A preparation method of degradation product of ganoderma lucidum polysaccharide and its application in cosmetics

Abstract

This invention discloses a method for preparing degradation products of Ganoderma lucidum var. tiger milk polysaccharides and their application in cosmetics, comprising the following steps: S1, extracting Ganoderma lucidum var. tiger milk polysaccharides; S2, preparing an aqueous solution of Ganoderma lucidum var. tiger milk polysaccharides by adding H2O2 and V to the aqueous solution of Ganoderma lucidum var. tiger milk polysaccharides. C The solution was prepared to obtain a mixed solution; S3, the tiger milk Ganoderma lucidum polysaccharide was subjected to ultrasonic degradation treatment using an ultrasonic-assisted H2O2 / Vc system to obtain the tiger milk Ganoderma lucidum polysaccharide degradation product. This invention effectively reduces the molecular weight and improves the water solubility of tiger milk Ganoderma lucidum polysaccharide by using an ultrasonic-assisted H2O2 / Vc system to degrade the polysaccharide. In vitro experiments show that tiger milk Ganoderma lucidum polysaccharide and its degradation products have good hygroscopic and moisturizing properties, antioxidant activity, inhibition of tyrosinase activity, anti-ultraviolet ability, and anti-inflammatory activity, providing a scientific basis for the development of cosmetics with multiple skin care effects. The preparation method of this invention is simple to operate, environmentally friendly, time-saving, and efficient.

Description

Technical Field

[0001] The invention belongs to the technical field of deep processing of edible fungus polysaccharides, and particularly relates to a preparation method of a degradation product of ganoderma lucidum polysaccharide and its application in cosmetics. Background Art

[0002] Lignosus rhinoceros (Cooke) Ryvarden belongs to Eumycota, Basidiomycotin, Hymenomycetes, Aphyllophorales, Polyporaceae. Lignosus rhinoceros is also called tiger milk. Its sclerotium is its medicinal part. It has the effects of relieving cough and asthma, strengthening the spleen and removing dampness. It is commonly used in folk treatment of asthma and cough with excellent efficacy. Polysaccharide is the main bioactive substance of Lignosus rhinoceros, which has the activities of immunomodulation, anti-tumor, anti-inflammatory, and promoting intestinal mucosal wound healing.

[0003] The existing alkaline solution extraction method obtains the polysaccharide of tiger milk ganoderma lucidum with high purity and yield. However, the large molecular weight and low water solubility of the polysaccharide of tiger milk ganoderma lucidum affect its biological activity and application to a certain extent. In order to obtain low molecular weight polysaccharide of tiger milk ganoderma lucidum with ideal biological activity, it needs to be properly degraded.

[0004] Ultrasound is considered to be one of the most effective "green" methods for the degradation of natural polysaccharides. Its degradation mechanism is mainly through the cavitation effect of ultrasound and the active free radicals generated by water or oxygen molecules. However, previous studies have shown that ultrasound is not ideal for the degradation of Ganoderma lucidum polysaccharides, and it can only partially depolymerize the aggregates of Ganoderma lucidum polysaccharides.

[0005] H 2 O 2 As a non-metallic Fenton chemical reaction system, the degradation efficiency of polysaccharides by the catalytic Fenton system is comparable to that of the metal-catalyzed Fenton system, and it is widely used due to its low cost, short time and environmental protection. However, it has not been applied to the degradation of Ganoderma lucidum polysaccharide.

[0006] In recent years, natural extracts have become a hot topic in cosmetic research due to their green, environmentally friendly, high safety, and biodegradability. Polysaccharides are widely available and are potential active ingredients in cosmetics as a macromolecular substance with natural activity. Compared with synthetic cosmetics, polysaccharide cosmetics have better application prospects. At present, hyaluronic acid, chitosan, animal and plant polysaccharides, fungal polysaccharides, etc. have been widely used in the field of cosmetics. However, there have been no reports on the skin care effects of tiger milk ganoderma polysaccharides and their degradation products and their application in the field of cosmetics. Summary of the invention

[0007] In view of the problems existing in the prior art, the present invention aims to provide a method for preparing degradation products of ganoderma lucidum polysaccharide and its application in cosmetics. 2 O 2 The degradation of alkali-extracted Ganoderma lucidum polysaccharide by the / Vc system not only reduces the molecular weight of Ganoderma lucidum polysaccharide and increases the water solubility of Ganoderma lucidum polysaccharide, but also improves the skin care effect of Ganoderma lucidum polysaccharide.

[0008] In order to achieve the above object, the present invention adopts the following technical solutions:

[0009] The first aspect of the present invention provides a method for preparing a degradation product of Ganoderma lucidum polysaccharide, comprising the following steps:

[0010] S1. extracting tiger milk ganoderma polysaccharide from tiger milk ganoderma;

[0011] S2, prepare a water solution of ganoderma lucidum polysaccharide, add H 2 O 2 and V C solution, and mixing to obtain a mixed solution;

[0012] S3, using ultrasound-assisted H 2 O 2 The polysaccharide in the mixed solution was subjected to ultrasonic degradation by the chloranthide / Vc system, and after the treatment, the degradation product of the polysaccharide was obtained by centrifugation, alcohol precipitation, dialysis and freeze-drying.

