CN108743433B

CN108743433B - Skin care lotion containing water-soluble crude polysaccharide of Lasiosphaera gigantea and alcohol extract of Lasiosphaera gigantea and preparation method thereof

Info

Publication number: CN108743433B
Application number: CN201810952822.8A
Authority: CN
Inventors: 俞苓; 薛儒康; 黄怡雯; 蔡梦婷; 刘新琦; 何先喆; 刘洁纯
Original assignee: Shanghai Institute of Technology
Current assignee: Shanghai Institute of Technology
Priority date: 2018-08-21
Filing date: 2018-08-21
Publication date: 2021-08-03
Anticipated expiration: 2038-08-21
Also published as: CN108743433A

Abstract

The invention discloses a skin care cream containing bald puffball water-soluble crude polysaccharide and bald puffball alcohol extract and a preparation method thereof, wherein the raw materials used for preparation comprise, by mass percent, 3.00-10.00% of emulsifier, 0.20-3.00% of cetostearyl alcohol, 2.00-20.00% of liquid grease, 0.01-1.00% of 2, 6-di-tert-butyl-4-methylphenol, 0.10-3.00% of xanthan gum, 0.01-10.00% of bald puffball water-soluble crude polysaccharide, 5.00-15.00% of absolute ethyl alcohol, 0.01-10.00% of bald puffball alcohol extract, 0.10-1.00% of preservative and the balance of deionized water; the preparation method comprises the steps of firstly preparing an oil phase, a water phase and an alcohol phase, then adding the oil phase into the water phase, adding the alcohol phase into the water phase, adding the preservative into the mixed phase, and uniformly stirring to obtain the skin care emulsion with strong moisturizing, antioxidant and anti-inflammatory effects.

Description

Skin care lotion containing water-soluble crude polysaccharide of Lasiosphaera gigantea and alcohol extract of Lasiosphaera gigantea and preparation method thereof

Technical Field

The invention relates to a skin care lotion containing water-soluble crude polysaccharide of Lasiosphaera gigantea and alcohol extract of Lasiosphaera gigantea and a preparation method thereof, belonging to the field of cosmetics.

Background

Lasiosphaera gigantea belonging to Abdominal class, order of Lasiosphaera, family of Lasiosphaera, genus Lasiosphaera, widely distributed around the world, and produced mainly in provinces such as inner Mongolia, Liaoning, Anhui, Gansu, Jiangsu, Yunnan, etc. in China. The Lasiosphaera gigantea is a large fungus used as both medicine and food, and when young, the fruiting body is white inside and outside, so that the Lasiosphaera gigantea can be eaten and is not used as a traditional Chinese medicine. With the growth of the Lasiosphaera gigantea, the fruiting body grows and the internal color changes into light brown, at the moment, the Lasiosphaera gigantea is mature, generally does not eat, is mainly used as a traditional Chinese medicine, and has the effects of reducing swelling, stopping bleeding, detoxifying and the like after entering lung channels according to records of famous medical records.

In addition, the separation and purification of polysaccharide from Lasiosphaera Seu Calvatia and its biological effectiveness record that Lasiosphaera Seu Calvatia sporophore in mature period contains bioactive substances such as polysaccharide, Lasiosphaera Seu Calvatia extract, and Lasiosphaera Seu Calvatia acid. Optimization of fermentation conditions and bacteriostasis of Lasiosphaera Calvatia mycelium polysaccharide (0254-5071(2016)08-0120-04) records that Lasiosphaera Calvatia in mature period contains various bioactive substances such as amino acids, ergosterol, lipid, Lasiosphaera Calvatia, ascorbic acid, inorganic salts and the like.

At present, the Lasiosphaera gigantea extract, and the preparation method and the application thereof are related to the following documents: in the analysis of the antibacterial activity of the Lasiosphaera gigantea fruiting body extract in different maturation periods and the volatile oil component thereof, the Lasiosphaera gigantea fruiting body volatile oil is extracted by adopting a steam distillation method, and the chloroform extract of the Lasiosphaera gigantea with stronger inhibition effect on escherichia coli ATCC8099 and staphylococcus aureus ATCC6538 is proved to be the chloroform extract of the Lasiosphaera gigantea. In the separation and purification of the puffball polysaccharide and certain biological effects thereof, the puffball polysaccharide in the mature period is prepared by a boiling water bath extraction-ethanol precipitation method, and then the single polysaccharide is obtained by separation and purification, and is proved to have the functions of resisting inflammation and easing pain. In the fermentation condition optimization and bacteriostasis of Calvatia gigantea mycelium polysaccharide, the mycelium is leached by hot water to obtain the Calvatia gigantea mycelium crude polysaccharide, and experiments prove that the Calvatia gigantea mycelium crude polysaccharide has obvious inhibition effect on gram-positive bacteria. Liver injury protection effect and in-vitro antioxidant activity of Lasiosphaera Seu Calvatia liquid fermentation mycelium crude polysaccharide₄Induction ofThe Calomelas has certain protection effect on acute liver injury of mice, and the Calomelas fir mycelium polysaccharide has the effects of eliminating DPPH free radical, superoxide anion free radical and hydroxyl free radical, and has certain antioxidation effect.

At present, only one of fat-soluble extract or water-soluble extract can be obtained by the extraction method of the Lasiosphaera gigantea, so that bioactive substances cannot be fully extracted, resource waste is caused, and systematic research and comparison on the bioactivity of the Lasiosphaera gigantea fat-soluble extract and the water-soluble extract are not available. The extraction rate of crude polysaccharide of fruiting bodies of other puffball varieties in puffball family is below 4%, and the optimal extraction rate is reported without optimization of extraction conditions when crude polysaccharide is extracted by using puffball fruiting bodies as raw materials. The extraction of the Lasiosphaera gigantea alcohol extract is not reported.

At present, Lasiosphaera gigantea is used as a medicine, is mainly applied to trauma hemostasis, detumescence and analgesia by external application, can be used as a heat-clearing and detoxifying medicine by internal application, can be used for treating tonsillitis, laryngitis and cough, and has the efficacies of clearing lung, inhibiting tumor and inhibiting bacteria. No reports on the application of Lasiosphaera gigantea in the cosmetic field are found.

Disclosure of Invention

The invention aims to provide the skin care cream containing the water-soluble crude polysaccharide of the Lasiosphaera gigantea and the alcohol extract of the Lasiosphaera gigantea, and the skin care cream containing the water-soluble crude polysaccharide of the Lasiosphaera gigantea and the alcohol extract of the Lasiosphaera gigantea has the advantages of strong moisture retention, oxidation resistance, inflammation resistance and the like because of containing the alcohol extract of the Lasiosphaera gigantea and the water-soluble crude polysaccharide of the Lasiosphaera gigantea.

The invention also aims to provide a preparation method of the skin care cream containing the crude water-soluble polysaccharide of the Lasiosphaera gigantea and the alcohol extract of the Lasiosphaera gigantea, and the method enables the crude water-soluble polysaccharide of the Lasiosphaera gigantea and the alcohol extract of the Lasiosphaera gigantea to be well fused with cosmetic matrix raw materials, so that the system is uniform and stable.

The invention also aims to provide the crude water-soluble polysaccharide of the puffball and the alcohol extract of the puffball, which are used in the skin care milk containing the crude water-soluble polysaccharide of the puffball and the alcohol extract of the puffball.

The invention also aims to provide a preparation method of the crude water-soluble polysaccharide of the Lasiosphaera gigantea, which solves the defect that the prior extraction method of the Lasiosphaera gigantea can only obtain one of fat-soluble extracts or water-soluble extracts, so that bioactive substances cannot be fully extracted, and resources are wasted. The yield of the Lasiosphaera gigantea water-soluble crude polysaccharide is 12.62 percent, is more than 200 percent higher than that of the Lasiosphaera gigantea crude polysaccharide reported at present, has the moisture retention, oxidation resistance and anti-inflammatory activity, the yield of the Lasiosphaera gigantea alcohol extract is 14.05 percent, the experiment proves that the Lasiosphaera gigantea alcohol extract also has the moisture retention and oxidation resistance activity, and the content of flavone in the alcohol extract is 5.35 percent. Therefore, the bioactive substances in the Lasiosphaera gigantea are fully extracted, so that the Lasiosphaera gigantea resource can be fully utilized.

