Preparation and application of mink gallbladder compound

Abstract

The invention discloses preparation and application of a mink gallbladder compound. The preparation method comprises the following steps: preparing a reaction solution from mink gallbladder dry powder, sodium hydroxide, ethyl acetate and ethanol; reacting the reaction solution under a heating condition; standing and layering the reacted solution, and filtering out precipitates; hydrolyzing the obtained precipitate under an acidic condition, and filtering, extracting, washing and drying the separated precipitate to obtain a crude total cholic acid extract; removing bilirubin to obtain a total cholic acid extract; putting the total cholic acid extract and adenosine into an ethanol water solution, fully mixing, and drying to obtain the mink bile compound. The mink gallbladder compound with high bioavailability is simply and reliably obtained from the mink gallbladder dry powder, and the anti-hepatitis activity of the mink gallbladder compound is effectively verified.

Description

Technical Field

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

[0002] Mink is a general term for mammals of the Mustelidae family, also known as sable. It has a slender body, similar in size to a domestic cat, relatively large ears, slightly triangular in shape, short limbs, and its whole body is covered with brown hair with sparse white guard hairs, and is mostly used for producing mink fur coats. However, although the subcutaneous fat of mink (mink oil) and the like have been commercially developed and utilized, such as being used as sunscreen skin care products, etc., mink bile is mostly discarded, so there is still room for further biological development and utilization. Summary of the Invention

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

[0004] According to one aspect of the present invention, there is provided a method for preparing a mink bile complex, comprising: Form a reaction solution with 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); React the reaction solution under heating conditions, wherein the reaction temperature is 80°C - 90°C and the reaction time is 1 - 3 h; Let the reacted solution stand for stratification and then filter out the precipitate; Hydrolyze the obtained precipitate under acidic conditions with a pH value of 1 - 2, and filter, extract and wash and dry the precipitated precipitate to obtain a crude total bile acid extract; Dissolve UDP-glucuronosyltransferase recombinant protein and glucuronic acid in PBS buffer to form a solution, wherein the concentration of UDP-glucuronosyltransferase recombinant protein is 5 - 15 ug / mL and the concentration of glucuronic acid is 50 - 150 mg / mL; Fully dissolve the obtained crude total bile acid extract in the solution and then separate to obtain a total bile acid extract after removing bilirubin; and Place the obtained total bile acid extract and adenosine in an aqueous ethanol solution for sufficient mixing and drying to obtain a mink bile complex, wherein the mass ratio of the total bile acid extract to adenosine is 10:1 - 20:1.

[0005] According to the preparation method of the present invention, the volume fraction of the aqueous ethanol solution is 20% - 40%. The molar fraction of adenosine in the aqueous ethanol solution and all bile acids (molecular weight calculated as cholic acid) contained in the total bile acid extract can be basically equal.

[0006] According to another aspect of the present invention, there is also provided a mink bile complex prepared according to the above method.

[0007] According to still another aspect of the present invention, there is also provided the use of the above-mentioned mink bile complex in the preparation of anti-hepatitis drugs or liver-protecting health products.

[0008] 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. The preparation method of the present invention is simple and the process conditions are mild, which is particularly suitable for industrial scale production. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1a 、 Figure 1b and Figure 1c are respectively the FESEM electron micrographs of the total bile acid extract, adenosine and mink bile complex; Figure 1d 、 Figure 1e and Figure 1f are respectively the TEM scanning diagrams of the total bile acid extract, adenosine and mink bile complex; Figure 2a and Figure 2b are respectively the expression levels of ALT and AST in the serum of the acute liver injury model mice under the intervention of different groups; Figure 3 is the H&E staining diagram of the pathological tissue section of acute liver injury under the intervention of different groups; Figure 4 is the H&E staining diagram of the pathological tissue section of alcoholic liver injury under the intervention of different groups. DETAILED DESCRIPTION OF THE INVENTION

