Tremella polysaccharide and application thereof

Abstract

The present invention belongs to the technical field of polysaccharides, and specifically relates to a Tremella polysaccharide, whose structural formula is: #imgabs0# The present invention also provides the use of the Tremella polysaccharide in the preparation of skin care products, the use of the Tremella polysaccharide in the preparation of drugs for inhibiting the generation and / or growth of melanoma, etc. The Tremella polysaccharide of the present invention has good whitening activity and / or inhibiting elastase activity, can be used in skin care products, can also inhibit the growth of melanoma, and can be used to prepare drugs.

Description

Technical Field

[0001] The invention belongs to the technical field of polysaccharides, and in particular relates to tremella polysaccharide and application thereof. Background Art

[0002] In recent years, people's living standards have been continuously improving, people have higher requirements for a better life, and women are paying more and more attention to skin care. Driven by market demand, the skin care industry is increasingly focusing on anti-aging, whitening and other effects. Studies have shown that polysaccharides are often added to skin care products as substances with skin care effects because of their good anti-oxidation, whitening, moisture absorption, moisturizing and other biological activities.

[0003] Tremella polysaccharide is a relatively safe natural active ingredient with antioxidant, anti-aging, anti-tumor, blood pressure lowering, blood lipid lowering, immune enhancement and other functions. Tremella polysaccharide can be widely used in many fields such as medical treatment, health care, food processing, skin care and beauty, animal husbandry and breeding. Moreover, the wide source of raw materials can bring greater application space for Tremella polysaccharide, which is of great significance for further research on the physicochemical properties and biological activity of Tremella polysaccharide. With the development of polysaccharide biological activity, more and more research is devoted to the effect of polysaccharide on skin moisturizing, anti-aging or whitening. However, most of the research on Tremella polysaccharide stays in the optimization of polysaccharide extraction process, structural characterization, antioxidant, anti-cancer, blood lipid lowering, blood sugar lowering and enhancing the body's immune activity. There are few or insufficient studies on the skin care activity of Tremella polysaccharide.

[0004] The Chinese patent with application number CN201410475033.1 discloses a preparation method and application of Tremella functional polysaccharide, which uses commercial Tremella as raw material. The preparation method includes the steps of mixing, water extraction, alkali extraction, dialysis, concentration, precipitation, rinsing and drying, which maximizes the yield of Tremella polysaccharide, and the Tremella polysaccharide prepared according to the method can maintain a high antioxidant activity and can be used in antioxidant foods and drugs. This method also only mentions the application of Tremella polysaccharide in antioxidant foods and drugs. There is also a Chinese patent with application number CN201910829061.1 that discloses a method for extracting Tremella functional components, which uses ethanol aqueous solution to extract Tremella, centrifuge, concentrate and dry to obtain flavonoid compounds; the Tremella filter residue obtained after centrifugation is enzymatically hydrolyzed and filtered to obtain a mixed solution containing a small amount of small molecule polysaccharides, proteins and polyacetyl glucosamine, as well as broken Tremella residue. The tremella residue is repeatedly extracted twice with a buffered aqueous solution and filtered. The filtrate is treated with a composite enzyme to remove protein, and inorganic salts are removed by a semipermeable membrane. The ultrafiltration membrane is concentrated, and the feed liquid is precipitated with alcohol in batches to obtain polyacetyl glucosamine and tremella polysaccharide, respectively. The text mentions that the process is simple, mainly using biological enzyme action technology, with mild process conditions, and segmented extraction to obtain flavonoid compounds, polyacetyl glucosamine, and tremella polysaccharide. The obtained tremella functional ingredients are of various types and have a wide range of applications. Tremella is fully and effectively utilized, which is conducive to the full development of tremella resources. The applications mentioned are also general, and no specific application or effect is mentioned.

[0005] Therefore, it is very necessary to study and develop more properties of Tremella polysaccharides, broaden the application fields of Tremella polysaccharides, and provide assistance for the development and utilization of Tremella resources. Summary of the invention

[0006] The present invention aims to solve the above technical problems and provides a Tremella polysaccharide, which has good efficacy in the preparation of skin care products and drugs for inhibiting melanoma growth.

[0007] The technical solution of the present invention is:

[0008] A Tremella polysaccharide, the structural formula of which is:

[0009]

[0010] The preparation method of Tremella polysaccharide of the present invention comprises the following steps:

[0011] (1) Crush dried Tremella fuciformis fruiting bodies and pass them through a 60-mesh sieve to obtain Tremella fuciformis dry powder; stir the Tremella fuciformis dry powder and distilled water at a solid-liquid ratio of 1:60 g / ml (m / V) to obtain a crude polysaccharide extract; after the crude extract is cooled to room temperature, centrifuge at a speed of 5000 r / min for 5 minutes, take the supernatant and rotary evaporate to 1 / 10 of the original supernatant volume; then add a 95% ethanol solution with a volume concentration of 4 times the volume of the concentrate, stir for 5 minutes, and then stand at 4°C for 12 hours, and freeze-dry the precipitate to obtain Tremella fuciformis crude polysaccharide CTP (with an extraction rate of 14.27%, a total sugar content of 82.19%, and a protein content of 3.36%);

