Preparation and application of mink gallbladder complex

Abstract

Preparation and application of a mink bile complex. The preparation comprises: forming a reaction solution from mink bile dry powder, sodium hydroxide, ethyl acetate, and ethanol; reacting the reaction solution under heating conditions; allowing the reaction solution to stand for stratification and filtering out the precipitate; hydrolyzing the resulting precipitate under acidic conditions, filtering, washing, and drying the precipitate to obtain a crude total bile acid extract; removing bilirubin to obtain a total bile acid extract, and thoroughly mixing the extract with adenosine in an ethanol-water solution and drying to obtain the mink bile complex. The present invention simply and reliably obtains a mink bile complex with high bioavailability from mink bile dry powder and effectively verifies its anti-hepatitis activity.

Description

Technical Field

[0001] The present invention generally relates to biological development and utilization of mink gallbladder. Background Art

[0002] Mink, a collective term for mammals in the family Mustelidae, also known as martens, have slender bodies similar in size to domestic cats, large, slightly triangular ears, short limbs, and a brownish-brown coat with sparse white guard hairs. Mink fur is primarily used in the production of mink coats. However, while mink subcutaneous fat (mink oil) has been commercially exploited, for example in sunscreen and skincare products, mink bile has largely been discarded, and thus further bioresource development is needed. Summary of the Invention

[0003] The object of the present invention is to provide a mink bile extract product which at least has an effective anti-hepatitis effect.

[0004] According to one aspect of the present invention, a method for preparing a mink gallbladder complex is provided, comprising:

[0005] A reaction solution is prepared by adding mink bile powder, sodium hydroxide, ethyl acetate and ethanol, wherein the concentration of mink bile powder is 15-25 g / l, the concentration of sodium hydroxide is 70-90 g / l, and the volume ratio of ethyl acetate to ethanol is 1:(8-12);

[0006] The reaction solution is reacted under heating conditions, wherein the reaction temperature is 80° C. to 90° C. and the reaction time is 1 to 3 hours;

[0007] The reaction solution was allowed to stand for stratification and the precipitate was filtered out;

[0008] The obtained precipitate is hydrolyzed under acidic conditions with a pH value of 1 to 2, and the precipitated precipitate is filtered, washed and dried to obtain a crude total bile acid extract;

[0009] Dissolve the UDP glucuronyltransferase recombinant protein and glucuronic acid in PBS buffer to form a dissolution solution, wherein the concentration of the UDP glucuronyltransferase recombinant protein is 5-15 ug / mL and the concentration of glucuronic acid is 50-150 mg / mL;

[0010] Fully dissolving the obtained crude total bile acid extract in a dissolving solution and then separating to obtain a total bile acid extract after removing bilirubin; and

[0011] The obtained total bile acid extract and adenosine are placed in an ethanol aqueous solution, fully mixed and dried to obtain a mink gallbladder complex, wherein the mass ratio of the total bile acid extract to adenosine is 10:1 to 20:1.

[0012] According to the preparation method of the present invention, the volume fraction of the ethanol aqueous solution is 20% to 40%, and the molar fraction of adenosine in the ethanol aqueous solution and the molar fraction of all bile acids (molecular weight calculated as bile acid) contained in the total bile acid extract can be substantially equal.

[0013] According to another aspect of the present invention, a mink gallbladder-containing complex prepared according to the above method is also provided.

[0014] According to another aspect of the present invention, there is also provided the use of the mink gallbladder complex in the preparation of anti-hepatitis drugs or liver protection health products.

[0015] The present invention simply and reliably obtains a mink gallbladder complex with high bioavailability from mink gallbladder dry powder and effectively verifies its anti-hepatitis activity. The preparation method of the present invention is simple, the process conditions are mild, and it is particularly suitable for industrial-scale production. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1a 、 Figure 1b and Figure 1c These are the FESEM images of total bile acid extract, adenosine and mink bile complex; Figure 1d 、 Figure 1e and Figure 1f These are the TEM scans of total bile acid extract, adenosine, and mink bile complex;

[0017] Figure 2a and Figure 2b These are the expression levels of ALT and AST in the serum of mice with acute liver injury model under different intervention groups;

[0018] Figure 3 H&E staining of pathological tissue sections of acute liver injury under different intervention groups;

