Preparation method of black garlic polysaccharide with high content and application thereof in anti-HBV drug

Abstract

The present invention relates to the technical field of polysaccharide extraction, and particularly to a preparation method of high-content black garlic polysaccharide and its application in anti-HBV drugs. Through ultrasonic extraction, impurity removal by ceramic membrane, and retention by a suitable organic membrane, the content of black garlic polysaccharide in the obtained black garlic polysaccharide solution is much higher than that of the ordinary water extraction and alcohol precipitation method; after the black garlic polysaccharide solution is dried, the content of black garlic polysaccharide is greater than 85%. In addition, the black garlic polysaccharide prepared by using the preparation method provided by the present invention can reduce the levels of HBsAg and HBeAg and inhibit the replication of HBV-DNA, that is, the black garlic polysaccharide can be used for anti-HBV infection or for the preparation of anti-HBV infection drugs.

Description

Technical Field

[0001] The present invention relates to the technical field of polysaccharide extraction, and particularly to a preparation method of high-content black garlic polysaccharide and its application in anti-HBV drugs. Background Art

[0002] Black garlic is a deep-processed garlic product formed by controlling temperature and humidity and undergoing high-temperature ripening of fresh garlic, which removes the garlic odor and enhances its antioxidant physiological activity. It has a soft texture, is sweet and sour without irritation, and emits a strong aroma that can arouse appetite, and is deeply welcomed by consumers.

[0003] Polysaccharide, also known as polyglycan, is a class of natural macromolecular substances condensed by more than 10 monosaccharide molecules, formed by the connection of aldose and / or ketose through glycosidic bonds, and is one of the four basic substances constituting life. Polysaccharides have a wide range of biological effects such as antioxidant, hypoglycemic, hypolipidemic, and anti-radiation.

[0004] At present, the extraction process for black garlic polysaccharide is mostly the water extraction and alcohol precipitation method, and the content of the extracted black garlic polysaccharide is relatively low. Summary of the Invention

[0005] In order to solve the above problems, the present invention provides a preparation method of high-content black garlic polysaccharide and its application in the preparation of anti-HBV (hepatitis B virus) drugs. The content of black garlic polysaccharide prepared by using the preparation method provided by the present invention is greater than 85%, which is much higher than that of the ordinary water extraction and alcohol precipitation method.

[0006] In order to achieve the above object, the present invention provides the following technical solutions:

[0007] The present invention provides a preparation method of high-content black garlic polysaccharide, comprising the following steps:

[0008] Crush black garlic and mix it with water, and perform ultrasonic extraction to obtain an extract;

[0009] Filter the extract through a filter membrane with a pore size of 0.1 - 0.5 μm to obtain a purified extract;

[0010] Concentrate the purified extract to an extract paste to obtain a concentrated solution;

[0011] Mix the concentrated solution with absolute ethanol, let it stand and then centrifuge to obtain a precipitate;

[0012] Dissolve the precipitate and filter it through an organic membrane with a molecular weight cut-off of 5000 - 20000 Daltons to obtain a filtrate as a black garlic polysaccharide solution.

[0013] Preferably, the particle size of the crushed black garlic is 5 - 200 μm.

[0014] Preferably, the mass-volume ratio of the pulverized black garlic to water is 1 g:(10 - 200) mL.

[0015] Preferably, the power of the ultrasonic extraction is 200 - 1000 W, the temperature of the ultrasonic extraction is 40 - 90 °C, and the time of the ultrasonic extraction is 15 - 120 min.

[0016] Preferably, the volume ratio of the concentrated solution to the purified extract is 1:(5 - 8).

[0017] Preferably, the volume ratio of the concentrated solution to absolute ethanol is 1:(3 - 6).

[0018] Preferably, the mass ratio of the precipitate to the water for dissolving the precipitate is 1:(1 - 10).

[0019] The present invention also provides a black garlic polysaccharide prepared by the preparation method described in the above technical solution.

[0020] The present invention also provides the application of the black garlic polysaccharide described in the above technical solution in the preparation of antiviral drugs, and the viruses include hepatitis B virus.

[0021] Preferably, the antiviral action targets include: HBeAg and / or HBsAg.