[0012] (2) The crude polysaccharide CTP was redissolved with distilled water at a solid-liquid ratio of 1:30 g / ml (m / V), and a 10% TCA (trichloroacetic acid method) solution of 10% by weight was added to the redissolved solution, and the pH value was adjusted to 2.5. Finally, the solution was placed at 4°C and allowed to stand for 12 hours to allow the protein to precipitate. Then, the solution was centrifuged at a speed of 5000 r / min for 10 minutes to remove the protein precipitate, and the supernatant was taken for later use. 1 / 4 volume of AB-8 macroporous resin was added to the supernatant, and the solution was placed in an electromagnetic stirrer at 50°C and stirred at a speed of 1000 r / min for 2 hours for decolorization. After the decolorization was completed, the macroporous resin was removed by suction filtration, and the supernatant was dialyzed in a 8000-14000Da dialysis bag at 4°C for 3 days, and the water was changed every 6 hours. The dialyzed supernatant was concentrated, precipitated with alcohol, and freeze-dried to obtain Tremella fuciformis polysaccharide RTP.

[0013] (3) The refined polysaccharide RTP was prepared into a 5 mg / mL polysaccharide solution, which was separated by an ion exchange column after passing through a 0.45 μm aqueous filter membrane. The filler was DEAE-52 cellulose. The filler was first soaked in distilled water and then acid-washed, then alkaline-washed and loaded into the column, and finally washed with ultrapure water until neutral; ultrapure water was first used to balance the contact surface between the filler and the eluent according to the elution flow rate; ultrapure water and 0.1 M, 0.3 M, and 0.5 M NaCl solutions were used as mobile phases for gradient elution at a flow rate of 1.5 mL / min, 10 mL per tube, and the eluent was collected using an automatic fraction collector. The sugar content of the eluent was determined by the phenol-sulfuric acid method, and the corresponding elution curve was drawn. The eluents of each tube under the same elution peak were combined, dialyzed with distilled water for 3 days, concentrated, and freeze-dried to obtain a polysaccharide sample with a single elution peak;

[0014] (4) The single elution peak component obtained by DEAE-52 cellulose column chromatography was reconstituted with ultrapure water to a 5 mg / mL polysaccharide sample solution and filtered through a 0.45 μm aqueous filter membrane; the absorbance of each tube was measured at 490 nm using the phenol-sulfuric acid method, and an elution curve was plotted, as shown in FIG. Figure 1As shown, a unique elution peak is obtained, and the eluent concentration is 0.5M NaCl solution. The eluents collected under the peak are combined, concentrated, dialyzed, and freeze-dried to obtain the separated and purified Tremella polysaccharide TP component; the single elution peak Tremella polysaccharide TP component obtained by DEAE-52 cellulose column chromatography is continuously eluted with Sephadex G-200 gel filtration column, and 0.5M NaCl solution is used for continuous elution at a flow rate of 0.5mL / min; each tube is collected for 20min; similarly, the single elution peak components are combined and the eluents are collected, and the Tremella homogeneous polysaccharide TP1 component (with a molecular weight of 568385Da, a total sugar content of 86.38%, and a uronic acid content of 34.35%) is obtained after dialyzing and freeze-drying.

[0015] The present invention also provides the use of the Tremella polysaccharide in the preparation of skin care products. Preferably, the Tremella polysaccharide of the present invention is used in the preparation of skin care products with whitening activity and / or elastase activity inhibition function. Through the tyrosinase inhibition experiment, Tremella polysaccharide exhibits good tyrosinase inhibition activity, indicating that Tremella polysaccharide is a biomacromolecule raw material with whitening potential. In the elastase inhibition experiment, Tremella polysaccharide also exhibits a strong inhibitory ability.

[0016] The present invention also provides the use of the Tremella polysaccharide in the preparation of a drug for inhibiting the generation and / or growth of melanoma cells, wherein the melanoma cells include melanoma cells B16-F10, and the Tremella polysaccharide of the present invention is also provided for the preparation of a drug for inhibiting the generation of melanin in melanoma cells, and the present invention also provides the use of the Tremella polysaccharide in the preparation of a drug for inhibiting the activity of tyrosinase in melanoma cells. Through experiments, in exploring the effect of Tremella polysaccharide on melanoma cells, the applicant found that Tremella polysaccharide does not affect the growth morphology of B16-F10 cells, and has a significant effect on the proliferation of B16-F10 cells. Further experiments also show that Tremella polysaccharide has an inhibitory effect on the generation of melanin in B16-F10 cells and has a significant inhibitory effect on the tyrosinase activity in B16-F10 cells.

[0017] Due to the adoption of the above technical solution, the beneficial effects of the present invention are:

[0018] The tremella polysaccharide of the present invention can be used to prepare skin care products, has good effects such as whitening, can also inhibit the growth of melanoma, inhibit the production of melanin in melanoma, can be used to prepare drugs for inhibiting the growth of melanoma, is conducive to the development of tremella polysaccharide products in the field of skin care, broadens the application field of tremella polysaccharide, and provides help for the development and utilization of tremella resources. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1This is the elution curve of a single elution peak component obtained by DEAE-52 cellulose column chromatography separation in Example 1 of the present invention.

[0020] Figure 2 The invention relates to the high performance gel permeation chromatography of the Tremella fuciformis homogeneous polysaccharide TP1 of the invention.

[0021] Figure 3 This is a Fourier infrared spectrum of the Tremella fuciformis uniform polysaccharide TP1 of the present invention.

[0022] Figure 4 This is an atomic force micrograph of the homogeneous Tremella fuciformis polysaccharide TP1 of the present invention.

