CN112442095B

CN112442095B - Refining method of vitamin B12 and application of obtained product

Info

Publication number: CN112442095B
Application number: CN202011400144.8A
Authority: CN
Inventors: 张红夺; 金峰; 金涛; 王雯; 金成福; 李仙洛
Original assignee: Shanghai Kedai Bio Tech Co ltd
Current assignee: Shanghai Kedai Bio Tech Co ltd
Priority date: 2020-12-01
Filing date: 2020-12-01
Publication date: 2022-07-22
Anticipated expiration: 2040-12-01
Also published as: CN112442095A

Abstract

The invention discloses a refining method of vitamin B12. The invention relates to a refining method of vitamin B12, which comprises the following steps: mixing the crude vitamin B12 with an organic solvent, cooling to 0-5 ℃, adding a reducing agent, stirring at room temperature for 1-2 hours, filtering, and drying at 40-60 ℃ in vacuum to obtain a refined pure vitamin B12 product, wherein the purity of the pure vitamin B12 product is more than 99%. The vitamin B12 is creatively used as the BNCT medicament auxiliary medicament, the enrichment effect of the BNCT medicament in tumor cells is more obvious through synergistic use, and meanwhile, the vitamin B12 has the effects of protecting cells, improving the immunity of organisms and the like.

Description

Refining method of vitamin B12 and application of obtained product

Technical Field

The invention relates to the technical field of chemistry, in particular to a refining method of vitamin B12 and application of a product obtained by the method.

Background

Vitamin B12, also called cobalamin, is a polycyclic compound containing 3-valent cobalt, and 4 reduced pyrrole rings are linked together to become 1 corrin macrocycle (similar to porphyrin), which is the only vitamin containing metal elements. The vitamin B12 is red crystal powder, has no smell and taste, is slightly soluble in water and ethanol, is most stable under the condition of pH 4.5-5.0 and weak acid, and can be decomposed in strong acid (pH <2) or alkaline solution to a certain extent when being heated. Higher animals and plants cannot produce vitamin B12, and vitamin B12 in nature is synthesized by microorganisms. Vitamin B12 is the only vitamin that needs the help of intestinal secretions (endogenous factors) to be absorbed, and is involved in the production of bone marrow red blood cells, prevention of pernicious anemia, and prevention of damage to the cerebral nerves. The physiological effects include the following: b12 is known to be a coenzyme for several mutases, such as methylaspartic mutase, which catalyzes the conversion of Glu to methyl Asp, methylmalonyl CoA mutase, which catalyzes the conversion of methylmalonyl CoA to succinyl CoA. The B12 coenzyme is also involved in the transfer of methyl and other one-carbon units. B12 mainly exists in meat, soybean and some herbs in plants also contain B12, intestinal bacteria can be synthesized, so that the B12 is not deficient in general, but B12 is a vitamin which is easy to be deficient by digestive tract disease patients and is an essential element indispensable for erythropoiesis, and if the B12 is seriously deficient, pernicious anemia can be caused. Vitamin B12 is widely present in animal food. And the form cannot be absorbed by human body. In addition, vitamin B12 is also the only vitamin containing essential minerals, and is red due to cobalt, also known as red vitamin, which is a few colored vitamins. Although vitamin B12 belongs to B group vitamin, it can be stored in liver, and its deficiency symptoms can only appear after more than half a year. The vitamin B12 is required in a small amount and is not deficient as long as the diet is normal. A small number of people with poor absorption need special attention.

Boron Neutron Capture Therapy (Boron Neutron Capture Therapy) is abbreviated as BNCT, namely is applied to thermal Neutron irradiation to target Boron gathered at tumor or cancer parts, and heavy particle alpha particles (helium nuclei) and high-energy Li nuclei are generated after the Boron captures neutrons so as to selectively kill tumor and cancer cells.

The vitamin B12 as BNCT type drug adjuvant is not reported in the literature, and is a great breakthrough if reasonable use can be realized. Meanwhile, since crude vitamin B12 has many impurities due to a complicated preparation process and cannot be directly applied, a refining method thereof needs to be studied.

Disclosure of Invention

Aiming at the defects in the prior art, the invention provides a refining method of vitamin B12, and the pure vitamin B12 obtained by the method can be used as BNCT type medicine auxiliary medicines. The effect of high concentration enrichment of boron ions in tumor cells is enhanced.

The technical scheme is as follows: the invention relates to a refining method of vitamin B12, which comprises the following steps: mixing the crude vitamin B12 product with an organic solvent, cooling to 0-5 ℃, adding a reducing agent, stirring at room temperature for 1-2 hours, filtering, and drying at 40-60 ℃ in vacuum to obtain a refined vitamin B12 pure product, wherein the purity of the pure vitamin B12 product is more than 99%.

Specifically, the organic solvent is: methanol and ethanol.

Specifically, the reducing agent is sodium cyanoborohydride.

Specifically, the addition amount of the reducing agent is 10-15% of the mass of the crude vitamin B12.

The purity of the vitamin B12 pure product obtained by the method is within the range of 99.4-99.7%.

Furthermore, the invention also provides the application of the refining method of the vitamin B12, and the pure product of the vitamin B12 obtained by the method of the invention and the o-carborane derivative have synergistic effect to increase the selectivity of the tumor cells to boron ions, thereby enhancing the effect of high-concentration enrichment of the boron ions in the tumor cells.

Specifically, the structure of the o-carborane derivative is as follows:

specifically, the mass ratio of the pure vitamin B12 to the o-carborane derivative is as follows: 1: 10-12. Preferably 1: 10.

Further, the resulting application may ultimately inhibit tumor cells, or kill tumor cells by irradiation with external atomic energy.

Has the advantages that: the vitamin B12 is creatively used as the BNCT medicament auxiliary medicament, the enrichment effect of the BNCT medicament in tumor cells is more obvious through synergistic use, and meanwhile, the vitamin B12 has the effects of protecting cells, improving the immunity of organisms and the like.

Drawings

FIG. 1 is a liquid chromatogram of vitamin B12 crude product

FIG. 2 is a liquid chromatogram of a pure vitamin B12 product obtained in example 1

FIG. 3 is a liquid chromatogram of pure vitamin B12 obtained in example 2

FIG. 4 is a liquid chromatogram of a blank experiment of vitamin B12 obtained in example 3

Detailed Description

The following examples illustrate the invention in more detail, but the scope of the invention is not limited to the examples.

Example 1

Adding 10 g of crude vitamin B12 (with the content of 96.512%) and 100 g of methanol into a 500 ml round-bottom flask, cooling to 0-5 ℃, adding 1 g of sodium cyanoborohydride for 3 times, stirring at room temperature for 1 hour, filtering, and vacuum-drying at 50 ℃ for 12 hours to obtain 8.6 g of pure vitamin B12, wherein the yield of the pure vitamin B12 is 86%; the purity is greater than 99.674%. The chromatogram is shown in FIG. 2, and the data are shown in the following table

Detector A214nm

Peak#	Ret.Time	Area	Height	Area%	Conc.	Unit
							1	3.600	44751	5051	0.180	0.180
2	3.876	3081	414	0.019	0.019
							3	4.226	15886874	1606650	99.674	99.674
4	6.080	4291	385	0.027	0.027
							5	6.863	3605	287	0.023	0.023
6	7.361	1221	95	0.008	0.008
							7	19.341	10634	434	0.067	0.067
8	22.669	10720	467	0.067	0.067
							9	22.875	3862	352	0.024	0.024
10	24.247	2392	182	0.015	0.015
							11	24.492	1081	159	0.007	0.007
Total of		15972511	1614477	100.000

Example 2

Adding 10 g of crude vitamin B12 (with the content of 96.512%) and 100 g of ethanol into a 500 ml round-bottom flask, cooling to 0-5 ℃, adding 1 g of sodium cyanoborohydride for 3 times, stirring at room temperature for 2 hours, filtering, and vacuum-drying at 50 ℃ for 12 hours to obtain a pure vitamin B12 product with the yield of 8.9 g of 89%; the purity is greater than 99.464%. The chromatogram is shown in FIG. 3, and the data are shown in the following table

Detector A214nm

Peak#	Ret.Time	Area	Height	Area%	Conc.	Unit
							1	3.600	44751	5051	0.280	0.280
2	3.876	3081	414	0.019	0.019
							3	4.226	15886874	1606650	99.464	99.464
4	6.080	4291	385	0.027	0.027
							5	6.863	3605	287	0.023	0.023
6	7.361	1221	95	0.008	0.008
							7	19.341	10634	434	0.067	0.067
8	22.669	10720	467	0.067	0.067
							9	22.875	3862	352	0.024	0.024
10	24.247	2392	182	0.015	0.015
							11	24.492	1081	159	0.007	0.007
Total of		15972511	1614477	100.000