The fifth purpose of the present invention is to provide a preparation method of the Lasiosphaera gigantea alcohol extract, which solves the problem that the prior extraction method of Lasiosphaera gigantea can only obtain one of fat-soluble extract and water-soluble extract, so that bioactive substances cannot be fully extracted, and resource waste is caused. The yield of the Lasiosphaera gigantea water-soluble crude polysaccharide is 12.62 percent, is more than 200 percent higher than that of the Lasiosphaera gigantea crude polysaccharide reported at present, has the moisture retention, oxidation resistance and anti-inflammatory activity, the yield of the Lasiosphaera gigantea alcohol extract is 14.05 percent, the experiment proves that the Lasiosphaera gigantea alcohol extract also has the moisture retention and oxidation resistance activity, and the content of flavone in the alcohol extract is 5.35 percent. Therefore, the bioactive substances in the Lasiosphaera gigantea are fully extracted, so that the Lasiosphaera gigantea resource can be fully utilized.

Technical principle of the invention

The Lasiosphaera gigantea contains bioactive substances such as polysaccharide, flavone, amino acid, ergosterol, lipidoid, Lasiosphaera gigantea acid, ascorbic acid, inorganic salt and the like, and the active extract is applied to skin care products to fully play the moisturizing, antioxidant and anti-inflammatory properties of the active substances such as polysaccharide, flavone and the like.

Technical scheme of the invention

A skin care lotion containing water-soluble crude polysaccharide of Lasiosphaera gigantea and alcohol extract of Lasiosphaera gigantea is prepared from the following raw materials in percentage by mass:

the skin care lotion containing the water-soluble crude polysaccharide of the Lasiosphaera gigantea and the alcohol extract of the Lasiosphaera gigantea is prepared from the following raw materials in percentage by mass:

the emulsifier consists of stearyl alcohol polyoxyethylene ether-2 and stearyl alcohol polyoxyethylene ether-21 according to the mass ratio of 4: 5;

the liquid grease is composed of dimethyl silicone oil, caprylic/capric triglyceride and white oil according to the mass ratio of 6:8: 7;

the preservative is composed of caprylyl glycol, ethylhexyl glycerin and phenoxyethanol according to the mass ratio of 1:1: 4.

The Lasiosphaera gigantea alcohol extract is prepared by the following steps:

(1) drying Lasiosphaera gigantea with hot air at 45-60 ℃, and pulverizing with a pulverizer to obtain Lasiosphaera gigantea powder of 10 meshes;

(2) and (2) taking the Lasiosphaera gigantea powder obtained in the step (1), and mixing the Lasiosphaera gigantea powder: the absolute ethyl alcohol is 1 g: adding 5-40 mL of Lasiosphaera Calvatia powder into absolute ethyl alcohol, heating and refluxing at 60-95 ℃ for ethanol extraction for 1-3 times, and each time for 0.5-3 hours to obtain an ethanol extract;

(3) carrying out reduced pressure suction filtration on the alcohol extract obtained in the step (2) under the control of the pressure of 0.070-0.090MPa, collecting the obtained filtrate, carrying out rotary evaporation and concentration on the obtained filtrate under the pressure of 0.092-0.098MPa at the temperature of 45-50 ℃, and drying at the temperature of 60 ℃ to obtain the Lasiosphaera gigantea alcohol extract;

the bald puffball water-soluble crude polysaccharide is prepared by the following steps:

(3) controlling the pressure of the alcohol extract obtained in the step (2) to be 0.070-0.090MPa, performing reduced pressure suction filtration, and collecting the obtained filter residue;

(4) and (3) filtering residues obtained in the step (3) according to the filtering residues: disodium hydrogen phosphate-citric acid buffer at pH 4-6 was 1 g: adding the filter residue into a disodium hydrogen phosphate-citric acid buffer solution with the pH value of 4-6 according to the proportion of 30-80mL to obtain a filter residue solution;

(5) adding degrading enzyme into the filter residue solution obtained in the step (4), and controlling the temperature to be 25-60 ℃ to carry out enzymolysis reaction for 20-120min to obtain an enzymolysis solution;

the degrading enzyme is cellulase, pectinase or mixed enzyme formed by mixing cellulase and pectinase according to a certain proportion, and the addition amount is calculated according to the mass ratio, namely the degrading enzyme: the bald puffball fungus powder used for preparing the filter residue solution is 0.1-10: 100, respectively;

the activity of the cellulase is 10000-50000U/g, and the activity of the pectinase is 5000-30000U/g;

(6) heating the enzymolysis liquid obtained in the step (5) to 80-100 ℃, then preserving heat for 1-5h, and carrying out hot water leaching, wherein the obtained extraction liquid is subjected to vacuum filtration at the control pressure of 0.070-0.090MPa, and the obtained filtrate is the Lasiosphaera gigantea leaching liquor;

(7) carrying out rotary evaporation concentration on the Calvatia gigantea leaching liquor obtained in the step (6) at the temperature of 75 ℃ until the volume of the Calvatia gigantea leaching liquor is 1/5 of the volume of the Calvatia gigantea leaching liquor to obtain a concentrate, then adding an ethanol water solution with the volume percentage concentration of 60-90% which is 3-5 times of the volume of the Calvatia gigantea leaching liquor into the concentrate, carrying out alcohol precipitation for 3-10h at the temperature of 4-10 ℃, and collecting the obtained precipitate;

(8) and (4) controlling the temperature of the precipitate obtained in the step (7) to be-55 ℃ and carrying out freeze drying to obtain the Lasiosphaera gigantea water-soluble crude polysaccharide.

The preparation method of the skin care cream containing the Lasiosphaera gigantea extract specifically comprises the following steps:

(1) sequentially adding an emulsifier, hexadecanol, liquid oil and 2, 6-di-tert-butyl-4-methylphenol into an oil phase pot, heating to 75-95 ℃, controlling the rotation speed to be 300-;

(2) sequentially adding xanthan gum, the water-soluble crude polysaccharide of the Lasiosphaera Calvatia and deionized water into a water phase pot, heating to 75-95 ℃, controlling the rotation speed to be 300-700rpm, and uniformly stirring to obtain a water phase;

(3) sequentially adding absolute ethyl alcohol and the Lasiosphaera gigantea alcohol extract into a premixing pot, heating to 25-50 ℃, controlling the rotation speed to be 300-700rpm, and stirring and uniformly mixing to obtain an alcohol phase;

(4) under the condition that the rotating speed is 300-700rpm, the dropping speed is controlled to be 120-190mL/min, the oil phase obtained in the step (1) is gradually added into the water phase obtained in the step (2), after the addition is finished, the rotating speed is controlled to be 10000-30000rpm, homogenization is carried out for 2-5min, then the alcohol phase obtained in the step (3) is added, and a mixed phase is obtained after uniform mixing;

(5) and (3) controlling the rotation speed of the mixed phase obtained in the step (4) to be 300-95 ℃ at 700rpm, stirring for 10-40min, then controlling the rotation speed to be 300-95 ℃ at 700rpm, naturally cooling to 45-60 ℃, adding a preservative, and continuing controlling the rotation speed to be 300-700rpm, stirring for 15-50min to obtain the skin care emulsion containing the water-soluble crude polysaccharide of the Lasiosphaera gigantea and the alcohol extract of the Lasiosphaera gigantea.

Furthermore, the preparation method of the skin care cream containing the crude water-soluble polysaccharide of the Lasiosphaera gigantea and the alcohol extract of the Lasiosphaera gigantea is suitable for preparing other cosmetics of the skin care cream containing the crude water-soluble polysaccharide of the Lasiosphaera gigantea and the alcohol extract of the Lasiosphaera gigantea, such as skin care cream containing the crude water-soluble polysaccharide of the Lasiosphaera gigantea and the alcohol extract of the Lasiosphaera gigantea and the like.

The invention has the beneficial technical effects

The comprehensive extraction method of Lasiosphaera Calvatia improves the effective utilization rate of active ingredients of Lasiosphaera Calvatia, and skin care product containing the extract has effects of keeping moisture, resisting oxidation and resisting inflammation.

