CN116574197A - Tremella polysaccharide and application thereof - Google Patents
Tremella polysaccharide and application thereof Download PDFInfo
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- CN116574197A CN116574197A CN202310535082.9A CN202310535082A CN116574197A CN 116574197 A CN116574197 A CN 116574197A CN 202310535082 A CN202310535082 A CN 202310535082A CN 116574197 A CN116574197 A CN 116574197A
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- polysaccharide
- tremella
- tremella polysaccharide
- cells
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Abstract
The invention belongs to the technical field of polysaccharide, and in particular relates to tremella polysaccharide, which has the structural formula:the invention also provides application of the tremella polysaccharide in preparing skin care products, application in preparing medicines for inhibiting melanoma formation and/or growth, and the like. The tremella polysaccharide has good whitening activity and/or elastase activity inhibition, can be applied to skin care products, can inhibit melanoma growth, and can be used for preparing medicines.
Description
Technical Field
The invention belongs to the technical field of polysaccharide, and particularly relates to tremella polysaccharide and application thereof.
Background
In recent years, the living standard of people is continuously improved, people have higher requirements on good life, women pay more attention to skin care, and the skin care industry is increasingly focused on the effects of resisting aging, whitening and the like under the promotion of market demands. The research shows that the polysaccharide has good biological activities such as oxidation resistance, whitening, moisture absorption, moisture preservation and the like, and is often added into skin care products as a substance with skin care efficacy.
The tremella polysaccharide is a natural active ingredient with the functions of resisting oxidation, resisting aging, resisting tumors, reducing blood pressure, reducing blood fat, enhancing immunity and the like, and is safer. The tremella polysaccharide can be widely used in the fields of medical treatment, health care, food processing, skin care and beauty treatment, livestock breeding and the like. The raw materials are widely available, so that a larger application space can be brought to tremella polysaccharide, and the tremella polysaccharide has important significance in further researching the physicochemical properties and biological activities of tremella polysaccharide. As the bioactivity of polysaccharides is developed, more and more research is being devoted to the moisturizing, anti-aging or whitening effect of polysaccharides on skin. However, most researches on tremella polysaccharide are remained in the aspects of polysaccharide extraction process optimization, structural characterization, antioxidation, anticancer, blood lipid reduction, blood sugar reduction, organism immunity enhancement and the like, and the research on the skin care activity of tremella polysaccharide is less or not deep enough.
The Chinese patent with the application number of CN201410475033.1 discloses a preparation method and application of tremella functional polysaccharide, wherein commercial tremella is used as a raw material, and the preparation method comprises the following steps: mixing, water extraction, alkali extraction, dialysis, concentration, precipitation, rinsing and drying, the yield of tremella polysaccharide is improved to the greatest extent, and tremella polysaccharide prepared by the method can keep high antioxidant activity, and can be applied to antioxidant foods and medicines. The method also only refers to the application of tremella polysaccharide in antioxidant foods and medicines. Also, chinese patent application No. CN201910829061.1 discloses a method for extracting functional components of tremella, which comprises extracting tremella with ethanol aqueous solution, centrifuging, concentrating, and drying to obtain flavonoid compound; and (3) carrying out enzymolysis and filtration on the tremella filter residue obtained after centrifugation to obtain a mixed solution containing a small amount of small molecular polysaccharide, protein and poly-acetylglucosamine, and wall-broken tremella residues. Repeatedly leaching Tremella residue with buffer water solution twice, filtering, removing protein from the filtrate by complex enzyme action, removing inorganic salt by semipermeable membrane, concentrating by ultrafiltration membrane, and precipitating with ethanol to obtain polyacetyl glucosamine and Tremella polysaccharide. The process is simple, mainly adopts a biological enzyme action technology, has mild process conditions, obtains flavonoid compounds, the polyacetyl glucosamine and the tremella polysaccharide by segmented extraction, has multiple functional components and wide application range, fully and effectively utilizes tremella, and is favorable for fully developing tremella resources. The applications mentioned therein are also broadly speaking, and specific applications and effects are not specifically mentioned.
Therefore, research and development of more characteristics of tremella polysaccharide, widening of the application field of tremella polysaccharide, and providing help for development and utilization of tremella resource are very necessary.
Disclosure of Invention
The invention aims to solve the technical problems and provides tremella polysaccharide which has good efficacy when applied to preparing skin care products and medicines for inhibiting melanoma growth.
The technical scheme of the invention is as follows:
a tremella polysaccharide has a structural formula:
the preparation method of the tremella polysaccharide comprises the following steps:
(1) Pulverizing dried tremella fruit body, and sieving with 60 mesh sieve to obtain tremella dry powder; uniformly stirring tremella dry powder and distilled water according to the ratio of 1:60 g/ml (m/V), and heating and boiling for 2 hours to obtain polysaccharide crude extract; after the crude extract is cooled to room temperature, centrifuging for 5min at a rotation speed of 5000r/min, and taking supernatant to be rotationally evaporated to 1/10 of the volume of the original supernatant; adding 95% ethanol solution with volume concentration of 4 times to the concentrated solution, stirring for 5min, standing at 4deg.C for 12 hr, precipitating, and lyophilizing to obtain tremella crude polysaccharide CTP (with extraction rate of 14.27%, total sugar content of 82.19%, and protein content of 3.36%);
(2) Re-dissolving crude polysaccharide CTP and distilled water according to the ratio of 1:30 g/ml (m/V), adding 10% TCA (trichloroacetic acid method) solution with the mass concentration of 10% into the re-solution, adjusting the pH value to 2.5, and finally placing the mixture in an environment of 4 ℃ for 12 hours to precipitate protein; then centrifuging the solution at a rotation speed of 5000r/min for 10min, removing protein precipitate, and taking supernatant for later use; adding 1/4 volume of AB-8 macroporous resin into the supernatant, placing into a 50 ℃ electromagnetic stirrer, and stirring at a speed of 1000r/min for 2 hours for decolorization; filtering after decolorizing, removing macroporous resin, dialyzing supernatant for 3d at 4deg.C in 8000-14000Da dialysis bag, and changing water every 6 h; concentrating the dialyzed supernatant, precipitating with ethanol, and lyophilizing to obtain tremella refined polysaccharide RTP;
(3) Preparing refined polysaccharide RTP into polysaccharide solution of 5mg/mL, separating the polysaccharide solution by using an ion exchange column after passing through a water-based filter membrane of 0.45 mu m, wherein the filler is DEAE-52 cellulose, soaking the filler in distilled water, then carrying out acid washing, then carrying out alkaline washing, loading the column, and then washing with ultra-pure water to be neutral; firstly, balancing the contact surface of the filler and the eluent by using ultrapure water according to the elution flow rate; gradient eluting with ultrapure water and 0.1M, 0.3M and 0.5M NaCl solution as mobile phase at flow rate of 1.5mL/min, collecting eluate with automatic part collector for 10mL per tube, measuring sugar content of eluate with phenol-sulfuric acid method, drawing corresponding elution curve, mixing the eluate of each tube under the same elution peak, dialyzing with distilled water for 3d, concentrating, and lyophilizing to obtain polysaccharide sample with single elution peak;
(4) Re-dissolving the single eluting peak component obtained by DEAE-52 cellulose column chromatography with ultrapure water to obtain polysaccharide sample solution of 5mg/mL, and passing through 0.45 μm water-based filter membrane; measuring absorbance of each tube at 490nm by phenol-sulfuric acid method, and drawing an elution curve, as shown in figure 1, to obtain a unique elution peak, wherein the concentration of the elution is 0.5M NaCl solution, combining the eluents collected under the peak, concentrating, dialyzing, and freeze-drying to obtain a separated and purified tremella polysaccharide TP component; continuously eluting the single eluting peak tremella polysaccharide TP component obtained by DEAE-52 cellulose column chromatography by using a Sephadex G-200 gel filtration column, and continuously eluting by using 0.5M NaCl solution at a flow rate of 0.5 mL/min; collecting each tube for 20min; similarly, the single eluting peak components are combined, and the eluent is collected, dialyzed and freeze-dried to obtain tremella uniform polysaccharide TP1 component (the molecular weight of which is 568385Da, the total sugar content is 86.38 percent, and the uronic acid content is 34.35 percent).
The invention also provides application of the tremella polysaccharide in preparing skin care products, preferably application of the tremella polysaccharide in preparing skin care products with whitening activity and/or elastase activity inhibiting functions. Through tyrosinase inhibition experiments, tremella polysaccharide shows good tyrosinase inhibition activity, which indicates that tremella polysaccharide is a biological macromolecule raw material with whitening effect potential. In the elastase inhibition experiment, tremella polysaccharide also shows a strong inhibition capability.
The invention also provides application of the tremella polysaccharide in preparing a medicament for inhibiting generation and/or growth of melanoma cells, wherein the melanoma cells comprise melanoma cells B16-F10, and application of the tremella polysaccharide in preparing a medicament for inhibiting generation of melanin in the melanoma cells. Through experiments, in researching the influence of tremella polysaccharide on melanoma cells, the applicant finds that tremella polysaccharide does not influence the growth form of B16-F10 cells, has a remarkable effect on proliferation of B16-F10 cells, and further experiments also show that tremella polysaccharide has an inhibiting effect on melanin generation in B16-F10 cells and has a remarkable inhibiting effect on tyrosinase activity in B16-F10 cells.
By adopting the technical scheme, the invention has the beneficial effects that:
the tremella polysaccharide can be used for preparing skin care products, has good effects of whitening and the like, can also inhibit the growth of melanoma and inhibit the generation of melanin in the melanoma, can be used for preparing medicines for inhibiting the growth of the melanoma, is beneficial to the development of tremella polysaccharide products in the field of skin care, widens the application field of tremella polysaccharide, and provides assistance for the development and utilization degree of tremella resources.