[0013] Preferably, in step S1, alkaline solution extraction is used to extract the ganoderma lucidum polysaccharide, specifically, 0.1-1.0 mol / L NaOH is used to extract the ganoderma lucidum polysaccharide from the ganoderma lucidum powder.

[0014] Preferably, in step S2, the concentration of the aqueous solution of Ganoderma lucidum polysaccharide is 1-5 mg / mL.

[0015] Preferably, the addition of H 2 O 2 and V C The volume ratio of the solution is 1:1-2:1, so that the H 2 O 2 and V C The final concentration of the solution is 10-50mmol / L.

[0016] Preferably, in step S3, the ultrasonic degradation conditions are: temperature of 4-25° C., ultrasonic power of 100-600 W, and ultrasonic degradation time of 10-60 min.

[0017] Preferably, in step S3, the centrifugation conditions are: 4°C, 6000-10000 r / min, 15-20 min.

[0018] Preferably, in step S3, 95% ethanol is used for the alcohol precipitation, and the amount added is 3-4 times the volume of the supernatant obtained after centrifugation, and the alcohol precipitation is carried out at 4° C. for 8-12 hours.

[0019] Preferably, in step S3, the molecular weight cut-off of the dialysis bag used for dialysis is 6-8 kDa.

[0020] The second aspect of the present invention provides a degradation product of Ganoderma lucidum polysaccharide obtained by the above preparation method.

[0021] The third aspect of the present invention provides a cosmetic comprising ganoderma lucidum polysaccharide or a degradation product of ganoderma lucidum polysaccharide, which has multiple functions of moisturizing, anti-oxidation, whitening, anti-ultraviolet and anti-inflammatory.

[0022] Preferably, the cosmetic is in the form of solvent dispersion or suspension, or in the form of emulsified particles, or in the form of polymer encapsulation.

[0023] Preferably, the cosmetics are skin care cream, skin care lotion, skin care gel, skin care essence, and skin care water.

[0024] Preferably, the cosmetic is a facial mask, eye cream, body lotion or sunscreen.

[0025] The present invention has the following beneficial effects:

[0026] (1) The present invention adopts ultrasound-assisted H 2 O 2 The / Vc system achieves effective degradation of alkali-extracted tiger milk ganoderma lucidum polysaccharide, which is simple to operate, green and environmentally friendly, time-saving and efficient. Compared with tiger milk ganoderma lucidum polysaccharide, the degradation product of tiger milk ganoderma lucidum polysaccharide obtained by this method has significantly lower weight average molecular weight and intrinsic viscosity, and significantly improved water solubility and biological activity.

[0027] (2) In vitro tests showed that both the polysaccharide from tiger milk ganoderma and its degradation products have good moisture absorption and moisturizing properties, antioxidant activity, tyrosinase inhibition, UV resistance, and anti-inflammatory activity, providing a scientific basis for the development of cosmetics based on tiger milk ganoderma polysaccharide with multiple skin care effects.

[0028] (3) The ganoderma lucidum polysaccharide and the degradation products of ganoderma lucidum polysaccharide obtained by the present invention are safe and non-toxic, have significant efficacy, and have broad application development prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0030] Figure 1 The infrared spectra of the ganoderma lucidum polysaccharide and its degradation products prepared in Examples 1-4;

[0031] Figure 2 This is a graph showing the variation of the maximum absorption wavelength of the complex of the ganoderma lucidum polysaccharide and its degradation products obtained from Congo red-Example 1-4 with the concentration of NaOH;

[0032] Figure 3 The results of the analysis of the hygroscopicity and moisture retention of the ganoderma lucidum polysaccharide and its degradation products prepared in Examples 1-4 are shown in FIG.

[0033] Figure 4 The antioxidant activity results of the ganoderma lucidum polysaccharide and its degradation products prepared in Examples 1-4 are as follows: (A) scavenging ability of DPPH free radical; (B) scavenging ability of ABTS free radical;

[0034] Figure 5 This is a graph showing the results of inhibiting tyrosinase activity of the ganoderma lucidum polysaccharide and its degradation products obtained in Examples 1-4;

[0035] Figure 6 The results of the anti-ultraviolet ability of the ganoderma lucidum polysaccharide and its degradation products prepared in Examples 1-4 are shown in FIG.

[0036] Figure 7 The anti-inflammatory activity results of the ganoderma lucidum polysaccharide and its degradation products prepared in Examples 1-4 are as follows: (A) cell viability result graph; (B) phagocytic ability result graph; (C) NO release result graph. DETAILED DESCRIPTION

[0037] In the following description, specific details such as specific system structures, technologies, etc. are provided for the purpose of illustration rather than limitation, so as to provide a thorough understanding of the embodiments of the present invention. However, it should be clear to those skilled in the art that the present invention may be implemented in other embodiments without these specific details. In other cases, detailed descriptions of well-known systems, devices, circuits, and methods are omitted to prevent unnecessary details from obstructing the description of the present invention.