The invention relates to a skin care cream containing crude water-soluble polysaccharide of Lasiosphaera gigantea and alcohol extract of Lasiosphaera gigantea, because the crude water-soluble polysaccharide of Lasiosphaera gigantea used as a raw material has the activities of moisturizing, oxidation resistance and inflammation resistance, the finally obtained skin care cream containing the extract of Lasiosphaera gigantea has the advantages of moisturizing, oxidation resistance and inflammation resistance, furthermore, because the alcohol extract of Lasiosphaera gigantea used as a raw material has the activities of moisturizing and oxidation resistance, the finally obtained skin care cream containing the extract of Lasiosphaera gigantea has the advantages of moisturizing and oxidation resistance, and the characteristics of moisturizing, oxidation resistance, inflammation resistance and the like of the skin care cream containing the crude water-soluble polysaccharide of Lasiosphaera gigantea and the alcohol extract of Lasiosphaera gigantea are not the simple superposition of the characteristics of moisturizing, oxidation resistance, inflammation resistance and alcohol extract of Lasiosphaera gigantea used as the crude water-soluble polysaccharide of Lasiosphaera gigantea used, due to the synergistic effect of the crude water-soluble polysaccharide of the Lasiosphaera gigantea, the alcohol extract of the Lasiosphaera gigantea and other components in the skin care cream, such as xanthan gum, caprylic/capric triglyceride, cetostearyl alcohol and the like, the finally obtained skin care cream containing the crude water-soluble polysaccharide of the Lasiosphaera gigantea and the alcohol extract of the Lasiosphaera gigantea has the advantages of better moisture retention, oxidation resistance, inflammation resistance and the like.

Furthermore, the preparation method of the skin care cream containing the crude water-soluble polysaccharide of the Lasiosphaera gigantea and the alcohol extract of the Lasiosphaera gigantea has strong operability and mild reaction conditions in the preparation process, and can well fuse the crude water-soluble polysaccharide of the Lasiosphaera gigantea and the alcohol extract of the Lasiosphaera gigantea with the base materials of cosmetics, so that the system is uniform and stable, and has better industrial application prospect.

Drawings

FIG. 1 is a graph showing the ability of alcohol extracts of Lasiosphaera gigantea to scavenge hydroxyl radicals, and the water-soluble crude polysaccharide of Lasiosphaera gigantea obtained in example 3;

FIG. 2 is a graph showing the ability of the water-soluble crude polysaccharide of Lasiosphaera gigantea and the alcohol extract of Lasiosphaera gigantea to scavenge hydrogen peroxide obtained in example 3;

FIG. 3 shows a reduction map of the water-soluble crude polysaccharide and alcohol extract of Lasiosphaera gigantea obtained in example 3;

FIG. 4 is a graph showing the ability of the crude water-soluble polysaccharide of Lasiosphaera gigantea obtained in example 3 to scavenge DPPH free radicals;

FIG. 5 is a graph showing the anti-inflammatory activity of the crude water-soluble polysaccharide of Lasiosphaera gigantea obtained in example 3;

fig. 6 and a comparison graph of the moisture retention of the skin care cream containing the water-soluble crude polysaccharide of the bald puffball and the alcohol extract of the bald puffball obtained in example 3 with the skin care cream containing the water-soluble crude polysaccharide of the bald puffball alone, the skin care cream containing the alcohol extract of the bald puffball alone and a pure glycerin sample.

Detailed Description

The invention is further illustrated, but not limited, by the following specific examples in connection with the accompanying drawings.

The content determination methods of the water-soluble crude polysaccharide of the Calvatia gigantea and the content determination methods of the total flavone, the total phenol, the total amino acid, the polypeptide and the protein in the alcohol extract of the Calvatia gigantea obtained in the embodiments of the invention are respectively as follows:

the method for measuring the content of the total flavonoids comprises the following steps:

taking 25mg of rutin standard product dried at 120 ℃ to constant weight, adding 200mL of distilled water, ultrasonically dissolving, and then fixing the volume to 250mL to prepare 0.1mg/mL solution. Precisely measuring rutin

standard substance solution

0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10mL, respectively placing into 25mL volumetric flasks, adding 5% NaNO respectively₂Shaking the solution 1mL, standing for 6min, and adding 10% Al (NO)₃)₃Shaking the solution 1mL, standing for 6min, adding 4% NaOH solution 10.0mL, shaking, adding distilled water to constant volume to scale mark, shaking, standing for 15min, and measuring absorbance at 510nm with the solution without rutin standard as blank. Each sample was assayed in triplicate and the average was taken. With reedAnd (3) drawing a standard curve by taking the butyl mass (mg) as an abscissa and the absorbance (A) as an ordinate, and performing regression on the absorbance by using the mass to obtain a regression equation.

Sucking 1mL of sample liquid with proper concentration, placing the sample liquid in a 25mL volumetric flask, measuring the absorbance value according to the steps, and calculating the content of the total flavonoids according to a standard curve. Each sample was assayed in triplicate and the average was taken.

The method for measuring the total phenol content comprises the following steps:

weighing 0.020g of gallic acid, diluting to 100mL with distilled water to obtain 200 μ g/mL gallic acid solution, respectively placing 0.1, 0.2, 0.3, 0.4, 0.5mL gallic acid solution in 25mL volumetric flask, respectively adding 5mL of distilled water, and shaking. Respectively adding 0.8mL of Folin phenol color developing agent, shaking up, respectively adding 1mL of 20% Na2C03 solution, shaking up, fixing the volume to 25mL with distilled water, shaking up, placing in a constant temperature water bath kettle at 55 ℃ for color development for 1.5h, after color development, taking a sample without gallic acid as a blank, and measuring the absorbance at the wavelength of 760 nm. Each sample was assayed in triplicate and the average was taken. And (3) drawing a standard curve by taking the mass (mug) of the gallic acid as a horizontal coordinate and the absorbance (A) as a vertical coordinate, and regressing the absorbance by the mass to obtain a regression equation.

0.1mL of sample solution with an appropriate concentration was aspirated, the solution was placed in a 25mL volumetric flask, the absorbance value was measured according to the above procedure, and the total phenol content was calculated from the standard curve. Each sample was assayed in triplicate and the average was taken.

The method for measuring the content of the total amino acid comprises the following steps:

the method is used for determining the content of amino acid in honey and high fructose corn syrup by adopting an indetrione method (1009) -7848(2013) 02-0171-06).

The method for measuring the content of the polypeptide comprises the following steps:

adopts the method used in the research on the antioxidant activity, the molecular weight and the amino acid composition characteristics of the walnut polypeptide (1002-jar 0306(2018) 13-0001-08).

The method for measuring the protein content comprises the following steps:

the method used in the method for measuring the content of the protein in the stauntonia chinensis polysaccharide by the Coomassie brilliant blue method is adopted.

The preparation method of the disodium hydrogen phosphate-citric acid buffer solution with the pH value of 4-6 comprises the following steps:

preparing 0.2mol/L Na₂HPO₄And (3) adjusting the addition ratio of the solution and 0.1mol/L citric acid solution to obtain a disodium hydrogen phosphate-citric acid buffer solution with the pH value of 4-6.

Example 1

The Lasiosphaera gigantea alcohol extract is prepared by the following steps:

(1) drying Lasiosphaera gigantea by hot air at 50 ℃, and pulverizing by a pulverizer to obtain Lasiosphaera gigantea powder of 10 meshes;

(2) and (2) taking the Lasiosphaera gigantea powder obtained in the step (1), and mixing the Lasiosphaera gigantea powder: the absolute ethyl alcohol is 1 g: adding 100g of Lasiosphaera Calvatia powder into 500mL of absolute ethanol at a ratio of 5mL, and performing heating reflux at 70 ℃ for 3 times of ethanol extraction for 3 hours each time to obtain an ethanol extract;

(3) carrying out vacuum filtration on the alcohol extract liquid obtained in the step (2) at the pressure of 0.080MPa, collecting the obtained filtrate, carrying out rotary evaporation and concentration on the obtained filtrate at the temperature of 48 ℃ and under the pressure of 0.092-0.098MPa, and drying at the temperature of 60 ℃ to obtain 12.57g of Lasiosphaera gigantea alcohol extract;

the content of total flavone, total phenol, total amino acid, polypeptide and protein in the obtained Lasiosphaera gigantea alcohol extract is determined, and the result is shown in the following table:

the contents of total flavone, total phenol, total amino acids, polypeptide and protein in the alcohol extract of Lasiosphaera gigantea

From the above table, it can be seen that the alcohol extract of Lasiosphaera gigantea contains abundant total flavonoids, total phenols, total amino acids, polypeptides and proteins, thereby showing that the alcohol extract of Lasiosphaera gigantea has strong antioxidant activity.