[0022] Beneficial effects:

[0023] The present invention provides a preparation method of high-content black garlic polysaccharide, which includes the following steps: pulverize black garlic and mix it with water, perform ultrasonic extraction to obtain an extract; filter the extract through a 0.1 - 0.5 μm filter membrane to obtain a purified extract; concentrate the purified extract to an extract paste to obtain a concentrated solution; mix the concentrated solution with absolute ethanol, let it stand and then centrifuge to obtain a precipitate; dissolve the precipitate and filter it through an organic membrane with a molecular weight cut-off of 5000 - 20000 Daltons to obtain a filtrate which is a black garlic polysaccharide solution. The black garlic polysaccharide solution obtained by the present invention through ultrasonic extraction, ceramic membrane impurity removal and appropriate organic membrane retention has a much higher black garlic polysaccharide content than the ordinary water extraction and alcohol precipitation method; after the black garlic polysaccharide solution is dried, the black garlic polysaccharide content is greater than 85%.

[0024] In addition, the black garlic polysaccharide prepared by the preparation method provided by the present invention can reduce the levels of HBsAg and HBeAg and inhibit the replication of HBV-DNA, that is, the black garlic polysaccharide can be used for anti-HBV infection or for the preparation of anti-HBV infection drugs. Specific embodiments

[0025] The present invention provides a preparation method of high-content black garlic polysaccharide, which includes the following steps:

[0026] Crush black garlic and mix it with water, then perform ultrasonic extraction to obtain an extract;

[0027] Filter the extract through a 0.1 - 0.5 μm filter membrane to obtain a purified extract;

[0028] Concentrate the purified extract to a paste - like state to obtain a concentrated solution;

[0029] Mix the concentrated solution with absolute ethanol, let it stand and then centrifuge to obtain a precipitate;

[0030] Dissolve the precipitate and filter it through an organic membrane with a molecular weight cut - off of 5000 - 20000 Daltons to obtain a filtrate, which is a black garlic polysaccharide solution.

[0031] In the present invention, it is preferred to dry black garlic to obtain dried black garlic. In the present invention, the drying method preferably includes drying black garlic to a water content < 5%, and more preferably drying black garlic to a water content < 5% by microwave drying.

[0032] After obtaining the dried black garlic, it is preferred to crush the dried black garlic in the present invention to obtain black garlic powder. In the present invention, the particle size of the black garlic powder is preferably 5 - 200 μm, further preferably 5 - 100 μm, more preferably 5 - 50 μm, and most preferably 5 μm. By crushing black garlic to an appropriate particle size in the present invention, the extraction rate of black garlic polysaccharide can be increased, that is, the content of the prepared black garlic polysaccharide can be increased.

[0033] After obtaining the black garlic powder, the present invention mixes the black garlic powder with water and performs ultrasonic extraction to obtain an extract. In the present invention, the mass - to - volume ratio of the black garlic powder to water is preferably 1 g:(10 - 200) mL, further preferably 1 g:(10 - 100) mL, more preferably 1 g:(10 - 50) mL, and most preferably 1 g:10 mL.

[0034] In the present invention, the power of the ultrasonic extraction is preferably 200 - 1000 W, further preferably 200 - 500 W, more preferably 200 - 300 W, and most preferably 200 W; the temperature of the ultrasonic extraction is preferably 40 - 90 °C, further preferably 40 - 70 °C, more preferably 40 - 60 °C, and most preferably 40 °C; the time of the ultrasonic extraction is preferably 15 - 120 min, further preferably 15 - 80 min, more preferably 15 - 50 min, and most preferably 15 min.

[0035] After obtaining the extract, the present invention filters the extract through a filter membrane with a pore size of 0.1 - 0.5 μm to obtain a purified extract. In the present invention, the pore size of the filter membrane is 0.1 - 0.5 μm, preferably 0.1 - 0.3 μm, and more preferably 0.2 μm; the filter membrane preferably comprises a ceramic membrane. By removing impurities through a ceramic membrane with an appropriate pore size, the present invention can remove impurities such as black garlic powder in the extract, thereby improving the purity of black garlic polysaccharide in the extract.

[0036] After obtaining the purified extract, the present invention concentrates the purified extract to a paste-like state to obtain a concentrated solution. In the present invention, the paste-like state means that the volume ratio of the concentrated solution to the purified extract is preferably 1:(5 - 8), and more preferably 1:6. In the present invention, the concentration method preferably includes vacuum concentration and / or organic membrane concentration. By concentrating the purified extract to a paste-like state, the present invention can reduce the amount of absolute ethanol used subsequently, thereby reducing the preparation cost.

[0037] After obtaining the concentrated solution, the present invention mixes the concentrated solution with absolute ethanol, stands still and then centrifuges to obtain a precipitate. In the present invention, the volume ratio of the concentrated solution to absolute ethanol is preferably 1:(3 - 6), further preferably 1:(3 - 5), more preferably 1:(3 - 4), and most preferably 1:3; the standing time is preferably 24 h.

