A kind of bear bile conversion substrate and preparation method thereof

Abstract

The present invention belongs to the technical field of traditional Chinese medicine, and discloses a transformed bear bile powder substrate and a preparation method thereof. The preparation method comprises the following steps: (1) enzymolyzing avian bile with protease to obtain a crude paste 1; (2) alcohol precipitation and extraction of the crude paste 1 to obtain a crude paste 2; (3) using octadecylsilane bonded silica gel as a filler of a chromatography column, using acetonitrile aqueous solution as a mobile phase, eluting the crude paste 2 with a column chromatography technique, concentrating and drying the target solution, and obtaining a transformed bear bile powder substrate. The preparation method can effectively remove taurocholic acid while retaining taurochenodeoxycholic acid and other nutrients, thereby obtaining a high-quality transformed bear bile powder substrate.

Description

Technical Field

[0001] The invention belongs to the technical field of traditional Chinese medicines, and in particular relates to a transformed bear bile powder substrate and a preparation method thereof. Background Art

[0002] Bear bile powder is a precious Chinese medicinal material obtained from the dried gallbladder bile of black bears or brown bears. It has a history of more than 2,000 years of medicinal use, and a large number of prescriptions contain bear bile ingredients. Bear bile powder is known to have antispasmodic, anticonvulsant, anti-inflammatory and cholelith-dissolving effects, and is a good medicine for protecting the liver and gallbladder. Various modern clinical practice applications have also shown that bear bile can treat a variety of liver and gallbladder diseases. Due to the limited resources of wild bears, the artificial breeding "drainage and bile extraction" method is now commonly used to produce bear bile powder (i.e. natural bear bile powder). At present, my country's annual production of natural bear bile powder is only about 30 tons, which can no longer meet people's growing health needs.

[0003] In order to solve the problem of shortage of natural bear bile powder, other animal bile is usually used as raw material, and transformed bear bile powder with similar overall composition to natural bear bile powder is obtained through impurity removal and chemical / biological transformation. It is known that the content of taurocholic acid (TCA) in natural bear bile powder is low, and TCA is highly toxic and has an inhibitory effect on the heart and central nervous system. Therefore, TCA needs to be impurity-removed in the process of preparing the transformed bear bile powder substrate. However, the existing impurity removal and purification methods can only remove some non-bile acid impurities, and the physical and chemical properties of taurocholic acid (TCA) and taurochenodeoxycholic acid (TCDCA) are very similar, making it difficult to separate the two. Taurochenodeoxycholic acid (TCDCA) can be partially converted into tauroursodeoxycholic acid (TUDCA), the key component in natural bear bile powder, by chemical or biological methods.

[0004] Therefore, the art hopes to develop a method for removing taurocholic acid (TCA) while retaining taurochenodeoxycholic acid (TCDCA) and other nutrients, so as to prepare high-quality converted bear bile powder substrate and provide high-quality raw materials for preparing converted bear bile powder products. Summary of the invention

[0005] The present invention aims to solve at least one of the technical problems existing in the above-mentioned prior art. To this end, the present invention provides a transformed bear bile powder substrate and a preparation method thereof, which can effectively remove taurocholic acid (TCA) while retaining taurochenodeoxycholic acid (TCDCA) and other nutrients, thereby preparing a high-quality transformed bear bile powder substrate.

[0006] The present invention provides a method for preparing a transformed bear bile powder substrate, comprising the following steps:

[0007] (1) enzymolyzing fowl bile with protease to obtain a crude paste 1;

[0008] (2) subjecting the crude paste 1 to alcohol precipitation and extraction to obtain a crude paste 2;

[0009] (3) Using octadecylsilane bonded silica gel as the filler of the chromatography column and acetonitrile aqueous solution as the mobile phase, the crude paste 2 is eluted by column chromatography technology, and the target solution is concentrated and dried to obtain the converted bear bile substrate.