(3) carrying out reduced pressure suction filtration at the pressure of 0.080MPa of the alcohol extract control obtained in the step (2), and collecting the obtained filter residue;

(4) and (3) filtering residue obtained in the step (3) is processed according to the proportion of the Lasiosphaera gigantea powder used for preparing the filtering residue: disodium hydrogen phosphate-citric acid buffer at pH 5.5 was 1 g: adding the filter residue obtained in the step (3) into 8L of disodium hydrogen phosphate-citric acid buffer solution with the pH value of 5.5 according to the proportion of 80mL to obtain a filter residue solution;

(5) adding 9g of degrading enzyme into the filter residue solution obtained in the step (4), and controlling the temperature to be 30 ℃ to perform enzymolysis reaction for 60min to obtain an enzymolysis solution;

the degrading enzyme is pectinase, and the addition amount of the degrading enzyme is calculated according to the mass ratio, namely the mass ratio of the pectinase: the bald puffball fungus powder used for preparing the filter residue solution is 9: 100, respectively;

the activity of the pectinase is 30000U/g;

(6) heating the enzymolysis liquid obtained in the step (5) to 95 ℃, preserving heat for 3 hours, and carrying out hot water leaching, wherein the control pressure of the obtained extraction liquid is 0.080MPa, and carrying out reduced pressure suction filtration to obtain filtrate, namely the Lasiosphaera gigantea leaching liquid;

(7) carrying out rotary evaporation concentration on the Calvatia gigantea leaching liquor obtained in the step (6) at the temperature of 75 ℃ until the volume of the Calvatia gigantea leaching liquor is 1/5, obtaining a concentrate, then adding an ethanol water solution with the volume percentage concentration of 75% which is 5 times of the volume of the Calvatia gigantea leaching liquor into the concentrate, carrying out alcohol precipitation for 4 hours at the temperature of 8 ℃, and collecting the obtained precipitate;

(8) and (3) controlling the temperature of the precipitate obtained in the step (7) to be-55 ℃ and carrying out freeze drying to obtain 11.01g of Lasiosphaera gigantea water-soluble crude polysaccharide.

The content of total flavone, total phenol, total amino acid, polypeptide and protein in the obtained crude water-soluble polysaccharide of Lasiosphaera gigantea is determined, and the results are shown in the following table according to the mass percentage:

the content of total flavone, total phenol, total amino acids, polypeptide and protein in Lasiosphaera gigantea water-soluble crude polysaccharide

From the above table, it can be seen that the crude water-soluble polysaccharide of Lasiosphaera gigantea contains a certain amount of total flavonoids, total phenols, total amino acids, polypeptides and proteins, thereby indicating that a part of the antioxidant and anti-inflammatory activities of the crude water-soluble polysaccharide of Lasiosphaera gigantea are derived from these active substances.

The preparation method of the skin care cream containing the water-soluble crude polysaccharide of the Lasiosphaera gigantea and the alcohol extract of the Lasiosphaera gigantea specifically comprises the following steps:

(1) sequentially adding an emulsifier, hexadecanol, liquid oil and 2, 6-di-tert-butyl-4-methylphenol into an oil phase pot, heating to 80 ℃, controlling the rotation speed to be 450rpm, and uniformly stirring to obtain an oil phase;

(2) sequentially adding xanthan gum, the water-soluble crude polysaccharide of the Lasiosphaera gigantea and deionized water into a water phase pot, heating to 80 ℃, controlling the rotating speed to be 450rpm, and uniformly stirring to obtain a water phase;

(3) sequentially adding absolute ethyl alcohol and the Lasiosphaera gigantea alcohol extract into a premixing pot, heating to 40 ℃, controlling the rotating speed to be 500rpm, and uniformly stirring to obtain an alcohol phase;

(4) under the condition that the rotating speed is 450rpm, controlling the dropping speed to be 150mL/min, gradually adding the oil phase obtained in the step (1) into the water phase obtained in the step (2), after the addition is finished, controlling the rotating speed to be 15000rpm for homogenizing for 4min, then adding the alcohol phase obtained in the step (3), and uniformly mixing to obtain a mixed phase;

(5) and (3) controlling the rotation speed of the mixed phase obtained in the step (4) to be 450rpm, stirring the mixed phase at the temperature of 80 ℃ for 35min, then controlling the rotation speed to be 450rpm, naturally cooling the mixed phase to the temperature of 55 ℃, adding a preservative, and continuously controlling the rotation speed to be 450rpm and stirring the mixed phase for 40min to obtain the skin care cream containing the water-soluble crude polysaccharide of the bald puffball and the alcohol extract of the bald puffball.

Example 2

The Lasiosphaera gigantea alcohol extract is prepared by the following steps:

(1) drying Lasiosphaera gigantea by hot air at 45 ℃, and crushing by a crusher to obtain Lasiosphaera gigantea powder of 10 meshes;

(2) and (2) taking the Lasiosphaera gigantea powder obtained in the step (1), and mixing the Lasiosphaera gigantea powder: the absolute ethyl alcohol is 1 g: adding 100g of Lasiosphaera Seu Calvatia powder into 1L of anhydrous ethanol at a ratio of 10mL, and heating and refluxing at 80 deg.C for 2 times of ethanol extraction, each time for 2 hr, to obtain ethanol extractive solution;

(3) carrying out vacuum filtration on the alcohol extract liquid obtained in the step (2) at the pressure of 0.070MPa, collecting the obtained filtrate, carrying out rotary evaporation and concentration on the obtained filtrate at 45 ℃ under the pressure of 0.092-0.098MPa, and then drying at 60 ℃ to obtain 12.98g of Lasiosphaera gigantea alcohol extract;

the content of total flavone, total phenol, total amino acid, polypeptide and protein in the obtained Lasiosphaera gigantea alcohol extract is determined, and the results are calculated according to the mass percentage as follows:

(3) carrying out vacuum filtration with the ethanol extraction liquid prepared in the step (2) at the pressure of 0.070MPa, and collecting the obtained filter residue;

(4) and (3) calculating the filter residue obtained in the step (3) according to the mass ratio to obtain the Lasiosphaera gigantea powder for preparing the filter residue: disodium hydrogen phosphate-citric acid buffer at pH 4.5 was 1 g: adding the filter residue obtained in the step (3) into 5L of disodium hydrogen phosphate-citric acid buffer solution with the pH value of 4.5 according to the proportion of 50mL to obtain a filter residue solution;

(5) adding 5g of degrading enzyme into the filter residue solution obtained in the step (4), and controlling the temperature to be 45 ℃ to carry out enzymolysis reaction for 45min to obtain an enzymolysis solution;

the degrading enzyme is cellulase, and the addition amount of the degrading enzyme is calculated according to the mass ratio, namely the cellulase: the bald puffball fungus powder used for preparing the filter residue solution is 5: 100, respectively;

the activity of the cellulase is 30000U/g;

(6) heating the enzymolysis liquid obtained in the step (5) to 90 ℃, preserving heat for 2 hours, and carrying out hot water leaching, wherein the pressure of the obtained extraction liquid is 0.070MPa, and carrying out reduced pressure suction filtration to obtain filtrate, namely the Lasiosphaera gigantea leaching liquid;

(7) carrying out rotary evaporation concentration on the Calvatia gigantea leaching liquor obtained in the step (6) at the temperature of 75 ℃ until the volume of the Calvatia gigantea leaching liquor is 1/5 of the volume of the Calvatia gigantea leaching liquor to obtain a concentrate, then adding an ethanol water solution with the volume percentage concentration of 80% which is 3 times of the volume of the Calvatia gigantea leaching liquor into the concentrate, carrying out alcohol precipitation for 5 hours at the temperature of 4 ℃, and collecting the obtained precipitate;

(8) and (3) controlling the temperature of the precipitate obtained in the step (7) to be-55 ℃ and carrying out freeze drying to obtain 12.07g of Lasiosphaera gigantea water-soluble crude polysaccharide.