[0038] Example 1

[0039] The polysaccharide of Ganoderma lucidum was extracted from the defatted Ganoderma lucidum powder using 0.5 mol / L NaOH solution.

[0040] Example 2

[0041] (1) extracting the polysaccharide of Ganoderma lucidum from the defatted Ganoderma lucidum powder with 0.5 mol / L NaOH solution;

[0042] (2) Weighing a quantity of Ganoderma lucidum polysaccharide and dissolving it in deionized water to prepare a 2 mg / mL aqueous solution of Ganoderma lucidum polysaccharide;

[0043] (3) Add H at a ratio of 1:1 (v / v) 2 O 2 and V C The solution was added to the aqueous solution of ganoderma lucidum polysaccharide in sequence, so that H 2 O 2 and V C The final concentration of the solution was 20 mmol / L, and the mixture was thoroughly mixed for 2 min to obtain a mixed solution;

[0044] (4) The mixed solution was heated at 4 °C and ultrasonic power was 130 W using ultrasound-assisted H 2 O 2 The polysaccharide from Ganoderma lucidum in the mixed solution was subjected to ultrasonic degradation for 30 min using the / Vc system, and then centrifuged at 4°C and 10000 r / min for 20 min to obtain the supernatant.

[0045] (5) 4 times the volume of supernatant was added with ethanol (95%), and the mixture was allowed to stand at 4°C for 12 h. The mixture was then centrifuged at 4°C and 10,000 r / min for 20 min. The precipitate was redissolved, dialyzed (6-8 kDa) for 48 h, and freeze-dried to obtain the degradation product of Ganoderma lucidum polysaccharide.

[0046] Example 3

[0047] The steps are basically the same as those in Example 2, except that the ultrasonic power is 325 W, and finally the degradation product of Ganoderma lucidum polysaccharide is obtained.

[0048] Example 4

[0049] The steps are basically the same as those in Example 2, except that the ultrasonic power is 520 W, and finally the degradation product of Ganoderma lucidum polysaccharide is obtained.

[0050] Comparative Example 1

[0051] (1) extracting the polysaccharide of Ganoderma lucidum from the defatted Ganoderma lucidum powder with 0.5 mol / L NaOH solution;

[0052] (2) Weighing a quantity of Ganoderma lucidum polysaccharide and dissolving it in deionized water to prepare a 2 mg / mL aqueous solution of Ganoderma lucidum polysaccharide;

[0053] (3) subjecting the polysaccharide aqueous solution of Ganoderma lucidum to ultrasonic degradation at 4° C. and an ultrasonic power of 130 W, and then centrifuging at 4° C. and 10,000 r / min for 20 min to obtain a supernatant;

[0054] (4) 4 times the volume of supernatant was added with ethanol (95%), and the mixture was allowed to stand at 4°C for 12 h. The mixture was then centrifuged at 4°C and 10,000 r / min for 20 min. The precipitate was redissolved, dialyzed (6-8 kDa) for 48 h, and freeze-dried to obtain the degradation product of Ganoderma lucidum polysaccharide.

[0055] Comparative Example 2

[0056] (1) extracting the polysaccharide of Ganoderma lucidum from the defatted Ganoderma lucidum powder with 0.5 mol / L NaOH solution;

[0057] (2) Weighing a quantity of Ganoderma lucidum polysaccharide and dissolving it in deionized water to prepare a 2 mg / mL aqueous solution of Ganoderma lucidum polysaccharide;

[0058] (3) Add H at a ratio of 1:1 (v / v) 2 O 2 and V C The solution was added to the aqueous solution of ganoderma lucidum polysaccharide in sequence, so that H 2 O 2 and V C The final concentration of the solution was 20 mmol / L. The mixture was thoroughly mixed for 2 min and then the mixture was stirred with H 2 O 2 / Vc system was used to degrade the polysaccharide from Ganoderma lucidum for 30 min, and then centrifuged at 4°C and 10000 r / min for 20 min to obtain the supernatant.

[0059] (4) 4 times the volume of supernatant was added with ethanol (95%), and the mixture was allowed to stand at 4°C for 12 h. The mixture was then centrifuged at 4°C and 10,000 r / min for 20 min. The precipitate was redissolved, dialyzed (6-8 kDa) for 48 h, and freeze-dried to obtain the degradation product of Ganoderma lucidum polysaccharide.

