Gynecological gel as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biology. The invention relates to the field of medicine, in particular to application of grifola frondosa fermentation liquor in preparation of medicine for treating cervicitis with high-risk human papilloma virus, gynecological gel for treating high-risk human papilloma virus infection and a preparation method and application of the gynecological gel. The grifola frondosa fermentation liquor is obtained by taking grifola frondosa as a fermentation strain and adding a phellinus igniarius extract into a fermentation substrate for liquid submerged fermentation. Tests prove that the grifola frondosa fermentation liquor can obviously inhibit the expression of E6mRNA in SiHa cells and HeLa cells; in a clinical test, the grifola frondosa fermentation liquor combined with the recombinant human interferon a2b has a remarkable curative effect on treating cervicitis with HPV infection, the HPV negative conversion rate can be effectively increased, the inflammatory factor secretion level can be reduced, the ecological restoration balance of the vagina microenvironment can be promoted, and yang recovery is not easy after treatment.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology. More specifically, it relates to a gel for treating gynecological diseases related to high-risk human papillomavirus infection, its preparation method and application. Background Art

[0002] Human papillomavirus (HPV) is a common pathogenic bacterium in the vagina. It belongs to spherical DNA virus and can increase the risk of cervical diseases. In particular, long-term persistent infection with high-risk HPV (HR-HPV) can induce cervical cancer. Currently, the most widely used drug for anti-cervical HPV infection in clinic is recombinant human interferon. Recombinant human interferon can bind to the surface receptor of target cells, thereby generating a variety of antiviral factors, inhibiting HPV transcription, regulating cellular immune function, and accelerating the clearance of HPV. However, since HPV infection is a long-term disease process, single drug treatment is prone to drug resistance and reduces the treatment effect. Moreover, HPV infection changes the vaginal mucosa metabolism and local immune response, strengthens the adhesion and invasion of pathogenic microbial flora, destroys the balance of vaginal microecology, increases the diversity of vaginal flora, and causes pH imbalance, which in turn enhances the immune escape of HPV and enables HPV to better colonize within cells. However, the current common anti-HPV infection drugs do not strengthen their function of repairing the vaginal microecological environment.

[0003] Grifola frondosa (Dicks, ex Fr) S.F. Gray is one of the rare edible and medicinal fungi developed in recent years. It belongs to Basidiomycotina, Hymenomycetes, Holobasidiomycetidae, Aphyllophorales, Polyporaceae, Grifola. It is also known as Polyporus frondosus, Grifola frondosa, Chestnut mushroom, Yun mushroom, Lotus mushroom, etc. A large number of studies have shown that the polysaccharides extracted from its fruiting bodies or mycelia have significant effects such as anti-tumor, hypoglycemic, anti-hepatitis, and improving immune system function. Currently, there is no research on using Grifola frondosa fermentation broth to treat HPV-related gynecological infectious diseases. Summary of the Invention

[0004] Aiming at the deficiencies of the prior art, the present invention aims to provide a Grifola frondosa fermentation broth, its use in the preparation of drugs for treating cervicitis accompanied by HPV infection, and a gynecological gel. The test results show that the Grifola frondosa fermentation broth prepared by the present invention has a significant anti-HPV virus effect and an enhanced ability to repair the balance of vaginal microecology. The combination of Grifola frondosa fermentation broth and recombinant human interferon a2b has a significant curative effect on treating cervicitis accompanied by HPV infection, and can effectively reduce the viral load of HPV while promoting the restoration of the balance of the vaginal microecological environment.

[0005] The first object of the present invention is to provide the use of Grifola frondosa fermentation broth in the preparation of a drug for treating cervicitis accompanied by HPV infection, wherein the Grifola frondosa fermentation broth is obtained by liquid deep fermentation using Grifola frondosa as a fermentation strain and adding Phellinus igniarius extract to a fermentation substrate.

[0006] Furthermore, the Phellinus igniarius extract is obtained by heating and refluxing Phellinus igniarius fruit body powder with ethanol as an extraction solvent and then extracting with n-butanol.

[0007] Furthermore, 3 - 10 g / L of Phellinus igniarius extract is added to the fermentation substrate.