(1) sequentially adding an emulsifier, hexadecanol, liquid oil and 2, 6-di-tert-butyl-4-methylphenol into an oil phase pot, heating to 95 ℃, controlling the rotation speed to be 700rpm, and uniformly stirring to obtain an oil phase;

(2) sequentially adding xanthan gum, the water-soluble crude polysaccharide of the Lasiosphaera gigantea and deionized water into a water phase pot, heating to 95 ℃, controlling the rotating speed to be 700rpm, and uniformly stirring to obtain a water phase;

(3) sequentially adding absolute ethyl alcohol and the Lasiosphaera gigantea alcohol extract into a premixing pot, heating to 50 ℃, controlling the rotating speed to be 700rpm, and uniformly stirring to obtain an alcohol phase;

(4) under the condition that the rotating speed is 700rpm, controlling the dropping speed to be 190mL/min, gradually adding the oil phase obtained in the step (1) into the water phase obtained in the step (2), after the addition is finished, controlling the rotating speed to be 30000rpm to homogenize for 5min, then adding the alcohol phase obtained in the step (3), and uniformly mixing to obtain a mixed phase;

(5) and (3) controlling the rotation speed of the mixed phase obtained in the step (4) to be 700rpm, stirring at the temperature of 95 ℃ for 40min, then controlling the rotation speed to be 700rpm, naturally cooling to the temperature of 60 ℃, adding a preservative, and continuously controlling the rotation speed to be 700rpm, and stirring for 50min to obtain the skin care cream containing the water-soluble crude polysaccharide of the bald puffball and the alcohol extract of the bald puffball.

Example 3

emulsifier 10.00%

3.00 percent of hexadecanol and octadecanol

20.00 percent of liquid grease

2, 6-di-tert-butyl-4-methylphenol 1.00%

0.30 percent of xanthan gum

5.00 percent of Lasiosphaera gigantea water-soluble crude polysaccharide

Absolute ethyl alcohol 15.00%

1.00 percent of Lasiosphaera gigantea alcohol extract

0.50 percent of preservative

The balance being deionized water (44.2%);

The Lasiosphaera gigantea alcohol extract is prepared by the following steps:

(1) drying Lasiosphaera gigantea by hot air at 60 ℃, and pulverizing by a pulverizer to obtain Lasiosphaera gigantea powder of 10 meshes;

(2) and (2) taking the Lasiosphaera gigantea powder obtained in the step (1), and mixing the Lasiosphaera gigantea powder: the absolute ethyl alcohol is 1 g: adding 100g of Lasiosphaera Calvatia powder into 3L of absolute ethyl alcohol at a ratio of 30mL, and performing heating reflux at the temperature of 80 ℃ to perform alcohol extraction for 2.5 hours to obtain alcohol extract;

(3) carrying out vacuum filtration on the alcohol extract liquid obtained in the step (2) at the pressure of 0.090MPa, collecting the obtained filtrate, carrying out rotary evaporation and concentration on the obtained filtrate at 50 ℃ under the pressure of 0.092-0.098MPa, and drying at 60 ℃ to obtain 13.27g of Lasiosphaera gigantea alcohol extract;

(3) carrying out reduced pressure suction filtration at the pressure of 0.090MPa of the alcohol extract obtained in the step (2), and collecting the obtained filter residue;

(4) and (3) filtering residue obtained in the step (3) is processed according to the proportion of the Lasiosphaera gigantea powder used for preparing the filtering residue: disodium hydrogen phosphate-citric acid buffer at pH 5 was 1 g: adding the filter residue obtained in the step (3) into 6L of disodium hydrogen phosphate-citric acid buffer solution with the pH value of 5 according to the proportion of 60mL to obtain a filter residue solution;

(5) adding 6g of degrading enzyme into the filter residue solution obtained in the step (4), and controlling the temperature to be 50 ℃ to perform enzymolysis reaction for 30min to obtain an enzymolysis solution;

the degrading enzyme is a mixture of pectinase and cellulose, and the addition amount of the degrading enzyme is calculated according to the mass ratio, namely the cellulose: and (3) pectinase: the bald puffball fungus powder used for preparing the filter residue solution is 4.5: 1.5: 100, respectively;

the vitality of the cellulase is 30000U/g, and the vitality of the pectinase is 10000U/g;

(6) heating the enzymolysis liquid obtained in the step (5) to 85 ℃, preserving heat for 1h, and carrying out hot water leaching, wherein the control pressure of the obtained extraction liquid is 0.090MPa, and carrying out reduced pressure suction filtration to obtain filtrate, namely the Lasiosphaera gigantea leaching liquor;

(7) carrying out rotary evaporation concentration on the Calvatia gigantea leaching liquor obtained in the step (6) at the temperature of 75 ℃ until the volume of the Calvatia gigantea leaching liquor is 1/5 of the volume of the Calvatia gigantea leaching liquor to obtain a concentrate, then adding an ethanol water solution with the volume percentage concentration of 90% which is 4 times of the volume of the Calvatia gigantea leaching liquor into the concentrate, carrying out alcohol precipitation for 10 hours at the temperature of 10 ℃, and collecting the obtained precipitate;

(8) and (4) controlling the temperature of the precipitate obtained in the step (7) to be-55 ℃ and carrying out freeze drying to obtain 11.98g of Lasiosphaera gigantea water-soluble crude polysaccharide.

(1) sequentially adding an emulsifier, hexadecanol, liquid oil and 2, 6-di-tert-butyl-4-methylphenol into an oil phase pot, heating to 85 ℃, controlling the rotation speed to be 500rpm, and uniformly stirring to obtain an oil phase;

(2) sequentially adding xanthan gum, the water-soluble crude polysaccharide of the Lasiosphaera gigantea and deionized water into a water phase pot, heating to 85 ℃, controlling the rotating speed to be 500rpm, and uniformly stirring to obtain a water phase;

(3) sequentially adding absolute ethyl alcohol and the Lasiosphaera gigantea alcohol extract into a premixing pot, heating to 30 ℃, controlling the rotating speed to be 300rpm, and uniformly stirring to obtain an alcohol phase;

(4) under the condition that the rotating speed is 500rpm, controlling the dropping speed to be 120mL/min, gradually adding the oil phase obtained in the step (1) into the water phase obtained in the step (2), after the addition is finished, controlling the rotating speed to be 10000rpm to homogenize for 3min, then adding the alcohol phase obtained in the step (3), and uniformly mixing to obtain a mixed phase;

(5) and (3) controlling the rotation speed of the mixed phase obtained in the step (4) to be 500rpm, stirring at 85 ℃ for 25min, then controlling the rotation speed to be 500rpm, naturally cooling to 50 ℃, adding a preservative, and continuously controlling the rotation speed to be 500rpm, and stirring for 30min to obtain the skin care cream containing the water-soluble crude polysaccharide of the bald puffball and the alcohol extract of the bald puffball.

The capacity of the water-soluble crude polysaccharide of the Calvatia gigantea and the hydroxyl radical scavenging capacity of the alcohol extract of the Calvatia gigantea obtained in example 3 is measured by a Clarity-2PC chemiluminescence apparatus (BIO TEX, USA) by a cuprous chloride-hydrogen peroxide-luminol system, and the specific experimental steps are as follows:

(1) and preparing a sample solution:

preparation of water-soluble crude polysaccharide sample solution of Lasiosphaera gigantea

Dissolving 4g of crude water-soluble polysaccharide of Lasiosphaera Seu Calvatia in 100mL of deionized water to prepare a 0.04g/mL sample solution of crude water-soluble polysaccharide of Lasiosphaera Seu Calvatia, and diluting to obtain a sample solution of crude water-soluble polysaccharide of Lasiosphaera Seu Calvatia with concentrations of 4.001mg/mL, 2.0005mg/mL, 1.00025mg/mL, 0.66683mg/mL, 0.50013mg/mL and 0.4001mg/mL in figure 1;

② preparation of Lasiosphaera gigantea alcohol extract sample solution

Dissolving 3.4g of Lasiosphaera gigantea alcohol extract in 100mL of absolute ethyl alcohol to prepare a Lasiosphaera gigantea alcohol extract sample solution with the concentration of 0.034g/mL, and diluting the Lasiosphaera gigantea alcohol extract sample solution with the concentrations of 94.5mg/mL, 81.88mg/mL, 62.94mg/mL, 57.63mg/mL, 49.52mg/mL and 31.09mg/mL in the figure 1 respectively;

(2) preparing a 6% hydrogen peroxide solution, 1 × 10^-3The mol/L pyrogallol solution adopts 1X 10^-3Preparing 1X 10 of mol/L hydrochloric acid solution^-3The mol/L cuprous chloride solution adopts 0.1mol/LNa₂CO₃ Solution preparation 1X 10^-3mol/L luminol, using 0.05mol/L NaHCO at pH 8.5₃-Na₂CO₃Buffer solution and 1X 10^-3And preparing a mixed solution of luminol and carbonate buffer solution by mol/L luminol in a ratio of 17: 1.