[0019] Figure 4 These are H&E staining images of pathological tissue sections of alcoholic liver injury under different intervention groups. DETAILED DESCRIPTION

[0020] Example: Preparation of mink gallbladder complex

[0021] 2 g of lyophilized mink gallbladder powder (provided by Baicheng Songmao Biotechnology Henan Co., Ltd.) was dissolved in 10 ml of ethyl acetate, then mixed with 100 ml of ethanol and 8 g of sodium hydroxide to form a reaction solution. The solution was refluxed at 85°C for 2 h, allowed to stand for stratification, and the precipitate was filtered. The precipitate was dissolved in water and the pH was adjusted to 1.5 with dilute hydrochloric acid. The precipitate was filtered, washed with distilled water, and then dried under vacuum at 60°C to obtain 1.45 g of a crude product. This crude product was added to 10 mL of PBS buffer, 100 μg of recombinant UDP-glucuronyltransferase protein, and 1.0 g of glucuronic acid. The mixture was stirred at 37°C for 24 h, filtered, and the precipitate was isolated and dried under vacuum to obtain 0.98 g of a bilirubin-removed mink gallbladder total bile acid extract.

[0022] Preparation of mink gallbladder complex by non-covalent assembly: Weigh 400 mg of the total bile acid extract (1 mM total bile acid; molecular weight based on bile acid) and gradually add 5 mL of 30% ethanol (volume fraction) to dissolve thoroughly. Weigh 27 mg of adenosine and dissolve thoroughly in 1.5 mL of deionized water. Add the completely dissolved total bile acid to the adenosine, stir, and sonicate to thoroughly mix. Once a viscous substance is initially formed on the stir bar, stop stirring and continue sonication for 3 minutes. Remove the extract and allow it to stand at room temperature. Observe the formation of a stable and uniform mink gallbladder complex using the "vial inversion method."

[0023] Characterization of mink gallbladder complexes

[0024] The mink bile compound prepared above was freeze-dried to obtain a powder (freeze-dried gel sample), which was evenly spread on a conductive tape attached to a sample carrier. Gold was sprayed on the sample carrier before testing. After being placed on the sample carrier, a cold field emission scanning electron microscope (FESEM) was used to scan and record the sample. The freeze-dried gel sample was diluted with 2 mL of 50% ethanol aqueous solution, dropped onto a copper mesh, dried in an oven, and observed using a transmission electron microscope (TEM). The FESEM electron microscope image showed that the total bile acid extract showed irregular blocks ( Figure 1a ); Adenosine is rod-shaped ( Figure 1b ); The mink gallbladder complex is dense and slender rod-shaped, with a small amount of lumps ( Figure 1c TEM scanning images show that the total bile acid extract is in the form of dots ( Figure 1d ); Adenosine appears fibrillar ( Figure 1e ); The mink gallbladder compound appears fibrous and burr-like ( Figure 1f ), which should be a new microscopic structure formed by the combination of total bile acid attached to adenosine fibers.

[0025] Evaluation of bioavailability of mink gallbladder complex

[0026] Wistar rats fasted for 12 hours were divided into two groups: a mink bile complex (the freeze-dried gel sample described above) and a total bile acid extract group. The two groups were controlled for each other. The dosage was calculated based on rat body weight (30 mg / kg). Blood was collected from the orbital cavity of each group at 1, 3, 5, 7, 14, and 28 hours after administration. The blood samples were placed in heparin-soaked centrifuge tubes and centrifuged at 4000 rpm for 10 minutes. The supernatant was aspirated and stored at -20°C until further use. Table 1 lists the bioavailability (blood concentrations) of the mink bile complex and total bile acid extract groups, measured as bile acid.

[0027]

[0028] As can be seen from Table 1, the mink bile complex groups at different time points can significantly improve the bioavailability of mink bile acid.

[0029] Anti-hepatitis activity application test

[0030] Method for establishing a mouse model of acute liver injury induced by CCL4 and grouping:

[0031] Male ICR mice (20-22 g) were randomly divided into six groups: a normal group, a model group, a bear bile powder group (250 mg / kg), a total bile acid extract group (250 mg / kg), a mink bile complex group (250 mg / kg), and a positive control group (250 mg / kg). Each treatment group was administered via gavage, with the normal and model groups receiving an equal volume of distilled water once daily for four consecutive days. One hour after the last administration, all mice, except the control group, received an intraperitoneal injection of 0.1% CCl₄ in vegetable oil (0.1 ml / 10 g). Following exposure, the mice were deprived of food but not water.