[0008] Furthermore, the Grifola frondosa fermentation broth is prepared by the following steps:

[0009] S1. Weigh Phellinus igniarius fruit body powder, add an ethanol solution with a volume fraction of 50 - 80% and a weight 5 - 10 times that of the powder, reflux extract at 55 - 65°C for 1 - 3 times, 2 hours each time, filter, combine the filtrates, recover ethanol and concentrate to an appropriate amount to obtain a concentrated solution;

[0010] S2. Add water with a weight 1 - 3 times that of the above concentrated solution to the concentrated solution, mix well, then add n-butanol with a weight 8 - 10 times that of the concentrated solution and extract 1 - 3 times, collect the extract, recover the solvent to obtain a Phellinus igniarius n-butanol extract;

[0011] S3. Inoculate the Grifola frondosa seed liquid into a fermentation substrate containing 3 - 10 g / L of Phellinus igniarius n-butanol extract at an inoculation amount of 5 - 10%, culture in a shaker at 25 - 27°C and a rotation speed of 100 - 200 r / min for 5 - 7 days, collect the fermentation broth, centrifuge and filter to obtain the product.

[0012] Another object of the present invention is to provide a gynecological gel, which contains the following components by weight:

[0013] 3 - 15% of the above-mentioned Grifola frondosa fermentation broth;

[0014] 0.5 - 20,000 IU / g of recombinant human interferon a2b;

[0015] 0.5 - 1% of a thickening agent;

[0016] 1 - 8% of a moisturizing agent; and

[0017] The balance is pure water.

[0018] Furthermore, the thickening agent is carbomer or poloxamer 188.

[0019] Furthermore, the moisturizing agent is one or a combination of two or more of glycerol, propylene glycol, 1,3 - butanediol and glycerin.

[0020] Another object of the present invention is to provide a method for preparing the gynecological gel, which is characterized by including the following steps:

[0021] Add a moisturizer and a thickener to pure water, homogenize for 3-4 minutes, slowly heat up to 80-85 °C, and after cooling to 40-45 °C, add the Grifola frondosa fermentation broth and recombinant human interferon a2b, and stir evenly to obtain the gynecological gel.

[0022] Through long-term research, it has been found that adding certain traditional Chinese medicines or certain components in traditional Chinese medicines to the fermentation substrate can promote the growth of Grifola frondosa and the production of certain special metabolites. It was unexpectedly found that the product obtained by fermenting with a specific solvent extract of Phellinus igniarius added to the fermentation substrate showed a significant effect of inhibiting the expression of E6 mRNA in SiHa cells and HeLa cells. And through comparative experiments, it was proved that the above characteristics shown by the product were related to the fermentation with the addition of Phellinus igniarius extract, because the above effects could not be observed or the effects were not good in the fermentation products without adding and adding other traditional Chinese medicine extracts.

[0023] Through further research, it was found that the combination of the Grifola frondosa fermentation broth and a lower concentration of recombinant human interferon a2b had a significant curative effect on treating HPV infection-related diseases, could effectively improve the negative conversion rate of HPV, promote the restoration of the vaginal microecological balance, reduce the secretion level of inflammatory factors, and was not easy to relapse after treatment. Detailed implementation mode

[0024] The following examples are further descriptions of the present invention, rather than limitations of the present invention. The components of the formulations in the following examples are all conventional commercially available products unless otherwise specified.

[0025] Example 1

[0026] Preparation of Grifola frondosa fermentation broth:

[0027] S1. Weigh the Phellinus igniarius fruit body powder, add an ethanol solution with a volume fraction of 70% that is 8 times its weight, heat and reflux for extraction 3 times at 65 °C for 2 h each time, filter, combine the filtrates, recover ethanol and concentrate to an appropriate amount to obtain a concentrated solution;

[0028] S2. Add water that is 2 times its weight to the above concentrated solution, mix evenly, and then extract with n-butanol that is 10 times the weight of the concentrated solution 2 times, collect the extract, recover the solvent, and obtain the Phellinus igniarius n-butanol extract;

[0029] S3. Cut the Grifola frondosa slant strain into small pieces and inoculate it into a liquid medium containing 100 mL (glucose 30 g / L, peptone 2 g / L, yeast extract 6 g / L, KH 2 PO 4 0.5 g / L, MgSO4 ·7H 2 In a 250 mL Erlenmeyer flask containing 0.5 g / L of O (with water as the solvent), cultured in a shaker at 25 °C and 150 r / min for 5 days to obtain a Grifola frondosa seed solution;

[0030] S4. Inoculate the Grifola frondosa seed solution into a fermentation substrate containing 5 g / L of Sanghuang butanol extract (50 g / L of glucose, 5 g / L of peptone, 10 g / L of yeast extract, KH 2 PO 4 2 g / L, MgSO 4 ·7H 2 O 2 g / L, with water as the solvent) at an inoculation amount of 8%, culture in a shaker at 25 °C and 150 r / min for 6 days, collect the fermentation broth, centrifuge at 3000 r / min for 1 min, and take the supernatant to pass through a 0.22 μm microporous filter membrane to obtain the Grifola frondosa fermentation broth.