Drawings
FIG. 1 is an elution profile of a single eluting peak component obtained by the column chromatography separation of DEAE-52 cellulose in example 1 of the present invention.
FIG. 2 shows the high performance gel permeation chromatography of tremella uniform polysaccharide TP1 according to the present invention.
Fig. 3 is a fourier infrared spectrogram of the tremella uniform polysaccharide TP1 according to the present invention.
Fig. 4 is an atomic force micrograph of tremella uniform polysaccharide TP1 according to the present invention.
FIG. 5 shows tremella uniform polysaccharides TP1 and I according to the present invention 2 -uv-visible spectrum of KI reactant.
FIG. 6 is a graph showing the reaction between tremella uniform polysaccharide TP1 and Congo red according to the present invention.
FIG. 7 shows a chromatogram of tremella uniform polysaccharide TP1 GC-MS.
FIG. 8 shows the tremella uniform polysaccharide TP1 of the present invention 1 HNMR spectra.
FIG. 9 shows the tremella uniform polysaccharide TP1 of the present invention 13 C NMR spectrum.
FIG. 10 shows HSQC spectrum of tremella uniform polysaccharide TP1 according to the present invention.
FIG. 11 is a partial enlarged view of HSQC spectrum of tremella uniform polysaccharide TP1 of the present invention.
FIG. 12 is an electron micrograph of B16-F10 cells of the invention at 100 Xmagnification, wherein A is a DMEM high-glucose medium group without TP as a control; b is a DMEM high-sugar culture medium group added with 10mg/mL TP 1; c is DMEM high-sugar culture medium group added with 10 mg/mLRTP; d is the DMEM high-sugar medium group added with 10mg/mL CTP, and other culture conditions are the same.
FIG. 13 shows the proliferation inhibitory activity of TP1 of the present invention on B16-F10 cells.
FIG. 14 shows the activity of TP in inhibiting tyrosinase in B16-F10 cells according to the present invention
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely, and it is apparent that the described embodiments are only some embodiments of the present invention, but not all embodiments. 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.
Example 1: preparation of tremella polysaccharide
(1) Pulverizing dried tremella fruit body, and sieving with 60 mesh sieve to obtain tremella dry powder; uniformly stirring tremella dry powder and distilled water according to the ratio of 1:60 g/ml (m/V), and heating and boiling for 2 hours to obtain polysaccharide crude extract; after the crude extract is cooled to room temperature, centrifuging for 5min at a rotation speed of 5000r/min, and taking supernatant to be rotationally evaporated to 1/10 of the volume of the original supernatant; adding 95% ethanol solution with volume concentration of 4 times to the concentrated solution, stirring for 5min, standing at 4deg.C for 12 hr, precipitating, and lyophilizing to obtain tremella crude polysaccharide CTP (with extraction rate of 14.27%, total sugar content of 82.19%, and protein content of 3.36%);
(2) Re-dissolving tremella polysaccharide CTP and distilled water according to the ratio of 1:30 (m/V), adding TCA (trichloroacetic acid method) solution with the mass of 10% into the re-solution, adjusting the pH value to 2.5, and finally placing the mixture in an environment of 4 ℃ for standing for 12 hours to separate out protein; then centrifuging the solution at a rotation speed of 5000r/min for 10min, removing protein precipitate, and taking supernatant for later use; adding 1/4 volume of AB-8 macroporous resin into the supernatant, placing into a 50 ℃ electromagnetic stirrer, and stirring at a speed of 1000r/min for 2 hours for decolorization; filtering after decolorizing, removing macroporous resin, dialyzing supernatant for 3d at 4deg.C in 8000-14000Da dialysis bag, and changing water every 6 h; concentrating the dialyzed supernatant, precipitating with ethanol, and lyophilizing to obtain tremella refined polysaccharide RTP;
(3) Preparing tremella refined polysaccharide RTP into polysaccharide solution of 5mg/mL, separating the polysaccharide solution by using an ion exchange column after passing through a water-based filter membrane of 0.45 mu m, wherein the filler is DEAE-52 cellulose, soaking the filler in distilled water, then carrying out acid washing, then carrying out alkali washing, loading the column, and then washing with ultra-pure water to be neutral; firstly, balancing the contact surface of the filler and the eluent by using ultrapure water according to the elution flow rate; gradient eluting with ultrapure water and 0.1M, 0.3M and 0.5M NaCl solution as mobile phase at flow rate of 1.5mL/min, collecting eluate with automatic part collector for 10mL per tube, measuring sugar content of eluate with phenol-sulfuric acid method, drawing corresponding elution curve, mixing the eluate of each tube under the same elution peak, dialyzing with distilled water for 3d, concentrating, and lyophilizing to obtain polysaccharide sample with single elution peak;
(4) Re-dissolving the single eluting peak component obtained by DEAE-52 cellulose column chromatography with ultrapure water to obtain polysaccharide sample solution of 5mg/mL, and passing through 0.45 μm water-based filter membrane; measuring absorbance of each tube at 490nm by phenol-sulfuric acid method, and drawing an elution curve, as shown in figure 1, to obtain a unique elution peak, wherein the concentration of the elution is 0.5M NaCl solution, combining the eluents collected under the peak, concentrating, dialyzing, and freeze-drying to obtain a separated and purified tremella polysaccharide TP component; continuously eluting the single eluting peak tremella polysaccharide TP component obtained by DEAE-52 cellulose column chromatography by using a Sephadex G-200 gel filtration column, and continuously eluting by using 0.5M NaCl solution at a flow rate of 0.5 mL/min; collecting each tube for 20min; similarly, the single eluting peak components are combined and the eluent is collected, the tremella uniform polysaccharide TP1 (the total sugar content is 86.38 percent, the uronic acid content is 34.35 percent) is obtained after dialysis and freeze-drying, the TP1 is re-dissolved to prepare 1mg/ml solution, no absorption peak is found through ultraviolet spectrum scanning within the wavelength range of 200-400nm, and the protein content is not detected by using a Coomassie brilliant blue method and Fu Lin Fenfa, so that the TP1 does not contain free proteins and nucleic acid substances, and the basic components of the tremella uniform polysaccharide TP1 are shown in the table 1.
TABLE 1 essential components of Tremella homogeneous polysaccharide TP1
| Total sugar content | Reducing sugar content | Uronic acid content | Protein content | Moisture content |
| 86.38±0.06％ | ND | 34.35±2.70％ | ND | 7.09±0.28％ |
Example 2: structural identification of tremella uniform polysaccharide TP1
The molecular weight and purity of tremella uniform polysaccharide TP1 of example 1 were determined by HPGPC. The tremella polysaccharide TP sample and the standard (dextran of different relative molecular masses) of example 1 were precisely weighed, prepared into solutions of 5mg/mL mass concentration, centrifuged at 12000r/min for 10min, the supernatant was taken and filtered with a 0.22 μm aqueous filter membrane, and then the standard and sample solutions were transferred into 1.8mL brown sample vials for use. The chromatographic column is as follows: BRT105-104-102 series gel column (8X 300 mm); the sample was taken at 20. Mu.L/min, eluted at a flow rate of 0.05M NaCl solution and a flow rate of 0.6mL/min, the column temperature was 40℃and detected using an RI-10A differential detector. The molecular mass measurement result of the tremella uniform polysaccharide TP1 is shown in FIG. 2, only a unique molecular weight peak exists, 46.5min is a peak of a mobile phase (0.05M NaCl solution), and the molecular weight of the tremella uniform polysaccharide TP1 is 568385Da according to the calculation of the relative retention time of a sample.
The monosaccharide composition of the TP samples was determined using IC. A5 mg TP sample was precisely weighed into an ampoule, 2mL of 3M TFA was added, and hydrolyzed at 120℃for 3h. Transferring the sample solution after acid hydrolysis into a centrifuge tube, blow-drying by a nitrogen blowing instrument, adding 5mL of ultrapure water, and vortex mixing uniformly. Next, 50. Mu.L of the well-mixed sample solution was aspirated, 950. Mu.L of ultrapure water was added, and the mixture was centrifuged at 12000r/min for 5min. The supernatant was analyzed by IC. The chromatographic conditions are as follows: dionex CarbopacTM PA20 (3X 150 mm) column; the mobile phase is A to H 2 O, B, 15mMNaOH,C:15mM NaOH and 100mM sodium peroxyacetate; flow rate: 0.3mL/min; sample injection amount: 5. Mu.L; column temperature: 30 ℃; a detector: an electrochemical detector. As shown in fig. 3, the ion chromatographic analysis of the tremella uniform polysaccharide TP1 shows that the molar ratio of the monosaccharides of the tremella uniform polysaccharide TP1 is calculated according to the peak area: mannose: fucose: xylose: glucose: glucuronic acid: galactose: mannuronic acid=0.367:0.229:0.217:0.127:0.046:0.007:0.007, wherein the ratio of glucuronic acid to mannose is 0.125.
