A nano drug preparation for treating hepatic encephalopathy and preparation method thereof

Abstract

The invention discloses a nano drug preparation for treating hepatic encephalopathy and a preparation method thereof. According to the pathogenesis of hepatic encephalopathy, a nano drug with a sustained-release effect is prepared, and a licorice extract is used to protect the liver, and ergothioneine is used to achieve antioxidant and detoxification treatment of the liver to protect liver cells; more importantly, aspartic acid obtained by dissociation of polyaspartic acid is used in combination with asparagine synthetase to absorb a large amount of blood ammonia to obtain asparagine. Asparagine is different from glutamine and will not cause accumulation of water in cells to cause cell edema.

Description

Technical Field

[0001] The invention belongs to the field of pharmaceuticals, and in particular relates to a nano drug preparation for treating hepatic encephalopathy and a preparation method thereof. Background Art

[0002] Hepatic encephalopathy (HE), also known as hepatic coma, is a central nervous system dysfunction syndrome caused by severe liver disease and based on metabolic disorders. Its main clinical manifestations are impaired consciousness, abnormal behavior and coma. To date, the pathogenesis of HE is still unclear, there is a lack of effective treatment, and the mortality rate is high.

[0003] Ammonia interferes with brain energy metabolism and causes HE mainly through the following pathways: ① Directly inhibiting the potential activity of nerve cell membranes. ② Combining with α-ketoglutarate and glutamate to form glutamine, interfering with the tricarboxylic acid cycle and oxidative metabolism; at the same time, glutamine is considered to have a permeable molecule effect. Increased intracellular glutamine can lead to the accumulation of intracellular water and cause cell edema. ③ Reducing the production of intracellular ATP and the storage of NADH. In recent years, many scholars have found that the effect of ammonia on neurotransmitters also plays a role in the onset of HE. How to reduce the blood ammonia level in the brain and prevent the accumulation of glutamine is the key to the treatment of hepatic encephalopathy. Summary of the invention

[0004] Purpose of the invention: The technical problem to be solved by the present invention is to provide a nano drug preparation for treating hepatic encephalopathy in order to reduce the blood ammonia level in the brain and prevent the accumulation of glutamine in view of the deficiencies of the prior art.

[0005] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0006] A method for preparing a nano drug preparation for treating hepatic encephalopathy comprises the following steps:

[0007] (1) dissolving liquorice extract, recombinant human asparagine synthetase, ergothioneine and sodium tripolyphosphate in deionized water to obtain a STPP solution;

[0008] (2) dissolving polyaspartic acid and povidone K30 in deionized water to prepare a PASP-PVP solution;

[0009] (3) adding chitosan to acetic acid solution to obtain CS solution;

[0010] (4) After the STPP solution in step (1) and the PASP-PVP solution obtained in step (2) are fully mixed, the mixture is slowly added dropwise to the CS solution obtained in step (3) under stirring, and the reaction is continued by stirring. After the reaction is completed, the reaction solution is centrifuged, and the precipitate is washed with water and freeze-dried to obtain the product.

[0011] Specifically, in step (1), the feeding ratio of the licorice extract, recombinant human asparagine synthetase, ergothioneine, sodium tripolyphosphate and deionized water is (3-5) g: (120-150) μg: (50-85) mg: (0.08-0.12) g: 50 ml; preferably 3.7 g: 130 μg: 68 mg: 0.10 g: 50 ml.

[0012] Specifically, in step (1), the licorice extract is a fine powder obtained by extracting dried root slices of the leguminous plant licorice with pure water, concentrating at low temperature and reducing pressure and spray drying; the specific activity of the recombinant human asparagine synthetase is 52-104 U / mg, preferably 95 U / mg.

[0013] Specifically, in step (2), the feeding ratio of the polyaspartic acid, povidone K30 and deionized water is (0.23-0.27) g: (0.08-0.12) g: 50 ml; preferably 0.26 g: 0.10 g: 50 ml.

[0014] Specifically, in step (2), the weight average molecular weight of the polyaspartic acid is 1.8-2 million Da, preferably 1.9 million Da.

[0015] Specifically, in step (3), in the CS solution, the concentration of chitosan is 0.02-0.08 wt%, preferably 0.06 wt%; and the acetic acid solution is a 1-2% v / v aqueous solution, preferably 1.5% v / v.