[0038] After obtaining the precipitate, the present invention dissolves the precipitate and filters it through an organic membrane with a molecular weight cut-off of 5000 - 20000 Da to obtain a filtrate as a black garlic polysaccharide solution. In the present invention, the mass ratio of the precipitate to the water for dissolving the precipitate is preferably 1:(1 - 10), further preferably 1:(3 - 8), and more preferably 1:5; the organic membrane is an organic membrane with a molecular weight cut-off of 5000 - 20000 Da, further preferably 8000 - 15000 Da, and more preferably 10000 Da. By filtering through an appropriate organic membrane, the present invention can improve the purity of black garlic polysaccharide in the black garlic polysaccharide solution.

[0039] After obtaining the black garlic polysaccharide solution, the present invention preferably dries the black garlic polysaccharide solution to obtain high-content black garlic polysaccharide.

[0040] Through ultrasonic extraction, impurity removal by a ceramic membrane, and retention by an appropriate organic membrane, the content of black garlic polysaccharide in the black garlic polysaccharide solution obtained by the present invention is much higher than that of the ordinary water extraction and alcohol precipitation method; in the solid obtained by drying the black garlic polysaccharide solution, the content of black garlic polysaccharide is greater than 85%.

[0041] The present invention also provides a black garlic polysaccharide prepared by the preparation method described in the above technical solution.

[0042] The present invention also provides the use of the black garlic polysaccharide as described in the above technical solution in the preparation of antiviral drugs, and the viruses include hepatitis B virus (HBV).

[0043] In the present invention, the antiviral action targets preferably include: HBeAg and / or HBsAg.

[0044] The black garlic polysaccharide prepared by using the preparation method provided by the present invention can reduce the levels of HBsAg and HBeAg and inhibit the replication of HBV-DNA. That is, the black garlic polysaccharide can be used for anti-HBV infection or for the preparation of anti-HBV infection drugs.

[0045] In order to further illustrate the present invention, the following examples are used to describe in detail a preparation method of a high-content black garlic polysaccharide provided by the present invention and its application in anti-HBV drugs, but they should not be construed as limiting the protection scope of the present invention.

[0046] Example 1

[0047] A high-content black garlic polysaccharide is prepared by the following method:

[0048] 1) The dried black garlic (the dried black garlic is prepared by microwave drying at a power of 800 W for 4 hours) is pulverized into a dry powder with a particle size of 5 μm by an ultrafine pulverizer;

[0049] 2) Water is added to the dry powder in step 1) to obtain a mixed solution; the mass-volume ratio of the dry powder to water is 1 g:10 mL;

[0050] 3) The mixed solution obtained in step 2) is extracted by ultrasonic extraction to obtain an extract; the ultrasonic power of the ultrasonic extraction is 200 W, the temperature of the ultrasonic extraction is 40 °C, and the time of the ultrasonic extraction is 15 min;

[0051] 4) The extract obtained in step 3) is purified by a 0.1-μm ceramic membrane to obtain a purified extract;

[0052] 5) The purified extract is concentrated under reduced pressure and dehydrated to an extract paste to obtain a concentrated solution; the volume ratio of the concentrated solution to the purified extract is 1:6;

[0053] 6) Three times the volume of absolute ethanol is added to the concentrated solution obtained in step 5) for precipitation. After standing for 24 h, centrifugation is carried out to collect the precipitate;

[0054] 7) The precipitate obtained in step 6) is dissolved in five times its mass of pure water, and after dissolution, it is filtered through a 5000-Dalton organic membrane to obtain a filtrate;

[0055] 8) The filtrate obtained in step 7) is concentrated and dried to obtain a high-content black garlic polysaccharide.

[0056] Example 2

[0057] A high-content black garlic polysaccharide prepared by a preparation method similar to that of Example 1, except that the pore size of the ceramic membrane in step 4) is 0.2 μm.

[0058] Example 3

[0059] A high-content black garlic polysaccharide prepared by a preparation method similar to that of Example 1, except that the organic membrane used for filtration in step 7) is an organic membrane with a molecular weight cut-off of 10,000 Da.

[0060] Example 4

[0061] A high-content black garlic polysaccharide prepared by a preparation method similar to that of Example 1, except that the pore size of the ceramic membrane in step 4) is 0.2 μm; the organic membrane used for filtration in step 7) is an organic membrane with a molecular weight cut-off of 10,000 Da.

