Application of combination of antrodia camphorata polysaccharide and forsythin in preparation of medicine for treating metabolic diseases related to insulin resistance

Abstract

The invention provides application of combination of antrodia camphorata polysaccharide and forsythin in preparation of drugs for treating metabolic diseases related to insulin resistance, and belongs to the technical field of biological medicines. The metabolic diseases related to insulin resistance include polycystic ovarian syndrome. Animal experiments find that the combination of the antrodia camphorata polysaccharide and forsythin can significantly reduce serum testosterone and luteinizing hormone levels of a rat with the polycystic ovarian syndrome, improve serum follicular hormone and estradiol levels, and achieve the effect of improving the polycystic ovarian syndrome.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly to the application of antrodia camphorata polysaccharide combined with forsythoside in the preparation of a medicament for treating metabolic diseases related to insulin resistance. Background Art

[0002] Polycystic ovary syndrome (PCOS) is an endocrine and metabolic disease characterized by hyperandrogenism, ovulation disorder, and insulin resistance, affecting 6 - 10% of reproductive-aged women globally. Its pathogenesis is complex, involving the interaction of multiple factors such as genetics, environment, lifestyle, and metabolic abnormalities. Currently, clinical treatments mainly include lifestyle interventions (such as diet adjustment) and medications (such as metformin), but there are problems such as limited efficacy, side effects, and large individual differences.

[0003] Studies have shown that the intestinal microbiota characteristics of PCOS patients are manifested as decreased diversity, increased conditional pathogenic bacteria, and abnormal short-chain fatty acid (SCFAs) metabolism. This imbalance may exacerbate insulin resistance, hyperandrogenism, and chronic inflammation. Akkermansia muciniphila (abbreviated as AKK bacterium), as a probiotic, has been found to be closely related to improving insulin sensitivity, regulating inflammatory responses, and metabolic homeostasis. The abundance of AKK bacteria in the intestines of PCOS patients is significantly reduced, suggesting that it may become a new therapeutic target.

[0004] Therefore, the purpose of the present invention is to provide a medicament that can treat PCOS by increasing the number of AKK bacteria in the intestine, providing a new idea for the treatment of PCOS. Summary of the Invention

[0005] The purpose of the present invention is to provide the application of antrodia camphorata polysaccharide combined with forsythoside in the preparation of a medicament for treating metabolic diseases related to insulin resistance.

[0006] To achieve the above-mentioned invention purpose, the present invention provides the following technical solutions:

[0007] The present invention provides the application of antrodia camphorata polysaccharide combined with forsythoside in the preparation of a product for promoting the proliferation of Akkermansia muciniphila in vivo.

[0008] The present invention provides the application of antrodia camphorata polysaccharide combined with forsythoside in the preparation of a medicament for treating metabolic diseases related to insulin resistance.

[0009] Preferably, the metabolic diseases related to insulin resistance include polycystic ovary syndrome.

[0010] The present invention provides a pharmaceutical composition, which comprises antrodia camphorata polysaccharide and forsythoside.

[0011] Preferably, the mass ratio of Antrodia camphorata polysaccharide to forsythin in the pharmaceutical composition is 3.5 - 4.5:0.8 - 1.2.

[0012] Preferably, the pharmaceutical composition has any one of the following uses:

[0013] (a) Preparing a product for promoting the proliferation of Akkermansia muciniphila in vivo;

[0014] (b) Preparing a drug for treating metabolic diseases related to insulin resistance;

[0015] (c) Preparing a drug for treating polycystic ovary syndrome.

[0016] Preferably, the preparation method of the Antrodia camphorata polysaccharide comprises the following steps:

[0017] (1) Crushing the Antrodia camphorata fruiting body, mixing it with water, adding cellulase for enzymatic hydrolysis to obtain a hydrolysate;

[0018] (2) Inactivating the enzyme in the hydrolysate, subjecting it to shearing treatment at 4000 - 6000 rpm for 15 - 25 min, performing solid - liquid separation, and collecting the liquid part to obtain a crude extract;

[0019] (3) Mixing the crude extract with an ethanol solution until the ethanol concentration in the mixture is 70 - 80%, standing for 40 - 80 min and then centrifuging, mixing the precipitate with water and dialyzing, and drying the dialysate to obtain Antrodia camphorata polysaccharide.

