A gold ear polysaccharide and its use in the preparation of pharmaceutical preparations

Abstract

The application discloses a gold ear polysaccharide and application thereof in preparation of a pharmaceutical preparation, wherein the gold ear polysaccharide is simply referred to as TAP-W, the total sugar content is 92.1%, the monosaccharide composition and molar percentage are as follows: mannose:glucuronic acid:glucose:galactose:arabinose = 37.39:6.73:3.57:1:12.22, and the relative molecular weight is 1.57*10 4 Da. Efficacy experiments show that the gold ear polysaccharide TAP-W has excellent tyrosinase inhibitory activity, can significantly reduce the tyrosinase activity in the skin of mice with ultraviolet-induced pigment deposition, and increase the content of skin collagen. The gold ear polysaccharide TAP-W can be used as a new type of natural active substance for whitening, and is widely applied in the fields of whitening and skin care.

Description

Technical Field

[0001] This invention belongs to the field of natural product development, specifically relating to a polysaccharide from *Auricularia auricula-judae* and its application in the preparation of pharmaceutical formulations. Background Technology

[0002] With the development of society and the economy and the improvement of people's living standards, healthy and fair skin has become the pursuit of more and more people. Genetics, air pollution, and ultraviolet radiation can all lead to abnormal production and accumulation of melanin in the skin, inducing skin diseases such as melasma and freckle-like moles, affecting human health and appearance. Existing skin whitening agents mainly achieve whitening effects by inhibiting tyrosinase activity, reducing inflammation, and anti-oxidation, but they all have certain drawbacks. For example, hydroquinone can cause vitiligo and skin cancer; kojic acid has nephrotoxicity and genotoxicity; natural arbutin is chemically unstable and easily converted into hydroquinone; L-ascorbic acid is sensitive to photothermal effects and its effects are not prominent. Some whitening products only involve simple extraction and mixing of raw materials, and their safety, sensitization, and the relationship between the active ingredient's material basis and efficacy urgently need to be clarified. Therefore, natural bioactive substances with clearly defined ingredients, high activity, and no side effects are highly sought after. In recent years, natural active polysaccharides have attracted increasing attention from researchers due to their lack of toxic side effects and broad pharmacological activities. Studies have shown that natural active polysaccharides such as Dendrobium and Camellia polysaccharides have whitening and beautifying effects.

[0003] Golden ear fungus (Tremella aurantialbau) is a rare and highly distinctive edible fungus in my country. Its fruiting body consists of a white or light yellow fibrous core composed of mycelia of *Trichoderma repens* and *Tremella aurantialbau*, surrounded by a golden or orange-yellow hymenium. Compared to silver ear fungus and wood ear fungus, golden ear fungus has a golden-yellow fruiting body, is rich in gelatinous substances, and has a more delicate, elastic, and refreshing texture. It contains abundant polysaccharides, proteins, and trace elements such as sulfur, manganese, iron, and magnesium. The dried product of golden ear fungus contains over 60% polysaccharides, which possess immunomodulatory, antioxidant, and blood sugar-lowering and lipid-reducing activities. Existing reports on the moisturizing and beautifying effects of golden ear fungus polysaccharides are mostly crude polysaccharides or combinations, with unclear functional components. Reports only address water absorption, water retention, and antioxidant properties, without optimizing and screening specific components of golden ear fungus polysaccharides to improve tyrosinase inhibition and reduce melanin content. Summary of the Invention

[0004] Therefore, this invention aims to provide a polysaccharide from *Auricularia auricula-judae* and its application in the preparation of pharmaceutical formulations. This invention utilizes ultrafiltration separation, DEAE-Sepharose Fast Flow ion exchange chromatography, and Superdex chromatography. TMTAP-W, an active polysaccharide component of Auricularia auricula-judae with a specific composition, was prepared by gel filtration chromatography. The results showed that TAP-W has excellent whitening and skin care effects and can be widely used in the fields of whitening, beautifying and skin care by evaluating indicators such as tyrosinase inhibition rate, DPPH free radical scavenging rate, skin tyrosinase activity and skin collagen content.

