Preparation method of red card wall skeleton polysaccharide, product and application thereof

Abstract

The application provides a preparation method of red card wall skeleton polysaccharide, a product and application thereof, and comprises the following steps: after fermentation of red Nocardia, the bacterial cells are subjected to impurity removal and degreasing treatment, and a step-by-step enzymatic hydrolysis method is used to extract the cell wall skeleton polysaccharide; then the red card wall skeleton polysaccharide is further separated and purified through a DEAE-52 cellulose anion exchange column or a dextran Sephadex G-15 and ultrafiltration; finally, HPLC, infrared spectroscopy and nuclear magnetic resonance hydrogen spectrum are used to perform structure characterization on the obtained red card wall skeleton polysaccharide. The red card wall skeleton polysaccharide prepared by the application has a complete structure, is low-toxic and safe, can prevent damage of a skin barrier, promote a repair process of the skin barrier damage, and has a good anti-allergic effect on sensitive skin with abnormal skin barrier function.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biotechnology, in particular to a preparation method of red karst skeleton polysaccharide and its product and application.

BACKGROUND

[0002] Among numerous natural products, polysaccharides are known for their complex structures, and their separation and purification are relatively difficult, and the in vivo metabolic evaluation technical system is lacking, which brings great challenges to the research and development of polysaccharide drugs. Polysaccharide compounds have specific monosaccharide composition and polyanion characteristics of glycosaminoglycans, and can bind to various functional proteins in the body, showing unique biological activities in antiviral, anticoagulation, antithrombosis, antitumor, hypoglycemic, etc. and have attracted widespread attention of researchers. In 2019, "Mannan oligosaccharide diacid" (GV-971) was listed, becoming the first specific drug for treating Alzheimer's disease, making polysaccharide innovative drug research and development with multiple pathways and multiple targets a research hotspot.

[0003] In the past decade, with the development of separation and purification, composition analysis and structure determination technology of polysaccharides and glycoconjugates, more and more sugar chains on glycoconjugates are found to participate in many life processes in living organisms, such as cell recognition, adhesion and fusion, signal transduction, cell differentiation, immune regulation and response, etc. Life phenomena cannot be separated from the participation of sugar chains. It is worth noting that different natural polysaccharide components show different biological activities due to the difference in monosaccharide composition and connection mode. Marudhupandi, T. et al. found that the antioxidant activity of brown algal polysaccharides depends on their structural properties, such as sulfate groups, molecular weight, monosaccharide composition, and stereochemical configuration, etc. Kirsten et al. separated the crude polysaccharide of Typha angustata by ion exchange chromatography and gel filtration into TL1-4 components, and the further purified TL1-TL3 components could significantly stimulate keratinocyte proliferation, thereby triggering keratinocytes into the initial differentiation state; TL4 could not stimulate keratinocyte proliferation, but could directly induce keratinocyte differentiation.

[0004] Nocardia rubra cell wall skeleton (N-CWS, referred to as red card wall skeleton) is a new type of immunomodulator, which is refined from the cell wall skeleton of Nocardia rubra prepared by fermentation, cell disruption, enzyme treatment and solvent extraction, and mainly contains polysaccharide, peptidoglycan, nocardic acid and mucin, etc. It is a new drug of national biological products of class II. In 1974 and 1976, Azuma et al. reported the anti-cancer immune adjuvant activity of red card wall skeleton. The main mechanism is not only to increase the levels of IL-1, IL-2, IL-6, IL-10, TNF, IFN (αβγ) and other cytokines, but also to promote the killing activity of macrophages, killer T cells, LAK cells and NK cells. It can enhance the activity of T cells, macrophages and natural killer cells in vivo, promote the production of cytokines, and has the characteristics of strong immune activity and small side effects. Pharmacological experiments show that red card wall skeleton has inhibitory effect on various tumors and inflammation, is effective for the treatment of chronic atrophic gastritis, and has significant inhibitory effect on hepatitis B virus. In particular, red card wall skeleton can effectively treat cervical diseases. Through the combination of TLRs on the surface of cervical epidermal immune cells-Langerhans cells, the ability of non-specific immunity, effector cells macrophages to recognize, kill, phagocytize and degrade HPV subtype viruses is enhanced; thereby further activating antigen-presenting Langerhans cells, promoting the proliferation and differentiation of cells, promoting the secretion of cytokines, and enhancing the ability of cytotoxic T cells to kill cells infected by HPV subtype viruses; rapidly eliminating local inflammation, allowing damaged tissues to repair and heal, thereby effectively eliminating HPV infection, reversing precancerous lesions of cervical cancer, and preventing and treating cervical-related diseases.

