume of pure water, and NaOH was added to a final concentration of 0.1%. The mixture was boiled for 20 min, and the precipitate was filtered through a 1 μm filter membrane. Finally, 0.2% sodium acetate and 3 times the volume of ethanol were added and stirred evenly. The precipitate was collected by centrifugation and dried to obtain the pure extracellular polysaccharide TDG3.

[0051] Example 2

[0052] Structural analysis of the exopolysaccharide TDG3

[0053] For the pure extracellular polysaccharide TDG3, infrared spectroscopy, high performance liquid chromatography, gas chromatography-mass spectrometry, nuclear magnetic resonance and other technologies were used to analyze and infer its structure. Its structural analysis is as follows:

[0054] The infrared scanning instrument NICOLETIS10 (Thermo Fisher Scientific) was used to test the pure exopolysaccharide TDG3 and analyze its functional groups. The specific test process is: put the pressed potassium bromide blank piece into the sample holder of the infrared scanner sample compartment, confirm the collection of the reference background spectrum, and then put the sample to be tested into the spectrometer and scan the sample. The sample dosage is 2 to 5 mg each time. The infrared test results are as follows: Figure 1 As shown, the pure exopolysaccharide TDG3 has the peaks at 3320, 2931, 1615, 1410 and 1023 cm -1 There is an obvious absorption peak at 3320cm -1 The broad absorption peak at 2931 cm belongs to the hydroxyl groups in various environments in the polysaccharide molecules; -1 The absorption peaks are derived from the stretching and bending vibrations of CH in the sugar ring; 1615 cm -1 The absorption peak at 1410 cm comes from the hydrogen bond between crystal water and sugar; -1 The absorption peak comes from the OCO bond in the acetal group; 1023 cm -1 The absorption peak comes from the CC bond in the pyranose ring.

[0055] High-performance liquid chromatography (HPLC) was used to quantitatively analyze the various monosaccharides present in the pure exopolysaccharide TDG3. The process was as follows: the exopolysaccharide TDG3 was completely hydrolyzed with trifluoroacetic acid, derivatized with PMP, extracted with chloroform, and the aqueous phase was filtered. The analysis was then performed on a Waters HPLC system (Waters Inc., USA) equipped with a Zorbax SB-Aq column (4.6 mm × 150 mm, Agilent Technologies, Inc., USA). Figure 2 Liquid chromatogram for determination of monosaccharide components, Figure 2 It can be seen that the extracellular polysaccharide TDG3 is composed of glucose, mannose, and galactose in a molar ratio of 3:2:1.

[0056] Gas chromatography-mass spectrometry was used to analyze the types of glycosidic bonds in the extracellular polysaccharide TDG3. The process was as follows: the exposed hydroxyl groups in the polysaccharide were labeled by methylation reaction, the polysaccharide was hydrolyzed, the glycosidic bonds were broken to expose the hydroxyl groups corresponding to the glycosidic bonds, and then these hydroxyl groups were modified by acetylation. After the above steps, the polysaccharide was converted into a variety of volatile and heat-stable monosaccharide derivatives. A gas chromatograph equipped with a TG-200MS column (Thermo Scientific ISQ LT, USA) was used to separate monosaccharide derivatives with different numbers of acetyl groups or spatial positions, thereby identifying the types of glycosidic bonds in the polysaccharide. The gas chromatography results are as follows: Figure 3 As shown in the figure, combined with the mass spectrometry data, it can be seen that the extracellular polysaccharide TDG3 is composed of T-Manp with a peak time of 18.5 min, 1,4-linked Glcp with a peak time of 21.7 min, 1,3-linked Manp with a peak time of 21.9 min and 1,2,4-linked Manp with a peak time of 24.1 min.