When the measurement is carried out, 10 mu L of the crude water-soluble polysaccharide sample solution of the Lasiosphaera gigantea and the alcohol extract sample solution of the Lasiosphaera gigantea obtained in the step (1) are respectively injected into a luminous cell for measurement, and then 10 mu L of aqueous hydrogen peroxide solution with the volume percentage concentration of 6 percent, 10 mu L of 1 multiplied by 10 are respectively injected into the luminous cell for measurement^-3mol/L cuprous chloride aqueous solution, 10. mu.L 1X 10^-3Reacting the mixed solution of the o-pyrogallol and the luminol-carbonate buffer solution of 150 mu L for 30s, recording experimental data every 2 seconds to obtain a peak value A₁And substituting deionized water or ethanol for the sample to obtain a peak value A₀The clearance rate calculation formula is: clearance (%) ═ a₀-A₁)/A₀*100％。

The determination result is shown in fig. 1, with the increase of the concentration of the crude water-soluble polysaccharide solution of the puffball, the clearance rate of each hydroxyl radical is continuously increased, and the IC50 values for clearing the hydroxyl radicals are 0.603mg/mL and 0.514mg/mL respectively, which indicates that the two solutions have stronger capacity of clearing the hydroxyl radicals, and the capacity of the alcohol extract of the puffball for clearing the hydroxyl radicals is stronger than that of the crude water-soluble polysaccharide of the puffball.

The capacity of the water-soluble crude polysaccharide of Lasiosphaera gigantea and the hydrogen peroxide-scavenging ability of the alcohol extract of Lasiosphaera gigantea obtained in example 3 was measured using a Clarity-2PC chemiluminescence apparatus (BIO TEX, USA) using a hydrogen peroxide-luminol system, and the specific experimental steps were as follows:

(1) and preparing a sample solution:

Dissolving 4g of crude water-soluble polysaccharide of Lasiosphaera Seu Calvatia in 100mL of deionized water to prepare a 0.04g/mL sample solution of the crude water-soluble polysaccharide of Lasiosphaera Seu Calvatia, and diluting to obtain a crude water-soluble polysaccharide sample solution of Lasiosphaera Seu Calvatia with concentrations of 0.8002mg/mL, 0.4001mg/mL, 0.20005mg/mL, 0.10003mg/mL, 0.06668mg/mL, 0.05001mg/mL and 0.04001mg/mL in figure 2;

② preparation of Lasiosphaera gigantea alcohol extract sample solution

Dissolving 3.4g of Lasiosphaera gigantea alcohol extract in 100mL of absolute ethyl alcohol to prepare a Lasiosphaera gigantea alcohol extract sample solution with the concentration of 0.034g/mL, and then diluting the Lasiosphaera gigantea alcohol extract sample solution with the concentrations of 0.6848mg/mL, 0.3424mg/mL, 0.1712mg/mL, 0.0856mg/mL, 0.05707mg/mL and 0.0428mg/mL in the figure 2 respectively;

(2) preparing 6 volume percent aqueous hydrogen peroxide solution by adopting 0.1mol/LNa₂CO₃Preparation of aqueous solution 1X 10^-3mol/L luminol, using 0.05mol/L NaHCO at pH 9.5₃-Na₂CO₃Buffer solution and 1X 10^-3Preparing a mixed solution of luminol and carbonate buffer solution by mol/L luminol according to the volume ratio of 2: 1;

during measurement, 10 mu L of the crude water-soluble polysaccharide sample solution of the Lasiosphaera gigantea and the alcohol extract sample solution of the Lasiosphaera gigantea obtained in the step (1) are injected into a luminous cell for measurement respectively, then 10 mu L of mixed solution of 6% aqueous hydrogen peroxide solution and 150 mu L of luminol-carbonate buffer solution in volume percentage concentration is injected into the luminous cell for reaction for 30s, experimental data are recorded every 2 seconds, and the peak value A is obtained₁And substituting deionized water or ethanol for the sample to obtain a peak value A₀The clearance rate calculation formula is: clearance (%) ═ a₀-A₁)/A₀*100％

The measurement results are shown in fig. 2, and it can be seen from fig. 2 that, as the concentration of the aqueous crude polysaccharide solution of puffball giganteum increases, the respective hydrogen peroxide removal rates increase continuously, and the IC50 values for removing hydrogen peroxide are 0.168mg/mL and 0.132mg/mL respectively, which indicates that the two solutions have stronger hydrogen peroxide removal capability, and the hydrogen peroxide removal capability of the alcoholic extract of puffball giganteum is stronger than that of the aqueous crude polysaccharide of puffball giganteum.

An experiment for respectively measuring the reducing power of the water-soluble crude polysaccharide of the Calvatia gigantea and the alcohol extract of the Calvatia gigantea obtained in example 3 by using an ultraviolet visible spectrophotometer (model UV759, Shanghai Yiyou electronic technology Co., Ltd.) comprises the following specific experimental steps:

(1) and preparing a sample solution:

Dissolving 4g of crude water-soluble polysaccharide of Lasiosphaera gigantea in 100mL of deionized water to prepare a 0.04g/mL crude water-soluble polysaccharide sample solution of Lasiosphaera gigantea, and then diluting the crude water-soluble polysaccharide sample solution of Lasiosphaera gigantea with concentrations of 10.512mg/mL, 7.2711mg/mL, 4.0010mg/mL, 2.0005mg/mL and 0.4001mg/mL in the figure 3 respectively;

② preparation of Lasiosphaera gigantea alcohol extract sample solution

Dissolving 3.4g of Lasiosphaera gigantea alcohol extract in 100mL of absolute ethyl alcohol to prepare a Lasiosphaera gigantea alcohol extract sample solution with the concentration of 0.034g/mL, and then diluting the Lasiosphaera gigantea alcohol extract sample solution with the concentrations of 17.120mg/mL, 13.696mg/mL, 8.560mg/mL, 6.848mg/mL and 3.424mg/mL in the graph 3;

(2) and (2) taking 1mL of the crude water-soluble polysaccharide sample solution of the Lasiosphaera gigantea and the alcohol extract sample solution of the Lasiosphaera gigantea obtained in the step (1) to respectively measure, adding 2.5mL of phosphate buffer solution with the pH value of 6.6 and 2.5mL of potassium ferricyanide aqueous solution with the mass percent concentration of 1%, uniformly mixing, placing in a constant-temperature water bath at 50 ℃ for 20min, taking out, adding 2.5mL of trichloroacetic acid aqueous solution with the volume percent concentration of 10%, uniformly mixing, placing in a centrifuge, centrifuging at 3000rpm for 10min, taking 2.5mL of supernate, adding 2.5mL of distilled water and 0.5mL of ferric trichloride aqueous solution with the mass percent concentration of 0.1%, reacting for 10min, shaking, measuring the absorbance value at 700nm by taking deionized water as a control, wherein the larger absorbance value is larger, and the larger reducing force is larger.

The measurement result is shown in fig. 3, and it can be seen from fig. 3 that both of the crude water-soluble polysaccharide solution of puffball giganteum and the alcohol extract solution of puffball giganteum have stronger reducing power, and the respective reducing power increases with the increase of the concentration of the crude water-soluble polysaccharide solution of puffball giganteum, and the reducing power of the crude water-soluble polysaccharide of puffball giganteum at the same concentration is stronger than that of the alcohol extract of puffball giganteum.

