Animal bile three-phase separation comprehensive development and utilization method

Abstract

The invention discloses a method for three-phase separation and comprehensive development and utilization of animal bile, and belongs to the technical field of extraction of bioactive products from animal bile. The method specifically comprises the following steps: step 1, filtering and impurity removal: filtering fresh animal bile; the method comprises the following steps: 1, filtering animal bile, 2, saponification: saponifying the filtered animal bile, and 3, three-phase extraction: adding a certain amount of dipotassium phosphate, sodium hydrogen sulfite and absolute ethyl alcohol into the saponified bile, and uniformly stirring. 4, three-phase layering: standing the extraction system for 1-5 hours to enable the extraction system to form three phases; step 5, separating bilirubin; and 6, separating deoxycholic acid. Materials used in the method are clean, green and basically free of pollution to the environment, operation steps are simple and convenient, expensive and complex instruments are not needed, reaction conditions are controllable and mild, the processing means related to the method can be used for extracting biological activity of bilirubin, deoxycholic acid and the like in animal bile at the same time, and the utilization rate of the raw materials is increased.

Description

Technical Field

[0001] The invention relates to the technical field of biologically active products extracted from animal bile, in particular to a method for comprehensive development and utilization of three-phase separation of animal bile. Background Art

[0002] Bezoar is the dried gallstone of cattle, a mammal of the Bovidae family. It has extremely high medicinal value and is a precious Chinese medicinal material. However, natural bezoar resources are scarce and very expensive. In order to alleviate the shortage of natural bezoar medicinal sources, my country's drug supervision and management department has approved artificial bezoar as one of the substitutes for bezoar, ensuring the urgent need for clinical medication. Bilirubin and hyodeoxycholic acid are the main raw materials for synthesizing artificial bezoar. Fresh animal bile is rich in bilirubin and deoxycholic acid and other pharmaceutical raw materials. At present, animal bile is mainly used for the extraction of high value-added products such as bilirubin and deoxycholic acid. Among them, the extraction methods of bilirubin mainly include calcium salt indirect extraction method, organic solvent direct extraction method and reductase preparation method. People mainly use the residual animal bile after extracting bilirubin to extract hyodeoxycholic acid or chenodeoxycholic acid. Deoxycholic acid is mainly extracted by barium salt precipitation method, calcium salt precipitation method, ion exchange resin method and a large amount of ethyl acetate. Among them, the ethyl acetate extract needs to be purified by acetone recrystallization, methanol (ethanol)-water recrystallization or directly by column chromatography.

[0003] Chinese patent CN201611033488.3 discloses a method for extracting bilirubin from bovine bile. The method pre-treats the bovine bile with high temperature heating and acid-base treatment multiple times, and then uses chloroform and ethanol to extract multiple times and activated carbon adsorption to obtain the bilirubin product. This process can effectively extract bilirubin from bovine bile, but a large amount of toxic chloroform is required in the extraction process of this process, and only bilirubin is extracted from bovine bile, and the raw material utilization rate is low. At present, there are few processes that can extract several components in animal bile at one time, the utilization rate of raw materials is low, the remaining raw materials are even discarded, and the cost of industrial production is high. Chinese patent number CN201911076648.6 discloses a co-production method for extracting bilirubin, hyodeoxycholic acid, and chenodeoxycholic acid from pig bile. The invention uses pig bile as raw material, heats the pig bile at 85°C to 95°C, filters the cooled pig bile using a molecular membrane, and then extracts the components in the bile using ethanol under negative pressure. Bilirubin is obtained by filtering the reflux liquid through a G3 sand core funnel. At the same time, hyodeoxycholic acid and chenodeoxycholic acid are obtained by multiple sodium bicarbonate and ethyl acetate treatments, thereby realizing the comprehensive utilization of pig bile. However, the invention requires high-temperature treatment in the pig bile treatment step, which increases energy input, and also requires the use of precision equipment such as molecular membranes. Moreover, the invention also requires the use of some toxic organic solvents in the separation process of bilirubin, hyodeoxycholic acid and chenodeoxycholic acid, which is not conducive to the green development of the product.

[0004] The biggest production bottlenecks encountered by the above-mentioned methods of extracting products are large energy and solvent consumption, toxic solvents, low product purity, cumbersome processes, and difficulty in large-scale implementation. Modern extraction and deep processing technology should meet the production requirements of "safety, efficiency, and greenness", improve the comprehensive utilization rate of by-products, and at the same time ensure the quality of the target product, so as to achieve zero emissions during the processing process, thereby achieving the purpose of saving resources and increasing product added value. Therefore, how to achieve the comprehensive utilization of bilirubin and deoxycholic acid in animal bile, so as to realize the process of "turning waste into treasure", is particularly important. Seeking a continuous production with high degree, green energy saving, and the simultaneous extraction of bilirubin and deoxycholic acid in animal bile has become a key problem to be solved in the large-scale production of high-value active ingredients of animal bile at home and abroad. Summary of the invention

[0005] The purpose of the present invention is to provide a method for comprehensive development and utilization of three-phase separation of animal bile, so as to solve the problems proposed in the above background technology, namely, large energy and solvent consumption, toxic solvent, low purity of the obtained product, complicated process and difficulty in large-scale implementation.

[0006] To achieve the above object, the present invention provides the following technical solution: a method for comprehensive development and utilization of three-phase separation of animal bile, specifically comprising the following steps:

[0007] Step 1: Filter and remove impurities: Filter the fresh animal bile to remove the residue;

[0008] Step 2: Saponification: Adjust the pH of the filtered animal bile to 10-12, and saponify at 50-100°C for 0.1-3h. Preferably, the saponification time is 0.2-1h and the temperature is 50-70°C.

[0009] Step 3: Three-phase extraction: Add a certain amount of dipotassium hydrogen phosphate, sodium bisulfite and anhydrous ethanol to the above saponified bile, and stir evenly at a temperature of 30 to 80°C; preferably, the temperature is adjusted to 45 to 60°C.

[0010] Step 4: Three-phase separation: The above extraction system is allowed to stand for 1 to 5 hours to allow the extraction system to form three phases;

[0011] Step 5: Bilirubin separation: remove the upper layer of the three-phase system, adjust the pH to neutral with hydrochloric acid, collect the precipitate, wash it twice with water and ethanol, dry the crystals, and obtain bilirubin;

[0012] Step 6: Separation of deoxycholic acid: remove the lower layer of the three-phase system, adjust the pH to neutral with hydrochloric acid, collect the precipitate, wash it twice with ethanol, dry the crystals, and obtain deoxycholic acid.

[0013] Preferably, the animal bile is cow bile, pig bile, snake bile, chicken bile, duck bile, fish bile or eel bile.

[0014] Preferably, the filtration in step 1 can be performed by simply filtering with qualitative filter paper or gauze material to remove particulate matter.

[0015] Preferably, the material used for pH adjustment in step 2 is an aqueous solution of sodium hydroxide, potassium hydroxide, sodium carbonate, barium hydroxide or calcium hydroxide.

[0016] Preferably, the added amount of dipotassium hydrogen phosphate and sodium bisulfite is between 5% and 50% of the liquid-solid ratio, and the added anhydrous ethanol is between 1:0.5 and 1:5 in volume ratio to the liquid.

[0017] The present invention also provides bilirubin and deoxycholic acid obtained by the processing method.

[0018] The yield of the bilirubin is higher than 0.03%, and the purity is higher than 95%; the yield of deoxycholic acid is higher than 3.5%, and the purity is higher than 85%.

[0019] The present invention also provides the application of the processing method or animal bile in the fields of raw material processing such as development and extraction of functional active components.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] (1) The materials used in the present invention are clean, green and basically non-polluting to the environment. The operation steps are simple and do not require the use of expensive and complex instruments. The reaction conditions are controllable and mild, and can be applied to the development and processing of all animal bile.

[0022] (2) The processing method of the present invention can simultaneously extract biological activities such as bilirubin and deoxycholic acid from animal bile, thereby enhancing the utilization rate of raw materials, making the extraction of bilirubin and deoxycholic acid more efficient, and reducing the extraction cost of bilirubin and deoxycholic acid.

[0023] Of course, any product implementing the present invention does not necessarily need to achieve all of the advantages described above at the same time. DETAILED DESCRIPTION

[0024] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0025] The materials used in the following examples are all available from commercial sources.

[0026] The detection method of bilirubin and deoxycholic acid yield in the embodiment is as follows:

[0027] (1) Yield of bilirubin (%) = 100% × mass of dried bilirubin / mass of bile;

[0028] (2) Deoxycholic acid yield (%) = 100% × mass of dried deoxycholic acid / mass of bile;

[0029] The detection method of bilirubin purity in the embodiment is as follows:

[0030] a. Accurately weigh 25ug of bilirubin reference substance (produced by Shanghai Institute of Biology) and dissolve it in 1mL of dichloromethane.

[0031] b. Accurately weigh 25 μg (ml) of the dried bilirubin sample and dissolve it in 1 mL of dichloromethane.

[0032] c. HPLC conditions: the chromatographic column was Boston Green ODS-C18 (150 mm × 4.6 mm, 5 μm), the mobile phase was acetonitrile:1% glacial acetic acid (95:5), the flow rate was 1.0 mL / min, the detection wavelength was 450 nm, the column temperature was 30°C, and the injection volume was 5 μL.

[0033] d. Determination: Using bilirubin (produced by Shanghai Institute of Biology) as the standard, the peak area was integrated to obtain the mass of bilirubin (m2).

[0034] d. Calculation: Bilirubin purity = 100% × m2 / m1

[0035] The detection method of deoxycholic acid purity in the embodiment is as follows:

[0036] a. Preparation of standard curve: Accurately measure 0mg, 0.1mg, 0.3mg, 0.5mg, 0.8mg, 1.0mg, and 1.2mg of deoxycholic acid standard (produced by Shanghai Institute of Biology) in a 25mL volumetric flask, add 2mL of glacial acetic acid, add H2SO4 to the scale, and place in a 70℃ water bath for 20min, followed by an ice bath for 5min. Using the reagent in the 0mL bottle as the blank, measure the OD380 of each solution to prepare a standard curve.

[0037] b. Accurately weigh 0.5 mg (m3) of the dried deoxycholic acid sample, perform the same operation as above, measure OD380, and calculate the content (m4).

[0038] c. Calculation: Deoxycholic acid purity = 100% × m4 / m3

[0039] Example 1

[0040] A method for comprehensive development and utilization of animal bile three-phase separation, comprising the following steps:

[0041] 1. Ox bile filtration: Take a certain amount of fresh ox bile and filter it with 8 layers of gauze to remove insoluble particulate matter.

[0042] 2. Bile saponification: Take 100 g of fresh filtered bovine bile, adjust the bile to pH = 10 with 2M NaOH solution, and then saponify at 60°C for 20 minutes.

[0043] 3. Establishment of three-phase separation system: Add 20 g of dimethyl hydrogen phosphate to the saponified ox bile, then add 100 mL of 80% pure ethanol solution, stir at 50°C for 30 min, and then stand for 1 h to wait for the formation of the three-phase separation system.

[0044] 4. Concentrate the upper layer of the three-phase system after stratification, adjust the pH to neutral with 1M hydrochloric acid to precipitate bilirubin, and wash and dry with anhydrous ethanol to obtain a bilirubin product.

[0045] 5. Concentrate the lower layer of the three-phase system after stratification, adjust the pH to neutral with 1M hydrochloric acid to precipitate deoxycholic acid, and wash and dry with anhydrous ethanol to obtain a deoxycholic acid product.

[0046] 6. The recovery rate of bilirubin was measured to be 0.038% and the purity was 98%; the recovery rate of deoxycholic acid was measured to be 4.1% and the purity was 86%;

[0047] Example 2

[0048] A method for comprehensive development and utilization of animal bile three-phase separation, comprising the following steps:

[0049] 1. Pig bile filtration: Take a certain amount of fresh pig bile and filter it with 8 layers of gauze to remove insoluble particulate matter.

[0050] 2. Saponification of pig bile: Take 5000 g of fresh pig bile after filtration, adjust the bile to pH = 11 with 2M Na(OH)2 solution, and then saponify at 70°C for 30 minutes.

[0051] 3. Establishment of three-phase separation system: Add 1000 g of dimethyl hydrogen phosphate to the saponified ox bile, then add 5000 mL of 95% pure ethanol solution, stir at 50°C for 30 min, and then stand for 5 h to form a three-phase separation system.

[0052] 4. Concentrate the upper layer of the three-phase system after stratification, adjust the pH to neutral with 1M hydrochloric acid to precipitate bilirubin, and wash and dry with anhydrous ethanol to obtain a bilirubin product.

[0053] 5. Concentrate the lower layer of the three-phase system after stratification, adjust the pH to neutral with 1M hydrochloric acid to precipitate deoxycholic acid, and wash and dry with anhydrous ethanol to obtain a deoxycholic acid product.

[0054] 6. The recovery rate of bilirubin was measured to be 0.038% and the purity was 98%; the recovery rate of deoxycholic acid was measured to be 4.1% and the purity was 86%;

[0055] Example 3

[0056] 1. Pig bile filtration: Take a certain amount of fresh pig bile and filter it with 8 layers of gauze to remove insoluble particulate matter.

[0057] 2. Saponification of pig bile: Take 500 g of fresh pig bile after filtration, adjust the bile to pH = 11 with 2M Ca(OH)2 solution, and then saponify at 70°C for 30 minutes.

[0058] 3. Establishment of three-phase separation system: Add 80 g of dimethyl hydrogen phosphate to the saponified ox bile, then add 500 mL of 100% pure ethanol solution, stir at 50°C for 30 min, and then stand for 5 h to form a three-phase separation system.

[0059] 4. Concentrate the upper layer of the three-phase system after stratification, adjust the pH to neutral with 1M hydrochloric acid to precipitate bilirubin, and wash and dry with anhydrous ethanol to obtain a bilirubin product.