[0060] Test and Inspection

[0061] 1. Molecular weight, water solubility and intrinsic viscosity of Ganoderma lucidum polysaccharide and its degradation products

[0062] Certainly

[0063] Determination of (weight average) molecular weight: The (weight average) molecular weight of the ganoderma lucidum polysaccharide and its degradation products obtained in Examples 1-4 was determined using a size exclusion chromatography-multi-angle laser scattering-differential detector combination. The mobile phase was 0.1 mol / L NaCl containing 0.02% Na3 N. The chromatographic columns were Shodex-OHpak SB-806 and SB-805. The mobile phase was used to prepare 1 mg / mL of 1 mg / mL of 1.45 μm aqueous membrane before injection. The injection volume and flow rate were set to 200 μL and 0.5 mL / min, respectively. Astra software (Version 6.1.2.84) was used to collect and analyze data.

[0064] Determination of water solubility: 50 mg of the ganoderma lucidum polysaccharide and the degradation product of ganoderma lucidum polysaccharide prepared in Examples 1-4 were weighed and dissolved in 5 mL of deionized water, stirred at room temperature for 24 h, centrifuged (10000 r / min, 20 min) to remove insoluble solids, and the supernatant was dried at 105° C. to constant weight. Water solubility = (mass of ganoderma lucidum polysaccharide or degradation product of ganoderma lucidum polysaccharide in the supernatant / mass of initial ganoderma lucidum polysaccharide or degradation product of ganoderma lucidum polysaccharide) × 100%.

[0065] Determination of intrinsic viscosity: The intrinsic viscosity of the ganoderma lucidum polysaccharide and its degradation products prepared in Examples 1-4 was measured at 25°C using an Ubbelohde viscometer (inner diameter 0.5-0.6 mm).

[0066] The (weight average) molecular weight, degradation rate, water solubility and intrinsic viscosity of the degradation products of the ganoderma lucidum polysaccharide prepared in Examples 1-4 and Comparative Examples 1-2 and the ganoderma lucidum polysaccharide were measured. The test results are shown in Table 1 below.

[0067] Table 1

[0068] sample Weight average molecular weight (Da) Degradation rate (%) Water solubility (%) Intrinsic viscosity (mL / g) Example 1 <![CDATA[7.838×10 5 ]]> - 18.41±0.54 181.78 Example 2 <![CDATA[5.197×10 5 ]]> 33.69 28.33±0.21 112.98 Example 3 <![CDATA[4.84×10 5 ]]> 38.25 33.53±0.43 90.47 Example 4 <![CDATA[4.326×10 5 ]]> 44.81 38.44±0.28 56.48 Comparative Example 1 <![CDATA[7.298×10 5 ]]> 6.89 22.98±0.26 169.82 Comparative Example 2 <![CDATA[6.86×10 5 ]]> 12.48 25.34±0.32 148.15

[0069] From the results in Table 1 above, it can be seen that compared with Example 1 and Comparative Examples 1-2, the degradation product of the polysaccharide from Ganoderma lucidum obtained in Example 4 has the smallest weight average molecular weight, the highest degradation rate and the best water solubility. This shows that the use of ultrasound-assisted H 2 O 2 The / Vc system can significantly reduce the molecular weight of the polysaccharide from tiger milk ganoderma lucidum, and improve the degradation rate and water solubility of the polysaccharide from tiger milk ganoderma lucidum. Compared with Example 1, the weight average molecular weight of the polysaccharide from comparative examples 1 and 2 is slightly reduced. Ultrasound degrades polysaccharides through cavitation effect and generated free radicals; while H 2 O 2 The hydroxyl radicals can react with Vc to generate hydroxyl radicals and react with hydrogen atoms on the polysaccharide, thereby degrading the polysaccharide. 2 O 2When the / Vc system is used to degrade tiger milk Ganoderma lucidum polysaccharide, ultrasound can disaggregate the tiger milk Ganoderma lucidum polysaccharide aggregates in a short time, making the polysaccharide chains more susceptible to free radical attack. The synergistic effect of the two achieves the purpose of significantly improving the degradation rate of tiger milk Ganoderma lucidum polysaccharide, thereby obtaining a tiger milk Ganoderma lucidum polysaccharide degradation product with a smaller weight average molecular weight.

[0070] The results of Examples 2-4 show that ultrasound-assisted H 2 O 2 The weight-average molecular weight and intrinsic viscosity of the degradation products of Ganoderma lucidum polysaccharide prepared by the treatment with the / Vc system decreased significantly, and the water solubility increased significantly with the increase of ultrasonic power, which indicated that the ultrasonic-assisted H 2 O 2 / Vc system can effectively degrade the polysaccharide of tiger milk ganoderma. Since the biological activity of polysaccharides is related to their water solubility, chemical structure, molecular weight, etc., polysaccharides with smaller molecular weight and lower viscosity are more conducive to their biological activity, so it is speculated that degradation treatment can enhance the biological activity of tiger milk ganoderma polysaccharide.

[0071] 2. Infrared spectroscopy analysis

[0072] The ganoderma lucidum polysaccharide and its degradation products prepared in Examples 1-4 were mixed with KBr at a mass ratio of 1:100, and the wavelengths at 400 cm were recorded using a Nicolet 670 FT-IR spectrometer. -1 ~4000cm -1 The spectrum of the results is shown in Figure 1 .