[0005] The polysaccharide from *Auricularia auricula-judae* of this invention, abbreviated as TAP-W, has a total sugar content of 92.1%. Its monosaccharide composition and molar percentage are mannose:glucuronic acid:glucose:galactose:arabinose = 37.39:6.73:3.57:1:12.22, with a relative molecular weight of 1.57 × 10⁻⁶. 4 Da.

[0006] The polysaccharide from *Auricularia auricula-judae* of this invention is prepared by a method comprising the following steps:

[0007] Step 1: Crush the fruiting bodies of Auricularia auricula-judae, pass them through an 80-mesh sieve, add double-distilled water at a ratio of 1g:80mL and mix well, then sonicate for 30 minutes.

[0008] Step 2: Extract the mixture obtained in Step 1 in a 90℃ water bath for 2 hours. Repeat the extraction 3 times and filter through 8 layers of gauze. Combine the extracts.

[0009] Step 3: Concentrate the extract obtained in Step 2 to 1 / 20 of its original volume, add anhydrous ethanol at a volume ratio of 1:4, stir thoroughly, let stand at 4°C for 12 hours, centrifuge at 6000 rpm for 10 minutes, collect the precipitate, and freeze dry.

[0010] Step 4: The precipitate obtained in Step 3 was subjected to protein removal by the Sevag method, dialysis, and freeze-drying to obtain crude polysaccharide TAP from Auricularia auricula-judae.

[0011] Step 5: The TAP over-retention amount of the crude polysaccharide from *Auricularia auricula-judae* obtained in Step 4 is 5 × 10⁻⁶. 4 Da's ultrafiltration membrane collects molecules with a molecular weight of less than 5 × 10⁻⁶. 4 The sample of Da was then separated and purified using a DEAE-Sepharose Fast Flow ion exchange chromatography column. The sample was eluted with deionized water at a flow rate of 2 mL / min, and the phenol-sulfuric acid method was used for monitoring and detection. The water washing fraction was collected in separate tubes, concentrated, and freeze-dried to obtain the water washing fraction of Auricularia auricula polysaccharide.

[0012] Step 6: Prepare a 100 mg / mL solution of the water-washed fraction of *Auricularia auricula-judae* polysaccharide obtained in Step 5 using Superdex. TMThe solution was further separated and purified using a 75 gel filtration column, eluted with water at a flow rate of 0.3 mL / min, collected in separate tubes, and freeze-dried. The eluent was detected by an Agilent high-performance liquid chromatograph with an evaporative light detector and identified as the homogeneous component of *Auricularia auricula-judae* polysaccharide, TAP-W.

[0013] The golden ear fungus (Tremella aurantialbau (Bandoni & M. Zang)) used in the preparation of this invention was obtained from Hefei Lixin Fungal Farm.

[0014] This invention relates to the application of *Auricularia auricula-judae* polysaccharide in the preparation of pharmaceutical formulations. The pharmaceutical formulation exhibits excellent tyrosinase inhibitory activity and antioxidant activity, effectively reducing melanin deposition and significantly increasing skin collagen content. Therefore, the pharmaceutical formulation not only has significant whitening effects but also effectively improves skin condition.

[0015] This invention is the first to obtain a whitening polysaccharide TAP-W from natural golden ear fungus. It has excellent tyrosinase inhibitory activity. UV-induced pigment deposition mouse experiments showed that this polysaccharide can reduce the activity of tyrosinase in mouse skin, reduce the content of melanin in the skin, and significantly increase the content of collagen in mouse skin. Attached Figure Description

[0016] Figure 1 This is the molecular weight distribution diagram of TAP-W polysaccharide from Auricularia auricula-judae;

[0017] Figure 2 This is a monosaccharide composition diagram of TAP-W polysaccharide from Auricularia auricula-judae; where A is the peak elution diagram of the standard monosaccharide, and B is the peak elution diagram of the monosaccharide of TAP-W (*-solvent peak, 1-mannose, 2-rhamnose, 3-glucuronic acid, 4-galacturonic acid, 5-glucose, 6-galactose, 7-arabinose, 8-fucose).