[0010] Example: Preparation of Mink Bile Complex Dissolve 2 g of mink bile freeze-dried powder (provided by "Bocheng Songmao Biotechnology Henan Co., Ltd.") in 10 ml of ethyl acetate, and then mix it with 100 ml of ethanol and 8 g of sodium hydroxide to form a reaction solution; reflux and heat at 85 °C for 2 h, let it stand for stratification, and filter out the precipitate. Dissolve the precipitate in water and adjust the pH to 1.5 with dilute hydrochloric acid, filter after precipitation, wash with distilled water, and then vacuum dry at 60 °C to obtain 1.45 g of crude product. Add the obtained crude product to 10 mL of PBS buffer solution, 100 μg of UDP glucuronosyltransferase recombinant protein, 1.0 g of glucuronic acid, stir and react at 37 °C for 24 h, then filter, separate the precipitate and vacuum dry to obtain 0.98 g of mink bile total bile acid extract without bilirubin.

[0011] Preparation of mink bile complex by non-covalent assembly: Weigh 400 mg of the above total bile acid extract (1 mM total bile acid; molecular weight calculated based on cholic acid), and gradually add it to 5 mL of 30% ethanol aqueous solution (volume fraction) while stirring slowly to fully dissolve it. Weigh 27 mg of adenosine and dissolve it in 1.5 mL of deionized water. Add the fully dissolved total bile acid to adenosine, stir and sonicate to fully mix the two. After observing that a viscous substance has initially formed on the stirring rod, stop stirring and continue sonication for 3 min. Take it out and let it stand at room temperature. Observe the formation of a stable and uniform mink bile complex through the "vial inversion method".

[0012] Characterization of Mink Gallbladder Complex The mink bile complex prepared above was freeze-dried to obtain a powder (lyophilized gel sample), which was evenly spread on the conductive tape attached to the sample stage. Before testing, it was sputter-coated with gold, and after placing it on the sample stage, it was scanned and observed using a cold field emission scanning electron microscope FESEM; after diluting the lyophilized gel sample with 2 mL of 50% ethanol aqueous solution, it was dropped on a copper grid and dried in an oven, and observed by transmission electron microscopy TEM. The FESEM electron micrograph shows that the total bile acid extract presents irregular blocks ( Figure 1a ); adenosine is rod-shaped ( Figure 1b ); the mink bile complex presents dense and slender rods, with a small amount of blocks attached ( Figure 1c ). The TEM scan shows that the total bile acid extract is round dot-shaped ( Figure 1d ); adenosine presents fibrous shape ( Figure 1e ); the mink bile complex presents fibrous burr shape ( Figure 1f ), which should be a new microscopic morphology structure formed by the combination and extension of total bile acid attached to adenosine fibers.

[0013] Evaluation of the Bioavailability of Mink Gallbladder Complex Using Wistar rats fasted for 12 h as experimental subjects, they were divided into 2 groups: the mink bile complex (the above lyophilized gel sample) group and the total bile acid extract group. The two groups were compared with each other, and the dosing dose was calculated according to the rat body weight (administered at 30 mg / kg). After administration, each group took blood from the orbital cavity at 1, 3, 5, 7, 14, and 28 h respectively. The obtained blood samples were placed in heparin-infiltrated centrifuge tubes, centrifuged at 4000 r / min for 10 min, the supernatant was aspirated and stored at -20 °C for later use. Table 1 lists the bioavailability (blood drug concentration) of the mink bile complex group and the total bile acid extract group calculated based on cholic acid.

[0014]

[0015] As can be seen from Table 1, the mink bile complex group can significantly improve the bioavailability of mink bile acid at different time points.

[0016] Application Test of Anti-Hepatitis Activity Method for establishing a mouse acute liver injury model induced by CCL4 and grouping: Male ICR mice (20 - 22 g) were randomly divided into 6 groups: normal group, model group, bear bile powder group (250 mg / kg), total bile acid extract group (250 mg / kg), mink bile complex group (250 mg / kg) dosage group, and bifendate (250 mg / kg) positive control group. Each administration group was given gavage administration, and the normal group and the model group were given an equal amount of distilled water once a day for 4 consecutive days. 1 h after the last administration, except for the control group, all were intraperitoneally injected with 0.1% CCl4 vegetable oil solution (0.1 ml / 10 g). After exposure to the toxin, food was withheld but water was available.