The ability of the water-soluble crude polysaccharide of the puffball giganteum obtained in example 3 and the ability of the alcohol extract of the puffball giganteum to remove DPPH free radicals are respectively determined, and the specific experimental steps are as follows:

(1) and preparing a sample solution:

Dissolving 4g of crude water-soluble polysaccharide of Lasiosphaera Seu Calvatia in 100mL of deionized water to prepare a 0.04g/mL crude water-soluble polysaccharide sample solution of Lasiosphaera Seu Calvatia, and diluting to obtain a crude water-soluble polysaccharide sample solution of Lasiosphaera Seu Calvatia with concentrations of 40.01mg/mL, 19.93097mg/mL, 11.23796mg/mL, 8.89145mg/mL, 6.88355mg/mL, 2.38707mg/mL and 1.65254mg/mL in FIG. 4;

② preparation of Lasiosphaera gigantea alcohol extract sample solution

Dissolving 3.4g of Lasiosphaera gigantea alcohol extract in 100mL of absolute ethyl alcohol to prepare a Lasiosphaera gigantea alcohol extract sample solution with the concentration of 0.034g/mL, and then diluting the Lasiosphaera gigantea alcohol extract sample solution with the concentrations of 34.103mg/mL, 30.645mg/mL, 21.780mg/mL, 10.324mg/mL and 5.937mg/mL in the graph 4;

(2) dissolving 4mg of DPPH by using 5mL of absolute ethyl alcohol, and then diluting the volume of the solution to 100mL by using an ethanol water solution with the volume percentage concentration of 50% to prepare a 0.1mmol/L DPPH solution;

taking 1mL of 0.1mmol/L DPPH solution, adding 3mL of the crude water-soluble polysaccharide sample solution of the Lasiosphaera gigantea and the alcohol extract sample solution of the Lasiosphaera gigantea in the step (1), reacting for 30min in a dark place, and respectively measuring the absorbance value As at 517 nm;

② taking 1mL of 0.1mmol/L DPPH solution, adding 3mL of ethanol aqueous solution with 50 percent of volume percentage concentration, reacting for 30min in a dark place, and measuring the absorbance value A at 517nm₀；

Thirdly, 1mL of ethanol water solution with the volume percentage concentration of 50 percent is taken and added into 3mL of the same sample solution in the step I to be reacted for 30min in a dark place, and the absorbance value A is measured at 517nm₁；

The clearance calculation formula is: clearance (%) ═ a₀-(As-A₁)]/A₀*100％；

The measurement result is shown in fig. 4, and it can be seen from fig. 4 that the alcohol extract of bobby turbob has no ability to scavenge DPPH free radicals, the crude water-soluble polysaccharide of bobby turbob has strong ability to scavenge DPPH free radicals, and the DPPH free radical scavenging rate increases with the increase of the concentration of the crude water-soluble polysaccharide solution of bobby turbob. The IC50 value for scavenging DPPH free radicals was 4.410 mg/mL.

According to the method in 'the influence of the new andrographolide on the mouse macrophage respiratory burst and lymphocyte proliferation' with slight modification, the inhibition effect of the crude water-soluble polysaccharide of the Lasiosphaera gigantea on the mouse macrophage respiratory burst stimulated by the fosaprepitant (PMA) is measured, the anti-inflammatory activity of the crude water-soluble polysaccharide is investigated, and the method comprises the following steps:

(1) and preparing experimental materials:

preparing a 0.04g/mL bald puffball water-soluble crude polysaccharide sample solution:

dissolving 4g of Lasiosphaera gigantea water-soluble crude polysaccharide in 100mL of deionized water to prepare a 0.04g/mL Lasiosphaera gigantea water-soluble crude polysaccharide sample solution;

culture of mouse RAW 264.7 macrophages (provided by shanghai cell research institute of chinese academy of sciences): subculturing with RPM I1640 culture solution (brand: Solarbio, manufactured by Beijing Solelaibao Tech Co., Ltd.), digesting with 0.25% pancreatin (Gibco Co.) during experiment, centrifuging the obtained suspension at 300 × g for 3min, collecting cells, adjusting cell concentration to 2 × 10 with RPM I1640 culture solution⁸Detecting the cell survival rate to be more than 95% by trypan blue staining method to obtain RAW 264.7 cell suspension for later use;

preparing Luminol luminescent liquid: adding 10mL of 0.01mol/L PBS aqueous solution and 10mL of triethylamine solution with the mass percent concentration of 0.8% into 20mg of Luminol, electromagnetically stirring for 5h, performing sterile filtration and subpackage to obtain a storage solution, and storing the storage solution at minus 20 ℃ in a dark place;

the Luminol luminescent solution is diluted by RPM I1640 culture solution before use, and the dilution ratio is that the Luminol luminescent solution: RPM I1640 culture solution volume ratio of 1: 10.

Preparing PMA working solution: PMA (ethyl phorbol myristate) is dissolved in DMSO (dimethyl sulfoxide) to prepare PMA working solution with the concentration of 125mg/L, and the PMA working solution is stored at the temperature of-20 ℃ in a dark place.

(2) And the specific experimental steps are as follows:

preheating the sample chamber to reach 37 +/-0.5 deg.c.

0.4mL of RAW 264.7 cell suspension is taken, 10 mu L of 0.04g/mL of Lasiosphaera gigantea water-soluble crude polysaccharide sample is added (equal amount of distilled water is added for blank control), 0.1mL of diluted luminol luminescent solution and 0.5 mu L of PMA working solution (final concentration is 125 mu g/L) are sequentially added, and the chemiluminescence intensity (cheminescence CL) is continuously measured for 20min and automatically recorded by a biochemical luminescence measuring instrument every 1min to obtain the peak value PV of the CL. The inhibition of PMA-stimulated respiratory burst of RAW 264.7 cells by the Lasiosphaera gigantea water-soluble crude polysaccharide sample is shown below: inhibition (%) × (1-sample PV value/blank PV value) × 100%.

The measurement result is shown in fig. 5, and it can be seen from fig. 5 that the crude water-soluble polysaccharide of Lasiosphaera gigantea has a significant inhibitory effect on the respiratory burst of PMA-stimulated macrophages, and the inhibition rate is 82.6%, that is, the crude water-soluble polysaccharide of Lasiosphaera gigantea has strong anti-inflammatory activity.

From the above, it can be seen that the crude water-soluble polysaccharide of Lasiosphaera gigantea has strong ability to scavenge hydroxyl radicals, strong ability to scavenge hydrogen peroxide, strong reducing power, strong ability to scavenge DPPH radicals and strong anti-inflammatory activity, and the IC50 value for scavenging DPPH radicals is 4.410 mg/mL. The Lasiosphaera gigantea alcohol extract also has strong capability of eliminating hydroxyl free radicals, strong capability of eliminating hydrogen peroxide and strong reducing power, but has no capability of eliminating DPPH free radicals. And the capacity of eliminating hydroxyl free radicals and hydrogen peroxide of the alcohol extract of the Lasiosphaera gigantea is stronger than that of the water-soluble crude polysaccharide of the Lasiosphaera gigantea. However, in terms of reducing power, the reducing power of the water-soluble crude polysaccharide of the Lasiosphaera gigantea is stronger than that of the alcohol extract of the Lasiosphaera gigantea under the same concentration. Therefore, the water-soluble crude polysaccharide of the Lasiosphaera gigantea can be used for a moisturizer, an antioxidant and an anti-inflammatory active substance of skin cosmetics, and the alcohol extract of the Lasiosphaera gigantea can be used for the moisturizer and the antioxidant of the skin cosmetics.

The skin care lotion containing the water-soluble crude polysaccharide of the Lasiosphaera gigantea and the alcohol extract of the Lasiosphaera gigantea obtained in the example 3 is evaluated for moisturizing performance, and the specific steps are as follows:

selecting 30 volunteers of 20-45 years old, cleaning arms of the volunteers, air drying, and balancing for 30min under constant temperature and humidity (20-24 deg.C and 40-60% humidity) to stabilize skin moisture. The method comprises the steps of respectively selecting 4 test areas of 2cm multiplied by 2cm on two arms, then smearing samples, smearing the skin care milk containing the crude water-soluble polysaccharide of the bald puffball and the alcohol extract of the bald puffball obtained in example 3 by the right hand as an experimental sample, smearing a control sample by the left hand (the control sample is a control sample 1, a control sample 2 and a control sample 3, wherein the control sample 1 is pure glycerol, and the control sample 2 is the alcohol extract of the bald puffball in the skin care milk containing the crude water-soluble polysaccharide of the bald puffball and the alcohol extract of the bald puffball obtained in example 3 is replaced by glycerol with the same amount, and the rest is the same as example 3), and smearing the control sample 3 is the skin care milk containing the crude water-soluble polysaccharide of the bald puffball and the alcohol extract of the bald puffball obtained in example 3 with the same amount of glycerol, and the rest is the same as example 3). Measuring skin moisture content of the test area before smearing as blank value, and then measuring the moisture content according to 2mg sample/cm²The experimental sample and each of the control samples 1, 2 and 3 are respectively smeared on the corresponding test areas, the skin moisture content is measured 0.5h, 1h, 2h, 3h and 4h after smearing, the skin moisture content increase rate is calculated, and the average value is taken.