[0010] The present invention enzymatically hydrolyzes the protein in the bile to release the fat in the lipoprotein complex, which is convenient for subsequent extraction; and the alcohol extraction can remove impurities such as proteases, macromolecular peptides, and inorganic salts insoluble in alcohol. Octadecylsilane bonded silica gel is selected as a filler, and acetonitrile aqueous solution is used as a mobile phase for elution, which can quickly and effectively separate TCA in the raw material, realize the removal of TCA, and retain TCDCA and other nutrients in the bile, so that the composition of the raw material is more similar to that of natural bear bile powder, which is more conducive to the subsequent preparation of high-quality converted bear bile powder.

[0011] Preferably, the protease in step (1) is papain.

[0012] Preferably, the enzymatic hydrolysis temperature in step (1) is 50-60°C, pH=6.0-7.0, and the enzyme amount is 0.01-0.02%. The enzyme amount means the mass fraction of protease in avian bile.

[0013] Preferably, ethanol is used for the alcohol precipitation in step (2).

[0014] Preferably, the temperature of the alcohol precipitation in step (2) is 5-15°C.

[0015] Preferably, the alcohol precipitation time in step (2) is ≥4h.

[0016] Preferably, in step (2), n-hexane is used for the extraction, and the number of extractions is ≥ 3 times.

[0017] Preferably, the sample loading amount of the chromatography column in step (3) is 12-18%. The sample loading amount is the mass ratio of the crude paste 2 to the filler.

[0018] Preferably, the elution step in step (3) is:

[0019] S1, adding acetonitrile to the crude paste 2 and loading it into a chromatography column to obtain a flow-through liquid;

[0020] S2, then eluting with 23-25% acetonitrile aqueous solution to obtain eluate 1;

[0021] S3, eluting with 80-90% acetonitrile aqueous solution to obtain eluate 2;

[0022] S4. Combine the flow-through solution and the eluent 2 to obtain the target solution.

[0023] The present invention also provides a transformed bear bile powder substrate, which is prepared by the above preparation method. The mass content of TCDCA in the transformed bear bile powder substrate is ≥60%, and the mass content of TCA is ≤1%.

[0024] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0025] The preparation method of the present invention can not only effectively remove protein and fat impurities, but also retain TCDCA and other nutrients in bile to the greatest extent while removing TCA, making its composition more similar to natural bear bile powder, and more conducive to the subsequent preparation of high-quality converted bear bile powder. The method is fast and convenient, saves time, and is easy to operate; and the mobile phase composition is simple, containing only one chemical reagent, acetonitrile, and can be repeatedly recycled. DETAILED DESCRIPTION

[0026] In order to make those skilled in the art more clearly understand the technical solution of the present invention, the following examples are listed for illustration. It should be pointed out that the following examples are only preferred embodiments of the present invention and do not constitute a limitation on the protection scope of the present invention. Any modification, substitution, and combination made without violating the spirit and principle of the present invention are included in the protection scope of the present invention.

[0027] Unless otherwise specified, the raw materials, reagents or devices used in the following examples can be obtained from conventional commercial sources or by existing known methods.

[0028] 1. Crude method for transforming bear bile powder

[0029] Example 1 provides a crude method for converting a bear bile powder substrate, comprising the following steps:

[0030] (1) 1 kg of frozen poultry bile was taken and thawed at room temperature, the bile was broken and the bile was obtained, and the bile was concentrated under reduced pressure to obtain about 200 g of bile (original bile);

[0031] (2) adding 0.015% papain to the obtained bile, adjusting the pH to 6.5, the temperature to 55° C., and performing enzymolysis for 2 h to obtain a crude paste 1;

[0032] (3) Add 95% ethanol to the crude paste 1 until the final ethanol concentration is 75%, and precipitate at 10°C for more than 4 hours; filter the precipitate through a filter with a pore size of 0.45 μm, collect the filtrate, concentrate it under reduced pressure until there is no alcohol taste, add an equal volume of n-hexane, extract three times, collect the aqueous phase, and concentrate it under reduced pressure to obtain a crude paste 2.

[0033] The crude method for converting the bear bile powder substrate in Examples 2-13 and Comparative Examples 1-6 is basically the same as that in Example 1, except for the selection of various parameters. The specific parameter selection is shown in Table 1.

[0034] Table 1 Parameter selection of Examples 2-13 and Comparative Examples 1-6

[0035]

[0036]

[0037] The crude paste 2 and original bile prepared in Examples 1-13 and Comparative Examples 1-6 were tested for lipid, TCDCA and TCA contents. The test results are shown in Table 2.

[0038] Table 2 Content determination results of Examples 1-13, Comparative Examples 1-6 and original bile

[0039]

[0040]

[0041] As shown in Table 2, enzymatic protein hydrolysis under the conditions of pH=6.0-7.0, temperature 50-60℃, and enzyme amount 0.01-0.02% can hydrolyze lipoprotein complexes, and the subsequent fat removal effect is equivalent, which is conducive to subsequent column chromatography. When the enzyme amount is too low, the enzymatic hydrolysis effect is not good, and the subsequent fat removal effect is poor, while the fat removal effect is equivalent to 0.01% when the enzyme amount is increased to 0.02%, and there is no significant improvement. Moreover, under the same enzyme amount conditions, it is difficult to completely enzymatically hydrolyze for 0.5h, and the effects of enzymatic hydrolysis for 1h and 4h are similar. When the alcohol precipitation temperature is too high and the time is too short, the precipitate and the clear liquid are suspended and difficult to separate; when the alcohol precipitation temperature is 5-15℃ and the alcohol precipitation time is 4-8h, the alcohol precipitation effect is equivalent. The fat removal effect of extraction 3 times and 4 times is equivalent, but the fat removal is not complete when extraction is 2 times, and there is still a lot of fat residue, so the number of extractions should not be less than 3 times.

[0042] 2. Refining method of transforming bear bile powder substrate

[0043] Example 14 provides a method for refining a transformed bear bile powder substrate, comprising the following steps:

[0044] S1. Using the crude paste 2 prepared in Example 1 as a raw material, acetonitrile was added to the crude paste 2 to a final concentration of 10% acetonitrile, and the crude paste 2 was loaded onto a C18 column (the filler was octadecylsilane bonded silica gel, the column length was 13.5 cm, and the inner diameter was 1.5 cm), the loading amount was 15% (i.e., the mass ratio of the crude paste 2 to the filler was 15%), the flow rate was 3 mL / min, and the flow-through liquid was collected;

[0045] S2, using 23% acetonitrile aqueous solution (i.e., the volume ratio of acetonitrile to water is 23:77) as the mobile phase, eluting for 1 column volume, and collecting eluent 1 (after testing, eluent 1 contains TCA and substantially does not contain TCDCA);

[0046] S3, using 80% acetonitrile aqueous solution (i.e., the volume ratio of acetonitrile to water is 80:20) as the mobile phase, eluting for 2 column volumes, and collecting the eluate 2;

[0047] S4. The flow-through liquid and the eluate 2 are combined, concentrated under reduced pressure, and dried in vacuo to obtain a transformed bear bile powder substrate.

[0048] The purification method of the transformed bear bile powder substrate in Examples 15-17 and Comparative Examples 7-10 is basically the same as that in Example 14, except for the selection of various parameters. The specific parameter selection is shown in Table 3.

[0049] Table 3 Parameter selection of Examples 15-17 and Comparative Examples 7-10

[0050]

[0051]

[0052] Comparative Example 11

[0053] Comparative Example 11 provides a method for refining a transformed bear bile powder substrate, comprising the following steps:

[0054] The crude paste 2 prepared in Example 1 was used as a raw material and loaded onto a silica gel G column (filler: silica gel G, column length: 12 cm, inner diameter: 1.2 cm) with a loading amount of 15%. Chloroform, isopropanol and ethanol in a volume ratio of 3:1:2 were used as mobile phases for elution at a flow rate of 0.4 mL / min. The liquid with an absorption value at 460 nm was collected and concentrated and dried into a powder.

[0055] Comparative Example 12

[0056] The crude paste 2 prepared in Example 1 was used as a raw material and loaded onto a silica gel G column (filler: silica gel G, column length: 3.5 cm, inner diameter: 2.2 cm), with a loading amount of 15% and a flow rate of 3 mL / min. Ethyl acetate, methanol and formic acid in a volume ratio of 80:20:1 were first used as mobile phases for elution, and 20 mL of the eluate was collected as 12-1; ethyl acetate, methanol and formic acid in a volume ratio of 75:25:1 were then used as mobile phases for elution, and 40 mL of the eluate was collected as 12-2. The eluate 12-1 and the eluate 12-2 were concentrated and dried into powders respectively.

[0057] The ingredients of the products obtained in Examples 14-17, Comparative Examples 7-12 and natural bear bile powder were tested, and the test results are shown in Table 4 (the remaining ingredients not listed are mainly inorganic salts and water).

[0058] Table 4 Content determination results of Examples 14-17 and Comparative Examples 7-12

[0059]

[0060] As shown in Table 4, when the sample load is 12-18%, TCDCA and TCA can be separated, but as the sample load increases, the separation effect becomes worse, and the recovery rate of TCDCA decreases. When the sample load is 25%, the two are difficult to separate, and a small sample load will increase the number of sample loads. When step S3 uses 60% acetonitrile aqueous solution for elution, TCA and TCDCA can be separated and TCDCA can be recovered, but other non-bile components are combined on the column and cannot be completely eluted, which not only affects the content of non-bile components in the final product but also affects continuous purification and column efficiency. When 90% acetonitrile aqueous solution is used for elution, the purity and recovery rate are equivalent to 80%, indicating that 80% acetonitrile has been completely eluted; step S2 uses 20% acetonitrile aqueous solution for elution, and the low acetonitrile concentration cannot effectively separate and remove TCA, resulting in a higher TCA content in the final product; when 28% acetonitrile aqueous solution is used for elution, the acetonitrile concentration is high, and some TCDCA will be removed together with TCA, resulting in a low recovery rate of TCDCA. When the methods of Comparative Examples 11 and 12 were used for purification, although the content of TCDCA in the collected liquid was high, TCA was not effectively separated, and the content of other non-bile components was low, which was only suitable for preparing pure compounds and was not suitable for preparing substrates for converting bear bile powder.

[0061] The embodiments of the present application are described in detail above, but the present application is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present application. In addition, the embodiments of the present application and the features in the embodiments can be combined with each other without conflict.

Claims

1. A method for preparing a transformed bear bile powder substrate, characterized in that: The following steps are involved: (1) enzymolyzing fowl bile with protease to obtain a crude paste 1; (2) subjecting the crude paste 1 to alcohol precipitation and extraction to obtain a crude paste 2; (3) using octadecylsilane bonded silica gel as a filler for a chromatography column and acetonitrile aqueous solution as a mobile phase, eluting the crude paste 2 by column chromatography, concentrating and drying the target solution to obtain a converted bear bile powder substrate; the enzymatic hydrolysis time in step (1) is 1, 2 or 4 hours; The enzymatic hydrolysis temperature in step (1) is 50-60°C, pH=6.0-7.0, and the enzyme amount is 0.01-0.02%; The protease in step (1) is papain; In step (2), ethanol is used for the alcohol precipitation; The temperature of the alcohol precipitation in step (2) is 5-15°C, and the alcohol precipitation time is ≥4h; In step (2), n-hexane is used for the extraction, and the number of extractions is ≥ 3 times; The sample loading amount of the chromatography column in step (3) is 12-18%; The elution step in step (3) is: S1, adding acetonitrile to the crude paste 2 and loading it into a chromatography column to obtain a flow-through liquid; S2, then eluting with 23-25% acetonitrile aqueous solution to obtain eluate 1; S3, eluting with 80-90% acetonitrile aqueous solution to obtain eluate 2; S4. Combine the flow-through solution and the eluent 2 to obtain the target solution.

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