[0017] Method for establishing a mouse alcoholic liver injury model and grouping: Healthy male ICR mice were randomly divided into 6 groups, namely normal group, model group, bear bile powder group (250 mg / kg), total bile acid extract group (250 mg / kg), mink bile complex group (250 mg / kg) dosage group, and bifendate (250 mg / kg) positive control group. Each administration group was given gavage administration respectively, and the normal group and the model group were given an equal amount of distilled water once a day for 4 consecutive days. 1 h after the last administration, 12 mL / kg of 56° Red Star Erguotou was given by gavage for modeling, and the normal group was given an equal amount of distilled water. Food was withheld but water was available.

[0018] Method for detecting serum ALT and AST levels 16 h after modeling, blood was collected by eye socket puncture. The blood was centrifuged at 3500 r / min for 10 min at 4 °C, and the supernatant was taken and aliquoted into EP tubes and stored at -80 °C. The activities of ALT and AST in the serum were detected by an automatic biochemical analyzer.

[0019] Method for measuring SOD, CAT, GSH-PX, GSH and ROS indexes in liver tissue 100 mg of liver tissue was taken and placed in a homogenizer, and 9 times the amount of normal saline was added. It was made into 10% tissue homogenate in an ice-water bath, centrifuged at 3500 r / min for 10 min at 4 °C, and the supernatant was taken and stored at -20 °C. According to the kit instructions, the protein content and the activities of CAT, SOD, GSH-PX, GSH and ROS in the liver tissue were detected.

[0020] Data Processing and Results The actual data are expressed as One-way ANOVA, t-test and other treatments were performed using the statistical software SPSS 21.0. A p value < 0.05 was considered statistically significant.

[0021] The results are shown in 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. From Figure 2a and Figure 2b it can be seen that in the test of the CCL4-induced acute liver injury model in mice, compared with the normal group, the levels of ALT and AST in the serum of the CCl4 model group were significantly increased (p < 0.01). Compared with the CCl4 model group, the levels of ALT and AST in the serum of the bear bile powder group, the total bile acid extract group, and the mink bile complex group were significantly decreased. Among them, the decrease in the mink bile complex group was particularly obvious, with ALT and AST decreasing by 97% and 84% respectively, while the positive drug bifendate decreased by 93% and 64% respectively. It is suggested that the assembled mink bile complex group can significantly reduce the activity of transaminase in the serum after CCl4 induction.

[0022] In the mouse alcoholic liver injury model, compared with the normal group, the activities of serum ALT and AST and the content of TG in the model group were significantly increased (p < 0.05, p < 0.01), and the difference was statistically significant, indicating that an acute alcoholic liver injury model was successfully established (Table 2). Compared with the model group, the activities of ALT and AST and the content of TG in the bear bile powder group, the total bile acid extract group, and the mink bile complex group were significantly decreased (p < 0.05, p < 0.01). Among them, the decrease in the activities of ALT and AST and the content of TG in the mink bile complex group was particularly significant (p < 0.01, p < 0.05), and it was better than the positive bifendate group. The positive bifendate group had no significant effect on the increase in the content of serum TG. It shows that the mink bile complex group can antagonize the increase of liver metabolic enzymes ALT, AST, and TG caused by excessive alcohol. Table 2 lists the effects of each administration group on serum ALT, AST, and TG in mice with alcohol-induced liver injury ( , n = 8). Table 3 lists the effects of each administration group on SOD, ADH, GSH, and GSH-Px in the liver tissue of mice with alcohol-induced acute liver injury ( , n = 8).

[0023]

[0024] 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), and the difference was statistically significant. Compared with the model group, the activities of SOD, ADH, GSH-Px and GSH in the liver tissue homogenate of the bifendate positive group were significantly increased (p<0.01, p<0.05). The activities of SOD, ADH, GSH-Px and GSH in the bear bile powder group, total bile acid extract group and mink bile complex group were also significantly increased (p<0.01, p<0.05), and the degree of increase was comparable to that of bifendate. The activities of GSH-Px and GSH in the mink bile complex group were significantly increased (p<0.01, p<0.05). This indicates that the mink bile complex group may achieve hepatoprotective effects through the antioxidant damage pathway.

