CN116254308A - Preparation method of red-calorie wall skeleton polysaccharide, product and application thereof - Google Patents
Preparation method of red-calorie wall skeleton polysaccharide, product and application thereof Download PDFInfo
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- CN116254308A CN116254308A CN202310094860.5A CN202310094860A CN116254308A CN 116254308 A CN116254308 A CN 116254308A CN 202310094860 A CN202310094860 A CN 202310094860A CN 116254308 A CN116254308 A CN 116254308A
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- red
- polysaccharide
- wall skeleton
- calorie
- protease
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Classifications
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- C—CHEMISTRY; METALLURGY
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- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
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Abstract
The invention provides a preparation method of red-calorie wall skeleton polysaccharide, a product and application thereof, and the shrinkage comprises the following steps: removing impurities and degreasing the thallus after fermenting the nocardia rubra, and extracting the muramida polysaccharide by adopting a step enzymolysis method; further separating and purifying by using DEAE-52 cellulose anion exchange column or dextran Sephadex G-15 and ultrafiltration to obtain a new red-kamural skeleton polysaccharide; and finally, carrying out structural characterization on the obtained red-calorie wall skeleton polysaccharide by using HPLC, infrared spectrum and nuclear magnetic resonance hydrogen spectrum. The polysaccharide of the red-calorie wall skeleton prepared by the method has complete structure, low toxicity and safety, can prevent damage of skin barrier, promote the repair process of the damage of the skin barrier, and has good anti-sensitization effect on sensitive skin with abnormal skin barrier function.
Description
[ field of technology ]
The invention relates to the technical field of biology, in particular to a preparation method of red-calorie wall skeleton polysaccharide, a product and application thereof.
[ background Art ]
Among the many natural products, polysaccharides are known to have complex structures, and their separation and purification are relatively difficult, and the in vivo metabolism evaluation technology system is lacking, which brings great challenges to the development of polysaccharide drugs. The polysaccharide compound has the polyanion characteristics of monosaccharide composition specificity and glycosaminoglycan, can be combined with various functional proteins of organisms, has unique biological activities in the aspects of antivirus, anticoagulation, antithrombotic, antitumor, blood sugar reduction and the like, and is widely focused by researchers. In 2019, "mannooligosaccharide diacid" (GV-971) is marketed and becomes a specific drug for treating Alzheimer's disease for the first time, so that the research and development of multi-path and multi-target saccharide innovative drugs become research hot spots.
In recent 10 years, with the development of separation, purification, composition analysis and structure measurement technologies of polysaccharide and its sugar complex, sugar chains on the sugar compounds, especially the sugar conjugates, are increasingly found to participate in many life processes in living bodies, such as cell recognition, adhesion and fusion, signal transduction, cell differentiation, immune regulation and response, and the like, without participation of sugar chains. Notably, different natural polysaccharide components exhibit different biological activities due to differences in monosaccharide composition and linkage. Marudhupandi, T. Et al found that fucoidan's antioxidant activity depends on their structural properties such as sulfate group, molecular weight, monosaccharide composition, and steric configuration; kirsten et al split typha crude polysaccharide into TL1-4 components by ion exchange chromatography and gel filtration, and the further purified TL1-TL3 components can significantly stimulate keratinocyte proliferation, thereby triggering keratinocytes to enter an initial differentiation state; TL4 does not stimulate keratinocyte proliferation but directly induces keratinocyte differentiation.
The Nocardia rubra cell wall skeleton (Nocardia rubra cell wall skeleton, N-CWS, abbreviated as Nocardia rubra skeleton) is a novel immunomodulator, which is prepared by taking the cell wall skeleton prepared by Nocardia rubra (Nocardia rubra) through fermentation, cell disruption, enzyme treatment, solvent extraction and the like as a main raw material, and mainly contains polysaccharide, peptidoglycan, nocardioic acid, mucin and other components, and belongs to a class of novel medicines of domestic biological products. The anti-cancer immunoadjuvant activity of the red karma skeleton was reported successively by Azuma et al in 1974 and 1976. The main action mechanism is that the level of a plurality of cytokines of IL-1, IL-2, IL-6, IL-10, TNF and IFN (alpha beta gamma) can be improved, and the killing activity of macrophages, killer T cells, LAK cells and NK cells can be promoted. 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 immunocompetence and small side effect. Pharmacological experiments show that the red karya skeleton has an inhibiting effect on various tumors and inflammations, is effective in treating chronic atrophic gastritis, and has a remarkable inhibiting effect on hepatitis B virus. Especially, the red karwall skeleton can effectively treat cervical diseases, and through combining with the cervical epidermic immune cell-Langerhans cell surface TLRs, the capacity of nonspecific immunity, recognition, killing, phagocytosis and degradation of HPV subtype viruses by effector cell macrophages is enhanced; thereby further activating antigen presenting Langerhans cells, promoting proliferation and differentiation of the cells, promoting secretion of cytokines and enhancing the capability of cytotoxic T cells to kill cells infected by HPV subtype viruses; can rapidly eliminate local inflammation, repair and heal damaged tissues, thereby effectively eliminating HPV infection, reversing cervical precancerous lesions, and preventing and treating cervical related diseases.
As the red-card wall skeleton is a complex mixture, the effective components are not exact, and the subsequent research on the action mechanism and action targets of the red-card wall skeleton is affected. Early studies showed that: the red karya skeleton is a mixture of polysaccharide, peptidoglycan and lipoid, wherein the soluble peptidoglycan has the function of promoting B cell mitosis, but no research on the biological activity of other components is reported. Studies have shown that in certain nocardia, mycobacteria, rhodococcus, cell wall polysaccharides have important immunological activities, and it is reasonable to speculate that polysaccharides in the karwall backbone also have important biological activities. However, the structure and activity of red-kamural skeleton polysaccharide have not been reported so far.
In view of this, studies on the chemical properties and activity of red-kamural skeleton polysaccharides are highly desired by practitioners. Therefore, the applicant researches to obtain high-purity red-calorie wall skeleton polysaccharide through enzymolysis extraction, further performs structural characterization through organic spectrum, and uses the red-calorie wall skeleton polysaccharide to prevent damage of skin barrier and promote repair of the damage of the skin barrier.
[ invention ]
The invention aims to solve the technical problem of providing a preparation method of red-calorie wall skeleton polysaccharide, a product and application thereof, wherein the method is used for obtaining the novel red-calorie wall skeleton polysaccharide, has a complete structure, has high purity and low toxicity, is safe, can be used for promoting the repair of skin barriers, and has good anti-sensitization effect on sensitive skin with abnormal skin barrier functions.
The invention is realized in the following way:
the preparation method of the red-kamural skeleton polysaccharide comprises the following operation steps:
(1) Red card thallus treatment: filtering the fermentation liquor after the fermentation of nocardia rubra is finished, taking thalli, adding deionized water with the same volume, stirring, filtering and washing, removing residual culture medium impurities in the fermentation liquor, and collecting thalli; soaking thallus in ethanol, stirring for a period of time, filtering to remove liposoluble impurities contained in thallus, blow-drying with air, and collecting thallus;
(3) Extracting polysaccharide of red-card wall skeleton: extracting red-kamural skeleton polysaccharide by glycosidase and protease: adding deionized water according to 10-20% (W/V) of thalli, heating to 70-80 ℃, fully stirring and uniformly mixing for a certain time, cooling to room temperature, regulating the pH value of the solution, adding glycosidase, controlling the enzyme adding amount and the enzymolysis temperature, and heating to 95 ℃ for reaction for 30 minutes after enzymolysis reaction for a certain time; cooling to room temperature, regulating pH value of the solution, adding protease, controlling enzyme adding amount and enzymolysis temperature, performing enzymolysis for a certain time, heating to 95 ℃ for reaction for 30 minutes, and cooling to room temperature; finally, collecting polysaccharide of the red-card wall skeleton and concentrating;
(3) Purifying polysaccharide of red-card wall skeleton: purifying the red card wall skeleton polysaccharide obtained in the step (2) by using a DEAE-52 cellulose anion exchange column or dextran Sephadex G-15, wherein the mobile phase is deionized water, the elution speed is 0.2-2 mL/min, collecting the eluent, concentrating and freeze-drying in vacuum; dissolving the eluted freeze-dried sample with deionized water, centrifuging and ultrafiltering for 30min at 5000rpm by using an ultrafiltration tube with a cutoff molecular weight of 3000Da, adding distilled water into the ultrafiltered upper layer, repeating the ultrafiltration operation for 2-5 times, collecting the sample of the ultrafiltered upper layer, and freeze-drying to obtain white uniform red Kazuku bone frame polysaccharide solid.
Further, in the step (1), the thalli are soaked in 95% ethanol with equal volume and stirred for 2 hours.
Further, in the step (2), the optimum pH of each enzyme is adjusted with acetic acid or ammonia water, and the pH range is 4.0 to 8.0.
Further, in the step (2), the glycosidase is any one or a combination of cellulase, hemicellulase, mannanase and pectinase; the protease is any one or a combination of papain, neutral protease, trypsin and acid protease.
Further, in the step (2), the glycosidase is any one or a combination of cellulase and mannitose polymerase; the protease is any one or a combination of neutral protease and trypsin.
Further, in the step (2), the added amounts of the glycosidase and the protease are 1.0 to 5.0% by mass of the cells, respectively.
Further, in the step (2), the enzymolysis temperature of the glycosidase and the protease is 35.0-60.0 ℃; the enzymolysis reaction time of the glycosidase and the protease is 2.0-12.0 hours.
