Preparation method of composite calcium bilirubin

Abstract

The invention relates to the technical field of medicine preparation, in particular to a preparation method of composite calcium bilirubin. The preparation method of the composite calcium bilirubin provided by the invention comprises the following steps: mixing calcium taurine, water, bilirubin and beta-glucuronidase, and carrying out catalytic reaction to obtain the composite calcium bilirubin, the mass ratio of the bilirubin to the calcium taurine is 1: 1.05; the mass of the water is 5 times of the sum of the mass of the bilirubin and the calcium taurine; the dosage ratio of the bilirubin to the beta-glucuronidase is 100g: 2ku; the temperature of the catalytic reaction is room temperature, and the time is 4 hours. According to the invention, the taurine calcium and the bilirubin are adopted as raw materials, the beta-glucuronidase is utilized to realize the preparation of the composite calcium bilirubin, and the process is optimized, so that the difference between batches of the product is smaller, and the quality is more stable. In addition, the method provided by the invention has simple steps and can be industrialized.

Description

Technical Field

[0001] The invention relates to the technical field of drug preparation, and in particular to a method for preparing composite calcium bilirubin. Background Art

[0002] Bezoar is a traditional precious medicinal material in my country, also known as "earth essence" and "ugly treasure". Bezoar comes from the dried liver and gallstones of bovine animals. It was first recorded in "Shennong's Herbal Classic" and is mainly used to treat fever, coma, epilepsy, sore throat, convulsions and other symptoms. In addition, bezoar is also an important formula for many Chinese patent medicines, but the natural bezoar source is in short supply and expensive, which makes it difficult to meet the clinical drug demand.

[0003] The existing in vitro bezoar cultivation production process uses bovine bile as raw material. After bacterial fermentation, a certain amount of calcium hydroxide, bilirubin, bile acid and other substances are added. According to the formation mechanism of natural bezoar, biochemistry, physical chemistry, fluid mechanics and other technical means are used to cultivate it in a simulated gallbladder and produce it through industrial equipment.

[0004] However, on the one hand, due to the different sources of ox bile, the content of conjugated bile acid in ox bile varies greatly, and the ox bile that can meet the requirements of in vitro cultivation of bezoar is limited, and the ox bile resources cannot be fully utilized. On the other hand, to control the difference of the finished product within a reasonable range, there are many control links in the industrial production process, and the control process is more complicated. With the continuous expansion of the application range of bezoar and the growing market demand, there is an urgent need for a new artificial synthesis method to be applied to the industrial production of imitation natural bezoar, so that the production control process is more optimized, the batch difference of the product is smaller, and the quality is more stable. Summary of the invention

[0005] In order to solve the problems existing in the prior art, the present invention provides a preparation method of composite bilirubin calcium. The composite bilirubin calcium prepared by the preparation method provided by the present invention has smaller batch differences, more stable quality, and a simple preparation method.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides a method for preparing composite calcium bilirubin, comprising the following steps:

[0008] Calcium taurine, water, bilirubin and β-glucuronidase are mixed and catalytically reacted to obtain composite bilirubin calcium;

[0009] The mass ratio of bilirubin to calcium taurine is 1:1.05;

[0010] The mass of the water is 5 times the sum of the masses of bilirubin and calcium taurine;

[0011] The dosage ratio of bilirubin to β-glucuronidase is 100g:2ku;

[0012] The catalytic reaction was carried out at room temperature for 4 hours.

[0013] Preferably, the mixing is:

[0014] Mixing calcium taurine and water for the first time to obtain a first mixed solution;

[0015] performing a second mixing of the first mixed solution and β-glucuronidase to obtain a second mixed solution;

[0016] The bilirubin is added to the second mixed solution in batches.

[0017] Preferably, after the catalytic reaction, the system obtained by the catalytic reaction is filtered, and the obtained precipitate is washed and dried in sequence.

[0018] Preferably, the drying is freeze-drying.