[0062] Example 5

[0063] A high-content black garlic polysaccharide prepared by a preparation method similar to that of Example 1, except that the pore size of the ceramic membrane in step 4) is 0.5 μm.

[0064] Comparative Example 1

[0065] A black garlic polysaccharide prepared by a preparation method similar to that of Example 1, except that the impurity removal by the ceramic membrane in step 4) is not carried out.

[0066] Comparative Example 2

[0067] A garlic polysaccharide prepared by a preparation method similar to that of Example 1, except that steps 7) and 8) are not carried out.

[0068] Comparative Example 3

[0069] A black garlic polysaccharide prepared by a preparation method similar to that of Example 1, except that the crushing particle size in step 1) is 210 μm.

[0070] Comparative Example 4

[0071] A black garlic polysaccharide prepared by a preparation method similar to that of Example 1, except that the pore size of the ceramic membrane in step 4) is 1 μm.

[0072] Comparative Example 5

[0073] A black garlic polysaccharide prepared by a preparation method similar to that of Example 1, except that the pore size of the ceramic membrane in step 4) is 0.05 μm.

[0074] Comparative Example 6

[0075] A black garlic polysaccharide prepared by a preparation method similar to that of Example 1, except that the organic membrane used for filtration in step 7) is an organic membrane with a molecular weight cut-off of 50,000 Da.

[0076] Comparative Example 7

[0077] A black garlic polysaccharide prepared by a preparation method similar to that of Example 1, except that the organic membrane used for filtration in step 7) is an organic membrane with a molecular weight cut-off of 3,000 Da.

[0078] Comparative Application Example 1

[0079] Using the method for the determination of garlic polysaccharide in T / JXDS 001-2020, the content of high-content black garlic polysaccharide or black garlic polysaccharide prepared in Examples 1-5 and Comparative Examples 1-7 was determined. Each example or comparative example was subjected to 3 parallel repeated determination experiments, and the average value was taken. The determination results are shown in Table 1.

[0080] Table 1 Content of black garlic polysaccharide in black garlic polysaccharide prepared by different methods

[0081] Group Process difference from Example 1 Polysaccharide content Example 1 - 70％±1.3％ Example 2 The ceramic membrane is 0.2 μm 92％±0.8％ Example 3 The molecular weight of the organic membrane is 10,000 93％±0.9％ Example 4 The ceramic membrane is 0.2 μm and the molecular weight of the organic membrane is 10,000 88％±1.5％ Example 5 The ceramic membrane is 0.5 μm 75％±0.6％ Comparative Example 1 Do not use the ceramic membrane 36％±0.3％ Comparative Example 2 Do not use the organic membrane 32％±0.6％ Comparative Example 3 The crushing particle size is 210 μm 66％±0.4％ Comparative Example 4 The ceramic membrane is 1 μm 32％±0.6％ Comparative Example 5 The ceramic membrane is 0.05 μm 37％±0.5％ Comparative Example 6 The molecular weight of the organic membrane is 50,000 54％±0.3％ Comparative Example 7 The molecular weight of the organic membrane is 3,000 48％±0.2％

[0082] As can be seen from Table 1, the black garlic polysaccharide prepared by the present invention has the highest content; excluding the method of impurity removal by ceramic membrane or organic membrane filtration, the content of black garlic polysaccharide prepared is much lower than that of the black garlic polysaccharide prepared by the present invention; the content of black garlic polysaccharide prepared with too large crushing particle size is also significantly lower than that of the black garlic polysaccharide prepared by the present invention; and if the pore size or molecular weight of the ceramic membrane or organic membrane is not selected appropriately, the content of black garlic polysaccharide prepared is also much lower than that of the black garlic polysaccharide prepared by the present invention.

[0083] Application Example 1

[0084] In this experiment, HepG2.2.15 cells obtained by transfecting human hepatoma cells with HBV-DNA were used as an experimental model for screening anti-HBV virus activity in vitro. The CPE method was used to observe the non-toxic concentration (TC0) of black garlic polysaccharide on HepG2.2.15, and on this basis, the ELISA method was used to detect the inhibitory effect of the drug on the expression of hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) in HepG2.2.15. The specific method is as follows:

[0085] 1. Determination of the cytotoxicity of black garlic polysaccharide on HepG2.2.15 cells. The determination method refers to the reference [Wu Qianhu, Xu Weidong, Li Weidong, et al. Effects of total flavonoids from Alternanthera philoxeroides on the expression of HBsAg and HBeAg in HepG2.2.15 cells [J]. China Medical Herald, 2018, 15(1): 4.], and the method is as follows:

[0086] The HepG2.2.15 cells digested with trypsin were inoculated into a 96-well culture plate at a density of 5×10 3 / well. After culturing at 37°C and 5% CO2 for 48 h, the culture medium was discarded. The high-content black garlic polysaccharide prepared in Example 1 (denoted as the drug) was serially diluted with 1640 medium containing 5% FBS into 6 series of concentrations, namely 500 μg / mL, 200 μg / mL, 100 μg / mL, 50 μg / mL, 25 μg / mL, and 10 μg / mL;

[0087] The diluted high-content black garlic polysaccharide was added to the 96-well plate respectively. Each dilution was set with 3 replicate wells, 100 μL / well. At the same time, the cells without adding the diluted high-content black garlic polysaccharide were set as the control group.

[0088] On the 4th day of drug treatment, the culture medium was replaced with a newly prepared medium or fresh medium containing the same drug concentration, and the cells were continuously cultured. The changes of the cells were observed every day. On the 8th day, the non-toxic concentration of the drug acting on the cells was recorded.

[0089] After observation, the maximum non-toxic concentration of black garlic polysaccharide was 200 μg / ml.

[0090] 2. Inhibition experiment of the drug on the expression of HBeAg and HBsAg in HepG2.2.15 cells. The measurement method refers to the reference [Wu Qianhu, Xu Weidong, Li Weidong, et al. Effects of total flavonoids from Alternanthera philoxeroides on the expression of HBsAg and HBeAg in HepG2.2.15 cells [J]. China Medical Herald, 2018, 15(1): 4.]. The method is as follows:

[0091] The HepG2.2.15 cells digested with trypsin were inoculated into a 24-well culture plate at a density of 3×10 4 / well. After culturing at 37°C and 5% CO2 in an incubator for 48 h, the culture medium was discarded. The high-content black garlic polysaccharide prepared in Example 1 (denoted as the drug) was serially diluted with 1640 medium containing 5% FBS into 3 series of concentrations, namely 25 μg / mL, 10 μg / mL, and 5 μg / mL;

[0092] The diluted high-content black garlic polysaccharide was added to the 24-well plate respectively. Each concentration was set with 4 parallel wells, 1 mL / well. At the same time, the cells without adding the diluted high-content black garlic polysaccharide were set as the control group (denoted as 0 μg / mL).

[0093] On the 3rd, 6th, and 9th days respectively, the culture medium was replaced with a newly prepared medium or fresh medium containing the same drug concentration, and the supernatant of each well was collected into a 1.5 mL centrifuge tube and stored frozen for later use.

[0094] Detection was carried out using an ELISA kit, and the operation was carried out according to the instructions of the kit. Finally, the absorbance value at a wavelength of 450 nm was read using an enzyme-linked immunosorbent assay reader; the ELISA kit was purchased from Apexbio Technology Co., Ltd. The data was analyzed using SPSS software, and the statistical description was expressed as mean±SD. After the homogeneity test of measurement data, analysis of variance was performed. The results are shown in Table 2.

[0095] Table 2 Inhibitory effects of black garlic polysaccharide on the expression of HBeAg and HBsAg in HepG2.2.15 cells

[0096]

[0097]

[0098] Note: ** in Table 2 indicates a highly significant difference compared with the control group, P < 0.01; * indicates a significant difference compared with the control group, P < 0.05.

[0099] As can be seen from Table 2, the black garlic polysaccharide prepared in Example 1 has a significant inhibitory effect on the expression of HBeAg and HBsAg in HepG2.2.15 cells. The inhibitory effects of black garlic polysaccharides with different concentrations on the expression levels of HBsAg and HBeAg show that the anti-hepatitis effect of black garlic polysaccharide is concentration-dependent.

[0100] Comparative Application Example 2

[0101] For the inhibitory experiment of the drug on the expression of HBeAg and HBsAg in HepG2.2.15 cells, the measurement method refers to the reference [Wu Qianhu, Xu Weidong, Li Weidong, et al. Effects of total flavonoids from Alternanthera philoxeroides on the expression of HBsAg and HBeAg in HepG2.2.15 cells [J]. China Medical Herald, 2018, 15(1): 4.], and the method is as follows:

[0102] After trypsin digestion, HepG2.2.15 cells were inoculated into a 24-well culture plate at a density of 3×10 4 / well, and cultured in an incubator at 37 °C and 5% CO2 for 48 h. Then, the culture medium was discarded, and the high-content black garlic polysaccharide prepared in Example 2 (denoted as the drug) was serially diluted into 3 series of concentrations, namely 25 μg / mL, 10 μg / mL, and 5 μg / mL, with 1640 medium containing 5% FBS;

[0103] The diluted high-content black garlic polysaccharide was added to the 24-well plate respectively, with 4 parallel wells for each concentration, 1 mL / well. At the same time, cells without the diluted high-content black garlic polysaccharide were set as the control group (denoted as 0 μg / mL).