The functional groups of the tremella uniform polysaccharide TP1 of example 1 were identified using Fourier transform infrared spectroscopy, and the results of the infrared spectroscopy analysis of the tremella uniform polysaccharide TP1 are shown in FIG. 3. The absorption band is 3600-3200cm -1 Is the telescopic vibration absorption peak of-OH, and the absorption peak of this region is the characteristic peak of saccharides. 3390cm -1 Is the stretching vibration absorption peak of O-H, which shows that the intermolecular and intramolecular hydrogen bonds exist, and is the characteristic peak of saccharides. At 2929cm -1 There is an absorption peak, probably attributed to C-H stretching vibration. At 1635cm -1 There is an absorption peak, probably attributed to the water of crystallization. At 1558cm -1 At 1540cm -1 There is an absorption peak, probably attributed to c=o stretching vibration. At 1455cm -1 There is an absorption peak, possibly attributed to C-H angular vibration. At 1417cm -1 At 1243cm -1 At 1133cm -1 Department, 1066cm -1 There is an absorption peak, possibly attributed to C-O stretching vibration. At 1338cm -1 There is an absorption peak, probably attributed to the symmetrical stretching vibration of c=o. Based on these several characteristic peaks, it can be preliminarily determined that TP1 is a polysaccharide compound. In addition, at 917cm -1 There is an absorption peak, possibly attributed to asymmetric ring stretching vibration of the pyran ring. At 894cm -1 There is an absorption peak, probably due to the beta-anomeric C-H angular vibration of the pyran ring. In summary, TP1 may be a polysaccharide with β -type glycosidic linkages.
Molecular morphology of tremella uniform polysaccharide TP1 was observed using an atomic force microscope (Atomic force microscope, AFM). mu.L of 10. Mu.g/mL TP sample solution was added dropwise to the surface of mica flakes. And naturally air-drying the sample solution for 12 hours, and then scanning the sample on the surface of the mica sheet by using an atomic force microscope. The resolution of the image is 256×256 pixels. As shown in FIG. 4, FIG. 4-A is an atomic force micrograph of TP1 at a sensing height of 5 μm, and it can be seen that TP1 molecules are in the form of smooth circular particles. Some irregular blocks are probably the aggregation of molecules due to the fact that TP1 contains uronic acid and is negatively charged, mica flakes are also negatively charged, or more flexible lines are wound in TP1 molecules due to too many branches of polysaccharide molecules, which is shown in FIG. 4-B (at a sensing height of 1 μm), and the existence of more branches in TP1 molecules is further proved by spider-web-like structures. The diameter size and surface roughness of the samples were calculated by AFM data analysis software nanoscope8.0 analysis. The diameter of TP1 is 6.44-33.70 nm, and the roughness is 0.34-0.92 nm. It is shown that TP1 is an acidic macromolecule that is aggregated from a plurality of smaller molecules and has multiple branches.
And (3) performing iodine-potassium iodide reaction analysis on tremella uniform polysaccharide TP 1. Preparing tremella homogeneous polysaccharide TP1 sample into 1mg/mL solution, mixing with iodine reagent (containing 0.02% I) 2 0.2% ki solution) and measuring the absorption spectrum thereof in the wavelength range of 300-600 nm. The results are shown in FIG. 5, TP1 and I 2 The maximum absorbance peak of the reaction for KI is 350.5nm and there is no maximum absorbance at 565nm, indicating that TP1 may have longer side chains and more branches. The method is similar to the method that TP1 monosaccharide has complex composition, more molecular bonds in infrared analysis and complex AFM microstructureSo that. In addition, TP1 and I 2 The KI reaction was negative, indicating that no starch branches were present.
The triple helix structure of TP1 was determined by the Congo red experiment. Uniformly mixing 2mL of TP1 solution with the mass concentration of 2.5mg/mL and 2mL of Congo red reagent with the concentration of 80 mu mol/L, then dropwise adding 4mol/L of NaOH solution into the mixed solution, sequentially enabling the NaOH concentration in the solution to be 0, 0.1, 0.2, 0.3, 0.4 and 0.5mol/L, respectively carrying out ultraviolet scanning in the wavelength range of 400-600nm, and measuring the maximum absorption wavelength of the sample under each alkaline condition. Equivalent distilled water was used as a blank instead of TP solution. As shown in fig. 6, as the concentration of NaOH increases, the maximum absorption wavelength of the complex formed by congo red and TP1 increases correspondingly, i.e., the maximum absorption wavelength is shifted relatively compared to congo red. And the complex formed by Congo red and TP1 deepens and turns into purple. TP1 is a polysaccharide having a triple helix structure and a complex structure.