[0019] The present invention provides a preparation method of composite bilirubin calcium, comprising the following steps: mixing calcium taurine, water, bilirubin and β-glucuronidase, and performing a catalytic reaction to obtain composite bilirubin calcium; the mass ratio of bilirubin to calcium taurine is 1:1.05; the mass of water is 5 times the mass sum of bilirubin and calcium taurine; the dosage ratio of bilirubin to β-glucuronidase is 100g:2ku; the temperature of the catalytic reaction is room temperature, and the time is 4h. The present invention uses calcium taurine and bilirubin as raw materials and uses β-glucuronidase to realize the preparation of composite bilirubin calcium, and through process optimization, the batch-to-batch difference of the product is smaller and the quality is more stable. In addition, the method provided by the present invention is simple in steps and can be industrialized. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 , Figure 2 The mass spectrum of different molecular ion peaks of the composite calcium bilirubin prepared in Example 1;

[0021] Figure 3 It is a schematic diagram of the structure of complex bilirubin calcium obtained by computer simulation;

[0022] Figure 4 Schematic diagram of the catalytic reaction equation of Example 1. DETAILED DESCRIPTION

[0023] The invention mixes calcium taurine, water, bilirubin and beta-glucuronidase, and performs catalytic reaction to obtain composite calcium bilirubin.

[0024] As an embodiment, the water is deionized water. As an embodiment, the mass of the water is 5 times the mass of calcium taurine and bilirubin.

[0025] As an embodiment, the mass ratio of bilirubin to calcium taurine is 1:1.05. As an embodiment, the dosage ratio of calcium taurine to β-glucuronidase can be 100g:2ku.

[0026] As an embodiment, the mixing is preferably:

[0027] Mixing calcium taurine and water for the first time to obtain a first mixed solution;

[0028] performing a second mixing of the first mixed solution and β-glucuronidase to obtain a second mixed solution;

[0029] The bilirubin is added to the second mixed solution in batches.

[0030] As an embodiment, the temperature of the catalytic reaction is room temperature.

[0031] As an embodiment, after the catalytic reaction, the catalytic reaction system is filtered, the obtained precipitate is redissolved in water, and then filtered again; the filter cake is washed with water 2 to 3 times and then dried.

[0032] As an embodiment, the drying is freeze-drying.

[0033] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0034] Example 1

[0035] Weigh 100 mg of calcium taurine and place it in a reactor, add 1 mL of deionized water, stir for 30 minutes until the reactant is completely dissolved, and obtain a first mixed solution; add 4 ku of β-glucuronidase to the first mixed solution, stir evenly, weigh 100 mg of bilirubin, add it to the reactor in batches, stir evenly, and catalyze the reaction at room temperature for 4 hours (at this time, take 5 mL of the reaction system and add 3 mL of dichloromethane for extraction, and the organic phase is colorless and transparent); then, filter the catalytic reaction system through a 300-mesh filter, discard the filtrate, add water to the precipitate, stir evenly, and filter again. After washing the filter cake with water twice, a solid precipitate is obtained, and the solid precipitate is placed in a freeze dryer, freeze-dried, and powdered to obtain composite bilirubin calcium.

[0036] The present invention also analyzes the structure of the composite calcium bilirubin prepared in Example 1, wherein the mass spectrometry analysis results show that Figures 1-2From the mass spectrometry results 1 to 2, we can know that the molecular weight of complex calcium bilirubin is 1205.85; 1206.34 [M+H] + , 1165.50[M-Ca] + ; The elemental analysis results show that the elemental composition of complex bilirubin calcium is: C, 60.17; H, 5.19; Ca, 3.69; N, 8.59; O, 20.67, which is consistent with the theoretical value and its molecular formula is C 66 H 68 CaN8O 12 The elemental composition of C, 65.76; H, 5.69; Ca, 3.32; N, 9.30; O, 15.93 is basically consistent.

[0037] The molecular formula of calcium bilirubin complex is: 66 H 68 CaN8O 12 , melting point 190°C, the schematic diagram of the complex calcium bilirubin structure obtained by computer simulation is shown in Figure 3 The catalytic reaction equation of Example 1 is shown in Figure 4 .

[0038] In addition, the mass spectrometry results of natural bezoar and in vitro cultured bezoar samples showed that 1205.47 and 1206.76 [M+H] + Molecular ion peak. This result shows that the structure of the composite bilirubin calcium synthesized in the present application is basically consistent with that of the composite bilirubin calcium contained in natural bezoar and in vitro cultured bezoar.

[0039] Comparative Example 1

[0040] Weigh 100 mg of Ca(OH)2, place it in a reactor, add 1 mL of dichloromethane, and stir for 30 min until the reactant is completely dissolved to obtain a first mixed solution; weigh 100 mg of bilirubin, add it to the first mixed solution in batches, stir evenly, and react at room temperature for 4 h.