[0023] Figure 5 It is the UV-visible spectrum of the reaction product of Tremella homogeneous polysaccharide TP1 and I2-KI of the present invention.

[0024] Figure 6 It is a reaction curve diagram of Tremella homogeneous polysaccharide TP1 of the present invention and Congo red.

[0025] Figure 7 GC-MS chromatogram of Tremella fuciformis uniform polysaccharide TP1 of the present invention.

[0026] Figure 8 The present invention Tremella uniform polysaccharide TP1 1 HNMR spectrum.

[0027] Fig. 9 The present invention Tremella uniform polysaccharide TP1 13 C NMR spectrum.

[0028] Fig.10 HSQC spectrum of Tremella homogeneous polysaccharide TP1 of the present invention.

[0029] Fig.11 A partial enlarged view of the HSQC spectrum of the Tremella fuciformis uniform polysaccharide TP1 of the present invention.

[0030] Fig.12 The electron microscope 4X photograph of B16-F10 cells of the present invention is magnified 100 times, wherein A is a DMEM high-glucose medium group without TP added as a control; B is a DMEM high-glucose medium group with 10 mg / mL TP1 added; C is a DMEM high-glucose medium group with 10 mg / mL RTP added; D is a DMEM high-glucose medium group with 10 mg / mL CTP added, and other culture conditions are the same.

[0031] Fig.13 This is the proliferation inhibitory activity of TP1 of the present invention on B16-F10 cells.

[0032] Fig.14 The inhibitory activity of TP on tyrosinase in B16-F10 cells DETAILED DESCRIPTION

[0033] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0034] Example 1: Preparation of Tremella polysaccharide

[0035] (1) Crush dried Tremella fuciformis fruiting bodies and pass them through a 60-mesh sieve to obtain Tremella fuciformis dry powder; stir the Tremella fuciformis dry powder and distilled water at a solid-liquid ratio of 1:60 g / ml (m / V) to obtain a crude polysaccharide extract; after the crude extract is cooled to room temperature, centrifuge at a speed of 5000 r / min for 5 minutes, take the supernatant and rotary evaporate to 1 / 10 of the original supernatant volume; then add a 95% ethanol solution with a volume concentration of 4 times the volume of the concentrate, stir for 5 minutes, and then stand at 4°C for 12 hours, and freeze-dry the precipitate to obtain Tremella fuciformis crude polysaccharide CTP (with an extraction rate of 14.27%, a total sugar content of 82.19%, and a protein content of 3.36%);

[0036] (2) The Tremella crude polysaccharide CTP is redissolved with distilled water at a solid-liquid ratio of 1:30 (m / V), and a 10% by weight TCA (trichloroacetic acid method) solution is added to the redissolved solution, and the pH value is adjusted to 2.5. Finally, the solution is placed at 4°C and allowed to stand for 12 hours to allow protein precipitation; then, the solution is centrifuged at a speed of 5000 r / min for 10 minutes to remove the protein precipitate, and the supernatant is taken for standby use; 1 / 4 volume of AB-8 macroporous resin is added to the supernatant, and the solution is placed in an electromagnetic stirrer at 50°C and stirred at a speed of 1000 r / min for 2 hours for decolorization; after the decolorization is completed, suction filtration is performed to remove the macroporous resin, and the supernatant is dialyzed in a 8000-14000Da dialysis bag at 4°C for 3 days, and the water is changed every 6 hours; the dialyzed supernatant is concentrated, alcohol precipitated, and freeze-dried to obtain Tremella refined polysaccharide RTP;

[0037] (3) The Tremella polysaccharide RTP was prepared into a 5 mg / mL polysaccharide solution, which was separated by an ion exchange column after passing through a 0.45 μm aqueous filter membrane. The filler was DEAE-52 cellulose. The filler was first soaked in distilled water and then acid-washed, then alkaline-washed and loaded into the column, and then washed with ultrapure water until neutral; ultrapure water was first used to balance the contact surface between the filler and the eluent according to the elution flow rate; ultrapure water and 0.1 M, 0.3 M, and 0.5 M NaCl solutions were used as mobile phases for gradient elution at a flow rate of 1.5 mL / min, 10 mL per tube, and the eluent was collected using an automatic fraction collector. The sugar content of the eluent was determined by the phenol-sulfuric acid method, and the corresponding elution curve was drawn. The eluents of each tube under the same elution peak were combined, dialyzed with distilled water for 3 days, concentrated, and freeze-dried to obtain a polysaccharide sample with a single elution peak;

[0038] (4) The single elution peak component obtained by DEAE-52 cellulose column chromatography was reconstituted with ultrapure water to a 5 mg / mL polysaccharide sample solution and filtered through a 0.45 μm aqueous filter membrane; the absorbance of each tube was measured at 490 nm using the phenol-sulfuric acid method, and an elution curve was plotted, as shown in FIG. Figure 1 As shown in the figure, a single elution peak was obtained, and the concentration of the eluent was 0.5M NaCl solution. The eluents collected under the peak were combined, concentrated, dialyzed, and freeze-dried to obtain the separated and purified Tremella polysaccharide TP component; the single elution peak Tremella polysaccharide TP component obtained by DEAE-52 cellulose column chromatography was further subjected to Sephadex The mixture was eluted with a G-200 gel filtration column using a 0.5 M NaCl solution at a flow rate of 0.5 mL / min; each tube was collected for 20 min; similarly, the single elution peak components were combined and the eluate was collected, and after dialysis and freeze-drying, Tremella fuciformis uniform polysaccharide TP1 (total sugar content was 86.38%, uronic acid content was 34.35%) was obtained. After TP1 was redissolved, it was prepared into a 1 mg / ml solution, and no absorption peak was found by ultraviolet scanning within the wavelength range of 200-400 nm, and the protein content was still not measured using the Coomassie brilliant blue method and the folin phenol method, indicating that TP1 no longer contained free protein and nucleic acid substances. The basic components of Tremella fuciformis uniform polysaccharide TP1 are shown in Table 1.