[0057] The linking sequence of the exopolysaccharide TDG3 was analyzed by nuclear magnetic resonance technology. The process was as follows: the exopolysaccharide TDG3 was partially hydrolyzed with trifluoroacetic acid, dissolved in D2O and deuterated, and finally TMSP was added as an internal standard. The NMR analysis was performed using a Bruker Avance 500 MHz analyzer (Bruker, Karlsruhe, Germany). The heteronuclear single quantum coherence (HSQC) and heteronuclear multiple quantum correlation (HMBC) spectra were as follows: Figure 4 As shown. And for TDG3 1 H NMR and 13 The results are shown in Table 1.

[0058] Table 1 TDG3 1 H NMR and 13 C NMR shift (ppm)

[0059]

[0060]

[0061] Example 3

[0062] Study on the effect of exopolysaccharide TDG3 on promoting collagen synthesis in mouse skin

[0063] 1. Establishment of Animal Skin Model

[0064] The experimental animals were male C57BL / 6 mice, 6-8 weeks old, weighing 20 ± 2 g, obtained from the Animal Model Research Center of Nanjing University. All animals were isolated for 7 days before treatment under stable conditions (free access to food and water, 20-22°C, 50-60% relative humidity, and a 12-hour light / dark cycle). The dorsal skin of the mice was depilated and observed for two days to confirm the absence of pre-existing skin lesions.

[0065] 1.1 Topical application

[0066] Mice were randomly divided into three groups: (1) control group (Ctrl group), which did not receive any sugar solution treatment; (2) low-dose TDG3 group (LG group), in which 0.5 mg / mL of TDG3 solution was applied to the back of the mice daily; and (3) high-dose TDG3 group (HG group), in which 1 mg / mL of TDG3 solution was applied to the back of the mice daily. Twenty-four hours after applying TDG3 on the seventh day, all mice were sacrificed. Skin tissue from the back of the mice was collected, one portion fixed with 4% paraformaldehyde and then subjected to pathological section analysis, while the other portion was quickly frozen in liquid nitrogen and stored at -80°C for subsequent use in other experiments.

[0067] 1.2 Subcutaneous injection on the back:

[0068] Mice were randomly divided into three groups: (1) control group (referred to as Ctrl group), which did not receive any treatment by injection of the extracellular polysaccharide TDG3 solution; (2) low-dose TDG3 group (referred to as LG3 group), which received a subcutaneous injection of 0.5 mg / mL of the extracellular polysaccharide TDG3 solution on the back of the mice every day; and (3) high-dose TDG3 group (referred to as HG3 group), which received a subcutaneous injection of 1 mg / mL of the extracellular polysaccharide TDG3 solution on the back of the mice every day. 24 hours after the subcutaneous injection of the extracellular polysaccharide TDG3 on the seventh day, all mice were sacrificed. Skin tissues from the back of the mice were collected, one part was fixed with more than 4% paraformaldehyde and then subjected to pathological section analysis, and the other part was quickly frozen in liquid nitrogen and stored at -80°C for subsequent use in other experiments.

[0069] 2. Histopathological Analysis of Mouse Skin

[0070] (1) Fixation: Spread the mouse back skin tissue flat and immerse it in 4% paraformaldehyde fixative for 24 hours;

[0071] (2) Dehydration: The prepared skin tissue samples were immersed in alcohol from low to high concentrations for gradient dehydration, and then placed in xylene until the tissue became transparent;

[0072] (3) Embedding: Place melted liquid paraffin in a container and place the transparent tissue into it, ensuring that it is completely covered by the paraffin;

[0073] (4) Sectioning: Use a cutting machine to cut the fixed wax block into thin slices of approximately 5 μm thick. Attach the slices to a glass slide in a 37°C water bath and then dry the slide in a 60°C oven. Next, soak it in xylene to remove the paraffin wax, then immerse it in a gradient of alcohol from low to high concentrations to remove the xylene, and finally soak it in deionized water to remove the alcohol.

[0074] (5) H&E staining: First, stain with hematoxylin solution for 15 minutes, then separate with 1% hydrochloric acid in alcohol and 1% ammonia in alcohol for 10-30 seconds, and finally stain with 1% eosin solution for 2-3 minutes;

[0075] (6) Masson staining: Masson trichrome staining kit was used for corresponding staining;

[0076] (7) Sealing: Dehydrate the slices with anhydrous ethanol and then place them in xylene until the tissue becomes transparent. Next, add a drop of neutral resin to the slice surface and cover with a coverslip for sealing. Once the resin solidifies, the slices can be observed under a microscope.

[0077] 3. Determination of Hydroxyproline Levels in Mouse Skin Tissue

[0078] After killing mice, approximately 0.2 g of fresh skin tissue was collected, accurately weighed, added to 9 volumes of deionized water, and homogenized for 40 seconds. The homogenate was used to measure hydroxyproline levels. Subsequent procedures were performed according to the instructions of the corresponding commercial assay kit.

[0079] 4. Data Analysis

[0080] One-way ANOVA was used for statistical analysis of multiple groups, and P < 0.05 was considered significant.

[0081] 5. Results Analysis

[0082] like Figure 5 The results of histopathological examination of mouse skin are shown. Figure 5 As shown in A, the H&E staining results show that the number of hair follicles on the back of mice in the TDG3-treated group increased compared with the control group, indicating that TDG3 accelerated the growth of hair follicles in mice. Figure 5 As shown in B, compared with the control group, the color of collagen fibers in the group treated with extracellular polysaccharide TDG3 deepened, indicating that local application of extracellular polysaccharide TDG3 promoted collagen synthesis.

[0083] like Figure 6 Histological evaluation of the dorsal skin of C57BL / 6 mice injected subcutaneously with the exopolysaccharide TDG3. Figure 6 As shown in A, the H&E staining results show that the number of hair follicles on the back of mice in the subcutaneous injection of exopolysaccharide TDG3 group increased compared with the control group, indicating that exopolysaccharide TDG3 accelerated the growth of hair follicles in mice. Figure 6 As shown in B, compared with the control group, the color of collagen fibers in the subcutaneous injection of extracellular polysaccharide TDG3 group deepened, indicating that subcutaneous injection of extracellular polysaccharide TDG3 can also promote skin collagen synthesis to a certain extent.

[0084] like Figure 7 As shown in the figure, the hydroxyproline content in the fresh skin samples of each group of mice was quantitatively analyzed, and the results also proved that the extracellular polysaccharide TDG3 can promote the synthesis of mouse skin collagen.

[0085] like Figure 8 As shown, qRT-PCR assay results showed that exopolysaccharide TDG3 could increase the expression of collagen genes (Col1α1, Col2α1 and Col3α1) in mouse skin, thereby maintaining the integrity of skin structure.

[0086] Example 4

[0087] Study on the effect of TDG3 polysaccharide on accelerating wound healing in mice

[0088] 1. Establishment of Animal Model and Experimental Grouping

[0089] The experimental animals were male C57BL / 6 mice, 6-8 weeks old, 20±2g, obtained from the Animal Model Research Center of Nanjing University. All animals were isolated for 7 days under stable conditions (free access to food and water, 20-22°C, 50-60% relative humidity, 12-hour natural light / dark cycle) before treatment. The back skin of the mice was depilated and observed for two days to confirm that there was no pre-existing skin damage. (1) The process of establishing mouse wounds: The back of the mice was depilated the day before the experiment. On the 0th day of the experiment, a wound with a diameter of Φ=1.5cm was created in the depilated area on the back of the mice using a scalpel. (2) The process of treating mouse wounds: Starting from the 0th day of the experiment, the wounds of the mice were treated every other day. The wounds of the control group mice were smeared with 0.9% normal saline (referred to as the Ctrl group), and the wounds of the experimental group mice were smeared with 2% extracellular polysaccharide TDG3 (referred to as the HG group). The size of the mouse wounds was recorded and counted every other day. The mice were killed after recording the data on the 10th day.