[0041] Comparative Example 2

[0042] Weigh 100 mg of Ca(OH)2, place it in a reactor, add 1 mL of dichloromethane, and stir for 30 min until the reactant is completely dissolved to obtain a first mixed solution; add 5.5 mg of tetrabutylammonium bromide (TBAB) to the first mixed solution, stir evenly, then weigh 100 mg of bilirubin, add it to the reactor in batches, stir evenly, and catalyze the reaction at room temperature for 4 hours.

[0043] Comparative Example 3

[0044] The only difference from Comparative Example 2 is that Ca(OH)2 is replaced by CaO.

[0045] Comparative Example 4

[0046] The only difference from Comparative Example 2 is that Ca(OH)2 is replaced by CaH2.

[0047] Comparative Example 5

[0048] Weigh 100 mg of calcium taurine, place it in a reactor, add 1 mL of deionized water, and stir for 30 min until the reactant is completely dissolved to obtain a first mixed solution; add 100 mg of bilirubin in batches to the first mixed solution, stir evenly, and catalyze the reaction at room temperature for 4 h.

[0049] Comparative Example 6

[0050] Weigh 100 mg of calcium taurine, 20 mg of CA (cholic acid in Chinese) and 10 mg of DCA (deoxycholic acid in Chinese), place in a reactor, add 1 mL of deionized water, stir for 30 min until the reactants are completely dissolved, and obtain a first mixed solution; add 100 mg of bilirubin in batches to the first mixed solution, stir evenly, and catalyze the reaction at room temperature for 4 h.

[0051] Comparative Example 7

[0052] Weigh 100 mg of calcium taurine, 20 mg of CA and 10 mg of DCA into a reaction kettle, add 1 mL of deionized water, and stir for 30 min until the reactants are completely dissolved to obtain a first mixed solution;

[0053] Add 4 ku of β-glucuronidase to the first mixed solution, stir evenly, weigh 100 mg of bilirubin, add it to the reactor in batches, stir evenly, and catalyze the reaction at room temperature for 4 hours (at this time, take 5 mL of the reaction system and add 3 mL of dichloromethane for extraction, and the organic phase is colorless and transparent); then, filter the catalytic reaction system through a 300-mesh filter, discard the filtrate, add water to the precipitate, stir evenly, and filter again. After washing the filter cake with water twice, a solid precipitate is obtained, and the solid precipitate is placed in a freeze dryer, freeze-dried, and powdered to obtain composite bilirubin calcium.

[0054] Comparative Example 8

[0055] Weigh 100 mg of calcium taurine and place it in a reactor, add 1 mL of deionized water, stir for 30 minutes until the reactant is completely dissolved, and obtain a first mixed solution; add 5.5 mg of TBAB to the first mixed solution, weigh 100 mg of bilirubin, add it to the reactor in batches, stir evenly, and catalyze the reaction at room temperature for 4 hours (at this time, take 5 mL of the reaction system and add 3 mL of dichloromethane for extraction, and the organic phase is colorless and transparent); then, filter the catalytic reaction system through a 300-mesh filter, discard the filtrate, add water to the precipitate, stir evenly, and filter again. After washing the filter cake with water twice, a solid precipitate is obtained, and the solid precipitate is placed in a freeze dryer, freeze-dried, and powdered to obtain composite bilirubin calcium.

[0056] The yields of the composite bilirubin calcium prepared in Example 1 of the present invention and Comparative Examples 1 to 8 are shown in Table 1.

[0057] Table 1 Yield of composite bilirubin calcium in Example 1 and Comparative Examples 1 to 8

[0058] serial number Product yield% Comparative Example 1 - Comparative Example 2 Trace Comparative Example 3 Trace Comparative Example 4 Trace Comparative Example 5 - Comparative Example 6 - Comparative Example 7 52.6 Example 1 85.7 Comparative Example 8 57.8

[0059] It can be seen from Table 1 that when calcium taurine and bilirubin are reacted with water under the catalysis of β-glucuronidase (Example 1), complex calcium bilirubin can be obtained with a yield of 85.7%.

[0060] In order to further study the effects of the amount of catalyst, the amount of reaction substrate, and the amount of reaction solvent on this scheme, further research was conducted, and the reaction substrates, β-G enzymes, solvents, and reaction times for 1 to 12 are shown in Table 2.