[0073] Depend on Figure 1 The results show that ultrasound-assisted H 2 O 2 The treatment of the polysaccharide with the Vc system did not change the basic chemical structure of the polysaccharide. -1 There are obvious absorption peaks nearby, indicating that β-D-glucan exists in the polysaccharide of Ganoderma lucidum and its degradation products.

[0074] 3. Congo red test analysis

[0075] The ganoderma lucidum polysaccharide and its degradation products prepared in Examples 1-4 were dissolved in deionized water to prepare a ganoderma lucidum polysaccharide solution and a ganoderma lucidum polysaccharide degradation product solution with a concentration of 2 mg / mL, and then mixed with equal volumes of 91 μmol / l Congo red, and then different volumes of NaOH solution (2 mol / L) were added to obtain a series of Congo red-polysaccharide (ganoderma lucidum polysaccharide and ganoderma lucidum polysaccharide degradation product) complex solutions with final NaOH concentrations of 0 mol / L, 0.05 mol / L, 0.10 mol / L, 0.15 mol / L, 0.20 mol / L, 0.25 mol / L, and 0.30 mol / L. The maximum absorption wavelength in the range of 400 to 700 nm was measured using an ultraviolet spectrophotometer. The measurement results are shown in Figure 2 .

[0076] Depend on Figure 2 The results show that compared with the pure Congo red solution, when the final concentration of NaOH is in the range of 0-0.20 mol / L, the maximum absorption wavelength (λ max ) first slowly increased and then sharply decreased with the increase of NaOH concentration, and a red shift occurred obviously, which indicated that there was an ordered helical structure in the polysaccharide solution of Ganoderma lucidum. When the final concentration of NaOH exceeded 0.20 mol / L, its λ max The results are similar to those of pure Congo red solution, indicating that the ordered structure of the polysaccharide solution of Ganoderma lucidum is completely destroyed under alkaline conditions. 2 O 2 / Vc treated with the degradation product solution of Ganoderma lucidum polysaccharide also undergoes red shift, and λ max The change trend of is similar to that in Example 1, indicating that ultrasound-assisted H 2 O 2 / Vc treatment will not completely destroy the ordered structure of the polysaccharide solution of Ganoderma lucidum, but the degree of red shift will decrease.

[0077] 4. Analysis of moisture absorption and moisture retention

[0078] (a) Determination of hygroscopicity at relative humidity (RH) of 43% and 81%: Accurately weigh 0.1 g of the ganoderma lucidum polysaccharide and its degradation product samples and glycerol obtained in Examples 1-4 and place them in a weighing bottle that has been dried to constant weight. Place the weighing bottle in a dryer with a relative humidity of 43% and 81% respectively with the bottle open. Seal the dryer and place it in a constant temperature box at 20°C. After leaving it for a period of time, take it out and weigh it. Hygroscopicity = (M t -M 0 ) / M 0 ×100%,M t is the mass of the sample after being placed for a period of time (g), M0 is the mass of the sample at the start (g). The results are shown in Figure 3 (A) and (B).

[0079] (b) Determination of moisture retention when relative humidity (RH) is 0: First, place 200g of dried silica gel in a desiccator. Accurately weigh 0.1g of the ganoderma lucidum polysaccharide and its degradation product samples and glycerol obtained in Examples 1-4 and place them in a weighing bottle that has been dried to a constant weight. Add 5mL of distilled water to each bottle and place it in a desiccator. After sealing, place it in a constant temperature box at 20°C, and take it out and weigh it after a period of time. Moisture retention = (M 0 -M t ) / M 0 ×100%,M t is the mass of the sample after being placed for a period of time (g), M 0 is the mass of the sample at the start (g). The results are shown in Figure 3 (C).

[0080] Depend on Figure 3 (A) The results show that when the relative humidity (RH) is 43%, the moisture absorption rate of each sample increases rapidly within the first 12 hours. When the placement time reaches 24 hours, the moisture absorption rate of each sample basically reaches saturation; when the placement time is 48 hours, the moisture absorption performance of each sample is as follows: glycerol> Example 4> Example 3> Example 2> Example 1. There is a certain difference in the moisture absorption rate of different molecular weight tiger milk ganoderma polysaccharides and their degradation products. The lower the molecular weight of the degradation product, the better its moisture absorption performance, which may be due to the ultrasound-assisted H 2 O 2 / Vc treatment destroys the structure of polysaccharides, shortens the long chains of polysaccharides, makes the structure loose, and increases the area in contact with water molecules, which is conducive to the binding of a large number of water molecules.

[0081] Depend on Figure 3 (B) The results show that when the relative humidity (RH) is 81%, the moisture absorption rate of Ganoderma lucidum polysaccharide and its degradation products is similar to that when the relative humidity (RH) is 43%, but both are higher than the water absorption rate when the relative humidity (RH) is 43%. This may be because when the relative humidity (RH) is 81%, there are relatively more water molecules in the air, which causes the moisture absorption rate of each sample to increase faster.