[0031] Example 2

[0032] Preparation of Grifola frondosa fermentation broth:

[0033] S1. Weigh the Sanghuang fruiting body powder, add an ethanol solution with a volume fraction of 75% that is 10 times its weight, heat and reflux for extraction 3 times at 55 °C, 2 h each time, filter, combine the filtrates, recover ethanol and concentrate to an appropriate amount to obtain a concentrated solution;

[0034] S2. Add 3 times the weight of water to the above concentrated solution, mix well, then extract with 8 times the weight of the concentrated solution of n-butanol once, collect the extract, recover the solvent to obtain the Sanghuang butanol extract;

[0035] S3. Cut the Grifola frondosa slant strain into small pieces and inoculate them into a 250 mL Erlenmeyer flask containing 100 mL of liquid medium (30 g / L of glucose, 2 g / L of peptone, 6 g / L of yeast extract, KH 2 PO 4 0.5 g / L, MgSO 4 ·7H 2 O 0.5 g / L, with water as the solvent), culture in a shaker at 25 °C and 150 r / min for 5 days to obtain a Grifola frondosa seed solution;

[0036] S4. Inoculate the Grifola frondosa seed solution into a fermentation substrate containing 3 g / L of Sanghuang butanol extract (50 g / L of glucose, 5 g / L of peptone, 10 g / L of yeast extract, KH 2 PO 4 2 g / L, MgSO 4 ·7H 2(in water with an O₂ concentration of 2 g / L), cultured in a shaker at 25°C with a rotation speed of 200 r / min for 5 days, the fermentation broth was collected, centrifuged at 3000 r / min for 1 min, and the supernatant was filtered through a 0.22-μm microporous filter membrane to obtain the Grifola frondosa fermentation broth.

[0037] Example 3

[0038] Preparation of Grifola frondosa fermentation broth:

[0039] S1. Weigh the fruiting body powder of Phellinus igniarius, add an ethanol solution with a volume fraction of 60% five times its weight, heat under reflux for extraction twice at 60°C, each time for 2 h, filter, combine the filtrates, recover the ethanol and concentrate to an appropriate amount to obtain a concentrated solution;

[0040] S2. Add three times the weight of water to the above concentrated solution, mix well, add n-butanol ten times the weight of the concentrated solution for extraction twice, collect the extraction solution, recover the solvent to obtain the n-butanol extract of Phellinus igniarius;

[0041] S3. Cut the slant strain of Grifola frondosa into small pieces and inoculate it into a 250-mL Erlenmeyer flask containing 100 mL of liquid medium (glucose 30 g / L, peptone 2 g / L, yeast extract 6 g / L, KH 2 PO 4 0.5 g / L, MgSO 4 ·7H 2 O 0.5 g / L, with water as the solvent), culture in a shaker at 25°C and 150 r / min for 5 days to obtain the Grifola frondosa seed liquid;

[0042] S4. Inoculate the Grifola frondosa seed liquid into the fermentation substrate containing 10 g / L of the n-butanol extract of Phellinus igniarius (glucose 50 g / L, peptone 5 g / L, yeast extract 10 g / L, KH 2 PO 4 2 g / L, MgSO 4 ·7H 2 O 2 g / L, with water as the solvent) at an inoculation amount of 5%, culture in a shaker at 25°C and a rotation speed of 100 r / min for 7 days, collect the fermentation broth, centrifuge at 3000 r / min for 1 min, and take the supernatant to filter through a 0.22-μm microporous filter membrane to obtain the Grifola frondosa fermentation broth.

[0043] Comparative Example 1

[0044] Preparation of Grifola frondosa fermentation broth: The difference from Example 1 is that steps S1 and S2 are omitted, and the n-butanol extract of Phellinus igniarius is not added during the fermentation process.

[0045] Comparative Example 2

[0046] Preparation of Grifola frondosa fermentation broth: Different from Example 1, gastrodia alcohol extract was used instead of Sanghuang n-butanol extract, and the other parameters remained unchanged; Preparation of gastrodia alcohol extract: Fresh gastrodia was washed, sliced, freeze-dried, crushed, passed through a 60-mesh sieve, 10 times the weight of gastrodia powder of 70% ethanol solution by volume was added, extracted for 48 h, the extract was collected, and ethanol was removed by reduced pressure concentration to obtain it.