[0016] Specifically, in step (3), the chitosan solution has a deacetylation degree of 85-95%, preferably 90%; and a weight average molecular weight of 200,000-500,000 Da, preferably 400,000 Da.

[0017] Specifically, in step (4), the feeding ratio of the STPP solution, PASP-PVP solution and CS solution is (40-50) ml: (30-50) ml: 100 ml, preferably 46 ml: 45 ml: 100 ml.

[0018] Specifically, in step (4), the stirring reaction temperature is 4-10°C, preferably 8°C; and the time is 24-48h, preferably 36h.

[0019] Furthermore, the nano drug preparation for treating hepatic encephalopathy prepared by the above preparation method is also within the protection scope of the present invention.

[0020] Beneficial effects:

[0021] (1) The present invention is based on the pathogenesis of hepatic encephalopathy, prepares a nano drug with a sustained release effect, protects the liver by licorice extract, and then uses ergothioneine to achieve antioxidant and detoxification treatment of the liver, protects liver cells, so that the transamination ability of liver cells and the ornithine cycle efficiency can be brought into play, and ammonia is promoted to be converted into urea to prevent ammonia accumulation; More importantly, aspartic acid obtained by dissociation of polyaspartic acid is combined with asparagine synthetase, and blood ammonia is continuously absorbed in large quantities to obtain asparagine, which is different from glutamine and will not cause the accumulation of intracellular water, resulting in cell edema. Reducing blood ammonia concentration and preventing cell edema can achieve the effect of treating hepatic encephalopathy.

[0022] (2) The present invention uses chitosan and polyaspartic acid as the main body of nanoparticles, can embed the active ingredients in the licorice extract, ergothioneine and recombinant human asparagine synthetase through electrostatic self-assembly, and then solidify into nanoparticles by sodium tripolyphosphate as a cross-linking agent, and polyvinyl pyrrolidone (polyvinylpyrrolidone K30) is used as a dispersant in this process to prevent the aggregation caused by the low surface charge density of the nanoparticles formed by electrostatic self-assembly. Ergothioneine also ensures its stability before forming nanoparticles in an alkaline STPP solution, and acid-base neutralization during the stirring reaction process, and ergothioneine will not be exposed to an acidic environment for a long time, resulting in oxidative inactivation. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a scanning electron micrograph of the nano drug preparation for treating hepatic encephalopathy prepared in Example 1. DETAILED DESCRIPTION

[0024] The present invention can be better understood with reference to the following examples.

[0025] Example 1

[0026] A nano drug preparation for treating hepatic encephalopathy and a preparation method thereof, comprising the following steps:

[0027] (1) 3.7 g of licorice extract, 130 μg of recombinant human asparagine synthetase with a specific activity of 95 U / mg, 68 mg of ergothioneine and 0.10 g of sodium tripolyphosphate were added to 50 ml of deionized water to prepare a STPP solution;

[0028] (2) 0.26 g of polyaspartic acid with a weight average molecular weight of 1.9 million Da and 0.10 g of povidone K30 were added to 50 ml of deionized water to prepare a PASP-PVP solution.

[0029] (3) chitosan with a deacetylation degree of 90% and a weight average molecular weight of 400,000 Da was added to a 1.5% v / v acetic acid solution to prepare a 0.06 wt% CS solution;

[0030] (4) After fully mixing 46 ml of the STPP solution obtained in step (1) and 45 ml of the PASP-PVP solution obtained in step (2), slowly dropwise add the mixture to 100 ml of the CS solution obtained in step (3), and continue stirring to react at a temperature of 8° C. for 36 h. After the reaction is completed, the reaction solution is centrifuged, the precipitate is washed with water and freeze-dried to obtain a nanopharmaceutical preparation for treating hepatic encephalopathy. Figure 1 shown.

[0031] Example 2

[0032] A nano drug preparation for treating hepatic encephalopathy and a preparation method thereof, comprising the following steps:

[0033] (1) 3 g of licorice extract, 120 μg of recombinant human asparagine synthetase with a specific activity of 52 U / mg, 50 mg of ergothioneine and 0.08 g of sodium tripolyphosphate were added to 50 ml of deionized water to prepare a STPP solution;

[0034] (2) 0.23 g of polyaspartic acid with a weight average molecular weight of 2 million Da and 0.08 g of povidone K30 were added to 50 ml of deionized water to prepare a PASP-PVP solution.