[0025] Liver Pathology Detection The left lobe of the sample was cut off and fixed in 10% formaldehyde solution, then paraffin-embedded, sectioned at 5 μm thickness, mounted on glass slides, and then dewaxed. For histological analysis, the sections were stained using the standard hematoxylin-eosin technique. The damage was observed and photographed under a microscope at magnifications of 40X, 100X and 400X.

[0026] Figure 3 A-F respectively show the liver pathological sections of mice (pathological tissue sections stained by H&E staining method, 400×). The liver pathological sections showed that the liver tissue structure of mice in the normal group was normal, the hepatocyte morphology was round, and the nucleus was located in the center (A); in the CCl4 liver injury model group, lymphocyte infiltration was visible around the liver lobules and blood vessels, necrosis of hepatocytes at the edge of the liver tissue formed necrosis foci, scattered necrosis of hepatocytes, nuclear pyknosis, and a large amount of amyloidosis of hepatocytes (B); in the bear bile powder group (C), total bile acid extract group (D), and mink bile complex group (E), the liver lobule structure was intact, the hepatocyte morphology was basically normal, and there were no obvious pathological changes, and the mink bile complex group was more complete; in the bifendate positive group, partial necrosis of hepatocytes and nuclear pyknosis were observed at the edge of the liver tissue (F). This suggests that the mink bile complex group can significantly improve the pathological changes of liver tissue.

[0027] Figure 4In A-F respectively, the pathological sections of mouse livers are shown. In the normal group, the liver tissue structure of mice is normal, the hepatocytes are round in shape, the nucleus is located in the center, and there is only a small amount of apoptosis (A); in the alcoholic liver injury model group, lymphocyte infiltration can be seen in the liver lobules of mice, hepatocyte apoptosis or necrosis occurs, and megakaryocytes appear (B); in the bear bile powder group (C), the total bile acid extract group (D), and the mink bile complex group (E), the liver lobule structure is complete, the hepatocyte morphology is basically normal, and there are no obvious pathological changes, and the mink bile complex group is more complete; in the bifendate positive group, the hepatocyte morphology has basically returned to normal and there are no obvious pathological changes (F). It shows that the mink bile complex group has an obvious improvement effect on acute liver injury caused by alcohol. The above results show that the mink bile complex prepared by the present invention has a certain protective effect on CCL4-induced acute liver injury compared with untreated bile, and has a good protective effect on acute alcoholic liver injury.

Claims

1. A preparation method of a mink gallbladder complex, comprising: Forming a reaction solution from mink gallbladder dry powder, sodium hydroxide, ethyl acetate and ethanol, wherein the concentration of the mink gallbladder 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); Reacting the reaction solution under heating conditions, wherein the reaction temperature is 80°C - 90°C and the reaction time is 1 - 3 h; Allowing the reacted solution to stand and separate layers, and then filtering out the precipitate; Hydrolyzing the obtained precipitate under acidic conditions with a pH value of 1 - 2, and filtering, extracting, washing and drying the precipitated precipitate to obtain a crude total cholic acid extract; Dissolving UDP-glucuronosyltransferase recombinant protein and glucuronic acid in PBS buffer to form a solution, wherein the concentration of UDP-glucuronosyltransferase recombinant protein is 5 - 15 μg / mL and the concentration of glucuronic acid is 50 - 150 mg / mL; Fully dissolving the obtained crude total cholic acid extract in the solution and then separating to obtain a total cholic acid extract after removing bilirubin; And Placing the obtained total cholic acid extract and adenosine in an aqueous ethanol solution, mixing well and drying to obtain a mink gallbladder complex, wherein the mass ratio of the total cholic acid extract to adenosine is 10:1 - 20:

1.

2. The preparation method according to claim 1, wherein the volume fraction of the aqueous ethanol solution is 20% - 40%.

3. A mink gallbladder complex prepared by the method according to claim 1 or 2.

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

Images