[0039] Table 1 Basic components of Tremella homogeneous polysaccharide TP1

[0040] Total sugar content Reducing sugar content Uric acid content Protein Content Moisture content 86.38±0.06％ ND 34.35±2.70％ ND 7.09±0.28％

[0041] Example 2: Structural Identification of Tremella Homogeneous Polysaccharide TP1

[0042] The molecular weight and purity of Tremella fuciformis homogeneous polysaccharide TP1 in Example 1 were determined by HPGPC. Accurately weigh the Tremella fuciformis polysaccharide TP sample and standard (dextran of different relative molecular mass) in Example 1, respectively prepare solutions with a mass concentration of 5 mg / mL, and centrifuge at 12000r / min for 10 min. Take the supernatant and filter it with a 0.22μm aqueous filter membrane, then transfer the standard and sample solutions to 1.8mL brown injection vials for later use. The chromatographic column is: BRT105-104-102 tandem gel column (8×300mm); the sample injection volume is controlled to 20μL / time, with 0.05M NaCl solution as the fluidity, elution at a flow rate of 0.6mL / min, the column temperature is 40°C, and detection is performed using an RI-10A differential detector. The molecular mass determination results of Tremella fuciformis homogeneous polysaccharide TP1 are shown in the figure. Figure 2 As shown, there is only one molecular weight peak, and 46.5 min is the peak of the mobile phase (0.05 M NaCl solution). According to the relative retention time of the sample, the molecular weight of Tremella homogeneous polysaccharide TP1 is calculated to be 568385 Da.

[0043] IC was used to determine the monosaccharide composition of the TP sample. Accurately weigh 5 mg of TP sample and place it in an ampoule, add 2 mL of 3M TFA, and hydrolyze at 120°C for 3 hours. Transfer the sample solution after acid hydrolysis to a centrifuge tube, blow dry with a nitrogen blower, then add 5 mL of ultrapure water and vortex to mix. Next, take 50 μL of the mixed sample solution, add 950 μL of ultrapure water, and centrifuge at 12000 r / min for 5 minutes. Take the supernatant for IC analysis. The chromatographic conditions are: Dionex CarbopacTM PA20 (3×150mm) chromatographic column; mobile phase is A: H2O, B: 15mM NaOH, C: 15mM NaOH and 100mM sodium peracetate mixed solution; flow rate: 0.3 mL / min; injection volume: 5 μL; column temperature: 30°C; detector: electrochemical detector. Ion chromatography analysis of Tremella homogeneous polysaccharide TP1 is as shown in Figure 3 As shown, the molar ratio of each monosaccharide in Tremella fuciformis uniform polysaccharide TP1 calculated according to the peak area is: 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.

[0044] The functional groups of Tremella fuciformis uniform polysaccharide TP1 of Example 1 were identified using Fourier transform infrared spectroscopy. The infrared spectroscopy analysis results of Tremella fuciformis uniform polysaccharide TP1 are as follows: Figure 3 As shown. The absorption band is at 3600-3200cm -1 It is the stretching vibration absorption peak of -OH. The absorption peak in this area is the characteristic peak of sugars.-1 It is the stretching vibration absorption peak of OH, indicating the existence of intermolecular and intramolecular hydrogen bonds, which is the characteristic peak of sugars. -1 There is an absorption peak at 1635cm, which may be attributed to CH stretching vibration. -1 There is an absorption peak at 1558cm -1 1540cm -1 There is an absorption peak at 1455cm -1 There is an absorption peak at 1417cm -1 1243cm -1 1133cm -1 1066cm -1 There is an absorption peak at 1338cm -1 There is an absorption peak at 917cm, which may be attributed to the symmetric stretching vibration of C=O. Based on these characteristic peaks, it can be preliminarily determined that TP1 is a polysaccharide compound. -1 There is an absorption peak at 894cm, which may be attributed to the asymmetric ring stretching vibration of the pyran ring. -1 There is an absorption peak at , which may be attributed to the CH angle vibration of the β-end diastereomer of the pyran ring. In summary, TP1 may be a polysaccharide with β-type glycosidic bonds.