[0082] Depend on Figure 3(C) The results show that under the absolute dry condition of relative humidity RH=0, the moisture retention rate of each sample continued to decrease within 72 hours. The moisture retention rates after 72 hours are as follows: Example 1> Example 2> Glycerin> Example 3> Example 4. There are certain differences in the moisture retention rates of different molecular weights of tiger milk ganoderma polysaccharides and their degradation products. The higher the molecular weight, the better the moisture retention performance. This may be because the higher molecular weight tiger milk ganoderma polysaccharides are conducive to forming a large network structure, making it difficult for the absorbed water molecules to be released, while the degradation products with small molecular weight are easy to lose water due to their loose structure.

[0083] 5. Antioxidant activity analysis

[0084] Determination of DPPH free radical scavenging ability: The ganoderma lucidum polysaccharide and its degradation products prepared in Examples 1-4 were dissolved in deionized water to prepare a series of concentrations (0.5 mg / mL, 1.0 mg / mL, 1.5 mg / mL, 2.0 mg / mL, 2.5 mg / mL) of ganoderma lucidum polysaccharide solutions and ganoderma lucidum polysaccharide degradation product solutions. 2 mL of the series of sample solutions were added with 2 mL of 0.1 mmol / L DPPH anhydrous ethanol solution, mixed, reacted in the dark for 30 minutes, and the absorbance at a wavelength of 517 nm was measured. V C As positive control. Clearance rate = [1-(A 1 -A 2 ) / A 0 ]×100%, where A 1 For sample + DPPH; A 2 A is sample + anhydrous ethanol; 0 is water + DPPH. See the results Figure 4 (A).

[0085] Determination of ABTS free radical scavenging ability: The ganoderma lucidum polysaccharide and its degradation products prepared in Examples 1-4 were dissolved in deionized water to prepare a series of concentrations (0.5 mg / mL, 1.0 mg / mL, 1.5 mg / mL, 2.0 mg / mL, 2.5 mg / mL) of ganoderma lucidum polysaccharide solutions and ganoderma lucidum polysaccharide degradation product solutions. 0.2 mL of the series of sample solutions were added to 2 mL of ABTS working solution respectively, and the reaction was carried out at room temperature in the dark for 10 minutes, and then the absorbance value at a wavelength of 734 nm was measured. VC was used as a positive control. Scavenging rate = [1-(A 1 -A 2 ) / A 0 ]×100%, where A 1 For sample + ABTS; A 2 A is sample + water; 0 is water + ABTS. See the results Figure 4 (B).

[0086] Depend on Figure 4 (A) The results show that, within the range of 0.5-2.5 mg / mL, the different molecular weights of the ganoderma lucidum polysaccharides and their degradation products and Vc prepared in Examples 1-4 have a certain ability to inhibit DPPH free radicals, and the effect of scavenging DPPH free radicals increases with the increasing sample concentration. At the same sample concentration, the order of scavenging DPPH free radicals is: Vc>Example 4>Example 3>Example 2>Example 1.

[0087] Depend on Figure 4 (B) The results show that as the sample concentration increases, its scavenging effect on ABTS free radicals also increases. When the concentration is 2.5 mg / mL, the degradation product solution sample of the ganoderma lucidum polysaccharide prepared in Example 4 has the greatest scavenging ability on ABTS free radicals (55.4%).

[0088] 6. Analysis of tyrosinase inhibition activity

[0089] Tyrosinase, also known as polyphenol oxidase, is a key enzyme in the synthesis of melanin. It can catalyze the hydroxylation of tyrosine into quinones, which are then converted into melanin through a series of reactions. If the tyrosinase activity in the body is high, it will accelerate the production and deposition of melanin, making the skin darker. Therefore, inhibiting the activity of tyrosinase can achieve the effect of skin care and whitening.

[0090] Specific steps: the ganoderma lucidum polysaccharide and its degradation product prepared in Examples 1-4 were dissolved in deionized water to prepare a series of ganoderma lucidum polysaccharide solutions and ganoderma lucidum polysaccharide degradation product solutions with concentrations of 1.0 mg / mL, 2.0 mg / mL, 4.0 mg / mL, 6.0 mg / mL and 8.0 mg / mL, 150 μL of the series of sample solutions (with phosphate buffer as blank) were mixed with 50 μL of 2.5 mmol / L DOPA reaction solution, incubated at 37°C for 5 min, 50 μL of tyrosinase (50 U) was added, reacted at 37°C for 30 min, absorbance was measured at a wavelength of 475 nm, and the tyrosinase activity inhibition rate was calculated, inhibition rate = [1-(CD)] / (AB)] × 100% (A is a mixed solution with tyrosinase added but without sample, B is a mixed solution with tyrosinase added but without sample, C is a mixed solution with sample and tyrosinase, and D is a mixed solution with sample but without tyrosinase). Results Figure 5 .