[0018] Figure 3 The determination of DPPH free radical scavenging activity of TAP-W polysaccharide from Auricularia auricula-judae;

[0019] Figure 4 The assay of tyrosinase inhibitory activity of TAP-W polysaccharide from Auricularia auricula-judae;

[0020] Figure 5 The effect of TAP-W polysaccharide on tyrosinase activity in the skin of mice with pigmentation.

[0021] Figure 6 The effect of TAP-W polysaccharide from Auricularia auricula-judae on melanin content in the skin of mice with pigmentation.

[0022] Figure 7 This study investigated the effect of TAP-W polysaccharide on collagen content in the skin of mice with pigmentation. Detailed Implementation

[0023] The preparation, whitening and skin care effects of the present invention are described below with reference to specific embodiments and accompanying drawings. The listed embodiments are only used to explain the present invention and are not intended to limit the scope of protection of the present invention.

[0024] The golden ear fungus (Tremella aurantialbau (Bandoni & M. Zang)) used in this invention was provided by Hefei Lixin Fungal Culture Farm.

[0025] Example 1: Preparation of TAP-W polysaccharide from Auricularia auricula-judae

[0026] (1) Extraction of polysaccharides from Auricularia auricula-judae

[0027] The fruiting bodies of *Auricularia auricula-judae* were pulverized and passed through an 80-mesh sieve. Double-distilled water was added at a material-to-liquid ratio of 1g:80mL and mixed well. The mixture was ultrasonically treated for 30 minutes and then extracted in a 90℃ water bath for 2 hours. The extraction was repeated 3 times and then filtered through 8 layers of gauze. The extracts were combined and concentrated to 1 / 20 of the original volume. Anhydrous ethanol was added to the concentrate at a volume ratio of 1:4. After thorough stirring, the mixture was allowed to stand at 4℃ for 12 hours and then centrifuged at 6000rpm for 10 minutes. The precipitate was collected and dried.

[0028] (2) Remove protein

[0029] The polysaccharide of Auricularia auricula-judae was prepared into a polysaccharide solution of 20 mg / mL with double-distilled water. An equal volume of Sevag reagent (n-butanol: chloroform = 1:4, V / V) was added, and the mixture was shaken vigorously for 15 min. The supernatant was then collected by centrifugation. The process was repeated until the protein was completely removed. The polysaccharide solution was then concentrated, dialyzed, and freeze-dried to obtain crude polysaccharide TAP from Auricularia auricula-judae.

[0030] (3) Ultrafiltration membrane retention

[0031] The crude polysaccharide TAP from *Auricularia auricula-judae* was prepared as a 100 mg / mL solution, treated with an ultrafiltration membrane with a 50 kDa cutoff, and fractions with molecular weights below 50 kDa were collected, concentrated, and lyophilized.

[0032] (4)DEAE-Sepharose Fast Flow and Superdex TM 75 Chromatographic separation and purification

[0033] Fractions with molecular weights below 50 kDa were reconstituted in double-distilled water to prepare a 20 mg / mL polysaccharide solution. This solution was then purified using a DEAE-Sepharose Fast Flow column on an AKTA Prime plus chromatographic purification system, eluting with deionized water at a flow rate of 2 mL / min. The washed fractions were combined, concentrated, and freeze-dried. Subsequently, the polysaccharide was reconstituted in double-distilled water to prepare a 100 mg / mL polysaccharide solution. This solution was then purified using a Superdex chromatography system on an AKTA Prime plus system. TM A 75 chromatography column was used for elution with water at a flow rate of 0.3 mL / min. The eluent fractions (2 mL / tube) were collected, and the elution curves were plotted using the phenol-sulfuric acid method. Fractions with the same elution peak were combined and lyophilized to obtain TAP-W polysaccharide from *Auricularia auricula-judae*. The total sugar content was determined to be 92.1% by the phenol-sulfuric acid method.