[0045] Atomic force microscope (AFM) was used to observe the molecular morphology of Tremella fuciformis uniform polysaccharide TP1. 5 μL of 10 μg / mL TP sample solution was dripped onto the surface of the mica sheet. After the sample solution was allowed to air dry for 12 hours, the sample on the surface of the mica sheet was scanned with an atomic force microscope. The resolution of the image was 256×256 pixels. Figure 4 As shown, Figure 4 -A is the atomic force micrograph of TP1 at a sensing height of 5μm. It can be seen that the TP1 molecules are smooth round particles. There are some irregular lumps, which may be caused by the molecular aggregation due to the negative charge of uronic acid contained in TP1 and the negative charge of mica itself, or by the excessive branching of polysaccharide molecules. Figure 4 -B (at 1μm sensing height) shows that there are many soft lines entangled in the TP1 molecule, which is like a spider web, further proving that there are many branches in the TP1 molecule. The diameter and surface roughness of the sample were analyzed and calculated by AFM data analysis software Nanoscope8.0. The diameter of TP1 is 6.44~33.70nm, and the roughness is 0.34nm~0.92nm. This shows that TP1 is composed of multiple smaller molecules and has a multi-branched acidic macromolecule.

[0046] The iodine-potassium iodide reaction analysis of Tremella fuciformis homogeneous polysaccharide TP1 was performed. The Tremella fuciformis homogeneous polysaccharide TP1 sample was prepared into a solution with a mass concentration of 1 mg / mL, and then mixed with an iodine reagent (0.2% KI solution containing 0.02% I2), and its absorption spectrum was measured in the wavelength range of 300-600nm. The results are as follows Figure 5 As shown in the figure, the maximum absorption peak of the reaction product of TP1 and I2-KI is at 350.5nm, while there is no maximum absorption at 565nm, indicating that TP1 may have longer side chains and more branches. This is consistent with the complex monosaccharide composition of TP1, more molecular bonds in infrared analysis, and complex microscopic morphological structure of AFM. In addition, the reaction of TP1 and I2-KI is negative, indicating that it does not contain starch branches.

[0047] The triple helix structure of TP1 was determined by the Congo red experiment. 2 mL of 2.5 mg / mL TP1 solution was mixed evenly with 2 mL of 80 μmol / L Congo red reagent, and then 4 mol / L NaOH solution was added dropwise to the mixed solution. The NaOH concentration in the solution was adjusted to 0, 0.1, 0.2, 0.3, 0.4, and 0.5 mol / L, respectively. UV scanning was performed in the wavelength range of 400-600 nm to determine the maximum absorption wavelength of the sample under various alkaline conditions. An equal amount of distilled water was used instead of TP solution as a blank control. Figure 6 As shown in the figure, as the concentration of NaOH increases, the maximum absorption wavelength of the complex formed by Congo red and TP1 increases accordingly, that is, the maximum absorption wavelength is relatively shifted compared with Congo red. And the color of the complex formed by Congo red and TP1 deepens and turns purple-red. This shows that TP1 has a triple helical structure and is a polysaccharide with a complex structure.

[0048] After the polysaccharide samples were derivatized by methylation, their connection mode was measured by GC-MS. 2 mg of TP sample was accurately weighed and placed in a glass reaction bottle, and then 1 mL of anhydrous dimethylsulfoxide (DMSO) was added, followed by the rapid addition of methylation reagent A (20 mg of NaOH powder), which was sealed and dissolved under ultrasonication, and then methylation reagent B solution (0.4 mL of CH3I) was added. The reaction was carried out at 30 °C in a magnetic stirring water bath for 60 min. Finally, 2 mL of ultrapure water was added to the above mixture to terminate the methylation reaction. Take the methylated polysaccharide, add 1 mL of 2M TFA for hydrolysis for 90 min, and then evaporate to dryness using a rotary evaporator. Add 2 mL of double distilled water to the residue, reduce 60 mg of sodium borohydride for 8 h, add glacial acetic acid for neutralization, rotary evaporate, dry in an oven at 101 °C, then add 1 mL of acetic anhydride for acetylation at 100 °C for 1 h, and cool. Then add 3 mL of toluene, concentrate and evaporate to dryness under reduced pressure, and repeat 4 to 5 times to remove excess acetic anhydride. The acetylated product was dissolved in 3mL CH2Cl2 and transferred to a separatory funnel. After adding a small amount of distilled water and shaking thoroughly, the upper aqueous solution was removed and this process was repeated 4 times. The CH2Cl2 layer was dried with an appropriate amount of anhydrous sodium sulfate, fixed to 10mL, and placed in a liquid phase vial. The acetylated product samples were analyzed using a Shimadzu GCMS-QP 2010 gas chromatograph-mass spectrometer; GC-MS conditions: RXI-5SIL MS column 30mm×0.25mm×0.25μm; program temperature conditions: starting temperature 120℃, heating at 3℃ / min to 250℃ / min; maintained for 5min; injection port temperature 250℃, detector temperature 250℃ / min, carrier gas helium, flow rate 1mL / min. The analysis results of the glycosidic bond composition of TP1 are shown in Figure 7 As shown. TP1 was first methylated and then hydrolyzed to form a sugar alcohol acetate derivative, and then analyzed by GC-MS, and the structure was identified based on the retention time and characteristic fragments in the mass spectrum. The methylation results of TP1 showed that it had three main residues, namely (1→4,6)-linked α-mannose (22.6%), (1→3)-linked α-fucose (22.6%) and (1→4)-linked β-glucose (21.6%). Its branched sugar residues mainly contained (1→3)-linked β-xylose (6.4%), (1→4)-linked mannose (1.5%) and (1→)-linked galactose (1.3%). Combined with the analysis of monosaccharide results, it was concluded that TP1 is an acidic heteropolysaccharide with (1→4,6)-linked α-mannose as the main chain, (1→3)-linked α-fucose, (1→4)-linked β-glucose, (1→3)-linked β-xylose as the main branches, and a small amount of galactose, mannuronic acid, and glucuronic acid branches.