Further, the red-calorie wall skeleton polysaccharide is prepared based on the preparation method of the red-calorie wall skeleton polysaccharide, and the average molecular weight distribution of the red-calorie wall skeleton polysaccharide solids is 1-50 KD; the monosaccharide contains mannose, arabinose, galactose, glucose and N-acetylglucosamine; the monosaccharide connection mode is mainly 2, 6) -alpha-D-Manp- (1-and the structure contains a branch structure.
Further, the molecular weight of the red-calorie wall skeleton polysaccharide solid is evenly distributed to be 4-10 KD; the main chain part of the polysaccharide composing the red-kamural skeleton comprises a connection mode of 2, 6) -alpha-D-Manp- (1-glycosidic bond, a branch structure is connected at a C-2 position, a branched chain part is formed by a 1,5-Ara (f) connection terminal t-Ara (f), and the peripheral part of the whole sugar ring structure is connected with 1,6-Gal (p), t-Gal (p) and alpha-D-GlcpNAC- (1-).
Further, the application of the red-calorie wall skeleton polysaccharide is characterized in that the red-calorie wall skeleton polysaccharide is prepared based on the preparation method of the red-calorie wall skeleton polysaccharide, and the red-calorie wall skeleton polysaccharide can be used for preventing and repairing human epidermis keratinocyte aging and collagen degradation.
The invention has the following advantages:
1) The invention utilizes the high specificity and mild characteristics of enzyme, and the erythrocyte wall is destroyed or degraded by enzymolysis to release the mural polysaccharide, thus greatly improving the extraction rate of the erythrocyte wall skeleton polysaccharide.
2) The method is simple to operate, mild in condition, high in purity, complete in structure, low in toxicity and safe, can be used for preventing and repairing human epidermal keratinocyte aging and collagen degradation, can prevent damage of skin barriers, promotes repair process of the damage of the skin barriers, and has good anti-sensitization effect on sensitive skin with abnormal skin barrier functions.
[ description of the drawings ]
The invention will be further described with reference to examples of embodiments with reference to the accompanying drawings.
FIG. 1 shows the structure of the red-kamural skeleton polysaccharide of example 1 in the present invention.
FIG. 2 is a graph showing the absolute molecular weight analysis of the polysaccharide from Red Ka-wall skeleton of example 2 of the present invention.
FIG. 3 is an HPLC analysis of monosaccharide composition of the red-card wall skeleton polysaccharide of example 3 of the present invention.
FIG. 4 is an infrared spectrum of the red-kamural skeleton polysaccharide of example 4 of the present invention.
FIG. 5 is a total ion flow chart of the methylation gas chromatography mass spectrometry analysis of the red Ka-wall skeleton polysaccharide of example 5 of the present invention.
FIG. 6 is a polysaccharide of example 6 Red Ka-wall skeleton in the present invention 1 H NMR spectrum.
FIG. 7 is a polysaccharide of example 6 Red Ka-wall skeleton in the present invention 13 C NMR spectrum.
FIG. 8 is a polysaccharide of example 6 Red Ka-wall skeleton in the present invention 1 H- 13 C HMQC spectrum.
FIG. 9 is a polysaccharide pair H of Red Ka-wall skeleton in example 7 of the present invention 2 O 2 Schematic of the effect of injured HaCaT cell activity.
[ detailed description ] of the invention
The invention relates to a preparation method of red-calorie wall skeleton polysaccharide, which comprises the following operation steps:
(1) Red card thallus treatment: filtering the fermentation liquor after the fermentation of nocardia rubra is finished, taking thalli, adding deionized water with the same volume, stirring, filtering and washing, removing residual culture medium impurities in the fermentation liquor, and collecting thalli; soaking thallus in ethanol, stirring for a period of time, filtering to remove liposoluble impurities contained in thallus, blow-drying with air, and collecting thallus;
(4) Extracting polysaccharide of red-card wall skeleton: extracting red-kamural skeleton polysaccharide by glycosidase and protease: adding deionized water according to 10-20% (W/V) of thalli, heating to 70-80 ℃, fully stirring and uniformly mixing for a certain time, cooling to room temperature, regulating the pH value of the solution, adding glycosidase, controlling the enzyme adding amount and the enzymolysis temperature, and heating to 95 ℃ for reaction for 30 minutes after enzymolysis reaction for a certain time; cooling to room temperature, regulating pH value of the solution, adding protease, controlling enzyme adding amount and enzymolysis temperature, performing enzymolysis for a certain time, heating to 95 ℃ for reaction for 30 minutes, and cooling to room temperature; finally, collecting polysaccharide of the red-card wall skeleton and concentrating;
(3) Purifying polysaccharide of red-card wall skeleton: purifying the red card wall skeleton polysaccharide obtained in the step (2) by using a DEAE-52 cellulose anion exchange column or dextran Sephadex G-15, wherein the mobile phase is deionized water, the elution speed is 0.2-2 mL/min, collecting the eluent, concentrating and freeze-drying in vacuum; dissolving the eluted freeze-dried sample with deionized water, centrifuging and ultrafiltering for 30min at 5000rpm by using an ultrafiltration tube with a cutoff molecular weight of 3000Da, adding distilled water into the ultrafiltered upper layer, repeating the ultrafiltration operation for 2-5 times, collecting the sample of the ultrafiltered upper layer, and freeze-drying to obtain white uniform red Kazuku bone frame polysaccharide solid.