[0020] Preferably, in step (1), the mass - to - volume ratio of the Antrodia camphorata fruiting body to water is 1 g:10 - 20 mL;

[0021] The concentration of the cellulase is 0.8 - 1.2%;

[0022] The pH during the enzymatic hydrolysis is 5.0 - 5.5, the temperature is 45 - 55 °C, and the time is 50 - 70 min.

[0023] Preferably, in step (2), the method for inactivating the enzyme is to treat it in a boiling water bath for 5 - 10 min;

[0024] The shearing is carried out at 30 - 50 °C;

[0025] The solid - liquid separation is centrifugation, the centrifugation rate is 3000 - 5000 rpm, and the time is 8 - 12 min.

[0026] Preferably, in step (3), the concentration of the ethanol solution is 90 - 95%;

[0027] The centrifugation rate is 3000 - 5000 rpm, and the time is 10 - 20 min;

[0028] The cut-off molecular weight of the dialysis bag used during dialysis is 3,000 to 4,000 Da;

[0029] Before drying, the dialysis solution is concentrated to 1 / 8 to 1 / 12 of the original volume and freeze-dried at -30 to -50 °C.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] Through animal experiments, the present invention found that polysaccharide from Antrodia cinnamomea combined with forsythin can significantly reduce the serum testosterone and luteinizing hormone levels in rats with polycystic ovary syndrome, and increase the serum follicle-stimulating hormone and estradiol levels, achieving the effect of improving polycystic ovary syndrome. At the same time, polysaccharide from Antrodia cinnamomea combined with forsythin can also significantly increase the number of Akkermansia muciniphila in the rat intestine and reduce the insulin resistance index, indicating that polysaccharide from Antrodia cinnamomea and forsythin may treat metabolic diseases related to insulin resistance, especially polycystic ovary syndrome, by synergistically increasing the number of Akkermansia muciniphila in the intestine.

[0032] The present invention provides a new idea for the treatment of polycystic ovary syndrome and has broad application prospects. Brief Description of the Drawings

[0033] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for description in the embodiments or the prior art. Obviously, the drawings in the following description are only the embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained according to the provided drawings without creative efforts.

[0034] Figure 1 It is a schematic diagram of vaginal smears in different estrous cycles in the experimental examples. Among them, (A) is proestrus, (B) is estrus, (C) is metestrus, and (D) is diestrus;

[0035] Figure 2 It is the change of the estrous cycle of rats in different treatment groups in the experimental examples;

[0036] Figure 3 It is the HE staining result of the ovarian morphology of rats in different treatment groups in the experimental examples. A to E represent the PCOS group, ACP group, ACP + Phr group, Met group, and control group in sequence;

[0037] Figure 4 It is the statistical result of the number of follicles and corpora lutea in the ovaries of rats in different treatment groups in the experimental examples;

[0038] Figure 5Results of the determination of the levels of sex hormones testosterone (T), luteinizing hormone (LH), follicle-stimulating hormone (FSH), estrogen (estradiol, E2) and the LH / FSH ratio in rats of different treatment groups in the experimental examples. Detailed implementation manners

[0039] The present invention provides the use of antrodia camphorata polysaccharide combined with forsythin in the preparation of a product for promoting the proliferation of Akkermansia muciniphila in vivo.

[0040] The present invention provides the use of antrodia camphorata polysaccharide combined with forsythin in the preparation of a drug for treating metabolic diseases related to insulin resistance.

[0041] In the present invention, the metabolic diseases related to insulin resistance include polycystic ovary syndrome.

[0042] The present invention provides a pharmaceutical composition, which comprises antrodia camphorata polysaccharide and forsythin.

[0043] In the present invention, the mass ratio of antrodia camphorata polysaccharide to forsythin in the pharmaceutical composition is 3.5-4.5:0.8-1.2, preferably 3.8-4.2:0.9-1.1, and more preferably 4:1.

[0044] In the present invention, the pharmaceutical composition has any one of the following uses:

[0045] (a) Preparing a product for promoting the proliferation of Akkermansia muciniphila in vivo;

[0046] (b) Preparing a drug for treating metabolic diseases related to insulin resistance;

[0047] (c) Preparing a drug for treating polycystic ovary syndrome.

[0048] In the present invention, the preparation method of the antrodia camphorata polysaccharide comprises the following steps:

[0049] (1) Crushing the antrodia camphorata fruiting body and mixing it with water, adding cellulase for enzymatic hydrolysis to obtain an enzymatic hydrolysate;

[0050] (2) Inactivating the enzymatic hydrolysate and performing shearing treatment at 4000-6000 rpm for 15-25 min, performing solid-liquid separation, collecting the liquid part to obtain a crude extract;

[0051] (3) Mixing the crude extract with an ethanol solution until the concentration of ethanol in the mixture is 70-80%, standing for 40-80 min and then centrifuging, mixing the precipitate with water and then dialyzing, and drying the dialysate to obtain antrodia camphorata polysaccharide.

[0052] In the present invention, the mass-to-volume ratio of Antrodia cinnamomea fruit body to water in step (1) is 1 g: 10 - 20 mL, preferably 1 g: 12 - 18 mL, more preferably 1 g: 14 - 16 mL, and even more preferably 1 g: 15 mL;

[0053] The concentration of the cellulase is 0.8 - 1.2%, preferably 0.9 - 1.1%, and more preferably 1%;

[0054] The pH during enzymatic hydrolysis is 5.0 - 5.5, preferably 5.2 - 5.4, and more preferably 5.3; the temperature is 45 - 55 °C, preferably 48 - 52 °C, and more preferably 50 °C; the time is 50 - 70 min, preferably 55 - 65 min, and more preferably 60 min.

[0055] In the present invention, step (2) is preferably to shear the enzymatic hydrolysate for 18 - 22 min at 4500 - 5500 rpm after inactivating the enzyme, and more preferably to shear the enzymatic hydrolysate for 20 min at 5000 rpm after inactivating the enzyme.

[0056] In the present invention, the method for inactivating the enzyme in step (2) is to treat it in a boiling water bath for 5 - 10 min, preferably for 8 min;

[0057] The shearing is carried out at 30 - 50 °C, preferably at 35 - 45 °C, and more preferably at 40 °C;

[0058] The solid-liquid separation is centrifugation. The centrifugation rate is 3000 - 5000 rpm, preferably 3500 - 4500 rpm, and more preferably 4000 rpm; the time is 8 - 12 min, preferably 9 - 11 min, and more preferably 10 min.

[0059] In the present invention, step (3) is preferably to mix the crude extract with an ethanol solution until the ethanol concentration in the mixed solution is 72 - 78%, and then centrifuge after standing for 50 - 70 min. More preferably, the crude extract is mixed with an ethanol solution until the ethanol concentration in the mixed solution is 75%, and then centrifuge after standing for 60 min.

[0060] In the present invention, the concentration of the ethanol solution in step (3) is 90 - 95%, preferably 95%;

[0061] The centrifugation rate is 3000 - 5000 rpm, preferably 3500 - 4500 rpm, and more preferably 4000 rpm; the time is 10 - 20 min, preferably 12 - 18 min, and more preferably 15 min;

[0062] The cut-off molecular weight of the dialysis bag used during dialysis is 3000 - 4000 Da, preferably 3500 Da;

[0063] Before drying, the dialysis solution is concentrated to 1 / 8 - 1 / 12 of the original volume, preferably concentrated to 1 / 10 of the original volume, and freeze-dried at -30 to -50 °C, preferably freeze-dried at -35 to -45 °C, and further preferably freeze-dried at -40 °C.

[0064] The technical solutions provided by the present invention will be described in detail below with reference to the examples, but they should not be construed as limiting the protection scope of the present invention. The CAS number of phillyrin used in the following examples is 487-41-2.

[0065] Example 1

[0066] A method for preparing polysaccharide from Antrodia cinnamomea, the steps are as follows:

[0067] (1) The Antrodia cinnamomea fruiting body is crushed and passed through an 80-mesh sieve. The material passing through the sieve is mixed with water at a ratio of 1 g:10 mL, and cellulase is added until its concentration is 0.8%. Enzymolysis is carried out at pH 5.0 and a temperature of 45 °C for 50 min to obtain an enzymolysis solution.

[0068] (2) The enzymolysis solution is placed in a boiling water bath for 5 min to inactivate the enzyme. The inactivated enzymolysis solution is sheared at 30 °C and 4000 rpm for 15 min. The sheared reaction solution is centrifuged at 3000 rpm for 8 min, and the supernatant is collected to obtain a crude extract.

[0069] (3) 95% ethanol (v / v) is added to the crude extract until the ethanol concentration in the mixed solution reaches 70%. After standing for 40 min, it is centrifuged at 3000 rpm for 10 min. The precipitate is mixed with water and dialyzed in a dialysis bag with a cut-off molecular weight of 3000 Da. The dialysis solution is concentrated to 1 / 8 of the original volume and then dried at -30 °C to obtain polysaccharide from Antrodia cinnamomea.

[0070] Example 2

[0071] A method for preparing polysaccharide from Antrodia cinnamomea, the steps are as follows:

[0072] (1) The Antrodia cinnamomea fruiting body is crushed and passed through an 80-mesh sieve. The material passing through the sieve is mixed with water at a ratio of 1 g:20 mL, and cellulase is added until its concentration is 1.2%. Enzymolysis is carried out at pH 5.5 and a temperature of 55 °C for 70 min to obtain an enzymolysis solution.

[0073] (2) Place the enzymolysis solution in a boiling water bath for 10 min to inactivate the enzyme. Subject the inactivated enzymolysis solution to shearing treatment at 50 °C and 6000 rpm for 25 min. Centrifuge the sheared reaction solution at 5000 rpm for 12 min, collect the supernatant, and obtain the crude extract.

[0074] (3) Add 95% ethanol (v / v) to the crude extract until the ethanol concentration in the mixture reaches 80%. Let it stand for 80 min and then centrifuge at 5000 rpm for 20 min. Mix the precipitate with water, place it in a dialysis bag with a molecular weight cut-off of 4000 Da for dialysis. Concentrate the dialysis solution to 1 / 12 of the original volume and then dry it at -50 °C to obtain the polysaccharide from Antrodia cinnamomea.

[0075] Example 3

[0076] A method for preparing the polysaccharide from Antrodia cinnamomea comprises the following steps:

[0077] (1) Crush the fruiting body of Antrodia cinnamomea and sieve it through a 80-mesh sieve. Mix the material passing through the sieve with water at a ratio of 1 g:15 mL, add cellulase until its concentration is 1%, and perform enzymolysis at pH 5.2 and 50 °C for 60 min to obtain the enzymolysis solution.

[0078] (2) Place the enzymolysis solution in a boiling water bath for 8 min to inactivate the enzyme. Subject the inactivated enzymolysis solution to shearing treatment at 40 °C and 5000 rpm for 20 min. Centrifuge the sheared reaction solution at 4000 rpm for 10 min, collect the supernatant, and obtain the crude extract.

[0079] (3) Add 95% ethanol (v / v) to the crude extract until the ethanol concentration in the mixture reaches 75%. Let it stand for 60 min and then centrifuge at 4000 rpm for 15 min. Mix the precipitate with water, place it in a dialysis bag with a molecular weight cut-off of 3500 Da for dialysis. Concentrate the dialysis solution to 1 / 10 of the original volume and then dry it at -40 °C to obtain the polysaccharide from Antrodia cinnamomea.

[0080] Experimental example: Study on the therapeutic effects of the polysaccharide from Antrodia cinnamomea (ACP) and phillyrin (Phr) on polycystic ovary syndrome (PCOS)

[0081] 1. Animal grouping

[0082] Twenty-five 3-week-old female SD rats weighing about 50 g were adaptively fed and quarantined for one week, and randomly divided into a control group and a PCOS model group, with 5 in the control group and 20 in the preliminary PCOS model group.

[0083] 2. Modeling

[0084] The rats in the model group were intragastrically administered letrozole (0.1 g / kg) every day. When intragastrically administered, letrozole was dissolved in 1% sodium carboxymethyl cellulose to make a suspension; the control group was intragastrically administered an equal amount of 1% sodium carboxymethyl cellulose. The intragastric administration was carried out for 21 days. From the 10th day of intragastric administration, vaginal smears were examined at the same time every day to monitor the estrous cycle, the body weights of the rats in each group were weighed, and the changes in their estrous cycles were observed. Continuous weight gain and disrupted estrous cycles indicated successful establishment of the PCOS model.

[0085] 3. Administration of drugs

[0086] Twenty rats with successful modeling were divided into a PCOS group, an ACP group, an ACP + Phr group, and a Met (metformin) group, with 5 rats in each cage. The rats in the ACP group were intragastrically administered the polysaccharide from Antrodia cinnamomea prepared in Example 3 (1 g / kg / d), the rats in the ACP + Phr group were simultaneously intragastrically administered the polysaccharide from Antrodia cinnamomea prepared in Example 3 (0.8 g / kg / d) and forsythin (0.2 g / kg / d), the rats in the Met group were intragastrically administered metformin (280 mg / kg / d), and the rats in the control group and the PCOS group were intragastrically administered an equal amount of normal saline; the continuous intragastric administration was carried out for 21 d.

[0087] 4. Observation of estrous cycle

[0088] The schematic diagram for judging vaginal smears in different estrous cycles is as Figure 1 shown. Among them, (A) is proestrus, mainly showing nucleated epithelial cells, and occasionally a small amount of keratinized cells; (B) is estrus, where the nucleated epithelium gradually transforms into keratinized epithelial cells; (C) is metestrus, with a decrease in keratinized epithelium, the appearance of nucleated epithelial cells, and an increase in white blood cells; (D) is diestrus, mainly consisting of white blood cells, and occasionally a small amount of keratinized epithelium.

[0089] The changes in the estrous cycles of rats in different treatment groups are as Figure 2 shown. It can be seen that as the modeling progressed, the estrous cycles of the rats in the PCOS group became disordered, mainly staying in diestrus and metestrus. After the intervention of ACP and Phr, the estrous cycles of the rats in the PCOS group gradually returned to normal, indicating that ACP and Phr can improve the estrous cycle disorders related to PCOS.

[0090] 5. Observation by HE staining

[0091] Paraffin sections of the ovaries of rats in the PCOS group, the control group, and the ACP + Phr group were prepared and subjected to HE staining. The morphology of the ovaries was observed under a microscope. The results are as Figure 3 shown. A - E represent the PCOS group, the ACP group, the ACP + Phr group, the Met group, and the control group in sequence; at the same time, the number of follicles and corpora lutea in the rats of each treatment group was counted, and the results are as Figure 4 shown.

[0092] It can be seen that compared with the control group, the number of cystic follicles in the PCOS group of rats was significantly increased, indicating the successful establishment of the PCOS rat model. Compared with the PCOS group, the number of cystic follicles in the ACP+Phr group of rats was significantly reduced, and the number of corpora lutea was increased, indicating that ACP combined with Phr could achieve the effect of treating polycystic ovary syndrome.

[0093] 6. Detection of serum hormone levels

[0094] After the administration was completed, 2 rats were randomly selected from each group for the detection of serum hormone levels. After anesthetizing the rats by intraperitoneal injection of sodium pentobarbital (2%, 0.2 mL / 100 g), blood was collected from the abdominal aorta and centrifuged (1000 rpm, 4 °C, 10 min). The levels of serum sex hormones testosterone (T), luteinizing hormone (LH), follicle-stimulating hormone (FSH), estrogen (estradiol, E2) and the LH / FSH ratio were detected using an ELISA kit.

[0095] The results are as Figure 5 shown. It can be seen that compared with the control group of rats, the levels of serum hormones T and LH in the PCOS group of rats were significantly increased, while the levels of FSH and E2 were significantly decreased, indicating the successful establishment of the polycystic ovary syndrome model rats. Compared with the PCOS group of rats, the levels of serum hormones T and LH in the ACP group, ACP+Phr group and Met group of rats were significantly decreased, and the levels of FSH and E2 were significantly increased, indicating that ACP, ACP combined with Phr, and Met could all significantly improve polycystic ovary syndrome, and the improvement effects from high to low were Met, ACP combined with Phr, and ACP.

[0096] 7. Detection of AKK bacteria abundance

[0097] The feces of rats in each treatment group were collected, and the abundance of AKK bacteria in the feces was detected. The detection results are shown in Table 1.

[0098] Table 1 Determination results of AKK bacteria abundance

[0099] Treatment group Abundance of Akkermansia muciniphila (%) Control group 7.462 PCOS group 1.573 ACP group 8.749 ACP + Phr group 10.262 Met group 7.297

[0100] As can be seen from Table 1, the abundance of AKK bacteria in the feces of the PCOS group of rats was significantly reduced, indicating a close correlation between AKK bacteria and the occurrence of PCOS. The abundance of AKK bacteria in the feces of the ACP+Phr group of rats was significantly increased compared with the ACP group and the Met group, indicating that ACP and Phr may achieve the effect of treating polycystic ovary syndrome by synergistically increasing the number of AKK bacteria in the intestine.

[0101] 8. Glucose tolerance and insulin release experiments

[0102] Two rats were randomly selected from each group for glucose tolerance and insulin release experiments. Before the experiment, the rats were fasted for 12 h without water deprivation, and then gavaged with glucose solution at a dose of 2 g / kg. Tail vein blood was collected before gavage and at 30, 60, 90, and 120 min after gavage to detect blood glucose levels. The insulin resistance index was calculated (insulin resistance index = fasting blood glucose (mmol / L) × fasting insulin (mIU / L) ÷ 22.5). The results are shown in Tables 2 and 3.

[0103] Table 2 Changes in fasting blood glucose levels of rats in each treatment group (mmol / L)

[0104] Treatment group Before gavage 30 min 60 min 90 min 120 min Control group 4.67 9.58 8.79 7.38 5.42 PCOS group 9.32 13.12 12.54 10.49 9.21 ACP group 7.68 11.95 11.20 9.83 8.13 ACP + Phr group 7.21 11.53 10.59 9.17 7.40 Met group 6.94 11.26 10.37 9.10 7.25

[0105] Table 3 Calculation results of fasting insulin levels and insulin resistance indices of rats in each treatment group

[0106] Treatment group Fasting insulin (mIU / L) Insulin resistance index Control group 9.25 2.23 PCOS group 36.28 14.85 ACP group 25.44 9.19 ACP + Phr group 23.67 7.78 Met group 21.52 6.93

[0107] It can be seen that ACP, ACP combined with Phr, and Met can reduce the insulin resistance index of rats, and the effect of ACP combined with Phr in reducing it is significantly better than that of ACP, indicating that ACP and Phr have a synergistic effect in reducing insulin resistance in rats.

[0108] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. Use of Antrodia cinnamomea polysaccharide combined with phillyrin in the preparation of a product for promoting the proliferation of Akkermansia muciniphila in vivo.

2. Use of Antrodia cinnamomea polysaccharide combined with phillyrin in the preparation of a drug for treating metabolic diseases related to insulin resistance.

3. The application according to claim 2, wherein The metabolic diseases related to insulin resistance include polycystic ovary syndrome.

4. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises Antrodia cinnamomea polysaccharide and phillyrin.

5. The pharmaceutical composition according to claim 4, wherein, The mass ratio of Antrodia cinnamomea polysaccharide to phillyrin in the pharmaceutical composition is 3.5 - 4.5:0.8 - 1.

2.

6. The pharmaceutical composition according to claim 4, characterized in that, The pharmaceutical composition has any one of the following uses: (a) Preparation of a product for promoting the proliferation of Akkermansia muciniphila in vivo; (b) Preparation of a drug for treating metabolic diseases related to insulin resistance; (c) Preparation of a drug for treating polycystic ovary syndrome.

7. The pharmaceutical composition according to any one of claims 4 to 6, characterized in that The preparation method of the Antrodia cinnamomea polysaccharide comprises the following steps: (1) Crushing the Antrodia cinnamomea fruiting body, mixing it with water, adding cellulase for enzymatic hydrolysis to obtain an enzymatic hydrolysate; (2) Inactivating the enzymatic hydrolysate, performing shearing treatment at 4000 - 6000 rpm for 15 - 25 min, separating the solid and liquid, collecting the liquid part to obtain a crude extract; (3) Mixing the crude extract with an ethanol solution until the concentration of ethanol in the mixed solution is 70 - 80%, standing for 40 - 80 min and then centrifuging, mixing the precipitate with water and then dialyzing, and drying the dialysate to obtain Antrodia cinnamomea polysaccharide.

8. The pharmaceutical composition according to claim 7, wherein In step (1), the mass - to - volume ratio of the Antrodia cinnamomea fruiting body to water is 1 g:10 - 20 mL; The concentration of the cellulase is 0.8 - 1.2%; The pH during enzymatic hydrolysis is 5.0 - 5.5, the temperature is 45 - 55 °C, and the time is 50 - 70 min.

9. The pharmaceutical composition according to claim 7, characterized in that, In step (2), the method for inactivating the enzyme is to treat it in a boiling water bath for 5 - 10 min; The shearing is carried out at 30 - 50 °C; The solid - liquid separation is centrifugation, and the centrifugation rate is 3000 - 5000 rpm, and the time is 8 - 12 min.

10. The pharmaceutical composition according to claim 7, characterized in that, In step (3), the concentration of the ethanol solution is 90 - 95%; The centrifugation rate is 3000 - 5000 rpm, and the time is 10 - 20 min; The cut - off molecular weight of the dialysis bag used during dialysis is 3000 - 4000 Da; Before drying, the dialysate is concentrated to 1 / 8 - 1 / 12 of the original volume and freeze - dried at - 30 - - 50 °C.

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