[0035] (3) chitosan with a deacetylation degree of 85% and a weight average molecular weight of 200,000 Da was added to a 1% v / v acetic acid solution to prepare a 0.02 wt% CS solution;

[0036] (4) After fully mixing 40 ml of the STPP solution obtained in step (1) and 30 ml of the PASP-PVP solution obtained in step (2), slowly dropwise add the mixture to 100 ml of the CS solution obtained in step (3), and continue stirring to react at a temperature of 4° C. for 48 h. After the reaction is completed, the reaction solution is centrifuged, the precipitate is washed with water and freeze-dried to obtain a nano drug preparation for treating hepatic encephalopathy.

[0037] Example 3

[0038] A nano drug preparation for treating hepatic encephalopathy and a preparation method thereof, comprising the following steps:

[0039] (1) 5 g of licorice extract, 150 μg of recombinant human asparagine synthetase with a specific activity of 104 U / mg, 85 mg of ergothioneine and 0.12 g of sodium tripolyphosphate were added to 50 ml of deionized water to prepare a STPP solution;

[0040] (2) 0.27 g of polyaspartic acid with a weight average molecular weight of 1.8 million Da and 0.12 g of povidone K30 were added to 50 ml of deionized water to prepare a PASP-PVP solution.

[0041] (3) chitosan with a deacetylation degree of 95% and a weight average molecular weight of 500,000 Da was added to a 2% v / v acetic acid solution to prepare a 0.08 wt% CS solution;

[0042] (4) After fully mixing 50 ml of the STPP solution obtained in step (1) and 50 ml of the PASP-PVP solution obtained in step (2), slowly dropwise add the mixture to 100 ml of the CS solution obtained in step (3), and continue stirring to react at a temperature of 10° C. for 24 h. After the reaction is completed, the reaction solution is centrifuged, the precipitate is washed with water and freeze-dried to obtain a nano drug preparation for treating hepatic encephalopathy.

[0043] Comparative Example 1 (without licorice extract)

[0044] A nano drug preparation for treating hepatic encephalopathy and a preparation method thereof, comprising the following steps:

[0045] (1) 130 μg of recombinant human asparagine synthetase with a specific activity of 95 U / mg, 68 mg of ergothioneine and 0.10 g of sodium tripolyphosphate were added to 50 ml of deionized water to prepare a STPP solution;

[0046] (2) 0.26 g of polyaspartic acid with a weight average molecular weight of 1.9 million Da and 0.10 g of povidone K30 were added to 50 ml of deionized water to prepare a PASP-PVP solution.

[0047] (3) chitosan with a deacetylation degree of 90% and a weight average molecular weight of 400,000 Da was added to a 1.5% v / v acetic acid solution to prepare a 0.06 wt% CS solution;

[0048] (4) After fully mixing 46 ml of the STPP solution obtained in step (1) and 45 ml of the PASP-PVP solution obtained in step (2), slowly dropwise add the mixture to 100 ml of the CS solution obtained in step (3), and continue stirring to react at a temperature of 8° C. for 36 h. After the reaction is completed, the reaction solution is centrifuged, the precipitate is washed with water and freeze-dried to obtain a nano drug preparation for treating hepatic encephalopathy.

[0049] Comparative Example 2 (without recombinant human asparagine synthetase)

[0050] A nano drug preparation for treating hepatic encephalopathy and a preparation method thereof, comprising the following steps:

[0051] (1) 3.7 g of liquorice extract, 68 mg of ergothioneine and 0.10 g of sodium tripolyphosphate were added to 50 ml of deionized water to prepare a STPP solution;

[0052] (2) 0.26 g of polyaspartic acid with a weight average molecular weight of 1.9 million Da and 0.10 g of povidone K30 were added to 50 ml of deionized water to prepare a PASP-PVP solution.

[0053] (3) chitosan with a deacetylation degree of 90% and a weight average molecular weight of 400,000 Da was added to a 1.5% v / v acetic acid solution to prepare a 0.06 wt% CS solution;

[0054] (4) After fully mixing 46 ml of the STPP solution obtained in step (1) and 45 ml of the PASP-PVP solution obtained in step (2), slowly dropwise add the mixture to 100 ml of the CS solution obtained in step (3), and continue stirring to react at a temperature of 8° C. for 36 h. After the reaction is completed, the reaction solution is centrifuged, the precipitate is washed with water and freeze-dried to obtain a nano drug preparation for treating hepatic encephalopathy.