Preferably, in the step (1), the cells are soaked in an equal volume of 95% ethanol and stirred for 2 hours.
Preferably, in the step (2), the optimum pH of each enzyme is adjusted with acetic acid or ammonia water, and the pH is in the range of 4.0 to 8.0.
Preferably, in the step (2), the glycosidase is any one or a combination of cellulase, hemicellulase, mannanase and pectinase; the protease is any one or a combination of papain, neutral protease, trypsin and acid protease.
Preferably, in the step (2), the glycosidase is any one or a combination of cellulase and mannitose polymerase; the protease is any one or a combination of neutral protease and trypsin.
Preferably, in the step (2), the glycosidase and the protease are added in an amount of 1.0 to 5.0% by mass of the cells, respectively.
Preferably, in the step (2), the enzymolysis temperature of the glycosidase and the protease is 35.0-60.0 ℃; the enzymolysis reaction time of the glycosidase and the protease is 2.0-12.0 hours.
The invention also relates to the red-calorie wall skeleton polysaccharide, which is prepared based on the preparation method of the red-calorie wall skeleton polysaccharide, wherein the average molecular weight distribution of the red-calorie wall skeleton polysaccharide solid is 1-50 KD; the monosaccharide contains mannose, arabinose, galactose, glucose and N-acetylglucosamine; the monosaccharide connection mode is mainly 2, 6) -alpha-D-Manp- (1-and the structure contains a branch structure.
Preferably, the molecular weight of the red-calorie wall skeleton polysaccharide solid is evenly distributed to be 4-10 KD; the main chain part of the polysaccharide composing the red-kamural skeleton comprises a connection mode of 2, 6) -alpha-D-Manp- (1-glycosidic bond, a branch structure is connected at a C-2 position, a branched chain part is formed by a 1,5-Ara (f) connection terminal t-Ara (f), and the peripheral part of the whole sugar ring structure is connected with 1,6-Gal (p), t-Gal (p) and alpha-D-GlcpNAC- (1-).
The invention also relates to application of the red-calorie wall skeleton polysaccharide, the red-calorie wall skeleton polysaccharide is prepared based on the preparation method of the red-calorie wall skeleton polysaccharide, and the red-calorie wall skeleton polysaccharide can be used for preventing and repairing human epidermal keratinocyte aging and collagen degradation
The technical solutions of the present invention will be clearly and completely described below with reference to fig. 1 to 9 and the detailed description. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
The invention adopts the sulfuric acid anthrone colorimetric method to detect the content and the extraction rate of the red-kamural skeleton polysaccharide, adopts a gel chromatography-differential-multi-angle laser light scattering system to detect the molecular weight of the red-kamural skeleton polysaccharide, adopts a PMP derivative high performance liquid chromatography to detect the monosaccharide composition and the proportion, and combines an infrared spectrum and a nuclear magnetic spectrum to carry out structural analysis, thereby fully knowing the effect and the product quality characteristics of the red-kamural skeleton polysaccharide extracted by an enzymatic method.
1. Preparation method of red-kamural skeleton polysaccharide
Example 1 preparation of Red Ka wall skeleton polysaccharide
After the red card fermentation is finished, adding deionized water into the fermentation liquid after filtering, stirring, filtering and washing, repeating for 2 times, and collecting thalli; adding 95% ethanol to the thallus, soaking, stirring for 2 hr, filtering to remove liposoluble impurities contained in thallus, repeating for 2 times, air drying, and collecting thallus. Adding deionized water according to 10% (W/V) of thalli, heating to 70 ℃, fully stirring and uniformly mixing for a certain time, cooling to room temperature, adjusting the pH value of the solution to 5.0, adding 3.0% of cellulase and 4.0% of mannose polymerase, performing enzymolysis at 50 ℃ for 60 minutes, and then heating to 95 ℃ for 30 minutes; cooling to room temperature, regulating the pH value of the solution to 7.2, adding 4% neutral protease, reacting for 24 hours at 35 ℃, heating to 95 ℃ for reacting for 30 minutes, and cooling to room temperature; filtering, collecting clear liquid and concentrating to obtain crude polysaccharide with red karwall skeleton. Purifying the crude polysaccharide with DEAE-52 cellulose anion exchange column or dextran Sephadex G-15, eluting with deionized water at a rate of 0.5mL/min, collecting eluate, concentrating, and vacuum freeze drying; dissolving the eluted freeze-dried sample with deionized water, centrifuging and ultrafiltering for 30min at 5000rpm with an ultrafiltration tube with a cutoff molecular weight of 3000Da, adding distilled water into the ultrafiltered upper layer, repeating the ultrafiltration operation for 3 times, collecting sample of the ultrafiltered upper layer, and lyophilizing to obtain white uniform polysaccharide solid with red Kaka wall skeleton, extraction rate of 16.5%, and total sugar content of 95.2%. The structure of the red-calorie wall skeleton polysaccharide is shown in figure 1.
2. Structural characterization: the structure research is carried out on the purity, the molecular weight, the composition proportion of monosaccharide, the connection sequence among the monosaccharide, the type of glycosidic bond, the configuration of anomeric carbon, the type and proportion of polysaccharide residues and the like of the purified red-kamural skeleton polysaccharide by adopting chemical and instrumental analysis means.
Example 2 determination of molecular weight of Red Caliper skeleton polysaccharide
The chromatographic system used was a gel chromatography-differential-multi-angle laser light scattering system, the differential detector was Optilab T-rEX (Wyatttechnology, CA, USA), the laser light scattering detector was DAWNHELEOSII (Wyatttechnology, CA, USA), a gel exclusion chromatographic column (Ohpak SB-805HQ (300X 8 mm), column temperature 45 ℃ C., sample injection amount 100. Mu.L, mobile phase A (0.1M NANO 3) flow rate 0.4ml/min, elution gradient: isocratic 100min was used according to the nature of the compound, the mobile phase A (0.1M NANO 3) flow rate 0.4ml/min, elution gradient: isocratic 100min was processed using software ASTRA 6.1, FIG. 2, the results were shown in FIG. 2, the obtained samples Mn: number average molecular weight 6.56KD, mw: weight average molecular weight 9.45KD.
Example 3 polysaccharide monosaccharide composition of Red Ka wall skeleton
And determining the composition and the proportion of the polysaccharide monosaccharides of the red-card wall skeleton by adopting a PMP pre-column derivatization high performance liquid chromatography (PMP-HPLC) method. Chromatographic conditions: agilent HPLC system, column kromasil C18 (4.6 mm. Times.150 mm,5 μm), mobile phase 82.0% PBS (0.05M, pH 7.0) and 18.0% acetonitrile (v/v), flow rate 1.0mL min-1, sample injection amount 10. Mu.L, detection wavelength 254nm.
Precisely weighing polysaccharide sample 2mg, respectively adding 0.5ml 2M trifluoroacetic acid, hydrolyzing at 120deg.C for 2 hr, adding small amount of methanol, evaporating in water bath at 45deg.C, and repeating for 3-5 times until trifluoroacetic acid is completely distilled off. 100ul of the prepared monosaccharide control and polysaccharide solution are respectively added with 100ul of 0.5M PMP methanol solution and 100ul of 0.3M NaOH solution, so that the sample is fully dissolved, and the mixture is heated in a constant-temperature water bath at 70 ℃ for 30min. Taking out, cooling to room temperature, adding 100ul of 0.3M hydrochloric acid solution, and vortex shaking and mixing uniformly. 0.5mL of chloroform was added, and after mixing, the remaining PMP reagent was extracted, the chloroform layer was removed by suction, and the aqueous layer was retained and repeated 2 times. Filtering with 0.22 μm filter membrane, diluting with distilled water 4 times, and analyzing by the above polysaccharide liquid chromatography method, specifically shown in FIG. 3. The results show that the red-kamural polysaccharide contains mainly mannose, but also arabinose, galactose, glucose and N-acetylglucosamine.
Example 4 Infrared Spectrometry
Taking 2mg of dried polysaccharide sample, tabletting with KBr, carrying out infrared spectrum scanning in the range of 4000-400 cm < -1 >, and recording infrared spectrum, as shown in figure 4.
As can be seen from an IR spectrogram, the bletilla striata polysaccharide has a characteristic absorption peak of polysaccharide, and the absorption peak at a position (3388.26 cm < -1 >) of 3600-3200 cm < -1 > is the stretching vibration of O-H; 2 absorption peaks are arranged at a position (2917.38 cm < -1 >) of 3000-2750 cm < -1 >, which indicate symmetrical and antisymmetric stretching vibration of C-H on the saccharide-CH 2; the absorption peak of the crystal water is ascribed to 1685.58cm-1, the telescopic vibration absorption peak of the C-O is ascribed to 1540cm-1, the bending vibration absorption peak of the C-H is ascribed to 1401.63cm-1, and the telescopic vibration of the C-OH on the alpha-type arabinofuranose is ascribed to 1082.73 cm-1. Absorption at 944.58cm-1 indicates the presence of a beta-type sugar residue. 787.55cm-1 is the characteristic absorption peak of the pyran ring.
Example 5 methylation analysis of polysaccharide
About 15mg of the acetylated (pyridine-acetic anhydride) treated sample was weighed, dried in a P2O5 dryer for 24 hours, dissolved in 3ml of anhydrous dimethyl sulfoxide (ultrasonic dissolution, optionally heated) at room temperature, added with 100mg of NaOH, sealed, and stirred for 30 minutes. About 1ml of methyl iodide was added dropwise over 0.5h in an ice bath, and after returning to room temperature (30 ℃ C.), the reaction was stopped by sealing and continuing stirring for 30min and adding a small amount of deionized water. Excess methyl iodide was distilled off under reduced pressure at room temperature, and the product was dialyzed in a dialysis bag for 24 hours and concentrated to dryness under reduced pressure.