[0005] Because red card wall skeleton is a complex mixture, its effective components are not exact, which affects the subsequent study of its mechanism of action and target. Early studies have shown that red card wall skeleton is a mixture composed of polysaccharide, peptidoglycan and lipid, and the soluble peptidoglycan has the effect of promoting B cell mitosis, but there is no research report on the biological activity of other components. Studies have shown that in some Nocardia, Mycobacterium and Rhodococcus, cell wall polysaccharide has important immunological activity, and it is reasonable to speculate that the polysaccharide in the card wall skeleton also has important biological activity. However, so far, there has been no report on the structure and activity of red card wall skeleton polysaccharide.

[0006] Therefore, it is urgent for practitioners to study the chemical properties and activity of red card wall skeleton polysaccharide. Therefore, the applicant studies the enzyme-extracted high-purity red card wall skeleton polysaccharide, further characterizes the structure by organic spectrum, and uses the red card wall skeleton polysaccharide to prevent the damage of skin barrier and promote the repair of skin barrier damage. SUMMARY

[0007] The technical problem solved by the present application is to provide a preparation method of red card wall skeleton polysaccharide, products and applications thereof, which obtains new red card wall skeleton polysaccharide with complete structure, high purity, low toxicity and safety, and can be used for promoting the repair of skin barrier and has good anti-allergic effect on sensitive skin with abnormal skin barrier function.

[0008] The present application is implemented as follows:

[0009] A preparation method of red card wall skeleton polysaccharide, the operation steps of the preparation method are as follows:

[0010] (1) Red card bacteria body treatment: after the fermentation of red nocardia is completed, the fermentation broth is filtered, the bacteria body is taken, an equal volume of deionized water is added and stirred, and the bacteria body is collected after the residual medium impurities in the fermentation broth are removed by filtration and washing; the bacteria body is soaked in ethanol, stirred for a period of time, and the liposoluble impurities contained in the bacteria body are removed by filtration, and the bacteria body is collected after air drying;

[0011] (3) Red card wall skeleton polysaccharide extraction: glycosidase and protease are used to extract the red card wall skeleton polysaccharide: deionized water is added according to 10-20% (W / V) of the bacteria body, heated to 70-80°C, fully stirred and mixed for a certain period of time, cooled to room temperature, the pH value of the solution is adjusted, glycosidase is added, the enzyme amount and enzyme hydrolysis temperature are controlled, and after a certain period of time of enzyme hydrolysis reaction, the temperature is increased to 95°C for 30 minutes; after cooling to room temperature, the pH value of the solution is adjusted, protease is added, the enzyme amount and enzyme hydrolysis temperature are controlled, and after a certain period of time of enzyme hydrolysis reaction, the temperature is increased to 95°C for 30 minutes, and after cooling to room temperature; finally, the red card wall skeleton polysaccharide is collected and concentrated;

[0012] (3) Red card wall skeleton polysaccharide purification: the red card wall skeleton polysaccharide obtained in step (2) is purified by DEAE-52 cellulose anion exchange column or dextran Sephadex G-15, the mobile phase is deionized water, the elution speed is 0.2 mL / min-2 mL / min, the eluate is collected, and then concentrated, vacuum freeze-dried; the freeze-dried sample is dissolved in deionized water, an ultrafiltration tube with a molecular weight cut-off of 3000 Da is used, centrifugal ultrafiltration is performed at a speed of 5000 rpm for 30 min, distilled water is added to the upper layer of the ultrafiltration, the above ultrafiltration operation is repeated 2-5 times, the sample in the upper layer of the ultrafiltration is collected, and freeze-dried to obtain white and uniform red card wall skeleton polysaccharide solid.

[0013] Further, in step (1), the bacteria body is soaked in an equal volume of 95% ethanol for 2 hours.

[0014] Further, in step (2), acetic acid or ammonia water is used to adjust the optimum pH of each enzyme, and the pH range is 4.0-8.0.

[0015] Further, in the step (2), the glycosidase is any one or complex of cellulase, hemicellulase, mannanase and pectinase; and the protease is any one or complex of papain, neutral protease, trypsin and acid protease.

[0016] Further, in the step (2), the glycosidase is any one or complex of cellulase, hemicellulase, mannanase and pectinase; and the protease is any one or complex of papain, neutral protease, trypsin and acid protease.

[0017] Further, in the step (2), the glycosidase and protease are added in an amount of 1.0-5.0% of the mass of the bacteria.

[0018] Further, in the step (2), the enzymolysis temperature of the glycosidase and protease is 35.0-60.0℃, and the enzymolysis reaction time of the glycosidase and protease is 2.0-12.0 hours.

[0019] Further, a red-kangaroo cell wall skeleton polysaccharide is prepared based on the preparation method of the red-kangaroo cell wall skeleton polysaccharide, the average molecular weight distribution of the red-kangaroo cell wall skeleton polysaccharide solid is 1-50KD, the monosaccharide contains mannose, arabinose, galactose, glucose and N-acetylglucosamine, the main connection mode of the monosaccharide is →2,6)-α-D-Manp-(1→6)-α-D-Manp-(1→, the structure contains a branch structure.

[0020] Further, the average molecular weight distribution of the red-kangaroo cell wall skeleton polysaccharide solid is 4-10KD, the main chain part of the red-kangaroo cell wall skeleton polysaccharide contains the connection mode of →2,6)-α-D-Manp-(1→6)-α-D-Manp-(1→ glycosidic bond, a branch structure is connected at the C-2 position, and 1,5-Ara(f) connected terminal t-Ara(f) constitutes a branched part, and 1,6-Gal(p), t-Gal(p) and α-D-GlcpNAC-(1→ are connected in the peripheral part of the whole sugar ring structure.

[0021] Further, the red-kangaroo cell wall skeleton polysaccharide prepared based on the preparation method of the red-kangaroo cell wall skeleton polysaccharide can be used for preventing and repairing human epidermal keratinocyte aging and collagen degradation.

[0022] The present application has the following advantages:

[0023] 1) The present application utilizes the high specificity and relatively mild characteristics of enzymes, and the red-kangaroo cell wall is destroyed or degraded by enzymolysis to release the cell wall polysaccharide, so that the extraction rate of the red-kangaroo cell wall skeleton polysaccharide can be greatly improved.

[0024] 2) The present application has simple operation, mild conditions, and prepared red card wall skeleton polysaccharide with high purity, complete structure, low toxicity and safety, which can be used for preventing and repairing human epidermal keratinocyte aging and collagen degradation, preventing damage to the skin barrier, promoting the repair process of the skin barrier damage, and having good anti-sensitivity effect on sensitive skin with abnormal skin barrier function. BRIEF DESCRIPTION OF DRAWINGS

[0025] The present application will be further described below with reference to the accompanying drawings and examples.

[0026] Figure 1 is the structure of the red card wall skeleton polysaccharide of Example 1 in the present application.

[0027] Figure 2 is the absolute molecular weight analysis diagram of the red card wall skeleton polysaccharide of Example 2 in the present application.

[0028] Figure 3 is the monosaccharide component HPLC spectrum analysis of the red card wall skeleton polysaccharide of Example 3 in the present application.

[0029] Figure 4 is the infrared spectrum of the red card wall skeleton polysaccharide of Example 4 in the present application.

[0030] Figure 5 is the total ion current diagram of the methylation gas chromatography mass spectrometry analysis of the red card wall skeleton polysaccharide of Example 5 in the present application.

[0031] Figure 6 is the red card wall skeleton polysaccharide of Example 6 in the present application 1 H NMR spectrum.

[0032] Figure 7 is the C NMR spectrum of the red card wall skeleton polysaccharide of Example 6 in the present application 13

[0033] Figure 8 is the H- of the red card wall skeleton polysaccharide of Example 6 in the present application 1 13 C HMQC spectrum.

[0034] Figure 9 is a schematic diagram showing the effect of the red card wall skeleton polysaccharide of Example 7 in the present application on the activity of H2O2 damaged HaCaT cells.

DETAILED DESCRIPTION

[0035] The present application relates to a preparation method of a red card wall skeleton polysaccharide, and the operation steps of the preparation method are as follows:

[0036] ​​(1) Red card bacteria body processing: after the fermentation of red nocardia is finished, the fermentation liquid is filtered, the bacteria body is taken, an equal volume of deionized water is added and stirred, filtered and washed to remove the residual culture medium impurities in the fermentation liquid, and the bacteria body is collected; the bacteria body is soaked in ethanol, stirred for a period of time, filtered to remove the liposoluble impurities contained in the bacteria body, air dried, and the bacteria body is collected;

[0037] (4) Red card wall skeleton polysaccharide extraction: glycosidase and protease are used to extract the red card wall skeleton polysaccharide: deionized water is added according to 10-20% (W / V) of the bacteria body, heated to 70-80°C, fully stirred and mixed for a certain period of time, cooled to room temperature, the pH value of the solution is adjusted, glycosidase is added, the enzyme amount and enzyme hydrolysis temperature are controlled, after a certain period of time of enzyme hydrolysis reaction, the temperature is increased to 95°C for 30 minutes; after cooling to room temperature, the pH value of the solution is adjusted, protease is added, the enzyme amount and enzyme hydrolysis temperature are controlled, after a certain period of time of enzyme hydrolysis reaction, the temperature is increased to 95°C for 30 minutes, and after cooling to room temperature; finally, the red card wall skeleton polysaccharide is collected and concentrated;

[0038] (3) Red card wall skeleton polysaccharide purification: the red card wall skeleton polysaccharide obtained in step (2) is purified by DEAE-52 cellulose anion exchange column or dextran Sephadex G-15, the mobile phase is deionized water, the elution speed is 0.2 mL / min-2 mL / min, the eluate is collected, then concentrated, vacuum freeze-dried; the above elution freeze-dried sample is dissolved with deionized water, an ultrafiltration tube with a molecular weight cut-off of 3000 Da is used, centrifugal ultrafiltration is carried out at a speed of 5000 rpm for 30 min, distilled water is added to the upper layer of the ultrafiltration, the above ultrafiltration operation is repeated 2-5 times, the sample in the upper layer of the ultrafiltration is collected, and freeze-dried to obtain white and uniform red card wall skeleton polysaccharide solid.

[0039] Preferably, in step (1), the bacteria body is soaked in an equal volume of 95% ethanol, and stirred for 2 hours.

[0040] Preferably, in step (2), acetic acid or ammonia water is used to adjust the optimum pH of each enzyme, and the pH range is 4.0-8.0.

[0041] Preferably, in step (2), the glycosidase is any one or a combination of cellulase, hemicellulase, mannanase and pectinase; and the protease is any one or a combination of papain, neutral protease, trypsin and acid protease.

[0042] Preferably, in step (2), the glycosidase is any one or a combination of cellulase and mannanase; and the protease is any one or a combination of neutral protease and trypsin.

[0043] Preferably, in step (2), the addition amount of the glycosidase and the protease is 1.0-5.0% of the mass of the bacteria body, respectively.

[0044] Preferably, in the step (2), the enzymolysis temperature of the glycosidase and the protease is 35.0-60.0℃; and the enzymolysis reaction time of the glycosidase and the protease is 2.0-12.0 hours.

[0045] The present application also relates to a Hongca wall skeleton polysaccharide, which is prepared by the method for preparing a Hongca wall skeleton polysaccharide described above, and has an average molecular weight distribution of 1-50KD; contains mannose, arabinose, galactose, glucose and N-acetylglucosamine; and has a main connection mode of →2,6)-α-D-Manp-(1→6)-α-D-Manp-(1→ and a branched structure in the structure.

[0046] Preferably, the Hongca wall skeleton polysaccharide has an average molecular weight distribution of 4-10KD; contains the main chain part of the Hongca wall skeleton polysaccharide in the connection mode of →2,6)-α-D-Manp-(1→6)-α-D-Manp-(1→ glycosidic bond, a branched structure at the C-2 position, and a branched part of 1,5-Ara(f) connected with the terminal t-Ara(f); and has 1,6-Gal(p), t-Gal(p) and α-D-GlcpNAC-(1→ connected with the peripheral part of the whole sugar ring structure.

[0047] The present application also relates to the application of a Hongca wall skeleton polysaccharide, which is prepared by the method for preparing a Hongca wall skeleton polysaccharide described above, and can be used for preventing and repairing human epidermal keratinocyte aging and collagen degradation.

[0048] The technical solutions of the present application will be described clearly and completely in combination with the accompanying drawings and specific embodiments. Figures 1-9 The technical solutions of the present application will be described clearly and completely in combination with the accompanying drawings and specific embodiments.

[0049] It should be noted that the content and extraction rate of the Hongca wall skeleton polysaccharide are detected by the sulfuric acid anthrone colorimetry, the molecular weight of the Hongca wall skeleton polysaccharide is detected by the gel chromatography-differential-multiple angle laser light scattering system, the monosaccharide composition and proportion are detected by the PMP derivative high performance liquid chromatography, and the structure is analyzed by the infrared spectrum and nuclear magnetic spectrum, so that the effect of the enzyme extraction Hongca wall skeleton polysaccharide and the product quality characteristics are fully understood.

[0050] I. Preparation method of Hongka wall skeleton polysaccharide

[0051] Example 1, Preparation of Hongka wall skeleton polysaccharide

[0052] After the fermentation of Hongka is completed, the fermentation broth is filtered, and the bacterial cells are added with deionized water for stirring, filtration and washing, which is repeated twice to collect the bacterial cells. The bacterial cells are soaked with an equal volume of 95% ethanol for 2 hours of stirring, and then filtered to remove the lipid-soluble impurities contained in the bacterial cells, which is repeated twice and air-dried to collect the bacterial cells. The bacterial cells are added with deionized water at 10% (W / V), and then stirred and mixed uniformly at 70°C for a certain period of time, and then cooled to room temperature. The pH of the solution is adjusted to 5.0, and then 3.0% cellulase and 4.0% mannanase are added. The enzymolysis temperature is 50°C, and the enzymolysis reaction is performed for 60 minutes. Then, the temperature is increased to 95°C for 30 minutes of reaction. After cooling to room temperature, the pH of the solution is adjusted to 7.2, and then 4% neutral protease is added for reaction at 35°C for 24 hours, and then the temperature is increased to 95°C for 30 minutes of reaction. After cooling to room temperature, the solution is filtered to collect the clear liquid, which is concentrated to obtain the crude Hongka wall skeleton polysaccharide. The crude polysaccharide is purified by DEAE-52 cellulose anion exchange column or Sephadex G-15, and the mobile phase is deionized water with an elution speed of 0.5 mL / min. The eluate is collected, concentrated, and vacuum freeze-dried. The freeze-dried sample is dissolved in deionized water, and then centrifuged at a speed of 5000 rpm for 30 minutes by using an ultrafiltration tube with a molecular weight cut-off of 3000 Da. Distilled water is added to the upper layer of the ultrafiltration, and the above ultrafiltration operation is repeated for 3 times. The sample in the upper layer of the ultrafiltration is collected and freeze-dried to obtain white and uniform Hongka wall skeleton polysaccharide solid, with an extraction rate of 16.5% and a total sugar content of 95.2%. The structure of the Hongka wall skeleton polysaccharide is shown in Figure 1 .

[0053] II. Structural characterization: The purity, molecular weight, composition ratio of monosaccharides, connection order between monosaccharides, type of glycosidic bond, configuration of anomeric carbon, and type and ratio of polysaccharide residues of the purified Hongka wall skeleton polysaccharide are studied by chemical and instrumental analysis methods.

[0054] Example 2, Determination of the molecular weight of Hongka wall skeleton polysaccharide

[0055] The chromatography system used was a gel chromatography-differential-multi-angle laser light scattering system, the differential detector was Optilab T-rEX (Wyatt technology, CA, USA), and the laser light scattering detector was DAWN HELEOS II (Wyatt technology, CA, USA). According to the properties of the compound, a gel exclusion chromatography column with a suitable molecular weight range was used (Ohpak SB-805HQ (300x8mm), column temperature 45℃, sample injection amount 100μL, mobile phase A (0.1M NaNO3). Flow rate 0.4ml / min, elution gradient: isocratic 100min. Chromatography data was processed using software ASTRA 6.1. The results are shown in the accompanying Figure 2 , the obtained sample Mn: number average molecular weight 6.56KD; Mw: weight average molecular weight 9.45KD.

[0056] Example 3, monosaccharide composition of Hongkabia skeleton polysaccharide

[0057] The monosaccharide composition and proportion of Hongkabia skeleton polysaccharide were determined by PMP pre-column derivatization high performance liquid chromatography (PMP-HPLC) method. Chromatography conditions: Agilent HPLC system, chromatography column kromasil C18 (4.6mmx150mm, 5μm), mobile phase was 82.0% PBS (0.05M, pH7.0) and 18.0% acetonitrile (v / v), flow rate was 1.0mL·min-1, sample injection amount was 10μL, and detection wavelength was 254nm.

[0058] Precisely take 2mg of polysaccharide sample, add 0.5ml of 2M trifluoroacetic acid, hydrolyze at 120℃ for 2 hours, add a small amount of methanol, and evaporate at 45℃ water bath until the trifluoroacetic acid is completely evaporated. Respectively 100ul prepared monosaccharide control and polysaccharide solution, add 100ul 0.5M PMP methanol solution and 100ul 0.3M NaOH solution, make the sample fully dissolved, heat in 70℃ constant temperature water bath for 30min. Take out, cool to room temperature, add 100ul 0.3M hydrochloric acid solution, vortex and mix well. Add 0.5mL chloroform, mix well and extract the remaining PMP reagent, suck off the chloroform layer, retain the water layer, repeat 2 times. Filter with 0.22μm filter membrane, dilute with appropriate amount of distilled water 4 times for detection, analyze according to the above polysaccharide liquid chromatography method, as shown in Figure 3 The results show that Hongkabia skeleton polysaccharide mainly contains mannose, and also contains arabinose, galactose, glucose and N-acetylglucosamine.

[0059] Example 4, infrared spectrum analysis

[0060] Take dry polysaccharide sample 2 mg, KBr tablet, infrared spectrum scanning in the range of 4000~400 cm-1, record the infrared spectrum chart, as shown in Figure 4 .

[0061] From the IR spectrum, it can be seen that the bletilla striata polysaccharide has the characteristic absorption peak of polysaccharide. The absorption peak at 3600~3200 cm-1(3388.26 cm-1) is the stretching vibration of O-H. There are two absorption peaks at 3000~2750 cm-1(2917.38 cm-1), indicating the symmetric and asymmetric stretching vibration of C-H on sugar-CH2. The absorption peak at 1685.58 cm-1 is attributed to the absorption peak of crystal water, the absorption peak at 1540 cm-1 is attributed to the stretching vibration absorption peak of C-O, the absorption peak at 1401.63 cm-1 is the bending vibration absorption peak of C-H, and the absorption peak at 1082.73 cm-1 is attributed to the stretching vibration of C-OH on α-type furan arabinose. There is absorption at 944.58 cm-1, indicating the existence of β-type sugar residue. 787.55 cm-1 is the characteristic absorption peak of pyran ring.

[0062] Example 5, polysaccharide methylation analysis

[0063] About 15 mg of acetylated (pyridine-acetic anhydride) treated sample was weighed and placed in a P2O5 dryer for 24 h, and then dissolved in 3 ml of anhydrous dimethyl sulfoxide at room temperature (ultrasonic dissolution, appropriate heating). 100 mg of NaOH was added, sealed, and stirring was continued for 30 min. About 1 ml of iodomethane was added dropwise in an ice bath within 0.5 h, and after the room temperature (30℃) was restored, the sealing was continued, and the stirring was continued for 30 min. A small amount of deionized water was added to terminate the reaction. The excess iodomethane was removed by distillation under reduced pressure at room temperature, and the product was placed in a dialysis bag for dialysis for 24 h, and then concentrated to dryness under reduced pressure.

[0064] An appropriate amount of completely methylated sample was taken and placed in an ampoule, 2 mol / L trifluoroacetic acid was added, and hydrolysis was carried out at 120℃ for 2 h. After cooling, methanol was added to remove excess trifluoroacetic acid by co-distillation several times. 2-3 ml of deionized water was added to dissolve the solid, and about 100 mg of NaBH4 was added at room temperature for reduction for 2 h. Acetic acid was added dropwise to neutralize to no bubbles, and a mixture of methanol / acetic acid (volume ratio 5:1) was added to co-distill for 3 times, and then methanol was added several times. The excess acetic acid was removed by concentration under reduced pressure. The obtained solid was dried in a 100℃ oven for 10 min, pyridine-acetic anhydride (1:1) 3 ml was added, and reaction was carried out at 100℃ for 100 min. Methyl alcohol was added, and excess acetic anhydride was removed by co-distillation several times. Extraction was carried out with chloroform-water system, the chloroform layer was recovered, anhydrous sodium sulfate was added to remove water, and after standing for 30 min, it was concentrated to dryness under reduced pressure. 0.5 ml of chloroform was added for dissolution, and after filtration through a 0.45 um organic filter membrane, GC-MS analysis was carried out.

[0065] GC-MS conditions: Chromatographic column: rtx-5ms column (30.0 mm x 0.25 mm, 0.25 μm); temperature program: 120 °C for 2 min, then raised to 250 °C at a rate of 5 °C / min, and held for 10 min; split injection, split ratio 3:1; injection volume 1 μL, injection port temperature 250 °C, ion source temperature 180 °C; ion source voltage 70 ev; interface temperature 200 °C.

[0066] Reference is made to Figure 5 According to the fragmentation rules of partially methylated sugar alcohol acetate derivatives, the primary and secondary fragments in the mass spectrum were assigned, and by analysis, the main components in the GC spectrum were 1,5,6-tri-O-acetyl-2,3,4-tri-O-methyl-D-mannitol (→6)-a-D-Manp-(1→), 1,4,5-tri-O-acetyl-2,3,6-tri-O-methyl-glucose (→4)-a-D-Glcp-(1→), 1,2,5,6-tetra-O-acetyl-3,4-di-O-methyl-D-mannitol (→2,6)-a-D-Manp-(1→), 1,5-di-O-acetyl-2-(acetylmethylamino)-2-deoxy-3,4,6-tri-O-methyl-D-glucitol (a-D-GlcpNAC-(1→), 1,4,5-tri-O-acetyl-2,3-di-O-methyl-arabinose (1,5-Ara(f)), 1,4-di-O-acetyl-2,3,5-tri-O-methyl-arabinose (t-Ara(f)), 1,4,5-tri-O-acetyl-l-2,3,6-tetra-O-methyl-galactose (1,4-Gal(p)), 1,5,6-tri-O-acetyl-2,3,4-tri-O-methyl-galactose (1,6-Gal(p)), 1,5-di-O-acetyl-2,3,4,6-tetra-O-methyl-galactose (t-Gal(p)), and 1,2,4,6-tetra-O-acetyl-3,5-di-O-methyl-D-mannitol (→2,6)-β-D-Galf-(1→). As shown in Table 1 below:

[0067] Table 1 Data of methylation analysis of Hongka wall skeleton polysaccharide

[0068]

[0069]

[0070] Example 6, analysis by nuclear magnetic resonance spectroscopy

[0071] The sample after freeze-drying was weighed 35 mg, dissolved in 1 ml D2O, centrifuged, and the precipitate was discarded. The liquid was freeze-dried, and the operation was repeated 4 times. The freeze-dried sample was dissolved in 0.5 ml D2O, 0.1 ml deuterated acetone was added, and the determination was carried out at 25°C.

[0072] The sample was further identified by one-dimensional nuclear magnetic resonance hydrogen spectrum (1H-NMR, spectrum as shown in Figure 6 Most of the hydrogen spectrum signals of the polysaccharide are in the range of δ3.0-5.5ppm, and the anomeric protons (H-1) usually resonate in the range of δ4.5-5.5ppm. A large number of proton resonance signals in the 1H-NMR spectrum of the sample are concentrated in the region of δ3.0-5.5ppm, and the signals are seriously overlapped. According to the HSQC spectrum (as shown in Figure 8 ) of the sample, 13 main coupling signals were found in the anomeric region, and the anomeric proton signals were δ5.40ppm, δ5.18ppm, δ5.09ppm, δ5.04ppm, δ5.05ppm, δ5.04ppm, δ5.04ppm, δ4.89ppm, δ5.03ppm, δ5.01ppm, δ5.05ppm, δ5.39ppm, δ5.02ppm, and the anomeric carbon signals were δ100.98ppm, δ110.45ppm, δ108.72ppm, δ109.03ppm, δ107.57ppm, δ103.58ppm, δ100.02ppm, δ100.71ppm, δ100.02ppm, δ98.07ppm, δ107.78ppm, δ100.98ppm, δ99.82ppm, indicating that there may be 13 kinds of monosaccharide residues. According to the COSY spectrum and HSQC spectrum of the sample, and combined with the methylation result of the sample, 10 kinds of monosaccharide residues were deduced, and according to the results, the corresponding 10 kinds of sugar residues were →4)-α-D-Glcp-(1→, α-L-Araf-(1→, →5)-α-L-Araf-(1→, →5)-α-L-Araf-(1→, →2,6)-β-D-Galf-(1→, α-D-Manp-(1→, →2,6)-α-D-Manp-(1→, →6)-α-D-Manp-(1→, →2)-α-D-Manp-(1→, α-D-GlcpNAC-(1→, →6)-β-D-Galf-(1→, →4,6)-α-D-Glcp-(1→, α-D-Glcp-(1→. Combined with the 13 CNMR, 1 H- 13 C HMQC spectrum (as shown in Figures 7-8 ), the main C-H correlation signals on the sugar ring can be given. According to the 1 H- 13C HMQC spectrum combined with two-dimensional 1 H- 1 HCOSY spectrum Figure 1 Generally, all carbon and hydrogen signals in the sugar ring can be assigned.

[0073] The structural analysis results show that the main chain part of the red card wall skeleton polysaccharide contains the connection mode of →2, 6)-α-D-Manp-(1→6)-α-D-Manp-(1→ glycosidic bond, has a branch structure connected at the C-2 position, and 1, 5-Ara(f) connected terminal t-Ara(f) constitutes a branched part, and 1, 6-Gal(p), t-Gal(p) and α-D-GlcpNAC-(1→ are connected in the peripheral part of the whole sugar ring structure. Therefore, the structure of the red card wall skeleton polysaccharide described in the application is as shown in Figure 1 .

[0074] III. Skin barrier damage prevention and repair effect evaluation: protective effect of red card wall skeleton polysaccharide on H2O2-induced human epidermal keratinocyte aging and collagen degradation.

[0075] Example 7, in vitro experiment to investigate the protective effect of red card wall skeleton polysaccharide on H2O2-induced HaCaT cell oxidative damage

[0076] Experimental method: human epidermal keratinocyte HaCaT cell strain was cultured with 10% FBS DMEM medium, normal control group, H2O2 treatment group, and H2O2+red card wall polysaccharide (different concentrations) experimental group were set up, MTT was used to detect cell viability, as shown in Figure 9 , it can be known from Figure 9 that the red card wall polysaccharide can promote the growth of human epidermal keratinocytes; the changes of epidermal keratinocyte aging markers β-galactosidase and collagen degradation enzyme MMP-1 were observed by cell immunohistochemical method, as shown in Table 2 below.

[0077] Table 2 Changes of β-galactosidase and MMP-1 (*P<0.05)

[0078] Experimental Groups Beta-galactosidase (%) MMP-1 (%) Control Group 8.34±1.08 7.45±1.24 [H2O2 group] 62.38±8.56* 56.72±6.94** H202 + polysaccharide 25.42±5.23** 22.28±3.46*

[0079] It can be known from Table 2 that the above-mentioned in vitro test proves that the red card wall skeleton polysaccharide has obvious protective effect on H2O2-induced human epidermal keratinocyte aging and collagen degradation.