[0061] Table 2: Reaction substrates, β-G enzymes, solvents and reaction times for 1 to 12

[0062]

[0063] Preparation methods of 1 to 12 (reaction substrate, β-G enzyme, solvent and reaction time are shown in Table 2):

[0064] Weigh calcium taurine and place it in a reactor, add deionized water, stir for 30 minutes until the reactant is completely dissolved, and obtain a first mixed solution; add β-glucuronidase to the first mixed solution, stir evenly, weigh bilirubin, add it to the reactor in batches, stir evenly, and catalyze the reaction at room temperature (at this time, take 5mL of the reaction system and add 3mL of dichloromethane for extraction, and the organic phase is colorless and transparent); then, filter the catalytic reaction system through a 300-mesh filter, discard the filtrate, add water to the precipitate, stir evenly, and filter again. After washing the filter cake with water twice, a solid precipitate is obtained, and the solid precipitate is placed in a freeze dryer, freeze-dried, and powdered to obtain composite bilirubin calcium.

[0065] From the experimental results in Table 2, it can be seen that when the amount of bilirubin is increased to 200g and the β-glucuronidase activity is 4ku, the yield of the product complex bilirubin calcium is 98.36% (No. 4); when the reaction substrate ratio is 1:1.05, the yield of the product is slightly improved (No. 7), but when the reaction substrate ratio is further increased to 1:1.2, the yield of the product remains basically unchanged; when the amount of the reaction solvent (H2O) is reduced to 5 times the mass sum of bilirubin and calcium taurine, the yield of the product is 98.66% (No. 9); as the reaction proceeds, when the reaction is carried out for 4h, the yield of the product is 98.17%, and when the reaction time is extended to 6h, the yield of the product remains basically unchanged.

[0066] In summary: the current optimal process for the synthesis of composite bilirubin calcium (explorations 1 to 12) is that the dosage ratio of bilirubin, calcium taurate and β-glucuronidase is 100g:1.05g:2ku (the above ratio is selected considering the loss of energy consumption and the scale-up of industrial production reaction equipment), the mass of water is 5 times the sum of the masses of bilirubin and calcium taurate, and the mixture is stirred at room temperature to form a uniform suspension and reacted for 4 hours. After the reaction is completed, the reaction system is filtered and the filtrate is discarded. The solid is added with water and stirred evenly, filtered again, the filtrate is discarded, and freeze-dried to obtain the product.

[0067] According to the above optimal process, three batches of parallel tests were carried out, and the test steps are as follows:

[0068] Weigh 500g of calcium taurine and place it in a reactor, add 5125mL of deionized water, stir for 30min until the reactant is completely dissolved to obtain a first mixed solution; add 10ku of β-glucuronidase to the first mixed solution, stir evenly, weigh 525mg of bilirubin, add it to the reactor in batches, stir evenly, and catalyze the reaction at room temperature for 4h; then, filter the catalytic reaction system through a 300-mesh filter, discard the filtrate, add water to the precipitate, stir evenly, and filter again. After washing the filter cake with water twice, a solid precipitate is obtained, and the solid precipitate is placed in a freeze dryer, freeze-dried, and powdered to obtain composite bilirubin calcium. The results of three parallel tests are shown in Table 3.

[0069] Table 3 Three batches of parallel test reaction results

[0070]

[0071] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. Other embodiments can be obtained based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for preparing composite bilirubin calcium, characterized in that: The following steps are involved: Calcium taurine, water, bilirubin and β-glucuronidase are mixed and catalytically reacted to obtain composite bilirubin calcium; The mass ratio of bilirubin to calcium taurine is 1:1.05; The mass of the water is 5 times the sum of the masses of bilirubin and calcium taurine; The dosage ratio of bilirubin to β-glucuronidase is 100g:2ku; The catalytic reaction was carried out at room temperature for 4 hours.

2. The preparation method according to claim 1, characterized in that The mixture is: Mixing calcium taurine and water for the first time to obtain a first mixed solution; performing a second mixing of the first mixed solution and β-glucuronidase to obtain a second mixed solution; The bilirubin is added to the second mixed solution in batches.

3. The preparation method according to claim 1, characterized in that: After the catalytic reaction, the system obtained by the catalytic reaction is filtered, and the obtained precipitate is washed and dried in sequence.

4. The preparation method according to claim 3, characterized in that: The drying is freeze drying.

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