Example 3

(blank experiment without adding sodium cyanoborohydride) 10 g of crude vitamin B12 (content 96.512%) and 100 g of ethanol are added into a 500 ml round-bottom flask, the temperature is reduced to 0-5 ℃, the mixture is stirred for 2 hours at room temperature, filtered and dried in vacuum at 50 ℃ for 12 hours to obtain a pure vitamin B12 product with the yield of 9.9 g and 99 percent; the purity was 96.912%. The chromatogram is shown in FIG. 4, and the data are shown in the following table

Detector A214nm

Peak#	Ret.Time	Area	Height	Area%	Conc.	Unit
							1	2.978	71287	5899	0.313	0.313
2	3.607	202610	22760	1.431	1.431
							3	4.228	13668896	1410020	96.912	96.912
4	7.237	5171	332	0.037	0.037
							5	16.184	148841	2561	1.051	1.051
6	19.283	7422	285	0.052	0.052
							7	22.698	43870	1228	0.310	0.310
8	24.200	14838	452	0.105	0.105
							Total of		14162934	1443537	100.000

Example 4

The test result of the in vitro cell activity of the o-carborane derivative and the vitamin B12 is shown.

The structure of the o-carborane derivative is as follows:

the determination of the related performance indexes of the compounds is a common method in the field.

1. Determination of IC50

At different sample concentrations, after 48h of administration, the growth inhibition of the compound on colon tumor cells CT26 was examined by the general MTT method, and finally IC50 was calculated so that the drug concentration required to inhibit cell survival was determined from half the lethal dose, and the results represent the average ±). .

MTT is tetramethyl azoazolium salt, which is a yellow dye capable of accepting hydrogen ions. Succinate dehydrogenase (succinate dehydrogenase) and cytochrome c (cytochromec) in mitochondria of living cells can crystallize blue-purple formazan (formazan) to precipitate MTT, and dead cells do not have this function. After the resulting formazan crystals were dissolved in dimethyl sulfoxide (DMSO), their absorbance at 490nM or 570nM was measured by an enzyme linked immunosorbent assay to monitor formazan production. Since formazan crystals are produced in an amount proportional to the number of living cells, the effect of the drug on the cell viability can be detected. The method is widely used for screening and testing the antitumor drugs due to the characteristics of high sensitivity, economy and the like.

The specific experimental method is as follows: various cells are prepared into 5 multiplied by 10 after centrifugation³Adding 100 mu L of cell suspension into each well of a 96-well plate, sucking out original culture solution after culturing for 24h conventionally, adding 200 mu L of prepared compound I, II or Boron Phenylalanine (BPA) samples, 5 concentrations of compound I and vitamin B12 and compound II and vitamin B12, wherein the final concentration of each sample is 0.137 mu M, 0.046 mu M, 1.235 mu M, 0.412 mu M and 3.704 mu M, each concentration is 4 multiple wells, wells around the 96-well plate are sealed by PBS, a negative control group and a blank control group are reserved, 20 mu L of MTT solution is added into each well after the compound acts for 72h, culturing is continued for 4h, carefully sucking away the culture medium in the wells, adding 150 mu L of DMSO, shaking for 10min, measuring the OD value of each well at 490nM of a microplate reader, and calculating the inhibition rate of the samples at different concentrations according to the following formula: the inhibition ratio (control well OD value-dosing well OD value)/control well OD value × 100%. Finally, the IC50 value of the sample was calculated using the relevant software.

The operation method comprises the following steps:

(1) colon tumor CT26 cell (5X 10)⁵Individual cells) were cultured aerobically and anaerobically in 96-well plates. Under the aerobic culture condition of 36.5-37 ℃, providing sterile deionized oxygen at the oxygen supply speed of 0.3 cm/minute²And sufficient administration time was 12 hours. The anaerobic culture condition is 36.5-37 ℃, the condition of a sterile anaerobic sealed drying oven is adopted, and the sufficient time is 12 hours.

(2) The o-carborane compounds I, II, compound I with vitamin B12, compound II with vitamin B12 and Borophenylalanine (BPA) were treated sequentially (3mM to 1mM) for 72 hours.

(3) Sequentially adding 3- (4, 5-dimethylthiazole-2) -2, 5-diphenyl tetrazolium bromide salt solution, and culturing for 4 hours.

(4) Shaking was performed after dilution with 150. mu.L of dimethyl sulfoxide.

(5) The protein content is determined by an enzyme linked immunosorbent assay device at 595 nm.

2. Boron absorption

P-35 cells (5X 10)³Cells) were treated in the presence of compounds I, II and Borophenylalanine (BPA) (uniform boron concentration of 10.8ppm) for 3 hours. Washing with 3 drops of phosphate buffer solution for three times;

(4) perchloric/peroxy acids were analyzed.