[0047] Comparative Example 3

[0048] Preparation of Grifola frondosa fermentation broth: Different from Example 1, chloroform was used instead of n-butanol for extraction to obtain Sanghuang chloroform extract, and the above extract was used instead of Sanghuang n-butanol extract in Example 1.

[0049] Comparative Example 4

[0050] Preparation of Grifola frondosa fermentation broth: Different from Example 1, the fermentation broth after microfiltration membrane filtration was extracted with ethanol to obtain Grifola frondosa extracellular polysaccharide. The specific preparation steps were as follows: 95% ethanol was added to the fermentation broth after microfiltration membrane filtration until the ethanol concentration reached 30%, adding was stopped, kept in a 4 °C refrigerator overnight, centrifuged at 3000 r / min for 1 min, and the precipitate was discarded; 95% ethanol was continuously added to the supernatant until the ethanol concentration reached 75%, kept in a 4 °C refrigerator overnight, centrifuged at 3000 r / min for 1 min, the precipitate was collected and dried to constant weight to obtain Grifola frondosa polysaccharide.

[0051] Experimental Example 1. In vitro anti-HPV test

[0052] The SiHa (HPV-16) cell line and HeLa (HPV-18) cell line (purchased from the Shanghai Cell Bank of the Chinese Academy of Medical Sciences) were respectively inoculated into 96-well plates. 2 mL of DMEM complete medium containing 15 μg / mL of the sample (containing 10% FBS and 1% penicillin-streptomycin) was added to the 96-well plates, and 6 replicates were set in each group. The blank control group was DMEM complete medium without adding the sample; Incubated in an incubator at 37 °C and 5% CO 2 for 24 h, the cells in each group were collected and resuspended with PBS buffer, and the total RNA of the cells in each group was extracted according to the method of the Trizol kit to detect the mRNA expression of the cells in each group. Using E6 as the target gene and GAPDH as the internal reference, three replicates were carried out respectively, the Ct value was read, and according to RQ = 2- △△Ct Calculate the mRNA expression level of the target molecule in the cells, and the results are shown in Table 1 below.

[0053] PCR primer information

[0054]

[0055] Reaction system

[0056]

[0057] Table 1: Statistics of E6 mRNA expression levels in SiHa cells and HeLa cells after different treatments

[0058]

[0059]

[0060] Note: Compared with the blank control group, * P < 0.05, ** P < 0.01, *** P < 0.001; compared with Example 1, # P < 0.05, ## P < 0.01.

[0061] The qRT-PCR test results showed that: through data analysis, it can be seen that compared with the blank control group, both Grifola frondosa fermentation broth and Grifola frondosa polysaccharide can affect the expression of E6 mRNA in the two types of cells to varying degrees. Among them, the Grifola frondosa fermentation broth prepared by adding Phellinus linteus extract showed a more obvious effect of inhibiting E6 mRNA expression, and the > Phellinus linteus n-butanol extract > Phellinus linteus chloroform extract, but there was no significant difference in the expression of E6 mRNA in the two types of cells in the Grifola frondosa fermentation broth group added with Gastrodia elata alcohol extract compared with the blank control group, and it was not statistically significant.

[0062] Examples 4 - 6 Gynecological Gel

[0063] Component Example 4 Example 5 Example 6 Grifola frondosa fermentation broth Example 1, 10% Example 2, 10% Example 3, 10% IFN-α2b 10,000 IU / g 5,000 IU / g 20,000 IU / g Carbomer 0.5％ 1％ 1％ Glycerol 5％ 3％ 2％ Glycerol 3％ 3％ 6％ Purified water The balance The balance The balance

[0064] Preparation process: Add glycerol, glycerin and carbopol to pure water, homogenize for 3 minutes, slowly heat up to 80 °C, cool down to 45 °C, then add Grifola frondosa fermentation broth and recombinant human interferon a2b, stir evenly, discharge, and fill according to the specification of 1 g / granule to obtain the gynecological gel.

[0065] Comparative Example 1

[0066] The difference from Example 4 is that it does not contain IFN-α2b.

[0067] Comparative Example 2

[0068] The difference from Example 4 is that it does not contain Grifola frondosa fermentation broth.

[0069] Comparative Example 3

[0070] The difference from Example 4 is that the Grifola frondosa fermentation broth is prepared in Comparative Example 1.

[0071] Comparative Example 4

[0072] The difference from Example 4 is that the Grifola frondosa polysaccharide prepared in Comparative Example 4 is used instead of the Grifola frondosa fermentation broth.

[0073] Experimental Example 2. Study on the treatment of cervicitis with HPV infection by Grifola frondosa fermentation broth combined with IFN-α2b

[0074] 1 Materials and methods

[0075] 1.1 General information: 225 voluntary patients with cervicitis complicated with HPV infection were selected and divided into 5 groups according to the random number table method, with 45 cases in each group. The average age was 35.28 ± 3.41 years old, and the average body mass index was 23.15 ± 1.62 kg / m 2 , and there was no significant difference in the general information between the two groups (P > 0.05).

[0076] 1.2 Inclusion and exclusion criteria: Inclusion criteria: ① Diagnosed as chronic cervicitis complicated with HPV infection by gynecological examination and pathological results; ② The patient gave informed consent to the study. Exclusion criteria: ① Pregnant or lactating women; ② Allergic to recombinant human interferon; ③ Co-existing other sexually transmitted diseases or severe systemic diseases; ④ Having used other antiviral drugs or antibiotics recently.

[0077] 1.3 Administration method: After 3 days of menstruation, the vulva of each group of patients was cleaned and the bladder was emptied. The patients took the supine position, and were asked to clench their fists and raise their buttocks. A disposable vaginal suppository injector equipped in the drug package was used to inject 1 g of the drug near the uterine orifice in the posterior fornix of the vagina. The patients were asked to lie flat for more than 20 minutes. The treatment was given every other day, 10 times for one course of treatment, and the drug was stopped during menstruation. Three courses of treatment were continued. Sexual intercourse, sitz bath and swimming were prohibited during the treatment.

[0078] 1.4 Observation indicators:

[0079] ① Efficacy evaluation: Clinical efficacy judgment criteria: Cure: The clinical symptoms improved significantly, the cervix was smooth, and there was no erosion; Marked effect: The clinical symptoms improved, the erosion area decreased by more than 50%, or decreased to grade I erosion; Effective: The clinical symptoms improved to a certain extent, the erosion area decreased by less than 50%, and the erosion degree was between grade I and grade II; Ineffective: The clinical symptoms did not improve significantly, and the erosion area remained unchanged or increased. The total effective rate = (cure + marked effect + effective) / total number of cases × 100%. The improvement of clinical symptoms included increased leucorrhea, low back pain, contact bleeding, dysuria, etc.

[0080] ②Vaginal microecological environment: Collect the secretions from the upper 1 / 3 of the vaginal fornix, detect the pH value of vaginal secretions with pH test paper, and record the number of patients with pH value ≤ 4.5 and pH value > 4.5. Smear and stain the vaginal secretions, observe under an oil immersion microscope (30-fold field of view), and randomly observe 5 fields of view to observe the vaginal ecological environment and judge the recovery of the flora: ①Recovery or normal: The pH value of the secretions < 4.5; both catalase and sialidase are negative; the flora density is normal, with diversity, and Lactobacillus is the dominant bacterium; ②Not recovered: The pH value of the secretions > 4.5; both catalase and sialidase are positive; the flora density is disordered, lacking diversity, and the dominant bacteria in the flora are miscellaneous bacteria such as cocci and short bacilli. Count the number of patients with normal recovery of the vaginal microecological environment in each group after treatment.

[0081] ③Inflammatory factors: Collect 5 mL of fasting venous blood from the patients before and after treatment, centrifuge at 2500 r / min for 10 min, collect the serum, and detect the expression levels of serum tumor necrosis factor-α (TNF-α) and IFN-γ by enzyme-linked immunosorbent assay.

[0082] ④Situation of turning negative and turning positive again: After the treatment, count the HPV negative conversion rate and HPV positive conversion rate in each group (recheck 2 months after the end of the treatment).

[0083] 1.5 Statistical analysis: Use SPSS 25.0 statistical software to analyze all data. Measurement data are expressed as and independent sample t-test is used; count data are expressed as rate, and x 2 test is used. P < 0.05 indicates that the difference is statistically significant.