[0055] Comparative Example 3 (without ergothioneine)

[0056] A nano drug preparation for treating hepatic encephalopathy and a preparation method thereof, comprising the following steps:

[0057] (1) 3.7 g of licorice extract, 130 μg of recombinant human asparagine synthetase with a specific activity of 95 U / mg, and 0.10 g of sodium tripolyphosphate were added to 50 ml of deionized water to prepare a STPP solution;

[0058] (2) 0.26 g of polyaspartic acid with a weight average molecular weight of 1.9 million Da and 0.10 g of povidone K30 were added to 50 ml of deionized water to prepare a PASP-PVP solution.

[0059] (3) chitosan with a deacetylation degree of 90% and a weight average molecular weight of 400,000 Da was added to a 1.5% v / v acetic acid solution to prepare a 0.06 wt% CS solution;

[0060] (4) After fully mixing 46 ml of the STPP solution obtained in step (1) and 45 ml of the PASP-PVP solution obtained in step (2), slowly dropwise add the mixture to 100 ml of the CS solution obtained in step (3), and continue stirring to react at a temperature of 8° C. for 36 h. After the reaction is completed, the reaction solution is centrifuged, the precipitate is washed with water and freeze-dried to obtain a nano drug preparation for treating hepatic encephalopathy.

[0061] Comparative Example 4 (Licorice Extract Only)

[0062] A nano drug preparation for treating hepatic encephalopathy and a preparation method thereof, comprising the following steps:

[0063] (1) Add 3.7 g of licorice extract and 0.10 g of sodium tripolyphosphate into 50 ml of deionized water to prepare a STPP solution;

[0064] (2) 0.26 g of polyaspartic acid with a weight average molecular weight of 1.9 million Da and 0.10 g of povidone K30 were added to 50 ml of deionized water to prepare a PASP-PVP solution.

[0065] (3) chitosan with a deacetylation degree of 90% and a weight average molecular weight of 400,000 Da was added to a 1.5% v / v acetic acid solution to prepare a 0.06 wt% CS solution;

[0066] (4) After fully mixing 46 ml of the STPP solution obtained in step (1) and 45 ml of the PASP-PVP solution obtained in step (2), slowly dropwise add the mixture to 100 ml of the CS solution obtained in step (3), and continue stirring to react at a temperature of 8° C. for 36 h. After the reaction is completed, the reaction solution is centrifuged, the precipitate is washed with water and freeze-dried to obtain a nano drug preparation for treating hepatic encephalopathy.

[0067] Comparative Example 5 (only ergothioneine)

[0068] A nano drug preparation for treating hepatic encephalopathy and a preparation method thereof, comprising the following steps:

[0069] (1) 68 mg of thioneine and 0.10 g of sodium tripolyphosphate were added to 50 ml of deionized water to prepare a STPP solution;

[0070] (2) 0.26 g of polyaspartic acid with a weight average molecular weight of 1.9 million Da and 0.10 g of povidone K30 were added to 50 ml of deionized water to prepare a PASP-PVP solution.

[0071] (3) chitosan with a deacetylation degree of 90% and a weight average molecular weight of 400,000 Da was added to a 1.5% v / v acetic acid solution to prepare a 0.06 wt% CS solution;

[0072] (4) After fully mixing 46 ml of the STPP solution obtained in step (1) and 45 ml of the PASP-PVP solution obtained in step (2), slowly dropwise add the mixture to 100 ml of the CS solution obtained in step (3), and continue stirring to react at a temperature of 8° C. for 36 h. After the reaction is completed, the reaction solution is centrifuged, the precipitate is washed with water and freeze-dried to obtain a nano drug preparation for treating hepatic encephalopathy.

[0073] Comparative Example 6 (recombinant human asparagine synthetase only)

[0074] A nano drug preparation for treating hepatic encephalopathy and a preparation method thereof, comprising the following steps:

[0075] (1) 130 μg of recombinant human asparagine synthetase with a specific activity of 95 U / mg and 0.10 g of sodium tripolyphosphate were added to 50 ml of deionized water to prepare a STPP solution;

[0076] (2) 0.26 g of polyaspartic acid with a weight average molecular weight of 1.9 million Da and 0.10 g of povidone K30 were added to 50 ml of deionized water to prepare a PASP-PVP solution.

[0077] (3) chitosan with a deacetylation degree of 90% and a weight average molecular weight of 400,000 Da was added to a 1.5% v / v acetic acid solution to prepare a 0.06 wt% CS solution;

[0078] (4) After fully mixing 46 ml of the STPP solution obtained in step (1) and 45 ml of the PASP-PVP solution obtained in step (2), slowly dropwise add the mixture to 100 ml of the CS solution obtained in step (3), and continue stirring to react at a temperature of 8° C. for 36 h. After the reaction is completed, the reaction solution is centrifuged, the precipitate is washed with water and freeze-dried to obtain a nano drug preparation for treating hepatic encephalopathy.

[0079] Comparative Example 7 (replacing recombinant human asparagine synthetase with asparagine)

[0080] A nano drug preparation for treating hepatic encephalopathy and a preparation method thereof, comprising the following steps:

[0081] (1) 3.7 g of liquorice extract, 68 mg of ergothioneine and 0.10 g of sodium tripolyphosphate were added to 50 ml of deionized water to prepare a STPP solution;

[0082] (2) 0.26 g of polyaspartic acid with a weight average molecular weight of 1.9 million Da and 0.10 g of povidone K30 were added to 50 ml of deionized water to prepare a PASP-PVP solution.

[0083] (3) chitosan with a deacetylation degree of 90% and a weight average molecular weight of 400,000 Da was added to a 1.5% v / v acetic acid solution to prepare a 0.06 wt% CS solution;

[0084] (4) After fully mixing 46 ml of the STPP solution obtained in step (1) and 45 ml of the PASP-PVP solution obtained in step (2), the mixture was slowly added dropwise to 100 ml of the CS solution obtained in step (3), and the mixture was continuously stirred for reaction at a temperature of 8° C. for 36 h. After the reaction, the reaction solution was centrifuged, the precipitate was washed with water and freeze-dried, and 0.26 g of asparagine was added and fully mixed to obtain a nano drug preparation for treating hepatic encephalopathy.

[0085] Construction and treatment of rat hepatic encephalopathy model

[0086] 8-week-old SD male rats were selected and given 300 mg / kg thioacetamide intraperitoneal injection for 4 consecutive days. After 3 days of modeling, the nano drug preparations of Examples 1-3 and Comparative Examples 1-7 were used, and 100 mg / kg was given by tail vein injection every 3 days for a total of 3 times. 1 week after the last administration, eyeball blood was taken to measure blood ammonia levels, and rats were killed by carbon dioxide asphyxiation, and brains were taken to measure brain ammonia levels and water content. The results are shown in Table 1.

[0087] Table 1 Results of blood ammonia level, brain ammonia level and water content measurement (n=10)

[0088]

[0089] As can be seen from Table 1, the nano drug preparations prepared in Examples 1-3 can significantly reduce blood ammonia and brain ammonia levels, while reducing the increased intracranial osmotic pressure caused by hepatic encephalopathy, thereby alleviating cerebral edema. The level after treatment is close to normal levels. Although the nano drug preparations prepared in the comparative examples have a certain degree of relief for hepatic encephalopathy, the effect is obviously not as good as that of the embodiments. Comparative Example 1 lacks licorice extract. Glycyrrhizic acid, glycyrrhetinic acid, glycyrrhizin, etc. in licorice extract have strong anti-inflammatory, hepatocyte membrane protection, and liver function improvement effects. Comparative Example 2 lacks recombinant human asparagine synthetase, and blood ammonia cannot be synthesized into asparagine with aspartic acid, so it does not have the effect of significantly reducing blood ammonia and brain ammonia, and cannot significantly improve the symptoms of hepatic encephalopathy; Comparative Example 3 lacks ergothioneine, which has antioxidant and detoxification capabilities, and is more stable than glutathione. It can increase the level of glutathione in the body and enhance liver cell vitality. Comparative Example 4 only has licorice extract, which can not effectively reduce the blood ammonia level in hepatic encephalopathy mice, so there is no difference compared with the model group; Comparative Example 5 only adds ergothioneine to improve liver cell viability, but it is still not enough to effectively remove blood ammonia, and the result is similar to Comparative Example 4. Comparative Example 6 only adds recombinant human asparagine synthetase to react dissociated aspartic acid with blood ammonia, and has a certain ability to reduce blood ammonia levels, but due to the lack of better liver function support, this effect cannot be sustained, and blood ammonia levels are also poorly controlled, and the symptoms of hepatic encephalopathy can not be fundamentally improved. It can be seen that licorice extract, recombinant human asparagine synthetase and ergothioneine, the three are directed to different targets for the treatment of hepatic encephalopathy, have synergistic effects, and are indispensable. The nano drug preparation of embodiment obtains a better effect of treating hepatic encephalopathy by the combined action of the three. In addition, Comparative Example 7 directly adds asparagine to replace the asparagine obtained by recombinant human asparagine synthetase, and its effect is similar to Comparative Example 2. The reason is that the synthesis of asparagine can continuously absorb a large amount of blood ammonia, which is an important part of treating hepatic encephalopathy. Directly adding asparagine cannot absorb blood ammonia, so the effect is not as good as in the embodiment.