[0034] Example 2: Molecular weight and monosaccharide composition of TAP-W polysaccharide from Auricularia auricula-judae

[0035] (1) Molecular weight determination: TAP-W and dextran standards (T10, T50, T500, T1000, T2000) were prepared into 2 mg / mL solutions with double-distilled water and determined using an Agilent high-performance liquid chromatography-evaporative light scattering detector. The chromatographic column was a TSKGel G6000 PWXL, the mobile phase was deionized water, the carrier gas was N2, the gas flow rate was 2.5 L / min, and the injection volume was 10 μL. A standard curve was plotted using the logarithm of the relative molecular mass (Lg Mw) and retention time (Rt) of the standards, and the molecular weight range of TAP-W was determined. The results are as follows: Figure 1 As shown. Based on the standard curves of dextran with different molecular weights (LgMw=-0.2409x+8.7788, R…),… 2 =0.9989), the molecular weight of TAP-W was calculated to be 15.7 kDa.

[0036] (2) Monosaccharide composition analysis:

[0037] Monosaccharide composition of samples derivatized by TAP-W acid hydrolysis and pre-column PMP (1-phenyl-3-methyl-5-pyrazolone) was determined using an Agilent high-performance liquid chromatography-diode array detector (DAD). Dissolve 8 mg of TAP-W in 5 mL of 2 mol / L trifluoroacetic acid, seal the tube with nitrogen, and incubate in an oil bath at 110 °C for 8 h. Add appropriate amounts of methanol several times and rotary evaporate to pH 7. Add 1 mL of deionized water for later use. Take 1 mL each of 3 mg / mL standard monosaccharide solution and acid-hydrolyzed AT-W solution, and add NaOH (0.5 mL, 0.3 mol / L) and PMP solution (0.5 mL, 0.5 mol / L) sequentially. After shaking and mixing, react in a water bath at 70 °C for 1 h for PMP pre-column derivatization. Then neutralize with HCl (0.5 mL, 0.3 mol / L) to neutral. Subsequently, extract three times with chloroform to remove excess PMP. Filter the aqueous phase through a 0.22 μm filter membrane and detect by HPLC-DAD.

[0038] HPLC-DAD conditions: Zorbox Eclipse XDB-C18 column, column temperature 30℃, wavelength 245nm; mobile phase A was acetonitrile, mobile phase B was 0.05mol / L phosphate buffer solution, gradient elution 0-60min, initial setting mobile phase A:mobile phase B = 17%:83%, final elution ratio was mobile phase A:mobile phase B = 20%:80%, injection volume was 10uL.

[0039] The results are as follows Figure 2 As shown, A is the peak chromatogram of the standard monosaccharide, and B is the peak chromatogram of the TAP-W monosaccharide; from Figure 2 It can be seen from the data that TAP-W is composed of 5 monosaccharides, and the monosaccharide composition and molar percentage are mannose:glucuronic acid:glucose:galactose:arabinose = 37.39:6.73:3.57:1:12.22.

[0040] Example 3: Determination of DPPH free radical scavenging activity of TAP-W polysaccharide from Auricularia auricula-judae

[0041] Preparation of DPPH ethanol solution: Weigh 1 mg DPPH, dissolve it in anhydrous ethanol and dilute to 25 mL in a volumetric flask. Store at 0-4℃ protected from light. Prepare and use immediately. Effective within 4 hours.