Skin moisture content (MMV) growth rate (moisture content of skin t period after application-moisture content of skin before application)/moisture content of skin before application × 100%.

As shown in fig. 6, it can be seen from fig. 6 that the skin care lotion containing crude water-soluble polysaccharide of calvatia pelagi and alcohol extract of calvatia pelagi obtained in example 3 has a lasting moisturizing ability significantly higher than that of control 1, control 2 and control 3, and the reason for this is probably due to the synergistic effect of crude water-soluble polysaccharide of calvatia pelagi and alcohol extract of calvatia pelagi and the components of xanthan gum, tricaprin caprylate and tricaprin, cetostearyl alcohol, etc. in the skin care lotion, so that the moisturizing effect of the skin care lotion containing the extract of calvatia pelagi is stronger than that of control 1, and also stronger than that of the products containing crude water-soluble polysaccharide of calvatia pelagi alone or the products of control 2 and control 3 containing alcohol extract of calvatia pelagi alone. For example, as seen in MMV growth data 1 hour after sample application, experimental (67%) > control 1 (61%) > control 2 (53%) > control 3 (49%).

Furthermore, the moisture retention of the skin care cream containing the crude water-soluble polysaccharide of the psiloside balia and the alcohol extract of the psiloside balia obtained in example 3 of the invention is not the superposition of the simple actions of the crude water-soluble polysaccharide of the psiloside balia and the alcohol extract of the psiloside balia, namely the crude water-soluble polysaccharide of the psiloside balia and the alcohol extract of the psiloside balia have strong moisture retention effect due to the synergistic action of the components of the xanthan gum, caprylic capric triglyceride, cetostearyl alcohol and the like in the skin care cream.

The skin care cream containing the water-soluble crude polysaccharide of the puffball giganteum and the alcohol extract of the puffball giganteum, which is obtained in the example 3, is subjected to a patch test to evaluate the potential skin irritation, and the specific steps are as follows:

30 volunteers aged 18-60 years without severe skin damage were randomly selected. 0.1g of the skin care cream containing the crude water-soluble polysaccharide of Lasiosphaera gigantea and the alcohol extract of Lasiosphaera gigantea obtained in example 3 was dropped on a filter paper sheet, and the filter paper sheet was placed in a well of a spot tester (Beijing billion Yida scientific and development Co., Ltd.) to obtain a test sample.

A blank control sample is set for each test sample, namely 0.1g of distilled water is dripped on a filter paper sheet, and then the filter paper sheet is placed in a hole of a spot tester (Beijing billion Yidao scientific and technological development Co., Ltd.) for comparison to obtain the control sample.

The flexed sides of the two forearms of the human body are selected as test areas, and the spot tester is fixedly pasted on the flexed sides of the two forearms of the volunteer by using a non-irritant adhesive tape. The test period was 24 h. For accurate, reliable and scientific results, the volunteer could not remove the plaque tester during the test and also did not expose the test area to water.

After 24h, the plaque tester was removed, and the skin reaction was observed after the indentation disappeared. If the test result is negative, the sample is not stimulated, and the test result of the patch is shown in Table 3

TABLE 3 Patch test results

In table 3: "-negative reaction;

only faint erythema in the response "+" ═ suspicious;

the erythema, infiltration, edema, and optionally pimple were reacted weakly positively "+";

the strong positive response rash reflects erythema, infiltration, edema, pimples, and rash beyond the test area.

As can be seen from table 3 above, the skin care lotion containing the crude water-soluble polysaccharide of calvatia gigantea and the alcohol extract of calvatia gigantea obtained in example 3 has no adverse reaction in the test, which indicates that the skin care lotion containing the crude water-soluble polysaccharide of calvatia gigantea and the alcohol extract of calvatia gigantea provided by the invention has higher safety.

In conclusion, the skin care lotion containing the crude water-soluble polysaccharide of the Lasiosphaera gigantea and the alcohol extract of the Lasiosphaera gigantea has higher safety, has the advantages of strong moisture retention, oxidation resistance, inflammation resistance and the like due to the containing of the crude water-soluble polysaccharide of the Lasiosphaera gigantea and the alcohol extract of the Lasiosphaera gigantea, and has strong moisture retention effect due to the synergistic effect of the crude water-soluble polysaccharide of the Lasiosphaera gigantea and the alcohol extract of the Lasiosphaera gigantea with the components of the skin care lotion, such as xanthan gum, caprylic capric triglyceride, cetostearyl alcohol and the like. The anti-inflammatory activity of the skin care lotion is mainly provided by the water-soluble crude polysaccharide of the Lasiosphaera gigantea contained in the skin care lotion; the anti-oxidation activity of the skin care milk is provided by the contained water-soluble crude polysaccharide of the Lasiosphaera gigantea and the alcohol extract of the Lasiosphaera gigantea, and the water-soluble crude polysaccharide of the Lasiosphaera gigantea and the alcohol extract of the Lasiosphaera gigantea respectively have long effects of removing different types of free radicals, and the common addition of the crude polysaccharide of the Lasiosphaera gigantea and the alcohol extract of the Lasiosphaera gigantea into the skin care milk can play roles of complementary advantages and synergistic interaction, so that the strength and the breadth of removing the free radicals of the skin care milk are enhanced, and the anti-oxidation performance of the skin care milk is improved; the moisturizing property of the skin care cream is the result of the synergistic effect of the contained bald puffball water-soluble crude polysaccharide and bald puffball alcohol extract and components such as xanthan gum, caprylic capric triglyceride, cetostearyl alcohol and the like, so the skin care cream has strong moisturizing effect.

The above description is only a basic description of the present invention, and any equivalent changes made according to the technical solution of the present invention should fall within the protection scope of the present invention.

Claims

1. The skin care lotion containing the water-soluble crude polysaccharide of the bald puffball and the alcohol extract of the bald puffball is characterized in that the skin care lotion containing the water-soluble crude polysaccharide of the bald puffball and the alcohol extract of the bald puffball is calculated by mass percent, and the raw materials for preparing the skin care lotion comprise the following components in percentage by mass:

The Lasiosphaera gigantea alcohol extract is prepared by the following steps:

(4) and (3) filtering residues obtained in the step (3) according to the filtering residues: disodium hydrogen phosphate-citric acid buffer solution with pH value of 4-6 is 1 g: adding the filter residue into a disodium hydrogen phosphate-citric acid buffer solution with the pH value of 4-6 according to the proportion of 30-80mL to obtain a filter residue solution;

2. The skin care lotion containing the crude water-soluble polysaccharide of the Lasiosphaera calvatia and the alcohol extract of the Lasiosphaera calvatia as claimed in claim 1, wherein the skin care lotion containing the crude water-soluble polysaccharide of the Lasiosphaera calvatia and the alcohol extract of the Lasiosphaera calvatia is prepared from the following raw materials in percentage by mass:

3. the skin care lotion containing the crude water-soluble polysaccharide of the Lasiosphaera calvatia and the alcohol extract of the Lasiosphaera calvatia as claimed in claim 2, wherein the skin care lotion containing the crude water-soluble polysaccharide of the Lasiosphaera calvatia and the alcohol extract of the Lasiosphaera calvatia is prepared from the following raw materials in percentage by mass:

4. the skin care lotion containing the crude water-soluble polysaccharide of Lasiosphaera gigantea and the alcohol extract of Lasiosphaera gigantea as claimed in claim 2, which is prepared from the following raw materials in percentage by mass:

5. the skin care lotion containing the crude water-soluble polysaccharide of Lasiosphaera gigantea and the alcohol extract of Lasiosphaera gigantea as claimed in claim 2, which is prepared from the following raw materials in percentage by mass:

6. the method for preparing the skin care cream containing the water-soluble crude polysaccharide of the bald puffball and the alcohol extract of the bald puffball as claimed in any one of claims 1 to 5, which comprises the following steps: