Method for extracting and identifying bile acid from animal bile
By using drying, crushing, ethanol extraction, sodium hydroxide reflux, pH adjustment and ethyl acetate extraction in animal gallbladder, the existing bile acid extraction process is complicated and low yield is solved, and an efficient, simple and environmentally friendly bile acid extraction effect is achieved.
Patent Information
- Application Number
- CN202510089973.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-21
- Publication Date
- 2025-05-23
AI Technical Summary
The existing methods for extracting bile acids from animal gallbladder are complicated and have a low yield.
After drying and crushing the bile paste, the purified bile acid was heated and refluxed in anhydrous ethanol, followed by reflux treatment with sodium hydroxide solution, then the pH was adjusted to acidity, and extracted by ethyl acetate, and finally the purified bile acid was obtained by rotary evaporation.
It achieves efficient extraction of bile acids, simple process, low cost, high extraction rate, and does not involve toxic chemicals, and has the advantages of green and environmental protection.
Smart Images
Figure CN120025391A_ABST
Abstract
Description
Technical field:
[0001] The invention belongs to the technical field of animal bile acid extraction and detection, and specifically relates to a method for extracting bile acid from animal bile and detecting its type and content. Background technology:
[0002] Bile acid is a series of steroidal substances produced during cholesterol metabolism in animals. It has multiple biological activities and is the main active component of bile. Bile acid molecules contain a steroid skeleton consisting of three 6-membered rings and one 5-membered ring, a carboxyl group and different numbers of hydroxyl groups. Its main functions in the body include: promoting the digestion and absorption of lipids, promoting liver circulation and inhibiting the precipitation of cholesterol in bile, inhibiting the growth of intestinal microorganisms, protecting the integrity of the bile duct, and reducing inflammation and sterilization.
[0003] In recent years, research on the types and composition of animal bile acids has continued to deepen. For example, when studying the effects of mammalian intestinal microorganisms on bile acid metabolism, the effects of microorganisms on bile acid metabolism can be evaluated by measuring the content of different bile acid types in animal bile. In addition, some studies have found that the types and content of animal bile acids may be related to the occurrence and development of some diseases, such as liver disease and metabolic diseases. Therefore, it is crucial to determine the types and content of bile acids in animal bile.
[0004] Bile acids mainly include cholic acid, hyodeoxycholic acid, chenodeoxycholic acid, etc., which are generally present in animal bile paste. At present, the conventional methods for extracting bile acids from animal bile paste mainly include ethanol extraction, acid precipitation and salting out. However, the above methods all have the problems of complicated extraction process and low yield. Summary of the invention:
[0005] In view of the above problems, the object of the present invention is to provide a method for extracting bile acid with high extraction rate, simple process and low cost.
[0006] The technical solution of the present invention is specifically as follows:
[0007] A method for extracting bile acid from animal bile, characterized by comprising the following steps:
[0008] 1) drying and crushing the animal bile paste to obtain bile paste powder;
[0009] 2) adding the bile paste powder to anhydrous ethanol, heating and reflux for 30 minutes, filtering after the bile paste powder is fully dissolved in the ethanol to obtain filtrate A;
[0010] 3) Filtrate A was evaporated to dryness and then cooled to room temperature, and the remaining material was added with 15% sodium hydroxide solution, heated under reflux for 2 hours, and then filtered while hot to obtain filtrate B;
[0011] 4) The pH of filtrate B is adjusted to acidic with dilute sulfuric acid to obtain a primary extract;
[0012] 5) adding ethyl acetate to the primary extract for extraction, and washing and filtering the obtained extract phase (containing the target extract) in sequence to obtain filtrate C;
[0013] 6) The filtrate C is subjected to rotary evaporation to obtain a purified bile acid product.
[0014] Furthermore, in the above step 2), the amount of the bile paste powder added to the anhydrous ethanol is in the ratio of 25 to 50 g of bile paste powder per liter of anhydrous ethanol; the heating reflux treatment is carried out in an electric heating jacket, the heating temperature of the electric heating jacket is 120 to 140°C, and the heating process is assisted by stirring, and the stirring speed is 600 to 900 rpm.
[0015] Furthermore, in the above step 3): the filtrate A is evaporated to dryness by vacuum rotary evaporation; the amount of the 15% sodium hydroxide solution is: according to the content of bile paste powder in the filtrate A, the amount of 15% sodium hydroxide solution corresponding to every 20 to 40 g of bile paste powder is 1 L.
[0016] Furthermore, in the above step 3), the heating reflux treatment is carried out in an electric heating jacket, the heating temperature of the electric heating jacket is 180° C., and the heating process is assisted by stirring, and the stirring speed is 900 rpm.
[0017] Furthermore, in the above step 5), the volume ratio of the primary extract to ethyl acetate is 2:1 to 3:1.
[0018] Furthermore, in the above step 4), the pH of the filtrate B is adjusted to 2.2-2.8 with dilute sulfuric acid.
[0019] Furthermore, in the above step 1), the animal bile paste is dried at 100-200° C. and then crushed to obtain bile paste powder.
[0020] The present invention further provides a method for detecting the type and content of bile acid obtained by the above-mentioned extraction method, characterized in that: the bile acid product obtained by the above-mentioned extraction method is weighed and dissolved in chromatographic grade methanol to obtain a sample to be tested, and then the prepared sample to be tested is subjected to HPLC-UV detection; wherein the addition ratio of the bile acid product in the chromatographic grade methanol is: 25 mg of the bile acid product is added to each milliliter of chromatographic grade methanol.
[0021] Furthermore, in the above-mentioned method for detecting the types and contents of bile acids, the HPLC-UV detection conditions are: detection wavelength is 192nm; chromatographic column: Agilent 5HC-C18 250×4.6mm, 5μm; mobile phase: acetonitrile:phosphoric acid (pH2.6)=50:50 (v / v); flow rate: 1mL / min; column temperature: 35°C; injection volume: 10μL.
[0022] The extraction and purification process of bile acid in animal bile paste of the present invention is simple and easy, and the bile acid yield is high; the extraction device and reagent involved are conventional and easy to obtain, and almost no toxic chemical substances are involved, which has the advantages of being green, environmentally friendly and low cost. In addition, the HPLC-UV detection method provided by the present invention can significantly improve the detection efficiency and accuracy, and at the same time, a suitable detector can be flexibly selected according to the sample requirements. Description of the drawings:
[0023] Figure 1 Schematic diagram of the reflux device structure.
[0024] Figure 2 Liquid chromatogram of a bile acid standard mixture.
[0025] Figure 3 Example 1 Liquid chromatogram of bile acid extraction sample in pig bile paste.
[0026] Figure 4 Example 2 Liquid chromatogram of bile acid extraction sample in chicken bile paste.
[0027] Figure 5 Example 3 Liquid chromatogram of bile acid extraction sample in duck bile paste.
[0028] Figure 6 Example 4 Liquid chromatogram of bile acid extraction sample in ox bile paste.
[0029] Figure 7 Example 5 Liquid chromatogram of bile acid extraction sample in sheep bile paste. Specific implementation method:
[0030] The technical scheme and effects of the present invention are further described below in conjunction with the embodiments and drawings. The protection scope of the present invention is not limited to the following embodiments.
[0031] Example 1
[0032] 1) drying the pig bile paste in an oven at 100° C. and then crushing the mixture to obtain bile paste powder;
[0033] 2) Add 0.9963 g (denoted as M) of bile paste powder to 20 mL of anhydrous ethanol, heat and reflux at 120° C. in an electric heating mantle for 30 min with a stirring speed of 600 rpm until the bile paste powder is fully dissolved in the ethanol; then filter to remove the ethanol insoluble matter to obtain filtrate A;
[0034] 3) The filtrate A was subjected to vacuum rotary evaporation, and after the liquid was evaporated to dryness, it was cooled to room temperature; the remaining material obtained was added with 30 mL of 15% sodium hydroxide solution, heated under reflux at 180° C. in an electric heating mantle for 2 h, and then filtered while hot to obtain filtrate B;
[0035] 4) The filtrate B was adjusted to pH = 2.8 with dilute sulfuric acid to obtain a primary extract;
[0036] 5) After the primary extract was cooled to room temperature, 0.5 times the volume of ethyl acetate was added for extraction, and the extraction was repeated 4 times. After each extraction, distilled water was added to the extract phase (containing the target extract) for washing, and the washed liquid was filtered to obtain filtrate C;
[0037] 6) The filtrate C was subjected to rotary evaporation, and the remaining material was weighed after the liquid was evaporated to dryness to obtain 0.7012 g (denoted as m) of a purified bile acid product.
[0038] Figure 1 The conventional structural design of the reflux device is simplified, and is composed of a distillation flask 1 and a condenser 2 on its upper portion, and is suitable for the heating reflux treatment in the above-mentioned step 2) and step 3).
[0039] Example 2
[0040] 1) drying the chicken gallbladder paste in an oven at 100° C. and then crushing the chicken gallbladder paste to obtain gallbladder paste powder;
[0041] 2) Add 0.9048 g (denoted as M) of bile paste powder to 20 mL of anhydrous ethanol, heat and reflux at 120° C. in an electric heating mantle for 30 min with a stirring speed of 800 rpm until the bile paste powder is fully dissolved in the ethanol; then filter to remove the ethanol insoluble matter to obtain filtrate A;
[0042] 3) The filtrate A was subjected to vacuum rotary evaporation, and after the liquid was evaporated to dryness, it was cooled to room temperature; the remaining material obtained was added with 30 mL of 15% sodium hydroxide solution, heated under reflux at 180° C. in an electric heating mantle for 2 h, and then filtered while hot to obtain filtrate B;
[0043] 4) The filtrate B was adjusted to pH = 2.3 with dilute sulfuric acid to obtain a primary extract;
[0044] 5) After the primary extract was cooled to room temperature, 0.5 times the volume of ethyl acetate was added for extraction, and the extraction was repeated 4 times. After each extraction, distilled water was added to the extract phase (containing the target extract) for washing, and the washed liquid was filtered to obtain filtrate C;
[0045] 6) The filtrate C was subjected to rotary evaporation, and the remaining material was weighed after the liquid was evaporated to dryness to obtain 0.2869 g (denoted as m) of a purified bile acid product.
[0046] Example 3
[0047] 1) drying the duck bile paste in an oven at 100° C. and then crushing the duck bile paste to obtain bile paste powder;
[0048] 2) 1.0219 g (denoted as M) of bile paste powder was added to 25 mL of anhydrous ethanol, and the mixture was heated under reflux at 120° C. for 30 min in an electric heating mantle with a stirring speed of 600 rpm until the bile paste powder was fully dissolved in the ethanol; then, the mixture was filtered to remove the ethanol insoluble matter to obtain filtrate A;
[0049] 3) The filtrate A was subjected to vacuum rotary evaporation, and after the liquid was evaporated to dryness, it was cooled to room temperature; the remaining material obtained was added with 30 mL of 15% sodium hydroxide solution, heated under reflux at 180° C. in an electric heating mantle for 2 h, and then filtered while hot to obtain filtrate B;
[0050] 4) The filtrate B was adjusted to pH = 2.6 with dilute sulfuric acid to obtain a primary extract;
[0051] 5) After the primary extract was cooled to room temperature, 0.5 times the volume of ethyl acetate was added for extraction, and the extraction was repeated 4 times. After each extraction, distilled water was added to the extract phase (containing the target extract) for washing, and the washed liquid was filtered to obtain filtrate C;
[0052] 6) The filtrate C was subjected to rotary evaporation, and the remaining material was weighed after the liquid was evaporated to dryness to obtain 0.6428 g (denoted as m) of a purified bile acid product.
[0053] Example 4
[0054] 1) drying the ox bile paste in an oven at 100° C. and then crushing the ox bile paste to obtain bile paste powder;
[0055] 2) Add 1.0021 g (denoted as M) of bile paste powder to 25 mL of anhydrous ethanol, heat and reflux at 120° C. in an electric heating mantle for 30 min with a stirring speed of 600 rpm until the bile paste powder is fully dissolved in the ethanol; then filter to remove the ethanol insoluble matter to obtain filtrate A;
[0056] 3) The filtrate A was subjected to vacuum rotary evaporation, and after the liquid was evaporated to dryness, it was cooled to room temperature; the remaining material obtained was added with 30 mL of 15% sodium hydroxide solution, heated under reflux at 180° C. in an electric heating mantle for 2 h, and then filtered while hot to obtain filtrate B;
[0057] 4) The filtrate B was adjusted to pH = 2.2 with dilute sulfuric acid to obtain a primary extract;
[0058] 5) After the primary extract was cooled to room temperature, 0.5 times the volume of ethyl acetate was added for extraction, and the extraction was repeated 4 times. After each extraction, distilled water was added to the extract phase (containing the target extract) for washing, and the washed liquid was filtered to obtain filtrate C;
[0059] 6) The filtrate C was subjected to rotary evaporation, and the remaining material was weighed after the liquid was evaporated to dryness to obtain 0.6487 g (recorded as m) of a purified bile acid product.
[0060] Example 5
[0061] 1) drying the sheep gall paste in an oven at 100° C. and then crushing the sheep gall paste to obtain gall paste powder;
[0062] 2) Add 0.9428 g (denoted as M) of bile paste powder to 20 mL of anhydrous ethanol, heat and reflux at 120° C. in an electric heating mantle for 30 min with a stirring speed of 600 rpm until the bile paste powder is fully dissolved in the ethanol; then filter to remove the ethanol insoluble matter to obtain filtrate A;
[0063] 3) The filtrate A was subjected to vacuum rotary evaporation, and after the liquid was evaporated to dryness, it was cooled to room temperature; the remaining material obtained was added with 30 mL of 15% sodium hydroxide solution, heated under reflux at 180° C. in an electric heating mantle for 2 h, and then filtered while hot to obtain filtrate B;
[0064] 4) The filtrate B was adjusted to pH = 2.4 with dilute sulfuric acid to obtain a primary extract;
[0065] 5) After the primary extract was cooled to room temperature, 0.5 times the volume of ethyl acetate was added for extraction, and the extraction was repeated 4 times. After each extraction, distilled water was added to the extract phase (containing the target extract) for washing, and the washed liquid was filtered to obtain filtrate C;
[0066] 6) The filtrate C was subjected to rotary evaporation, and the remaining material was weighed after the liquid was evaporated to dryness to obtain 0.5379 g (denoted as m) of a purified bile acid product.
[0067] Example 6
[0068] The specific steps for sample testing and determination of the types and contents of various bile acids are as follows:
[0069] 1) The bile acid products obtained in Examples 1 to 5 were weighed and dissolved in chromatographic grade methanol to prepare a 25 mg / ml methanol solution, which was passed through a disposable organic filter membrane (with a membrane pore size of 0.22 μm) to obtain a sample to be tested, which was then tested on a machine.
[0070] Before the machine test, prepare the standard regression curves of the concentration and peak area of the standards of bile acid, deoxycholic acid, chenodeoxycholic acid, hyodeoxycholic acid, and 7-ketolithocholic acid for high performance liquid chromatography determination in advance. Prepare at least 5 standard working solutions of bile acid, deoxycholic acid, chenodeoxycholic acid, hyodeoxycholic acid, and 7-ketolithocholic acid with a concentration gradient of 1 to 10 mg / mL, pass through a disposable organic filter membrane (pore size of 0.22 μm), and obtain each standard solution sample for machine test.
[0071] 2) The prepared standard solution samples and the samples to be tested were subjected to HPLC-UV detection. The external standard method was used for detection, and the test conditions were as follows: chromatographic column: Agilent 5HC-C18 250×4.6mm, 5μm; mobile phase: acetonitrile: phosphoric acid (pH2.6) = 50:50 (v / v); flow rate: 1mL / min; column temperature: 35℃; injection volume: 10μL; UV detector detection wavelength was 192nm.
[0072] The test results are analyzed as follows:
[0073] According to the corresponding relationship between the peak area and concentration of bile acid, deoxycholic acid, chenodeoxycholic acid, hyodeoxycholic acid, and 7-ketolithocholic acid measured by the standard solution, a regression standard curve was made to obtain the following results:
[0074] When the UV detector wavelength is 192nm:
[0075] The standard curve of bile acid is y = 475.74x-283.15R 2 =0.9996
[0076] The standard curve of deoxycholic acid is y = 354.83x-174.17R 2 =0.9991
[0077] The standard curve of chenodeoxycholic acid is y = 508.8x-277.94R 2 =0.9990
[0078] The standard curve of hyodeoxycholic acid is y = 235.61x + 114.1R 2 =0.9994
[0079] The standard curve of 7-ketolithocholic acid is y=2856.8x-299.63R 2 =0.9992
[0080] Wherein x is the concentration of cholic acid, deoxycholic acid, chenodeoxycholic acid, hyodeoxycholic acid or 7-ketolithocholic acid, and y is the peak area; the concentrations of cholic acid, deoxycholic acid, chenodeoxycholic acid, hyodeoxycholic acid and 7-ketolithocholic acid in the samples of Examples 1 to 5 are calculated according to the standard curve.
[0081] The corresponding extraction rate X is calculated based on the concentrations of bile acid, deoxycholic acid, chenodeoxycholic acid, hyodeoxycholic acid, and 7-ketolithocholic acid in the samples of Examples 1 to 5, and the specific calculation formula is as follows:
[0082] X=(C×m) / 25M×100%
[0083] Wherein: C is the concentration of bile acid, deoxycholic acid, chenodeoxycholic acid, hyodeoxycholic acid or 7-ketolithocholic acid in the sample obtained from the standard curve, in mg / mL; M is the mass of animal bile paste raw material; m is the mass of bile acid obtained after animal bile paste extraction.
[0084] The liquid chromatogram of the mixed sample of bile acid, deoxycholic acid, chenodeoxycholic acid, hyodeoxycholic acid and 7-ketolithocholic acid standard products measured by the above-mentioned high performance liquid chromatography (UV detector wavelength 192nm standard product concentration 5mg / ml) is as follows Figure 2 As shown in the chromatogram, it can be seen that the peak times of cholic acid, deoxycholic acid, chenodeoxycholic acid, hyodeoxycholic acid, and 7-ketolithocholic acid are 7.147 min (peak), 19.192 min (peak), 17.056 min (peak), 8.066 min (peak), and 11.330 min (peak), respectively. The liquid chromatograms of the samples of Examples 1 to 5 are shown in FIG. Figures 3 to 7 As shown, from the chromatogram of each sample, it can be intuitively judged whether the sample contains bile acid, deoxycholic acid, chenodeoxycholic acid, hyodeoxycholic acid, and 7-ketolithocholic acid, and the corresponding content can be calculated according to the peak area and the standard curve. Table 1 lists the different bile acid contents in the samples of each embodiment. In addition, it can also be judged according to known literature reports that the unknown bile acids in the pig bile paste of Example 1 and the duck bile paste of Example 3 are hyodeoxycholic acid and allocholic acid, respectively (as shown in Table 1).
[0085] Table 1 Types and contents of bile acids in the bile of animals in Examples 1 to 5
[0086]
[0087] *“%” in the table refers to mass percentage.
Claims
1. A method for extracting bile acid from animal bile, characterized in that The steps include: 1) drying and crushing the animal bile paste to obtain bile paste powder; 2) adding the bile paste powder to anhydrous ethanol, heating and reflux for 30 minutes, filtering after the bile paste powder is fully dissolved in the ethanol to obtain filtrate A; 3) Filtrate A was evaporated to dryness and then cooled to room temperature, and the remaining material was added with 15% sodium hydroxide solution, heated under reflux for 2 hours, and then filtered while hot to obtain filtrate B; 4) The pH of filtrate B is adjusted to acidic with dilute sulfuric acid to obtain a primary extract; 5) adding ethyl acetate to the primary extract for extraction, and washing and filtering the obtained extract phase (containing the target extract) in sequence to obtain filtrate C; 6) The filtrate C is subjected to rotary evaporation to obtain a purified bile acid product.
2. The bile acid extraction method according to claim 1, characterized in that: In the step 2), the bile paste powder is added to anhydrous ethanol in a ratio of 25 to 50 g of bile paste powder per liter of anhydrous ethanol; the heating reflux treatment is carried out in an electric heating jacket, the heating temperature of the electric heating jacket is 120 to 140° C., and stirring is performed during the heating process at a speed of 600 to 900 rpm.
3. The bile acid extraction method according to claim 1, characterized in that: In the step 3), the filtrate A is evaporated to dryness by vacuum rotary evaporation; the amount of the 15% sodium hydroxide solution is: according to the content of bile paste powder in the filtrate A, the amount of 15% sodium hydroxide solution corresponding to every 20 to 40 g of bile paste powder is 1 L.
4. The bile acid extraction method according to claim 3, characterized in that: In the step 3), the heating reflux treatment is carried out in an electric heating mantle, the heating temperature of the electric heating mantle is 180° C., and the heating process is assisted by stirring, and the stirring speed is 900 rpm.
5. The bile acid extraction method according to claim 1, characterized in that: In the step 5), the volume ratio of the primary extract to ethyl acetate is 2:1 to 3:
1.
6. The bile acid extraction method according to claim 1, characterized in that: In the step 4), the pH of the filtrate B is adjusted to 2.2-2.8 with dilute sulfuric acid.
7. The bile acid extraction method according to claim 1, characterized in that: In the step 1), the animal bile paste is dried at 100-200° C. and then crushed to obtain bile paste powder.
8. A method for detecting the type and content of a bile acid sample obtained by the bile acid extraction method of claim 1, characterized in that: The bile acid sample obtained by the bile acid extraction method of claim 1 is weighed and dissolved in chromatographic grade methanol to obtain a sample to be tested, and then the prepared sample to be tested is subjected to HPLC-UV detection; wherein the addition ratio of the bile acid product in the chromatographic grade methanol is: 25 mg of the bile acid product is added to each milliliter of chromatographic grade methanol.
9. The method for detecting the types and contents of bile acids according to claim 8, characterized in that: The HPLC-UV detection conditions are as follows: detection wavelength is 192 nm; chromatographic column: Agilent 5HC-C18 250×4.6 mm, 5 μm; mobile phase: acetonitrile:phosphoric acid (pH 2.6) = 50:50 (v / v); flow rate: 1 mL / min; column temperature: 35° C.; injection volume: 10 μL.