[0042] Take 2 mL of samples of different concentrations into test tubes, add 2 mL of DPPH ethanol solution, shake well, let stand at 25℃ for 30 min, and measure the absorbance Aa at 517 nm. At the same time, measure the absorbance A after mixing 2 mL of samples of different concentrations with 2 mL of anhydrous ethanol. b And the absorbance A0 after mixing 2 mL of DPPH ethanol solution with 2 mL of anhydrous ethanol.

[0043] The formula for calculating the DPPH free radical scavenging rate is: Scavenging rate (%) = [1 - (Aa - Ab) / A0] × 100%

[0044] The results are as follows Figure 3 TAP-W polysaccharide from Auricularia auricula-judae has certain antioxidant properties, and its antioxidant properties are better than those of crude polysaccharide TAP from Auricularia auricula-judae. When its concentration is 1 mg / mL, its DPPH free radical scavenging rate is 78.4%.

[0045] Example 4: Determination of the tyrosinase inhibition rate of TAP-W polysaccharide from Auricularia auricula-judae

[0046] Prepare tyrosinase solution (100 U / mL) using pH 6.8 disodium hydrogen phosphate-citrate buffer, and use immediately; prepare levodopa solution (1 mg / mL) using pH 6.8 disodium hydrogen phosphate-citrate buffer, and store protected from light.

[0047] Take 1 mL of samples of different concentrations into test tubes, add 0.5 mL of tyrosinase solution, incubate at 37℃ for 10 min, quickly add 2 mL of levodopa solution, and measure the absorbance T at 475 nm; mix 1 mL of different sample concentrations with 0.5 mL of disodium hydrogen phosphate-citrate buffer, incubate at 37℃ for 10 min, add 2 mL of levodopa solution, and measure the absorbance T0 at 475 nm; mix 1 mL of disodium hydrogen phosphate-citrate buffer with 0.5 mL of tyrosinase solution, incubate at 37℃ for 10 min, add 2 mL of levodopa solution, and measure the absorbance C at 475 nm; measure the absorbance C0 at 475 nm after mixing 1.5 mL of disodium hydrogen phosphate-citrate buffer with 2 mL of levodopa solution.

[0048] The formula for calculating the tyrosinase inhibition rate is: Inhibition rate (%) = [1 - (T - T0) / (C - C0)] × 100%

[0049] See results Figure 4 TAP-W polysaccharide from Auricularia auricula-judae exhibits good tyrosinase inhibitory activity, which is also superior to crude polysaccharide TAP from Auricularia auricula-judae. At a concentration of 1 mg / mL, the tyrosinase inhibition rate can reach 86.7%.

[0050] Example 5: Effects of TAP-W polysaccharide on UV-induced pigmentation in mouse skin

[0051] (1) Grouping and treatment of experimental animals

[0052] After a week of acclimatization, 56 female C57BL / 6 mice were randomly divided into 7 groups (n=8): normal group, pigmentation model group, glutathione positive control group, TAP group, and low, medium, and high dose TAP-W groups. All mice were treated as follows: hair was removed from their backs, and except for the normal group, all mice received UVA (14 J / cm²) three times a week.2 ) and UVB (40mJ / cm 2 The mice were irradiated for 8 weeks. Starting from week 4, the positive control group was administered commercially available Alzinc glutathione whitening pills at 300 mg / kg via gavage, the TAP group was administered 300 mg / kg TAP via gavage, and the low, medium, and high dose TAP-W groups were administered 75 mg / kg, 150 mg / kg, and 300 mg / kg TAP-W via gavage, respectively. The normal and model groups were administered an equal volume of physiological saline via gavage. After the experiment, the mice were euthanized by sodium pentobarbital anesthesia, and dorsal skin tissue was collected. The tyrosinase activity, melanin activity, and type I collagen content in the skin tissue of each group were measured. The obtained data were analyzed for significance using SPSS 27.0 software.