[0032] Method for establishing the mouse alcohol liver injury model and grouping:

[0033] Healthy male ICR mice were randomly divided into six groups: a normal group, a model group, a bear bile powder group (250 mg / kg), a total bile acid extract group (250 mg / kg), a mink bile complex group (250 mg / kg), and a bifendate (250 mg / kg) positive control group. Each treatment group was administered via oral gavage. The normal and model groups were given an equal volume of distilled water once daily for four consecutive days. One hour after the last dose, the mice were given 12 mL / kg of 56° Red Star Erguotou by oral gavage to establish the model. The normal group was given an equal volume of distilled water and fasted, but not water.

[0034] Serum ALT and AST level detection method

[0035] Sixteen hours after modeling, eyeballs were removed and blood was collected. The blood was centrifuged at 3500 rpm for 10 min at 4°C. The supernatant was collected, aliquoted into EP tubes, and frozen at -80°C. Serum ALT and AST activities were measured using an automatic biochemical analyzer.

[0036] Determination of liver tissue SOD, CAT, GSH-PX, GSH and ROS indicators

[0037] 100 mg of liver tissue was homogenized with a 9-fold volume of normal saline. The mixture was placed in an ice-water bath to prepare a 10% tissue homogenate. The homogenate was centrifuged at 3500 rpm for 10 min at 4°C. The supernatant was stored at -20°C. Liver tissue protein content and the activities of CAT, SOD, GSH-PX, GSH, and ROS were assayed according to the kit instructions.

[0038] Data processing and results

[0039] Actual data It indicates that the statistical software SPSS21.0 was used for one-way analysis of variance, t-test and other processing, and p<0.05 was considered to be statistically significant.

[0040] Results see Figure 2a and Figure 2b , where: I: normal group; II: CCl4 model group; III: bear bile powder group; IV: total bile acid extract group; V: mink bile complex group; VI: positive drug group. ## p < 0.01 compared with the normal group; * p < 0.05, ** p < 0.01 compared with the model group. Figure 2a and Figure 2b In a CCl4-induced acute liver injury model in mice, serum ALT and AST levels were significantly elevated in the CCl4 model group compared to the normal group (p < 0.01). Compared to the CCl4 model group, serum ALT and AST levels were significantly decreased in the bear bile powder, total bile acid extract, and mink bile complex groups. The decreases were particularly pronounced in the mink bile complex group, with ALT and AST decreasing by 97% and 84%, respectively. The positive drug bifendate decreased ALT and AST by 93% and 64%, respectively. This suggests that the assembled mink bile complex significantly reduces serum transaminase activity after CCl4 induction.

[0041] In the mouse alcohol liver injury model, compared with the normal group, the serum ALT, AST activity and TG content of the model group increased significantly (p<0.05, p<0.01), and the differences were statistically significant, indicating that the acute alcohol liver injury model was successfully established (Table 2). Compared with the model group, the ALT, AST activity and TG content of the bear bile powder group, the total bile acid extract group, and the marten bile complex group all decreased significantly (p<0.05, p<0.01), among which the reduction in ALT, AST activity and TG content in the marten bile complex group was particularly significant (p<0.01, p<0.05), and was better than that of the bifendate positive group. The bifendate positive group had no significant effect on the increase in serum TG content. This shows that the marten bile complex group can antagonize the increase in liver metabolic enzymes ALT, AST and TG caused by excessive alcohol. Table 2 lists the effects of each drug administration group on serum ALT, AST and TG in mice with alcohol-induced liver injury ( Table 3 lists the effects of each drug group on SOD, ADH, GSH and GSH-Px in liver tissue of mice with acute liver injury caused by alcohol ( , n=8).

[0042]

[0043] Compared with the normal group, the activities of SOD, ADH, GSH-Px, and GSH in the liver of the model group were significantly decreased (p < 0.01), with statistically significant differences. Compared with the model group, the activities of SOD, ADH, GSH-Px, and GSH in the liver homogenate of the bifendate-positive group were significantly increased (p < 0.01, p < 0.05). The activities of SOD, ADH, GSH-Px, and GSH were also significantly increased in the bear bile powder, total bile acid extract, and marten bile complex groups (p < 0.01, p < 0.05), with the degree of increase comparable to that of bifendate. The activities of GSH-Px and GSH were significantly increased in the marten bile complex group (p < 0.01, p < 0.05). This suggests that the marten bile complex may achieve its hepatoprotective effect by preventing oxidative damage.