[0049] The structural characteristics of TP were further analyzed by NMR. Weigh 50 mg of TP sample, dissolve it in 0.5 mL of heavy water and freeze-dry it. Then, the freeze-dried powder was dissolved in 0.5 mL of heavy water again and freeze-dried again. The above process was repeated several times until the active hydrogen was fully exchanged. Finally, the sample was dissolved in 0.5 mL of heavy water and placed in a 600 MHz nuclear magnetic resonance instrument for measurement. 1 HNMR spectrum, 13 C NMR spectrum, HH-COSY spectrum, HSQC spectrum, NOESY spectrum.

[0050] like Figure 8 As shown, 1 The H NMR spectrum signals are mainly concentrated between 3.0-5.5ppm. δ3.2-4.0ppm is the sugar ring proton signal, the main terminal proton peaks δ5.48, 5.31, 5.11, 5.09, 4.45, 4.42, 4.36 are concentrated in the 4.3-5.5ppm region, and the methyl proton peak of fucose is δ1.15ppm.

[0051] like Fig. 9 As shown in the carbon spectrum analysis 13 C NMR (201MHz, D2O): Nuclear magnetic carbon spectrum signals are mainly concentrated between 60-120ppm. By observing the carbon spectrum, it can be seen that the main anomeric carbon signal peaks are δ104.76, 103.48, 103.02, 102.94, 102.21, 101.1, 98.71ppm. The anomeric carbon region is mainly between δ93 and 105, while the main signal peaks of sugar non-anomeric carbon are distributed in the 60-85ppm region. The methyl carbon signal peak of fucose is at δ16.9ppm.

[0052] Derivation of the bonding method:

[0053] Taking D-glycosidic bond as an example, the HSQC spectrum (such as Fig. 9As shown), it can be observed that the anomeric carbon signal of the D glycosidic bond is δ98.71, and the corresponding anomeric hydrogen signal in the HSQC spectrum is δ5.48. Through HH-COSY, the signals of H1-2 are 5.48 / 3.69; the signals of H2-3 are 3.69 / 4.77; the signals of H3-4 are 3.77 / 3.53; the signals of H4-5 are 3.53 / 4.32; the signals of H5-6 are 4.32 / 1.32; we can infer that H1, H2, H3, H4, H5, and H6 are δ5.48, 3.69, 3.77, 3.53, 4.32, and 1.15, respectively, and the corresponding C1-6 are 98.71, 78.15, 70.01, 73.7, 67.54, and 16.9. Combined with the monosaccharide composition and methylation analysis structure, it is inferred that the signal of the glycosidic bond D should be attributed to the glycosidic bond →2)-α-L-Fucp-(1→.

[0054] According to similar rules and combined with NOESY spectra (such as Fig.10 ), HSQC spectra (such as Fig. 9 ), HSQC spectrum partial enlargement diagram (such as Fig.11 ), all glycosidic bond signals were assigned (see Table 2):

[0055] Table 2 Assignment of glycosidic bond signals of Tremella fuciformis homogeneous polysaccharide TP1

[0056]

[0057]

[0058] The connection mode of glycosidic bonds was analyzed based on HSQC and NOESY spectra. Main chain analysis:

[0059] First, in the BC spectrum, the anomeric hydrogen of glycosidic bond A has a correlated signal peak with its own C3; in the NOE, the anomeric hydrogen of glycosidic bond A has a correlated signal peak with its own H3, indicating the existence of a 3A1→3A1→ linkage mode.

[0060] Furthermore, in the BC spectrum, the anomeric hydrogen of glycosidic bond A and C3 of glycosidic bond B have related signal peaks; in the NOE, the anomeric hydrogen of glycosidic bond A and H3 of glycosidic bond B have related signal peaks, indicating the existence of a 3A1→3B1→ linkage mode.

[0061] At the same time, in the BC spectrum, the anomeric hydrogen of the glycosidic bond B has a correlated signal peak with its own C3; in the NOE, the anomeric hydrogen of the glycosidic bond B has a correlated signal peak with its own H3, indicating the existence of a 3B1→3B1→ linkage mode.

[0062] Finally, in the BC spectrum, the anomeric hydrogen of glycosidic bond B and C3 of glycosidic bond A have related signal peaks; in NOE, the anomeric hydrogen of glycosidic bond B and H3 of glycosidic bond A have related signal peaks, indicating the existence of a 3B1→3A1→ linkage mode.

[0063] The main chain is inferred to be: →3A1→3A1→3B1→3B1→

[0064] Branch chain analysis:

[0065] In NOE, the anomeric hydrogen of glycosidic bond C and its own H2 have related signal peaks, indicating the existence of a 2C1→2C1→ linkage mode; the anomeric hydrogen of glycosidic bond C and the H2 of glycosidic bond B have related signal peaks, indicating the existence of a 2C1→2B1→ linkage mode.

[0066] Furthermore, in NOE, the anomeric hydrogen of glycosidic bond G and H2 of glycosidic bond C have related signal peaks, indicating the existence of a G1→2C1→ linkage mode.

[0067] At the same time, it was found that in NOE, the anomeric hydrogen of glycosidic bond F and H4 of glycosidic bond D had a related signal peak, the anomeric hydrogen of glycosidic bond D and H3 of glycosidic bond E had a related signal peak, and the anomeric hydrogen of glycosidic bond E and H2 of glycosidic bond B had a related signal peak, indicating the existence of a linking mode of F1→4D1→3E1→2B1→.