[0084] 2 Results

[0085] 2.1 Comparison of clinical efficacy

[0086] Table 2: Comparison of clinical efficacy (cases, %, n = 45)

[0087] Group Cured Markedly effective Effective Ineffective Example 4 25(55.56％) 16(35.56％) 2(4.44％) 2(4.44％) Comparative Example 1 5(11.11％) 5(11.11％) 13(28.89％) 22(48.89％) Comparative Example 2 6(13.33％) 7(15.56％) 12(26.67％) 20(44.44％) Comparative Example 3 10(22.22％) 8(17.78％) 15(33.33％) 12(26.67％) Comparative Example 4 6(13.33％) 11(24.44％) 10(22.22％) 18(40.00％)

[0088] 2.2 Comparison of vaginal microecological environment

[0089] Table 3: Comparison of vaginal microecological environment (n = 45)

[0090]

[0091]

[0092] 2.3 Comparison of inflammatory factor levels before and after treatment

[0093] Table 4: Comparison of inflammatory factor levels before and after treatment ( pg / mL, n = 45)

[0094]

[0095] Note: Compared with before treatment, * P < 0.05, ** P < 0.01, *** P < 0.001.

[0096] 2.4 Comparison of HPV negative conversion rate and recurrence rate

[0097] Table 5: Comparison of HPV negative conversion rate and recurrence rate ( n = 45)

[0098] Group HPV negative conversion rate (cases / rate) Relapse rate (cases / rate) Example 4 41(91.11％) 0(0％) Comparative Example 1 18(40.00％) 1(5.56％) Comparative Example 2 22(48.89％) 4(18.18％) Comparative Example 3 31(68.89％) 5(16.13％) Comparative Example 4 26(57.78％) 5(19.23％)

[0099] The above is only the preferred embodiment of the present invention. It should be noted that the above preferred embodiment should not be regarded as a limitation of the present invention, and the protection scope of the present invention should be subject to the scope defined by the claims. For those of ordinary skill in the art, without departing from the spirit and scope of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as within the protection scope of the present invention.

Claims

1. Use of Grifola frondosa fermented liquid in the preparation of a drug for treating cervicitis associated with high-risk human papillomavirus, characterized in that: The Grifola frondosa fermented liquid is obtained by using Grifola frondosa as a fermentation strain and adding Phellinus linteus extract into a fermentation matrix for liquid deep fermentation.

2. The use according to claim 1, characterized in that The mulberry linterus extract is obtained by heating and refluxing the mulberry linterus fruiting body powder with ethanol as an extraction solvent and then extracting it with n-butanol.

3. The use according to claim 1 or 2, characterized in that The fermentation substrate is added with 3-10 g / L of Phellinus igniarius extract.

4. The use according to any one of claims 1 to 3, characterized in that: The Grifola frondosa fermented liquid is prepared by the following steps: S1. Weigh the Phellinus linteus fruiting body powder, add 5 to 10 times its weight of 50 to 80% ethanol solution by volume, extract under reflux at 55 to 65° C. for 1 to 3 times, each time for 2 hours, filter, combine the filtrate, recover ethanol and concentrate to an appropriate amount to obtain a concentrated solution; S2, adding 1 to 3 times the weight of water to the above concentrate, mixing, adding 8 to 10 times the weight of n-butanol to the concentrate, extracting 1 to 3 times, collecting the extract, recovering the solvent, and obtaining a n-butanol extract of Phellinus igniarius; S3. Inoculate 5-10% of the seed liquid of Grifola frondosa into a fermentation matrix containing 3-10 g / L of n-butanol extract of Phellinus linteus, culture the mixture in a shaker at 25-27° C. and a rotation speed of 100-200 r / min for 5-7 days, collect the fermentation liquid, centrifuge it, and filter it.

5. A gynecological gel, characterized in that: Contains the following components by weight: 3-15% of the Grifola frondosa fermented liquid according to any one of claims 1 to 4; Recombinant human interferon a2b 0.5-20,000 IU / g; Thickener 0.5-1%; Humectant 1-8%; and Pure water balance.

6. The gynecological gel according to claim 5, characterized in that The thickener is carbomer or poloxamer 188.

7. The gynecological gel according to claim 5 or 6, characterized in that: The moisturizing agent is one or a combination of two or more of glycerol, propylene glycol, 1,3-butylene glycol and glycerin.

8. A method for preparing the gynecological gel according to any one of claims 5 to 7, characterized in that: The following steps are involved: Add the moisturizer and thickener into pure water, homogenize for 3-4 minutes, slowly heat up to 80-85°C, cool down to 40-45°C, add Grifola frondosa fermentation liquid and recombinant human interferon a2b, stir evenly to obtain the gynecological gel.

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