[0080] In summary, the present application has the following advantages:

[0081] 1) The present application utilizes the high specificity and relatively mild characteristics of enzymes to destroy or degrade the red card cell wall through enzymatic hydrolysis, releasing the cell wall polysaccharide, which can greatly improve the extraction rate of red card wall skeleton polysaccharide (up to 16.5%). 2) The present application is simple to operate, the conditions are mild, the prepared red card wall skeleton polysaccharide has high purity (total sugar content 95.2%), complete structure, low toxicity and safety, and can be used for preventing and repairing human epidermal keratinocyte aging and collagen degradation, can prevent the damage of skin barrier, promote the repair process of skin barrier damage, and has good anti-sensitivity effect on sensitive skin with abnormal skin barrier function.

[0082] Although the specific embodiments of the present application are described above, those skilled in the art should understand that the specific examples described are only illustrative, and are not intended to limit the scope of the present application, and equivalent modifications and changes made by those skilled in the art in accordance with the spirit of the present application should be covered within the scope of the claims of the present application.

Claims

1. A process for the preparation of a red card wall skeleton polysaccharide, characterized by: The preparation method operation steps are as follows: (1) Red card bacteria body treatment: after the fermentation of red nocardia is finished, the fermentation liquor is filtered, the bacteria body is taken, an equal volume of deionized water is added and stirred, filtered and washed to remove the residual culture medium impurities in the fermentation liquor, and the bacteria body is collected; the bacteria body is soaked in ethanol, stirred for a period of time, filtered to remove the liposoluble impurities contained in the bacteria body, air-dried, and the bacteria body is collected; (2) Red card wall skeleton polysaccharide extraction: the red card wall skeleton polysaccharide is extracted by using glycosidase and protease: according to 10-20% (W / V) of the bacteria body obtained in step (1), deionized water is added, heated to 70-80°C, fully stirred and mixed for a certain period of time, cooled to room temperature, the pH value of the solution is adjusted, glycosidase is added, the enzyme amount and enzyme hydrolysis temperature are controlled, after a certain period of time of enzyme hydrolysis reaction, it is heated to 95°C for 30 minutes; after cooling to room temperature, the pH value of the solution is adjusted, protease is added, the enzyme amount and enzyme hydrolysis temperature are controlled, after a certain period of time of enzyme hydrolysis reaction, it is heated to 95°C for 30 minutes, and cooled to room temperature; finally, the red card wall skeleton polysaccharide is filtered and collected for concentration; wherein the glycosidase is composed of cellulase and mannanase, and the protease is neutral protease; (3) Red card wall skeleton polysaccharide purification: the red card wall skeleton polysaccharide obtained in step (2) is purified by DEAE-52 cellulose anion exchange column or dextran Sephadex G-15, the mobile phase is deionized water, the elution speed is 0.2 mL / min-2 mL / min, the eluate is collected, then concentrated, vacuum freeze-dried to obtain the freeze-dried sample; the freeze-dried sample is dissolved in deionized water, an ultrafiltration tube with a molecular weight cutoff of 3000 Da is used, centrifugal ultrafiltration is carried out at a speed of 5000 rpm for 30 min, distilled water is added to the upper layer of the ultrafiltration, the above ultrafiltration operation is repeated 2-5 times, the sample in the upper layer of the ultrafiltration is collected, and freeze-dried to obtain white and uniform red card wall skeleton polysaccharide solid.

2. A process for the preparation of a red card wall skeletal polysaccharide according to claim 1, characterized by: In step (1), the bacteria body is soaked in an equal volume of 95% ethanol for 2 hours.

3. The method of claim 1, wherein the red card wall skeleton polysaccharide is prepared by the following steps: In step (2), acetic acid or ammonia water is used to adjust the optimum pH of each enzyme, and the pH range is 4.0-8.

0. ​ 4. The method of claim 1, wherein the red card wall skeleton polysaccharide is prepared by the following steps: In step (2), the addition amount of cellulase, mannanase and neutral protease is 1.0-5.0% of the mass of the bacteria body, respectively. ​ 5. The method of claim 1, wherein the red card wall skeleton polysaccharide is prepared by the following steps: In step (2), the enzyme hydrolysis temperature of the glycosidase and the protease is 35.0-60.0°C; the enzyme hydrolysis reaction time of the glycosidase is 60 minutes; and the enzyme hydrolysis reaction time of the protease is 24 hours. ​ 6. Use of a red cardex skeletal polysaccharide in the preparation of a product for preventing and repairing aging and collagen degradation of human epidermal keratinocytes, characterized in that: The red card wall skeleton polysaccharide is prepared based on the preparation method of the red card wall skeleton polysaccharide in any one of claims 1-5.

Images