[0068] In summary, we can infer that the main glycosidic bond structure of this polysaccharide is:

[0069]

[0070] Example 3: Effect of Tremella polysaccharide on B16-F10 cells

[0071] 1. Test method:

[0072] (1) Cell morphology observation: The cells were dispersed into 1×10 5 The TP samples were then mixed with the culture medium and the B16-F10 cells were cultured for 24 h in the form of medium replacement. All cells were incubated in a humidified incubator at 37 °C with 5% CO2. The morphological changes induced by TP1 in B16-F10 cells were observed with an inverted microscope (XD-202, Caikong Optical Instrument Co., Ltd., Shanghai, China) and photographed with ScopeImage 9.0 software at 100x magnification.

[0073] (2) Determination of cell proliferation inhibition ability: The inhibitory activity of the samples on B16-F10 cell proliferation was measured using the CCK-8 assay. The cells were seeded in a 96-well plate (5×10 3cells) and placed in a 37°C, 5% CO2 cell culture incubator. After culturing for 24 hours, 10 μL of the sample of appropriate concentration was added and cultured for another 24 hours, then CCK-8 solution (10 μL) was added, the cells were cultured for another 4 hours, and finally the absorbance was measured at 450 nm. Kojic acid was used as a positive control. The cell proliferation inhibition ability was measured as IC 50 The values ​​are expressed as (mg / mL).

[0074] (3) Determination of the ability to inhibit intracellular melanin production: B16-F10 cells were dispersed in a 6-well plate at 2×10 per well. 4 The cells were cultured at a density of 100 cells. After culturing for 24 hours, the culture medium was removed. Then, a culture medium containing α-MSH (200nM) and different concentrations of TP was added and incubated for 72 hours. After the incubation, the cells were rinsed with ice-cold PBS and then 1mL of 0.25% trypsin was added for digestion. Finally, the digested cells were transferred to 500μL of sodium hydroxide (1N) containing 10% dimethyl sulfoxide and placed in an 80℃ water bath for 1h to promote the dissolution of melanin. Finally, the absorbance of the solution was recorded at 450nm (As). The cells cultured without polysaccharide samples were used as the blank group (A0), and the positive control was kojic acid. The melanin content was calculated using formula (1-1).

[0075]

[0076] (4) Determination of intracellular tyrosinase activity inhibition ability: B16-F10 cells were placed in a 96-well plate (100 μL / well) and the density was adjusted to 1×10 5 cells / mL. Then place in a 37°C incubator (containing 5% CO2, appropriate humidity) and culture for 12 hours. After the culture is completed, the cells are placed in a culture medium containing 200nM α-MSH and different concentrations of TP and incubated for 72 hours. After the incubation is completed, the culture medium is removed by rinsing with PBS, and then the cells are mixed with 1% Triton X-100 (90μL / well). The plates are frozen at -80°C for 30 minutes. After thawing, 10μL L-DOPA (15mM) is added to each well, and the plates are returned to the incubator for incubation for 120 minutes. Finally, the absorbance (As) is measured at 475nm. The cells cultured without polysaccharide samples are used as the blank group (A0), and the positive control is kojic acid. The intracellular tyrosinase activity is calculated according to formula (1-1).

[0077] 2. Test results

[0078] Melanoma is a common malignant tumor that begins with cancerous melanocytes or their precursors. It is highly immunogenic and invasive. Once it develops, it is malignant and generally occurs in the skin, mucous membranes, pigment membranes, etc., and migrates to various parts of the body through the blood and lymphatic vessels. Limited by the current traditional treatment techniques such as radiotherapy, chemotherapy, biological and immunotherapy, patients have poor treatment effects and poor prognosis. Therefore, exploring new high-efficiency and low-toxic drugs to improve the efficacy of melanoma is the focus of current research.

[0079] Depend on Fig.12 It can be seen that TP has no significant effect on the morphology of B16-F10 cells. The cells in the TP intervention group (B, C and D) and the control group (A) are regularly and evenly distributed in the field of vision, with similar cell sizes, oval or short spindle-shaped, clear cell periphery, strong three-dimensional sense and bright.

[0080] Depend on Fig.13 It can be seen that with the increase of TP concentration, its ability to inhibit the proliferation of B16-F10 cells increased. In particular, CTP showed the strongest inhibitory ability, with an IC 50 The value was 0.17 mg / mL. Secondly, RTP also showed an inhibitory ability (IC 50 In addition, TP1 also showed strong inhibitory ability (IC 50 Value = 0.84 mg / mL), but much weaker than (P < 0.05) the activity of RTP and CTP. Although the three TPs had weaker inhibitory effects on the proliferation of B16-F10 cells than the positive control group kojic acid (IC 50 value = 0.013 mg / mL), but it had a significant inhibitory effect on the proliferation of B16-F10 cells.

[0081] Depend on Fig.14 It can be seen that similar to the inhibitory activity of intracellular melanin production, the inhibitory activity of TP on intracellular tyrosinase in B16-F10 cells increased in a concentration gradient-dependent manner. CTP still showed the strongest inhibitory activity, with an IC 50 The value is 1.51mg / mL. The activity of RTP is slightly weaker than that of CTP. Its IC 50 The value was 3.77 mg / mL. TP1 had weak activity. Similarly, the activity of the three TPs was still weaker than that of the positive control group kojic acid (IC 50 value = 0.17 mg / mL).