Taking a proper amount of completely methylated sample, placing the sample in an ampoule bottle, adding 2mol/L trifluoroacetic acid, hydrolyzing for 2 hours at 120 ℃, cooling, adding methanol, co-evaporating for multiple times, and removing excessive trifluoroacetic acid; adding 2-3ml deionized water to dissolve solid, adding NaBH4 to about 100mg at room temperature for reduction for 2h, dripping acetic acid to neutralize until no bubble, adding methanol/acetic acid (volume ratio of 5:1) mixed solution for 3 times, adding methanol for several times, and concentrating under reduced pressure to remove excessive acetic acid. The resulting solid was dried in an oven at 100deg.C for 10min, 3ml of pyridine-acetic anhydride (1:1) was added, reacted at 100deg.C for 100min, methanol was added, and the excess acetic anhydride was removed by co-evaporation multiple times. Extracting with chloroform-water system, recovering chloroform layer, adding anhydrous sodium sulfate for dewatering, standing for 30min, concentrating under reduced pressure to dry, adding 0.5ml chloroform for dissolving, filtering with 0.45um organic filter membrane, and performing GC-MS analysis.
GC-MS conditions: the chromatographic column was a rtx-5ms column (30.0mm. Times.0.25 mm,0.25 μm); and (3) a temperature programming process: maintaining at 120deg.C for 2min; raising the temperature to 250 ℃ at a speed of 5 ℃/min, and keeping for 10min; split sample introduction, wherein the split ratio is 3:1; the sample injection amount is 1 mu L, the temperature of a sample injection port is 250 ℃, and the temperature of a mass spectrum ion source is 180 ℃; ion source voltage 70ev; the interface temperature was 200 ℃.
Referring to FIG. 5, according to the cleavage rules of the partially methylated sugar alcohol acetate derivatives, the primary and secondary fragments in the mass spectrum are attributed to 1,5, 6-tri-O-acetyl-2, 3, 4-tri-O-methyl-D-mannitol (→6) - α -D-Manp- (1→), 1,4, 5-tri-oxo-acetyl-2, 3, 6-tri-oxo-methyl-glucose (→4) - α -D-Glcp- (1→), 1,2,5, 6-tetra-O-acetyl-3, 4-di-O-methyl-D-mannitol (→2, 6) - α -D-Manp- (1→), 1, 5-di-O-acetyl-2- (acetylamino) -2-deoxy-3, 4, 6-tri-O-methyl-D-glucitol (α -D-Glc- (1→4), 1, 5-tri-oxo-3, 4-di-O-methyl-D-mannitol (1→), 1,2, 6-tetra-O-acetyl-3, 4-di-O-methyl-D-mannitol (1→), and Arf (1, 5-tri-O-acetyl-2, 6-Manp- (1→4)), also 1,4, 5-tri-oxo-acetyl-l-2, 3, 6-tetra-oxo-methyl-galactose (1, 4-Gal (p)), 1,5, 6-tri-oxo-acetyl-2, 3, 4-tri-oxo-methyl-galactose (1, 6-Gal (p)), 1, 5-di-oxo-acetyl-2, 3,4, 6-tetra-oxo-methyl-galactose (t-Gal (p)) and 1,2,4, 6-tetra-O-acetyl-3, 5-di-O-methyl-D-mannitol (. Fwdarw.2, 6) - β -D-Galf- (1. Fwdarw.). As shown in table 1 below:
TABLE 1 Red Ka wall skeleton polysaccharide methylation analysis data
Example 6 Nuclear magnetic resonance Spectrometry analysis
35mg of the freeze-dried sample was weighed and 1ml of D was added 2 O was dissolved, centrifuged, the precipitate was discarded, and the liquid was freeze-dried, and the operation was repeated 4 times. The resulting lyophilized sample was subjected to 0.5ml D 2 O was dissolved, 0.1ml of deuterated acetone was added, and the mixture was measured at 25 ℃.