[0091] Depend on Figure 5The results show that the polysaccharide from tiger milk ganoderma and its degradation products have a significant inhibitory effect on tyrosinase activity within a certain range, and as the sample concentration increases, the inhibition rate of the enzyme activity also increases. Compared with the polysaccharide from tiger milk ganoderma, the inhibition rate of the degradation products of the polysaccharide from tiger milk ganoderma is stronger, which may be related to the enhancement of its antioxidant activity. When the sample concentration is 6 mg / mL, the inhibition rates of the polysaccharide from tiger milk ganoderma and its degradation products prepared in Examples 1-4 are 17.64%, 25.53%, 32.12% and 48.1%, respectively.

[0092] 7. Analysis of UV resistance

[0093] The tiger milk ganoderma lucidum polysaccharide and its degradation product prepared in Examples 1-4 were dissolved in deionized water to prepare a 5 mg / mL tiger milk ganoderma lucidum polysaccharide solution and a tiger milk ganoderma lucidum polysaccharide degradation product solution, and a commercially available JM sunscreen spray of the same mass concentration was used as a control. The transmittance between the absorption wavelengths of 280 and 400 nm was measured using a spectrophotometer. Ultraviolet absorption rate = (1-transmittance) × 100%. The results are shown in Figure 6 .

[0094] Depend on Figure 6 The results show that within the wavelength range of 280-400nm, the corresponding ultraviolet absorption rates of the ganoderma lucidum polysaccharides and their degradation products obtained in Examples 1-4 all decrease as the wavelength increases; among them, the ultraviolet absorption rate of the ganoderma lucidum polysaccharide degradation product obtained in Example 4 is the highest, indicating that it has the best sunscreen effect. The above results show that ganoderma lucidum polysaccharides and their degradation products have a certain anti-ultraviolet effect and can be added to skin care products as sunscreen ingredients.

[0095] 8. Anti-inflammatory activity analysis

[0096] (1) RAW264.7 cell viability test:

[0097] The effects of Ganoderma lucidum polysaccharide and its degradation products and lipopolysaccharide (LPS) on macrophage proliferation activity were determined by MTT method. Ganoderma lucidum polysaccharide and its degradation products prepared in Examples 1-4 were dissolved in deionized water, and a series of Ganoderma lucidum polysaccharide solutions and Ganoderma lucidum polysaccharide degradation product solutions with different concentrations (100 μg / mL, 200 μg / mL, 400 μg / mL, 800 μg / mL) were prepared as sample solutions for later use; RAW264.7 cells in the logarithmic growth phase were taken and the cell concentration was adjusted to 1×10 5 100 μL was added to each well of a 96-well cell culture plate and incubated at 37°C with 5% CO 2 Under the same conditions, the cells were cultured overnight for 12 h, and 200 μL of the corresponding culture medium was added to the blank group, LPS group, and sample group. 37°C, 5% CO 2After 24 hours of culture, the culture medium was discarded, 150 μL of MTT solution was added to each well, and after 2 hours of reaction, 200 μL of DMSO was added to each well. The cells were shaken for 5 minutes using a microplate reader, and the absorbance was measured at a wavelength of 490 nm. Cell viability = absorbance of the treatment group / absorbance of the control group × 100%. The results are shown in Figure 7 (A).

[0098] (2) Detection of phagocytic ability of RAW264.7 cells:

[0099] Macrophages in the logarithmic growth phase were cultured at a rate of 1×10 4 The cells were inoculated at a density of 100 μg / mL / well in a 96-well plate, and cultured for 24 hours according to the experimental groups. Then, 1 mL of sample solution of different concentrations (the polysaccharide of tiger milk ganoderma and its degradation products prepared in Examples 1-4 were dissolved in deionized water to prepare a series of solutions of polysaccharide of tiger milk ganoderma and solutions of degradation products of polysaccharide of tiger milk ganoderma) of different concentrations (100 μg / mL, 200 μg / mL, 400 μg / mL, 800 μg / mL) as sample solutions) was added for pretreatment for 2 hours, and then 100 μL of LPS solution (1 μg / mL) was added for treatment for 24 hours. The supernatant was removed, and 100 μL of neutral red (0.1%) was added to each well. After incubation for 2 hours, the neutral red was discarded, the cells were washed 3 times with PBS solution, and 200 μL of cell lysis solution was added. The cells were lysed at room temperature for 30 minutes, and the microplate reader was oscillated for 5 minutes, and the absorbance value was measured at 540 nm. The results are shown in Figure 7 (B).

[0100] (3) Determination of NO content in RAW264.7 cells:

[0101] The increase of NO content is one of the signs of inflammatory response in RAW 264.7 cells. 0.5 mL of 10 × 10 5 / mL RAW 264.7 cells were incubated in an incubator for 24 hours, and then the culture medium was removed. Then, 1mL of sample solutions of different concentrations (the tiger milk ganoderma polysaccharide and its degradation products prepared in Examples 1-4 were dissolved in deionized water to prepare a series of tiger milk ganoderma polysaccharide solutions and tiger milk ganoderma polysaccharide degradation product solutions of different concentrations (100μg / mL, 200μg / mL, 400μg / mL, 800μg / mL) as sample solutions) were added for pretreatment for 2 hours, and then 100μL LPS solution (1μg / mL) was added for treatment for 24 hours. At the same time, a blank group (only culture medium) and a control group (only LPS) were set up. After the culture was completed, the supernatant was collected, and the NO content in the cell culture medium of each group was estimated according to the instructions of the NO kit. The results are shown in Figure 7 (C).