[0090] 2. Data Analysis

[0091] One-way ANOVA was used for statistical analysis of multiple groups, and P < 0.05 was considered significant.

[0092] 3. Results Analysis

[0093] Combine Figure 9 It can be seen that: on the 10th day, the wounds of the mice in the TDG3-treated group were basically healed, while the wounds of the mice in the control group had not yet healed, indicating that the TDG3-treated group significantly accelerated the wound healing of the mice. Figure 8 B) also confirms this conclusion.

[0094] Example 5

[0095] Effects of exopolysaccharide TDG3 on keratinocytes HaCaT and fibroblasts NIH3T3

[0096] 1. Cell Culture

[0097] Collect logarithmic phase cells, wash the cells 2-3 times with calcium-free and magnesium-free PBS, and pipette into a uniform cell suspension with complete culture medium. After counting the cells, add them to a 6-well plate and inoculate 2 mL (3.5×10 5 / mL) and placed in a constant temperature incubator for further culture.

[0098] 2. Experimental Grouping

[0099] Co-culture system: After HaCaT cells adhered for 24 hours, the extracellular polysaccharide TDG3 (100 and 200 μg / mL) was added for 3 hours. The HaCaT cell supernatant was then collected and co-cultured with NIH3T3 cells for 24 hours. The cells were divided into four groups, each with six replicates. The control group received only PBS (referred to as the PBS group); the positive control group received HaCaT cell supernatant and PBS (referred to as the PBS+Ha group); the low-dose TDG3 treatment group (referred to as the LG+Ha group) received TDG3 (100 μg / mL) and HaCaT cell supernatant; and the high-dose TDG3 treatment group (referred to as the HG+Ha group) received TDG3 (200 μg / mL) and HaCaT cell supernatant.

[0100] 3. Fluorescent Labeling of Extracellular Polysaccharide TDG3

[0101] Dissolve 10 mg of TDG3 in 100 mL of DMSO and add 3 drops of pyridine. Then, add 20 mg of FITC and 20 mg of dibutyltin dilaurate, mix, and react at 95°C for 2 hours. After the reaction, wash with ethanol to remove excess reagents. The labeled TDG3-FITC conjugate is dialyzed against a 10 kDa MW conjugate to remove unbound FITC. The dialyzed conjugate is freeze-dried for later use.

[0102] 4. Detection of the Binding of Extracellular Polysaccharide TDG3 to Cells

[0103] (1) Fluorescently labeled exopolysaccharide TDG3 was redissolved in PBS and sterilized by filtration through a 0.22 μm filter membrane;

[0104] (2) NIH3T3 cells and HaCaT cells were treated with labeled exopolysaccharide TDG3 (final concentration 100 μg / mL) and incubated in an incubator for 1 h. After incubation, the supernatant was discarded and the cells were washed three times with sterile PBS for 1 min each time.

[0105] (3) Fix the cells with paraformaldehyde fixative at room temperature for 20 min, then wash three times with PBS for 1 min each time;

[0106] (4) Permeabilize with 0.5% (v / v) PBS-Tritonx100 (PBST) at room temperature for 20 min (Tritonx100 is diluted to 0.5% with PBS).

[0107] (5) Wash the cells three times with sterile PBS, then counterstain with 50 μL of DAPI staining solution and incubate in the dark for 5-10 min;

[0108] (4) Wash with PBS three times to remove excess dye, then add a small amount of PBS solution to infiltrate the cells to keep them in a moist environment. Finally, observe the binding of the exopolysaccharide TDG3 to the cells under a confocal fluorescence microscope.

[0109] 5. Gene Expression Analysis

[0110] The co-cultured NIH3T3 cells were collected, RNA was extracted, and reverse transcribed into cDNA. The expression of related genes such as MMP-3, Col1α1 and Col3α1 was detected by fluorescence quantitative PCR.

[0111] 6. Data Analysis

[0112] One-way ANOVA was used for statistical analysis of multiple groups, and P < 0.05 was considered significant.