Further identifying glycosidic bond configuration of sample by one-dimensional nuclear magnetic resonance hydrogen spectrum (1H-NMR, spectrum is shown in figure 6), and hydrogen spectrum signal of polysaccharide is mostlyThe number is between delta 3.0 and 5.5ppm, and usually delta 4.5 and 5.5ppm is the resonance region of the hetero-head proton (H-1). The 1H-NMR spectrum of the sample was concentrated in the region of δ 03.0-5.5ppm with severe signal overlap, 13 major coupling signals were found in the region of the hetero-head according to the HSQC spectrum of the sample (FIG. 8), the hetero-head proton signals were δ15.40ppm, δ25.18ppm, δ35.09ppm, δ45.04ppm, δ 55.05ppm, δ 65.04ppm, δ 75.04ppm, δ 84.89ppm, δ 95.03ppm, δ5.01ppm, δ05.05ppm, δ15.39ppm, δ25.02ppm, the hetero-head carbon signals were δ 3100.98ppm, δ 4110.45ppm, δ 5108.72ppm, δ 6109.03ppm, δ 7107.57ppm, δ 8103.58ppm, δ100.02ppm, δ 100.71ppm, δ100.02ppm, δ98.07ppm, δ 107.78ppm, δ 100.98ppm, and δ99.82ppm, respectively, showing that 13 monosaccharide residues may be contained. The 10 monosaccharide residues were deduced from the sample COSY pattern and HSQC pattern, and the methylation results of the sample were combined, and the corresponding 10 saccharide residues were respectively → 4) - δ9-D-GlcP- (1 → δ0-L-Araf- (1 → 5) - δ1-L-Araf- (1 → 5) - δ2-L-Araf- (1 → 2, 6) - δ3-D-Galf- (1 → 4-D-Manp- (1 → 2, 6) - δ5-D-Manp- (1 → 6) - δ6-D-Manp- (1 → 2) - δ7-D-Manp- (1 → 8-D-GlcpNAC- (1 → 6) - δ9-D-Galf- (1 → 4, 6) - δ0-D-Manp- (1 → 2, 6) - δ5-D-Manp- (1 → 6) -GlcP- (1 → 1-GlcP 13 CNMR、 1 H- 13 The C HMQC spectrum (as shown in FIGS. 7-8) gives mainly a direct C-H-related signal on the sugar ring by 1 H- 13 C HMQC spectrogram recombination two-dimensional 1 H- 1 HCOSY spectra generally enable the assignment of all carbon and hydrogen signals in the sugar ring.
The results of structural analysis showed that the main chain portion constituting the polysaccharide of the red-calorie wall had a structure comprising → 2, 6) - α -D-Manp- (1 → glycosidic bond connection, a branched structure was attached at the C-2 position, and a 1,5-Ara (f) connection terminal t-Ara (f) constituted a branched part, and 1,6-Gal (p), t-Gal (p) and α -D-GlcpNAC- (1 → structure was attached at the peripheral portion of the whole sugar ring structure.
3. Evaluation of skin barrier injury prevention and repair effects: protection of H2O 2-induced human epidermal keratinocyte aging and collagen degradation by erythrocyte-wall scaffold polysaccharide.
Example 7 in vitro experiments discussing Red Ka wall skeleton polysaccharide vs H 2 O 2 Protection against oxidative damage induced in HaCaT cells
The experimental method comprises the following steps: culturing human epidermal keratinocyte HaCaT cell strain in DMEM medium containing 10% FBS to form normal control group and H 2 O 2 Treatment group, and H 2 O 2 In the +red kamuramyl polysaccharide (at various concentrations) experimental group, MTT was used to measure cell viability, as shown in FIG. 9, and as can be seen from FIG. 9: the red kamuramyl polysaccharide can promote the growth of human epidermal keratinocytes; changes in the epidermal keratinocyte senescence marker beta-galactosidase and collagen degrading enzyme MMP-1 were observed by cell immunohistochemistry as shown in Table 2 below.
Table 2 beta-galactosidase and MMP-1 changes (< 0.05P)
| Group of experiments | Beta-galactosidase (%) | MMP-1(％) |
| Control group | 8.34±1.08 | 7.45±1.24 |
| H 2 O 2 Group of | 62.38±8.56* | 56.72±6.94** |
| H 2 O 2 +polysaccharide group | 25.42±5.23** | 22.28±3.46* |
As can be seen from table 2: the in vitro test proves that the polysaccharide pair H of the red-card wall skeleton 2 O 2 The induced human epidermis keratinocyte aging and collagen degradation have obvious protective effect.
In summary, the invention has the following advantages:
1) The invention utilizes the high specificity and mild characteristic of enzyme, and the erythrocyte wall is destroyed or degraded by enzymolysis to release the mural polysaccharide, thus greatly improving the extraction rate (up to 16.5%) of the erythrocyte wall skeleton polysaccharide. 2) The method is simple to operate, mild in condition, high in purity (total sugar content is 95.2%), complete in structure, low in toxicity and safe, can be used for preventing and repairing human epidermal keratinocyte aging and collagen degradation, can prevent damage of skin barriers, can promote repair process of the damage of the skin barriers, and has good anti-sensitization effect on sensitive skin with abnormal skin barrier functions.
While specific embodiments of the invention have been described above, it will be appreciated by those skilled in the art that the specific embodiments described are illustrative only and not intended to limit the scope of the invention, and that equivalent modifications and variations of the invention in light of the spirit of the invention will be covered by the claims of the present invention.