[0090] The present invention provides a nano drug preparation for treating hepatic encephalopathy and a method for preparing the same. There are many methods and approaches to implement the technical solution. The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention. All components not specified in this embodiment can be implemented by existing technologies.

Claims

1. A method for preparing a nano drug preparation for treating hepatic encephalopathy, characterized in that: The steps include: (1) dissolving licorice extract, recombinant human asparagine synthetase, ergothioneine and sodium tripolyphosphate in deionized water to obtain a STPP solution; (2) dissolving polyaspartic acid and povidone K30 in deionized water to prepare a PASP-PVP solution; (3) adding chitosan to acetic acid solution to obtain CS solution; (4) After the STPP solution in step (1) and the PASP-PVP solution obtained in step (2) are fully mixed, the mixture is slowly added dropwise to the CS solution obtained in step (3) under stirring, and the reaction is continued by stirring. After the reaction is completed, the reaction solution is centrifuged, the precipitate is washed with water, and freeze-dried to obtain; In step (1), the feeding ratio of the licorice extract, recombinant human asparagine synthetase, ergothioneine, sodium tripolyphosphate and deionized water is (3-5) g: (120-150) μg: (50-85) mg: (0.08-0.12) g: 50 ml.

2. The method for preparing the nano drug preparation for treating hepatic encephalopathy according to claim 1, characterized in that: In step (1), the licorice extract is obtained by extracting dried root slices of the leguminous plant licorice with purified water, and then concentrating at low temperature and reduced pressure and spray drying to obtain a fine powder; the specific activity of the recombinant human asparagine synthetase is 52-104 U / mg.

3. The method for preparing the nano drug preparation for treating hepatic encephalopathy according to claim 1, characterized in that: In step (2), the feeding ratio of polyaspartic acid, povidone K30 and deionized water is (0.23-0.27) g: (0.08-0.12) g: 50 ml.

4. The method for preparing the nano drug preparation for treating hepatic encephalopathy according to claim 1, characterized in that: In step (2), the weight average molecular weight of the polyaspartic acid is 1.8-2 million Da.

5. The method for preparing the nano drug preparation for treating hepatic encephalopathy according to claim 1, characterized in that: In step (3), the concentration of chitosan in the CS solution is 0.02-0.08 wt %; and the acetic acid solution is a 1-2% v / v aqueous solution.

6. The method for preparing the nano drug preparation for treating hepatic encephalopathy according to claim 1, characterized in that: In step (3), the chitosan has a deacetylation degree of 85-95% and a weight average molecular weight of 200,000-500,000 Da.

7. The method for preparing the nano drug preparation for treating hepatic encephalopathy according to claim 1, characterized in that: In step (4), the feeding ratio of the STPP solution, PASP-PVP solution and CS solution is (40-50) ml: (30-50) ml: 100 ml.

8. The method for preparing the nano drug preparation for treating hepatic encephalopathy according to claim 1, characterized in that: In step (4), the stirring reaction temperature is 4-10°C and the time is 24-48 hours.

9. The nano drug preparation for treating hepatic encephalopathy prepared by the preparation method according to any one of claims 1 to 8.

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