After derivatization, such as methylation, of polysaccharide samples, the GC-MS measures the manner of attachment. Accurately weighing TP sample 2mg, placing in a glass reaction bottle, adding 1mL anhydrous dimethyl sulfoxide (DMSO), rapidly adding methylation reagent A (20 mg NaOH powder), sealing, dissolving under ultrasonic action, and adding methylation reagent B solution (0.4 mL CH) 3 I) A. The invention relates to a method for producing a fibre-reinforced plastic composite The reaction was carried out in a magnetic stirring water bath at 30℃for 60 min. Finally, 2mL of ultrapure water was added to the above mixture to terminate the methylation reaction. The methylated polysaccharide was taken, hydrolyzed with 1mL of 2M TFA for 90min, and evaporated to dryness on a rotary evaporator. 2mL of double distilled water and 60mg of sodium borohydride are added into the residue to reduce for 8 hours, glacial acetic acid is added to neutralize, rotary evaporation is carried out, a baking oven is used for drying at the temperature of 101 ℃, then 1mL of acetic anhydride is added to carry out acetylation at the temperature of 100 ℃ for reacting for 1 hour, and cooling is carried out. Then 3mL of toluene is added, the mixture is concentrated under reduced pressure and evaporated to dryness, and the mixture is repeated for 4 to 5 times to remove the redundant acetic anhydride. The acetylated product was reacted with 3mL of CH 2 Cl 2 After dissolution, the mixture was transferred to a separating funnel, and after adding a small amount of distilled water and shaking sufficiently, the upper aqueous solution was removed, and the above procedure was repeated 4 times. CH (CH) 2 Cl 2 The layers were dried over an appropriate amount of anhydrous sodium sulfate, fixed to a volume of 10mL, and placed in a liquid phase vial. Analysis Using Shimadzu GCMS-QP 2010 gas chromatograph-Mass SpectrometryA product; GC-MS conditions: RXI-5SIL MS chromatographic column 30mm×0.25mm×0.25μm; the temperature programming conditions are as follows: heating to a starting temperature of 120 ℃ at 3 ℃/min to 250 ℃/min; maintaining for 5min; the sample inlet temperature was 250deg.C, the detector temperature was 250deg.C/min, the carrier gas was helium, and the flow rate was 1mL/min. The analysis results of the glycosidic bond composition of TP1 are shown in FIG. 7. TP1 was methylated and then hydrolyzed to form a sugar alcohol acetate derivative, which was then analyzed by GC-MS for structural identification based on retention time and characteristic fragments in mass spectrometry. TP1 methylation results showed that it had 3 main residues, respectively (1.fwdarw.4, 6) -linked alpha mannose (22.6%), (1.fwdarw.3) -linked alpha fucose (22.6%) and (1.fwdarw.4) -linked beta glucose (21.6%), and branched sugar residues mainly containing (1.fwdarw.3) -linked beta xylose (6.4%), and (1.4) -linked mannose (1.5%) and (1.3%) linked galactose (1.3%). Combining monosaccharide results analysis, it was found that TP1 was an acidic heteropolysaccharide composed of (1- > 4, 6) -linked alpha-mannose as the main chain, (1- > 3) -linked alpha-fucose, (1- > 4) -linked beta-glucose, (1- > 3) -linked beta-xylose as the main branch chain, and a small amount of galactose, mannuronic acid, glucuronic acid branches.
The structural characteristics of TP were further analyzed by NMR. A TP sample (50 mg) was weighed, dissolved in 0.5mL of heavy water and freeze-dried. And then dissolving the freeze-dried powder into 0.5mL of heavy water again, continuing to freeze-dry, and repeating the above process for a plurality of times until active hydrogen is fully exchanged. Finally, the sample is dissolved in 0.5mL of heavy water and is placed in a nuclear magnetic resonance apparatus at 600MHz for measurement 1 HNMR spectrum, 13 C NMR spectrum, HH-COSY spectrum, HSQC spectrum, NOESY spectrum.
As shown in figure 8 of the drawings, 1 the H NMR spectrum signal is concentrated mainly between 3.0 and 5.5 ppm. Delta 3.2-4.0ppm is sugar ring proton signal, and signal peaks of main end group proton peaks delta 5.48, 5.31, 5.11, 5.09, 4.45, 4.42 and 4.36 are intensively distributed in a region of 4.3-5.5 ppm, and methyl proton peak delta 1.15ppm of fucose.
As shown in FIG. 9, the carbon spectrum analysis is that 13 C NMR(201MHz,D 2 O): the nuclear magnetic carbon spectrum signal is mainly concentrated between 60 and 120 ppm. By observing the carbon spectrum, the main anomeric carbon signal can be seenPeaks delta 104.76, 103.48, 103.02, 102.94, 102.21, 101.1, 98.71ppm of anomeric carbon domains are predominantly between delta 93 and 105, while the sugar non-anomeric carbon major signal peaks are distributed over the 60 to 85ppm domain. The methyl carbon signal peak for fucose was at delta 16.9ppm.
Deducing the bonding mode:
taking the D glycosidic bond as an example, the isocephalic carbon signal of the D glycosidic bond is delta 98.71, the corresponding isocephalic hydrogen signal in the HSQC spectrum is delta 5.48, and the signal of H1-2 is 5.48/3.69 through HH-COSY; the signal of H2-3 is 3.69/4.77; the signal of H3-4 is 3.77/3.53; the signal of H4-5 is 3.53/4.32; the signal of H5-6 is 4.32/1.32; we can infer that H1, H2, H3, H4, H5, H6 are δ5.48, 3.69, 3.77, 3.53, 4.32, 1.15, respectively, and the corresponding C1-6 is 98.71, 78.15, 70.01, 73.7, 67.54, 16.9. By combining the monosaccharide component and the methylation analysis structure, it is inferred that the signal of the glycosidic bond D is attributable to the glycosidic bond → 2) - α -L-Fucp- (1 → 2).