[0053] (2) Experimental Results

[0054] Ultraviolet radiation can trigger a series of molecular and cellular reactions in the skin, manifesting as atrophic wrinkles, loose nodules, and irregular pigmentation. Pigmentation is determined by the melanin content of the epidermis, and tyrosinase is the main catalyst for melanin deposition. Figure 5 , 6 As shown, after ultraviolet irradiation treatment, the skin tyrosinase activity and melanin content of the model group increased significantly, indicating that the pigmentation model was successfully established. After a 4-week intervention treatment, the tyrosinase activity and melanin content of the TAP-W treatment group decreased to a certain extent. Among them, the high-dose TAP-W group showed the most significant decrease in tyrosinase activity and melanin content, which was better than the positive control group and the same dose of TAP intervention. This indicates that TAP-W is a highly active component of Auricularia auricula polysaccharide and has excellent whitening effect.

[0055] Collagen is the main component of the dermis layer of human skin. Collagen I accounts for 85% of the dry weight of skin collagen and is the material basis for maintaining plump and full skin. Figure 7 As shown, ultraviolet irradiation leads to the degradation of Collagen I, while after TAP-W supplementation, the Collagen I content increased significantly in a dose-dependent manner. At low doses, the Collagen I content exceeded that of the positive group, indicating that TAP-W can not only inhibit tyrosinase activity and reduce pigmentation, but also effectively improve skin condition.

[0056] Note: Data values ​​are expressed as mean ± SD. # P<0.05, ## P < 0.01 is compared to the control group. *P < 0.05, **P < 0.01 is compared to the model group. a P<0.05, aa P<0.01 indicates a comparison between the same dose of TAP and TAP-W.

Claims

1. The application of *Auricularia auricula-judae* polysaccharide in the preparation of pharmaceutical formulations, characterized in that: The pharmaceutical preparation has excellent tyrosinase inhibitory activity, can effectively reduce melanin deposition, and significantly increase the collagen content of the skin. The polysaccharide from *Auricularia auricula-judae*, abbreviated as TAP-W, has a total sugar content of 92.1%. Its monosaccharide composition and molar ratio are mannose:glucuronic acid:glucose:galactose:arabinose = 37.39:6.73:3.57:1:12.22, with a relative molecular weight of 1.57 × 10⁻⁶. 4 Da; The polysaccharide from *Auricularia auricula-judae* was prepared by the following steps: Step 1: Crush the fruiting bodies of the golden ear fungus, pass them through an 80-mesh sieve, add double-distilled water and mix well, then sonicate for 30 minutes. Step 2: Extract the mixture obtained in Step 1 in a 90℃ water bath for 2 hours. Repeat the extraction 3 times and filter through 8 layers of gauze. Combine the extracts. Step 3: Concentrate the extract obtained in Step 2 to 1 / 20 of its original volume, add anhydrous ethanol, stir thoroughly, let stand at 4°C for 12 hours, centrifuge and collect the precipitate, and freeze dry. Step 4: The precipitate obtained in Step 3 was subjected to protein removal by the Sevag method, dialysis, and freeze-drying to obtain crude polysaccharide TAP from Auricularia auricula-judae. Step 5: The TAP over-retention amount of the crude polysaccharide from *Auricularia auricula-judae* obtained in Step 4 is 5 × 10⁻⁶. 4 Da's ultrafiltration membrane collects molecules with a molecular weight of less than 5 × 10⁻⁶. 4 The sample of Da was then separated and purified using a DEAE-Sepharose Fast Flow ion exchange chromatography column. The sample was eluted with deionized water, the water wash fraction was collected, concentrated, and freeze-dried to obtain the water wash fraction of Auricularia auricula polysaccharide. Step 6: Prepare a 100 mg / mL solution of the water-washed fraction of *Auricularia auricula-judae* polysaccharide obtained in Step 5 using Superdex. TM The polysaccharide was further separated and purified using a 75 gel filtration column, eluted with water, collected in separate tubes, and freeze-dried to obtain the homogeneous fraction TAP-W of *Auricularia auricula-judae* polysaccharide.

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