[0082] In summary, the tremella polysaccharide of the present invention has a certain inhibitory effect on the proliferation of melanoma cells B16-F10, the production of intracellular melanin, and the activity of intracellular tyrosinase, indicating that tremella polysaccharide can be used in the preparation of drugs for inhibiting the generation and / or growth of melanoma, wherein the melanoma cells include melanoma cells B16-F10, and can also be used in the preparation of drugs for inhibiting the production of melanin in melanoma cells, and can also be used in the preparation of drugs for inhibiting the activity of tyrosinase in melanoma cells.

[0083] It should be noted that the TP mentioned in the present invention is Tremella polysaccharide.

[0084] The above description is a detailed description of the preferred feasible embodiments of the present invention, but the embodiments are not intended to limit the scope of the patent application of the present invention. All equivalent changes or modified changes completed under the technical spirit suggested by the present invention should fall within the patent scope covered by the present invention.

Claims

1. An application of Tremella polysaccharide in the preparation of a drug for inhibiting the formation and / or growth of melanoma, characterized in that: Its structural formula is: The preparation method of Tremella polysaccharide comprises the following steps: (1) Crush dried Tremella fuciformis fruiting bodies and pass them through a 60-mesh sieve to obtain Tremella fuciformis dry powder; stir the Tremella fuciformis dry powder and distilled water at a solid-liquid ratio of 1:60 g / ml, heat and boil for 2 hours to obtain a crude polysaccharide extract; after the crude extract is cooled to room temperature, centrifuge at a speed of 5000 r / min for 5 minutes, take the supernatant and rotary evaporate to 1 / 10 of the original supernatant volume; then add a 95% ethanol solution with a volume concentration of 4 times the volume of the concentrate, stir for 5 minutes, and then stand at 4°C for 12 hours, and freeze-dry the precipitate to obtain Tremella fuciformis crude polysaccharide CTP; (2) The crude polysaccharide CTP is re-dissolved with distilled water at a solid-liquid ratio of 1:30 g / ml, and a 10% TCA solution of 10% by weight is added to the re-dissolved solution, and the pH value is adjusted to 2.

5. Finally, the solution is placed at 4°C and allowed to stand for 12 hours to allow the protein to precipitate. Then, the solution is centrifuged at a speed of 5000 r / min for 10 minutes to remove the protein precipitate, and the supernatant is taken for later use. 1 / 4 volume of AB-8 macroporous resin is added to the supernatant, and the solution is placed in an electromagnetic stirrer at 50°C and stirred at a speed of 1000 r / min for 2 hours for decolorization. After the decolorization is completed, the macroporous resin is removed by suction filtration, and the supernatant is dialyzed in a 8000-14000Da dialysis bag at 4°C for 3 days, and the water is changed every 6 hours. The dialyzed supernatant is concentrated, precipitated with alcohol, and freeze-dried to obtain Tremella polysaccharide RTP. (3) The refined polysaccharide RTP was prepared into a 5 mg / mL polysaccharide solution, which was separated by an ion exchange column after passing through a 0.45 μm aqueous filter membrane. The filler was DEAE-52 cellulose. The filler was first soaked in distilled water and then acid-washed, then alkaline-washed and loaded into the column, and finally washed with ultrapure water until neutral; ultrapure water was first used to balance the contact surface between the filler and the eluent according to the elution flow rate; ultrapure water and 0.1 M, 0.3 M, and 0.5 M NaCl solutions were used as mobile phases for gradient elution at a flow rate of 1.5 mL / min, 10 mL per tube, and the eluent was collected using an automatic fraction collector. The sugar content of the eluent was determined by the phenol-sulfuric acid method, and the corresponding elution curve was drawn. The eluents of each tube under the same elution peak were combined, dialyzed with distilled water for 3 days, concentrated, and freeze-dried to obtain a polysaccharide sample with a single elution peak; (4) The single elution peak component obtained by DEAE-52 cellulose column chromatography was reconstituted with ultrapure water to a 5 mg / mL polysaccharide sample solution, and passed through a 0.45 μm aqueous filter membrane; the absorbance of each tube was measured at 490 nm by the phenol-sulfuric acid method, and an elution curve was drawn to obtain a unique elution peak, and the eluent concentration was 0.5 M NaCl solution. The eluents collected under the peak were combined, concentrated, dialyzed, and freeze-dried to obtain the separated and purified Tremella polysaccharide TP component; the single elution peak Tremella polysaccharide TP component obtained by DEAE-52 cellulose column chromatography was further eluted with a Sephadex G-200 gel filtration column, and eluted continuously with a 0.5 M NaCl solution at a flow rate of 0.5 mL / min; each tube was collected for 20 minutes; similarly, the single elution peak components were combined and the eluents were collected, dialyzed, and freeze-dried to obtain Tremella polysaccharide.

2. The use according to claim 1, characterized in that The melanoma cells include melanoma cells B16-F10.

3. Use of Tremella polysaccharide as claimed in claim 1 in the preparation of a drug for inhibiting the production of melanin in melanoma cells.

4. The use of Tremella polysaccharide as claimed in claim 1 in the preparation of a drug for inhibiting tyrosinase activity in melanoma cells.

Images