[0104] On the 3rd, 6th, and 9th days respectively, replace the newly prepared medium or fresh medium containing the same drug concentration, and collect the supernatant of each well into a 1.5 mL centrifuge tube for freezing and standby.

[0105] Detection was carried out using an ELISA kit. The operation was carried out according to the instructions of the kit. Finally, the absorbance value at a wavelength of 450 nm was read with an enzyme-linked immunosorbent assay reader; the ELISA kit was purchased from Aimeijie Technology Co., Ltd. The data was analyzed using SPSS software. Statistical description was expressed as mean±SD. After the homogeneity test of measurement data, analysis of variance was carried out. The results are shown in Table 3.

[0106] Table 3 Inhibitory effects of black garlic polysaccharide on the expression of HBeAg and HBsAg in HepG2.2.15 cells

[0107]

[0108]

[0109] ** in Table 3 indicates extremely significant difference compared with the control group, P < 0.01; * indicates significant difference compared with the control group, P < 0.05.

[0110] As can be seen from Table 3, the black garlic polysaccharide prepared in Example 2 has a significant inhibitory effect on the expression of HBeAg and HBsAg in HepG2.2.15 cells; and the inhibitory effect of the black garlic polysaccharide prepared in Example 2 is stronger than that of the black garlic polysaccharide prepared in Example 1. The inhibitory effects of black garlic polysaccharides with different concentrations on the expression levels of HBsAg and HBeAg show that the anti-hepatitis effect of black garlic polysaccharide is concentration-dependent.

[0111] In summary, the content of black garlic polysaccharide prepared by the preparation method provided by the present invention is greater than 85%, much higher than that of the ordinary water extraction and alcohol precipitation method; at the same time, the prepared black garlic polysaccharide can reduce the levels of HBsAg and HBeAg and inhibit the replication of HBV-DNA, that is, black garlic polysaccharide can be used for anti-HBV infection or for the preparation of anti-HBV infection drugs.

[0112] Although the above embodiments have made a detailed description of the present invention, they are only a part of the embodiments of the present invention, not all embodiments. People can also obtain other embodiments according to these embodiments without creative efforts, and these embodiments all belong to the protection scope of the present invention.

Claims

1. A preparation method of black garlic polysaccharide with high content, characterized in that It includes the following steps: Crush black garlic and mix it with water, and perform ultrasonic extraction to obtain an extract; the particle size of the crushed black garlic is 5 - 200 μm; After filtering the extract through a 0.1 - 0.5 μm filter membrane, a purified extract is obtained; Concentrate the purified extract to an extract paste to obtain a concentrated solution; Mix the concentrated solution and absolute ethanol, let it stand and then centrifuge to obtain a precipitate; Dissolve the precipitate and filter it through an organic membrane with a molecular weight cut-off of 5000 - 20000 Daltons to obtain a filtrate which is a black garlic polysaccharide solution.

2. The preparation method according to claim 1, characterized in that, The mass-to-volume ratio of the crushed black garlic to water is 1 g : (10 - 200) mL.

3. The preparation method according to claim 1, characterized in that, The power of the ultrasonic extraction is 200 - 1000 W, the temperature of the ultrasonic extraction is 40 - 90 °C, and the time of the ultrasonic extraction is 15 - 120 min.

4. The preparation method according to claim 1, characterized in that, The volume ratio of the concentrated solution to the purified extract is 1 : (5 - 8).

5. The preparation method according to claim 1 or 4, characterized in that, The volume ratio of the concentrated solution to absolute ethanol is 1 : (3 - 6).

6. The preparation method according to claim 1, characterized in that, The mass ratio of the precipitate to the water for dissolving the precipitate is 1 : (1 - 10).

7. Use of the black garlic polysaccharide prepared by the preparation method according to any one of claims 1 - 6 in the preparation of an antiviral drug, wherein the virus is hepatitis B virus.

8. The application according to claim 7, wherein The antiviral action targets include: HBeAg and / or HBsAg.

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