(5) Aerobic and anaerobic cultures were performed at 70 ℃. Under the aerobic culture condition of 36.5-37 ℃, sterile deionized oxygen is provided, and the oxygen supply speed is per minute/0.3 cm²And sufficient time was given at 12 hours. The anaerobic culture condition is 36.5-37 ℃, the condition of a sterile anaerobic sealed drying oven is adopted, and the sufficient time is 12 hours.

(6) Plasma emission spectrometry testing.

The operation method comprises the following steps:

(1) colon tumor CT26 cell (5X 10)⁵Individual cells) were cultured aerobically and anaerobically in p-35 cell dishes. Under the aerobic culture condition of 36.5-37 ℃, sterile deionized oxygen is provided, and the oxygen supply speed is per minute/0.3 cm²And sufficient time was given at 12 hours. The anaerobic culture condition is 36.5-37 ℃, and the time is fully 12 hours under the condition of an aseptic anaerobic sealed drying oven.

(2) The o-carborane derivatives I and II, the compound I and the vitamin B12, and the compound II and the vitamin B12 are treated for 3 hours in sequence (the boron concentration is 10.8 ppm).

(3) And washed three more times with 3 drops of phosphate buffered saline.

(4) The activity assay was then performed with perchloric/peroxyacids.

(5) Aerobic and anaerobic cultures were performed at 70 ℃. Under the aerobic culture condition of 36.5-37 ℃, sterile deionized oxygen is provided, and the oxygen supply speed is per minute/0.3 cm²And sufficient time was given at 12 hours. Anaerobic cultureThe conditions are 36.5-37 ℃, and the time is 12 hours under the conditions of an aseptic oxygen-free sealed drying oven.

(6) Plasma emission spectrometry testing.

The following table: IC50 and boron accumulation concentration Table for various Compounds

As a result, the IC50 activities of BPA were 6.43X 10, respectively^-5(+/-0.22) and the aggregation concentration of boron is 0.083 +/-0.012 ppm, while the designed compounds I and II reach the high aggregation concentration which is 8-10 times that of BPA, and the IC50 activity of the compound I is 1.10 multiplied by 10 respectively^-5(± 0.22); the boron concentration is 0.682 +/-0.42 ppm respectively; the IC50 activities of the compound II were 1.89X 10^-5(± 0.73); the boron concentration was 0.754. + -. 0.43ppm, respectively, and the aggregation effect of the original design was sufficiently obtained. However, the IC50 activity of the compound I after addition of vitamin B12 was 1.21X 10^-5(± 0.13); the boron concentration is 0.814 plus or minus 0.21ppm respectively; the IC50 activity of compound II was 1.94X 10^-5(± 0.58); the boron concentration is 0.912 +/-0.32 ppm, the inhibition activity of tumor cells and tumor cells becomes more prominent, and the inhibition effect is increased by 1-3 times. The self structure of the borane medicament has certain inhibitory activity through in vitro experiments, the concentration of boron in cells is increased by additionally introducing borane, and the novel structure ensures that boron ions are easier to gather in tumor cells to achieve a high boron gathering effect. After high boron is gathered in the tumor cells, the tumor cells are selectively killed by irradiation of external atomic energy to generate heavy-particle alpha particles (helium nuclei) and high-energy Li nuclei.

The above description is only a preferred embodiment of the present application and is not intended to limit the present application.

Claims

1. A refining method of vitamin B12 is characterized by mixing a vitamin B12 crude product with an organic solvent, cooling to 0-5 ℃, adding a reducing agent, stirring at room temperature for 1-2 hours, filtering, and drying at 40-60 ℃ in vacuum to obtain a refined vitamin B12 pure product, wherein the purity of the vitamin B12 pure product is more than 99%; the reducing agent is sodium cyanoborohydride; the crude purity was 96.512%.

2. The method for refining vitamin B12, according to claim 1, wherein the organic solvent is: methanol and ethanol.

3. The method for refining vitamin B12 according to claim 1, wherein the addition amount of the reducing agent is 10-15% of the weight of crude vitamin B12.

4. The use of the refining method of vitamin B12 according to claim 1, wherein the use comprises preparing pure vitamin B12 by the refining method of claim 1, and using the pure vitamin B12 and the orthocarborane derivative as BNCT type auxiliary drugs in a synergistic effect;

the structure of the o-carborane derivative is as follows:

5. the use of the refining method of vitamin B12 according to claim 4, wherein the mass ratio of the pure vitamin B12 to the o-carborane derivative is as follows: 1: 10-12.