[0102] Depend on Figure 7(A) The results show that within the experimental concentration range (100-800 μg / mL), Examples 1-4 and LPS have no cytotoxicity. Compared with the blank control group, both can effectively promote the proliferation of macrophages.

[0103] from Figure 7 (B) The results show that LPS can significantly enhance the phagocytic ability of macrophages compared with the blank control group. After adding the ganoderma lucidum polysaccharide and its degradation products prepared in Examples 1-4, the phagocytic ability of macrophages showed different changes. The phagocytic ability of macrophages induced by LPS in Examples 1 and 2 first increased and then decreased with the increase of concentration, while the phagocytic ability of macrophages induced by LPS in Examples 3 and 4 decreased with the increase of concentration, but both were higher than the blank control group.

[0104] Depend on Figure 7 (C) The results show that LPS induces macrophages to secrete a large amount of NO. Within the experimental concentration range (100-800 μg / mL), the addition of the ganoderma lucidum polysaccharide and its degradation products prepared in Examples 1-4 can significantly inhibit the production of NO induced by LPS, and the inhibitory effect on NO release is concentration-dependent. When the concentration is 800 μg / mL, the ganoderma lucidum polysaccharide and its degradation products prepared in Examples 1-4 have LPS-induced NO release amounts of 8.64 μmol / L, 7.94 μmol / L, 6.31 μmol / L, and 5.88 μmol / L, respectively. Compared with Example 1, Example 4 significantly reduces the LPS-induced NO release amount, which indicates that the reduction in molecular weight can effectively improve the anti-inflammatory activity of ganoderma lucidum polysaccharide.

[0105] The present invention is not limited to the above-mentioned specific implementation modes. Various changes made by ordinary technicians in this field based on the above-mentioned concepts without creative work are all within the protection scope of the present invention.

Claims

1. A method for preparing a degradation product of Ganoderma lucidum polysaccharide, characterized in that: The following steps are involved: S1. extracting tiger milk ganoderma polysaccharide from tiger milk ganoderma; In step S1, the alkaline solution extraction method is used to extract the ganoderma lucidum polysaccharide, specifically, 0.1-1.0 mol / L NaOH is used to extract the ganoderma lucidum polysaccharide from the ganoderma lucidum powder; S2, prepare a water solution of ganoderma lucidum polysaccharide, and add H2O2 and V to the water solution of ganoderma lucidum polysaccharide in sequence C solution, and mixing to obtain a mixed solution; S3, using ultrasound-assisted H2O2 / Vc system to perform ultrasonic degradation on the polysaccharide in the mixed solution, and after the treatment, centrifugation, alcohol precipitation, dialysis, and freeze-drying are performed to obtain a degradation product of the polysaccharide; In step S2, the concentration of the polysaccharide aqueous solution of Ganoderma lucidum is 1-5 mg / mL; the addition of H2O2 and V C The volume ratio of the solution is 1:1-2:1, so that the H2O2 and V C The final concentration of the solution is 10-50 mmol / L; The ultrasonic degradation conditions are as follows: temperature is 4-25° C., ultrasonic power is 100-600 W, and ultrasonic degradation time is 10-60 min.

2. The method for preparing the degradation product of Ganoderma lucidum polysaccharide according to claim 1, characterized in that: In step S3, the centrifugation conditions are: 4°C, 6000-10000 r / min, 15-20 min; 95% ethanol is used for the alcohol precipitation, and the added amount is 3-4 times the volume of the supernatant obtained after centrifugation, and the alcohol precipitation is carried out at 4°C for 8-12 h; the molecular weight cutoff of the dialysis bag used for dialysis is 6-8 kDa.

3. The degradation product of Ganoderma lucidum polysaccharide obtained by the preparation method as claimed in claim 1 or 2.

4. A cosmetic, characterized in that: The cosmetics contain the degradation product of the ganoderma lucidum polysaccharide according to claim 3, and have multiple functions of moisturizing, anti-oxidation, whitening, anti-ultraviolet and anti-inflammatory.

5. The cosmetic according to claim 4, characterized in that: The cosmetic is in the form of solvent dispersion or suspension, or in the form of emulsified particles, or in the form of polymer encapsulation.

6. The cosmetic according to claim 4, characterized in that: The cosmetics are skin care cream, skin care lotion, skin care gel, skin care essence and skin care water.

7. The cosmetic according to claim 4, characterized in that: The cosmetics are facial masks, eye creams, body lotions or sunscreen lotions.

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