[0113] 7. Results Analysis

[0114] like Figure 10 The results show that when the successfully labeled FITC-TDG3 conjugate was incubated with HaCaT cells and NIH3T3 cells, respectively, and observed and photographed under a confocal fluorescence microscope, the fluorescently labeled TDG3 was surrounded by the HaCaT cell surface, while no surrounding fluorescence was observed around the NIH3T3 cells, indicating that the exopolysaccharide TDG3 likely exerts its effect by specifically binding to receptors on the surface of keratinocytes.

[0115] like Figure 11 It can be seen that compared with the control group, the mRNA level of MMP-3 in the group to which only HaCaT cell supernatant was added increased slightly, but there was no significance. After treatment with the extracellular polysaccharide TDG3, the mRNA level of MMP-3 was significantly reduced, indicating that the extracellular polysaccharide TDG3 reduced the degradation of collagen. Similarly, compared with the control group, the mRNA levels of Col1α1 and Col3α1 in the group to which only HaCaT cell supernatant was added increased slightly, but there was no significance. After treatment with the extracellular polysaccharide TDG3, the collagen gene levels (Col1α1 and Col3α1) increased significantly and were dose-dependent. These results indicate that the extracellular polysaccharide TDG3 can stimulate the skin HaCaT cells to secrete some immune or growth factors, thereby promoting fibroblasts to synthesize more collagen.

[0116] In summary, the exopolysaccharide TDG3 can specifically bind to receptors on the keratinocyte membrane, thereby secreting cytokines and regulating the proliferation of fibroblasts and collagen synthesis.

Claims

1. Use of extracellular polysaccharide TDG3 in the preparation of a product that promotes skin collagen synthesis, characterized in that: The extracellular polysaccharide TDG3 is composed of glucose, mannose and galactose in a molar ratio of 3:2:1, and the connection mode of its glycosidic bonds is: 4) -β-D-Glc p - (1→4)[α-D-Man p - (1→2)]-β-D-Man p - (1→4)-β-D-Glc p - (1→3)-β-D-Gal p - (1→4)-β-D-Glc p -(1→, its structural formula is: , where n=100~100000.

2. The use according to claim 1, characterized in that: The extracellular polysaccharide TDG3 is prepared from the Dugong bacteria ( Duganella sp.) G3 produced.

3. The use according to claim 2, characterized in that: The extracellular polysaccharide TDG3 is prepared by the following steps: The fermentation broth of D. dulcis G3 was mixed with twice the volume of pure water, and NaOH was added to make the final concentration 0.1%~0.2%. The mixture was boiled for 10~20 min, and then the precipitate was filtered through a 1 μm filter membrane. Finally, 0.1%~0.3% sodium acetate and 3 times the volume of ethanol were added and stirred evenly. The precipitate was collected by centrifugation and dried to obtain the pure extracellular polysaccharide TDG3.

4. The use according to claim 1, characterized in that: The product is a composition or a product.

5. The use according to claim 4, characterized in that: The composition is a skin care product or a pharmaceutical composition; the product is a medical dressing, a medical device, a biomaterial or a tissue engineering product.

6. The use according to claim 5, characterized in that: The pharmaceutical composition can be administered by topical application or subcutaneous injection.

7. The use according to claim 5, characterized in that: The administration object of the pharmaceutical composition is human or non-human animal.

8. The use according to claim 5, characterized in that In the pharmaceutical composition, the administration concentration of the extracellular polysaccharide TDG3 is 0.5 mg / mL~20 mg / mL, and the dosage is 0.5 mg / kg~2 g / kg.

9. The use according to claim 5, characterized in that: The pharmaceutical carrier used in the pharmaceutical composition is an isotonic physiological saline solution, an isotonic glucose solution, or an isotonic solution containing a buffer system.

10. The use according to claim 9, characterized in that: The solution containing the buffer system is a PBS buffer solution.

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