[0044] Liver pathology testing

[0045] The left lobe of the specimen was excised and fixed in 10% formaldehyde, then embedded in paraffin and sectioned at 5 μm thickness, mounted on slides, and then deparaffinized. For histological analysis, sections were stained with hematoxylin-eosin using standard techniques. Lesions were observed and photographed under a microscope at 40x, 100x, and 400x magnifications.

[0046] Figure 3Figures A to F show liver pathological sections of mice (H&E staining, 400×). Liver pathological sections show normal liver tissue structure in the normal group, with round hepatocytes and centrally located nuclei (A). In the CCl4 liver injury model group, lymphocyte infiltration was observed in the hepatic lobules and around blood vessels. Hepatocytes at the edges of the liver tissue underwent necrosis, forming necrotic foci. Hepatocytes showed scattered necrosis, pyknosis of the nuclei, and extensive amyloidosis (B). In the bear bile powder group (C), the total bile acid extract group (D), and the mink bile complex group (E), the hepatic lobules were intact, with normal hepatocyte morphology and no significant pathological changes. The mink bile complex group showed more complete liver lobule structure. In the bifendate-positive group, some hepatocytes showed necrosis at the edges of the liver tissue, with pyknosis of the nuclei (F). This suggests that the mink bile complex group significantly improved liver tissue pathological changes.

[0047] Figure 4 Figures A to F show pathological sections of mouse livers, respectively. The liver tissue structure of mice in the normal group was normal, with round hepatocytes and centrally located nuclei, and only a small amount of apoptosis (A). Lymphocyte infiltration, hepatocyte apoptosis or necrosis, and the presence of megakaryocytes were observed in the hepatic lobules of mice in the alcoholic liver injury model group (B). The hepatic lobules of the bear bile powder group (C), the total bile acid extract group (D), and the mink bile complex group (E) were intact, with essentially normal hepatocyte morphology and no significant pathological changes, with the mink bile complex group showing more intact hepatocytes. The hepatocyte morphology of the bifendate-positive group was essentially restored to normal, with no significant pathological changes (F). This indicates that the mink bile complex group significantly improved alcohol-induced acute liver injury. These results demonstrate that the mink bile complex prepared in this invention has a certain protective effect against CCL4-induced acute liver injury compared to untreated bile, and exhibits a strong protective effect against acute alcoholic liver injury.

Claims

1. A method for preparing a mink gallbladder complex, comprising: A reaction solution is prepared by combining mink bile dry powder, sodium hydroxide, ethyl acetate and ethanol, wherein the concentration of mink bile dry powder is 15-25 g / l, the concentration of sodium hydroxide is 70-90 g / l, and the volume ratio of ethyl acetate to ethanol is 1:(8-12); The reaction solution is reacted under heating conditions, wherein the reaction temperature is 80° C. to 90° C. and the reaction time is 1 to 3 hours; The reaction solution was allowed to stand for stratification and the precipitate was filtered out; The obtained precipitate is hydrolyzed under acidic conditions with a pH value of 1 to 2, and the precipitated precipitate is filtered, washed and dried to obtain a crude total bile acid extract; Dissolve the UDP glucuronyltransferase recombinant protein and glucuronic acid in PBS buffer to form a dissolution solution, wherein the concentration of the UDP glucuronyltransferase recombinant protein is 5-15 ug / mL and the concentration of glucuronic acid is 50-150 mg / mL; The crude total bile acid extract is fully dissolved in a dissolving solution and then separated to obtain a total bile acid extract after bilirubin removal; as well as The obtained total bile acid extract and adenosine are placed in an ethanol aqueous solution, fully mixed and dried to obtain a mink gallbladder complex, wherein the mass ratio of the total bile acid extract and adenosine is 10:1~20:1, and the volume fraction of the ethanol aqueous solution is 20%~40%.

2. A mink gallbladder complex prepared according to the method of claim 1.

3. Use of the mink gallbladder complex according to claim 2 in the preparation of anti-hepatitis drugs or liver protection health products.

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