All glycosidic bond signals were assigned according to a similar law in combination with the NOESY spectrum (see fig. 10), HSQC spectrum (see fig. 9), HSQC spectrum local amplification (see fig. 11):
table 2 tremella homogeneous polysaccharide TP1 glycosidic bond signal to home list
And analyzing the connection mode of the glycosidic bond according to the HSQC and NOESY maps. Backbone analysis:
firstly, in BC spectrum, the hydrogen at the different head of the glycosidic bond A has a relevant signal peak with the C3 of the hydrogen at the different head; in NOE, the anomeric hydrogen of glycosidic bond A has a related signal peak with its own H3; indicating that there is a 3a1→3a1→link.
Furthermore, in BC spectra, the anomeric hydrogen of glycosidic bond a and C3 of glycosidic bond B have a relevant signal peak; in NOE, the anomeric hydrogen of glycosidic bond A and H3 of glycosidic bond B have related signal peaks; indicating that there is a 3 A1-3B 1-linkage.
Meanwhile, in BC spectrum, the hydrogen at the different head of the glycosidic bond B has a related signal peak with the C3 of the hydrogen at the different head; in NOE, the hydrogen at the different head of the glycosidic bond B has a relevant signal peak with H3 of the hydrogen at the different head; indicating that there is a 3b1→3b1→link.
Finally, in BC spectrum, the isocephalic hydrogen of glycosidic bond B and C3 of glycosidic bond A have related signal peaks; in NOE, the anomeric hydrogen of glycosidic bond B has a relevant signal peak with H3 of glycosidic bond A; indicating that there is a 3b1→3a1→link.
The backbone is inferred as: 3A1 3B1
Branch analysis:
in NOE, the hydrogen at the different head of the glycosidic bond C has a related signal peak with H2 of the hydrogen at the different head, which indicates that a linkage mode of 2C 1-exists; the isosorbide hydrogen of glycosidic bond C has a related signal peak with H2 of glycosidic bond B, indicating that there is a linkage mode of 2C1.fwdarw.2B1.fwdarw..
In NOE, the hetero-head hydrogen of the glycosidic bond G and H2 of the glycosidic bond C have a signal peak, which indicates that the linkage mode of G1.fwdarw.2C1.fwdarw.exists.
Meanwhile, it was found that in NOE, the hetero-head hydrogen of glycosidic bond F has a related signal peak with H4 of glycosidic bond D, the hetero-head hydrogen of glycosidic bond D has a related signal peak with H3 of glycosidic bond E, and the hetero-head hydrogen of glycosidic bond E has a related signal peak with H2 of glycosidic bond B, indicating that there is a linking mode of F1→4D1→3E1→2B1.
From the above, we can infer that the main glycosidic bond structure of the polysaccharide is as follows:
example 3: effect of Tremella polysaccharide on B16-F10 cells
1. The test method comprises the following steps:
(1) Cell morphology observation: dispersing cells to 1X 10 5 Cells were cultured in 6-well plates per well. Then mixing TP sample with culture medium, and performing liquid exchangeB16-F10 cells were subjected to an intervention culture for 24h. All cells were at 37℃and contained 5% CO 2 Incubation was performed in humidified incubator. The morphological changes induced in B16-F10 cells by TP1 were observed with an inverted microscope (XD-202, shanghai, air-light instruments, inc. of China) and photographed at 100-fold magnification with Scomeimage 9.0 software.
(2) Measurement of cell proliferation inhibition ability: measurement of inhibitory Activity of samples on proliferation of B16-F10 cells Using CCK-8 assay cells were seeded in 96-well plates (5X 10 per well) 3 Individual cells) and placed at 37℃with 5% CO 2 In a cell incubator. After incubation for 24h, 10. Mu.L of the appropriate concentration of sample was added and incubation was continued for 24h, then CCK-8 solution (10. Mu.L) was added, the cells were incubated for an additional 4h, and finally absorbance was measured at 450 nm. Kojic acid was used as positive control. Cell proliferation inhibition ability by IC 50 Values (mg/mL) are expressed.
(3) Measurement of intracellular melanogenesis inhibitory ability: B16-F10 cells were dispersed in 6-well plates at 2X 10 per well 4 Density culture of individual cells. After 24h of incubation, the medium was removed. After addition of medium containing alpha-MSH (200 nM) and TP at various concentrations, incubation was carried out for 72 hours. After the incubation, the cells were rinsed with ice-cold PBS and then digested with 1mL of 0.25% trypsin. Finally, the digested cells were transferred to 500. Mu.L of sodium hydroxide (1N) containing 10% dimethyl sulfoxide for soaking, and placed in a 80℃water bath for 1 hour to promote melanin dissolution. Finally, the absorbance (As) of the solution was recorded at 450nm, with cells cultured without polysaccharide sample As blank (A0), positive control being kojic acid. And the melanin content was calculated by using the formula (1-1).