[0060] 5. Concentrate the lower layer of the three-phase system after stratification, adjust the pH to neutral with 1M hydrochloric acid to precipitate deoxycholic acid, and wash and dry with anhydrous ethanol to obtain a deoxycholic acid product.

[0061] 6. The recovery rate of bilirubin was measured to be 0.031% and the purity was 95%; the recovery rate of deoxycholic acid was measured to be 3.6% and the purity was 91%;

[0062] The following comparative example 1 uses a calcium salt organic solvent direct extraction method to extract bilirubin, and the specific example is as follows:

[0063] 1. Pig bile filtration: Take a certain amount of fresh cow bile and filter it with 8 layers of gauze to remove insoluble particulate matter.

[0064] 2. Bilirubin extraction: Take 100g of filtered pig bile, add 2M NaOH solution, adjust the pH to 12, heat to boiling, add calcium chloride solution, heat to boiling again, cool, filter, and obtain bilirubin calcium salt.

[0065] 3. Bilirubin purification: Add 1M hydrochloric acid to adjust the pH to 2 in bilirubin calcium salt, add 300mL of chloroform to fully dissolve, then centrifuge at 5000rpm and 5℃ for 10min, take the supernatant, evaporate the chloroform, and dry to obtain the bilirubin product. The yield of bilirubin is 0.026% and the purity is 98%. Deoxycholic acid cannot be extracted at the same time.

[0066] It can be seen from the above embodiments and comparative examples that in embodiments 1 to 3 of the present invention, the method of the present invention can be used to simultaneously extract bilirubin and deoxycholic acid, the recovery rate of bilirubin is more than 0.031%, the purity is more than 95%, and the recovery rate of deoxycholic acid is more than 3.5%, and the purity is more than 86%. Compared with the comparative example, the method of the present invention can significantly simultaneously extract bilirubin and deoxycholic acid, which is beneficial to the comprehensive utilization of raw materials. The present invention has another obvious advantage, the extraction process is mild, the solvent used is cheap, non-toxic and harmless, the operation is simple, the instrument performance requirements are low, and it is easy to expand the scale to industrial production.

[0067] In the description of this specification, the description with reference to the terms "one embodiment", "example", "specific example", etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner.

[0068] The preferred embodiments of the present invention disclosed above are only used to help illustrate the present invention. The preferred embodiments do not describe all the details in detail, nor do they limit the invention to the specific implementation methods described. Obviously, many modifications and changes can be made according to the content of this specification. This specification selects and specifically describes these embodiments in order to better explain the principles and practical applications of the present invention, so that those skilled in the art can understand and use the present invention well. The present invention is limited only by the claims and their full scope and equivalents.

Claims

1. A method for comprehensive development and utilization of three-phase separation of animal bile, characterized in that: The specific steps include: Step 1: Filter and remove impurities: Filter the fresh animal bile to remove the residue; Step 2: Saponification: adjust the pH of the filtered animal bile to 10-12 and saponify at 50-100°C for 0.1-3h; Step 3: Three-phase extraction: Add a certain amount of dipotassium hydrogen phosphate, sodium bisulfite and anhydrous ethanol to the above saponified bile, and stir evenly at a temperature of 30-80°C; Step 4: Three-phase separation: The above extraction system is allowed to stand for 1 to 5 hours to allow the extraction system to form three phases; Step 5: Bilirubin separation: remove the upper layer of the three-phase system, adjust the pH to neutral with hydrochloric acid, collect the precipitate, wash it twice with water and ethanol, dry the crystals, and obtain bilirubin; Step 6: Separation of deoxycholic acid: remove the lower layer of the three-phase system, adjust the pH to neutral with hydrochloric acid, collect the precipitate, wash it twice with ethanol, dry the crystals, and obtain deoxycholic acid.

2. The method for comprehensive development and utilization of three-phase separation of animal bile according to claim 1, characterized in that: The animal bile is cow bile, pig bile, snake bile, chicken bile, duck bile, fish bile or eel bile.

3. The method for comprehensive development and utilization of animal bile three-phase separation according to claim 1, characterized in that: The filtration in step 1 can be performed by simply filtering with qualitative filter paper or gauze material to remove particulate matter.

4. The method for comprehensive development and utilization of animal bile three-phase separation according to claim 1, characterized in that: The material used for pH adjustment in step 2 is an aqueous solution of sodium hydroxide, potassium hydroxide, sodium carbonate, barium hydroxide or calcium hydroxide.

5. The method for comprehensive development and utilization of animal bile three-phase separation according to claim 1, characterized in that: The added amount of dipotassium hydrogen phosphate and sodium bisulfite is between 5% and 50% of the liquid-solid ratio, and the added anhydrous ethanol is between 1:0.5 and 1:5 of the liquid volume ratio.