Claims (10)
1. A preparation method of red-calorie wall skeleton polysaccharide is characterized by comprising the following steps: the preparation method comprises the following operation steps:
(1) Red card thallus treatment: filtering the fermentation liquor after the fermentation of nocardia rubra is finished, taking thalli, adding deionized water with the same volume, stirring, filtering and washing, removing residual culture medium impurities in the fermentation liquor, and collecting thalli; soaking thallus in ethanol, stirring for a period of time, filtering to remove liposoluble impurities contained in thallus, blow-drying with air, and collecting thallus;
(2) Extracting polysaccharide of red-card wall skeleton: extracting red-kamural skeleton polysaccharide by glycosidase and protease: adding deionized water according to 10-20% (W/V) of thalli, heating to 70-80 ℃, fully stirring and uniformly mixing for a certain time, cooling to room temperature, regulating the pH value of the solution, adding glycosidase, controlling the enzyme adding amount and the enzymolysis temperature, and heating to 95 ℃ for reaction for 30 minutes after enzymolysis reaction for a certain time; cooling to room temperature, regulating pH value of the solution, adding protease, controlling enzyme adding amount and enzymolysis temperature, performing enzymolysis for a certain time, heating to 95 ℃ for reaction for 30 minutes, and cooling to room temperature; finally, collecting polysaccharide of the red-card wall skeleton and concentrating;
(3) Purifying polysaccharide of red-card wall skeleton: purifying the red card wall skeleton polysaccharide obtained in the step (2) by using a DEAE-52 cellulose anion exchange column or dextran SephadexG-15, wherein the mobile phase is deionized water, the elution speed is 0.2mL/min-2mL/min, collecting the eluent, concentrating and freeze-drying in vacuum; dissolving the eluted freeze-dried sample with deionized water, centrifuging and ultrafiltering for 30min at 5000rpm by using an ultrafiltration tube with a cutoff molecular weight of 3000Da, adding distilled water into the ultrafiltered upper layer, repeating the ultrafiltration operation for 2-5 times, collecting the sample of the ultrafiltered upper layer, and freeze-drying to obtain white uniform red Kazuku bone frame polysaccharide solid.
2. The method for preparing the red-calorie wall skeleton polysaccharide according to claim 1, which is characterized in that: in the step (1), the thalli are added with 95% ethanol with the same volume for soaking, and stirred for 2 hours.
3. The method for preparing the red-calorie wall skeleton polysaccharide according to claim 1, which is characterized in that: in the step (2), the optimal pH of each enzyme is regulated by acetic acid or ammonia water, and the pH range is 4.0-8.0.
4. The method for preparing the red-calorie wall skeleton polysaccharide according to claim 1, which is characterized in that: in the step (2), the glycosidase is any one or a combination of cellulase, hemicellulase, mannanase and pectinase; the protease is any one or a combination of papain, neutral protease, trypsin and acid protease.
5. The method for preparing the red-calorie wall skeleton polysaccharide according to claim 4, which is characterized in that: in the step (2), the glycosidase is any one or a combination of cellulase and mannitose polymerase; the protease is any one or a combination of neutral protease and trypsin.
6. The method for preparing the red-calorie wall skeleton polysaccharide according to claim 1, which is characterized in that: in the step (2), the added amounts of the glycosidase and the protease are respectively 1.0-5.0% of the mass of the thalli.
7. The method for preparing the red-calorie wall skeleton polysaccharide according to claim 1, which is characterized in that: in the step (2), the enzymolysis temperature of the glycosidase and the protease is 35.0-60.0 ℃; the enzymolysis reaction time of the glycosidase and the protease is 2.0-12.0 hours.
8. A red card wall skeleton polysaccharide, characterized in that: the red-calorie wall skeleton polysaccharide is prepared based on the preparation method of the red-calorie wall skeleton polysaccharide of any one of claims 1-7, and the average molecular weight distribution of the red-calorie wall skeleton polysaccharide solid is 1-50 KD; the monosaccharide contains mannose, arabinose, galactose, glucose and N-acetylglucosamine; the monosaccharide connection mode is mainly 2, 6) -alpha-D-Manp- (1-and the structure contains a branch structure.
9. A red card wall skeleton polysaccharide according to claim 8, wherein: the molecular weight of the red-calorie wall skeleton polysaccharide solid is evenly distributed to be 4-10 KD; the main chain part of the polysaccharide composing the red-kamural skeleton comprises a connection mode of 2, 6) -alpha-D-Manp- (1-glycosidic bond, a branch structure is connected at a C-2 position, a branched chain part is formed by a 1,5-Ara (f) connection terminal t-Ara (f), and the peripheral part of the whole sugar ring structure is connected with 1,6-Gal (p), t-Gal (p) and alpha-D-GlcpNAC- (1-).
10. An application of red-calorie wall skeleton polysaccharide, which is characterized in that: the red-calorie wall skeleton polysaccharide is prepared based on the preparation method of the red-calorie wall skeleton polysaccharide of any one of claims 1-7, and can be used for preventing and repairing human epidermal keratinocyte aging and collagen degradation.
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