(4) Measurement of intracellular tyrosinase activity inhibitory ability: B16-F10 cells were placed in 96-well plates (100. Mu.L/well) and the density was adjusted to 1X 10 5 Individual cells/mL. Then placed in a 37 ℃ incubator (containing 5% CO) 2 Suitable humidity) for 12h. After the completion of the culture, the cells were placed in a culture medium containing 20Incubation was performed for 72h in medium with 0 nM. Alpha. -MSH and varying concentrations of TP. After incubation, the medium was removed by washing with PBS, after which the cells were mixed with 1% Triton X-100 (90. Mu.L/well). The plates were frozen at-80℃for 30min. After thawing, 10. Mu. L L-DOPA (15 mM) was added to each well, the mixture was returned to the incubator for further incubation for 120min, and finally absorbance (As) was measured at 475nm, and cells cultured without polysaccharide sample were used As a blank (A 0 ) The positive control was kojic acid. Intracellular tyrosinase activity was calculated with reference to formula (1-1).
2. Test results
Melanoma is a malignant tumor disease which is initiated by cancerous melanocytes or precursors thereof and has high immunogenicity, strong invasiveness and quite common, once the malignant tumor is malignant, the malignant tumor is generally developed at skin mucous membrane, pigment membrane and the like, and the malignant tumor is migrated to the body all around through blood and lymphatic vessels. The traditional treatment technical means such as radiotherapy, chemotherapy, biological treatment and immunotherapy are limited, the treatment effect of patients is poor, and prognosis is poor, so that the search for novel high-efficiency low-toxicity medicines for improving the curative effect on melanoma is the key direction of current researches.
From FIG. 12, it can be seen that TP has no significant effect on the morphology of B16-F10 cells. The cells of TP intervention group (B, C and D) and control group (A) are uniformly distributed in visual field regularly, and the cells are similar in size, elliptical or short, and are strong in extracellular Zhou Qingxi and third dimension and shiny.
As is clear from FIG. 13, the proliferation inhibitory ability of B16-F10 cells was enhanced with increasing TP concentration. In particular CTP exhibits the strongest inhibitory ability, IC thereof 50 The value was 0.17mg/mL. Second, RTP also exhibits no differentiation from CTP (P>0.05 Inhibition ability (IC) 50 Value = 0.19 mg/mL). In addition, TP1 also exhibits a strong inhibitory ability (IC 50 Value = 0.84 mg/mL), but much weaker than (P)<0.05 RTP and CTP activity. Although the proliferation inhibitory ability of three TPs on B16-F10 cells was weaker than that of positive control group kojic acid (IC 50 Value = 0.013 mg/mL), but it has a significant inhibitory effect on proliferation of B16-F10 cells.
As is clear from FIG. 14, TP is effective for B16-like the intracellular melanogenesis inhibitory activityF10 intracellular tyrosinase inhibitory activity was concentration gradient dependent enhanced. CTP still shows the strongest inhibitory activity, IC thereof 50 The value was 1.51mg/mL. RTP activity is slightly weaker than CTP, its IC 50 The value was 3.77mg/mL. TP1 activity was weak. Similarly, the activity of the three TPs was still weaker than that of positive control group kojic acid (IC 50 Value = 0.17 mg/mL).
In conclusion, the tremella polysaccharide has certain inhibition effects on proliferation of melanoma cells B16-F10, generation of intracellular melanin and intracellular tyrosinase activity, and the tremella polysaccharide can be applied to preparation of drugs for inhibiting generation and/or growth of melanoma cells including the melanoma cells B16-F10, can be applied to preparation of drugs for inhibiting generation of melanin in the melanoma cells, and can be applied to preparation of drugs for inhibiting the activity of tyrosinase in the melanoma cells.
It should be noted that TP mentioned in the present invention is tremella polysaccharide.
The foregoing description is directed to the preferred embodiments of the present invention, but the embodiments are not intended to limit the scope of the invention, and all equivalent changes or modifications made under the technical spirit of the present invention should be construed to fall within the scope of the present invention.
Claims (7)
1. The tremella polysaccharide is characterized by having the structural formula:
2. the use of tremella polysaccharide as claimed in claim 1 for the preparation of skin care products.
3. The use of the tremella polysaccharide as claimed in claim 1 for preparing skin care products with whitening activity and/or elastase activity inhibition function.
4. The use of the tremella polysaccharide as claimed in claim 1 for the preparation of a medicament for inhibiting melanogenesis and/or growth.
5. The use of claim 4, wherein the melanoma cells comprise melanoma cells B16-F10.
6. The use of the tremella polysaccharide as defined in claim 4 for the preparation of a medicament for inhibiting the production of melanin in melanoma cells.
7. The use of tremella polysaccharide as claimed in claim 4 for the preparation of a medicament for inhibiting tyrosinase activity in melanoma cells.
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