JPH036153B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention provides a cobaltichlorophyllin complex compound 1
This invention relates to a method for producing a novel thiamine cobaltichlorophyllin complex compound in which one molecule of thiol-type thiamine is coordinated. The thiamine cobaltichlorophyllin complex compound obtained by the present invention is a substance useful as a medicine, and is expected to have many physiological effects in addition to having medical effects on gastrointestinal ulcers, for example. Conventional thiamin cobaltichlorophyllin complex compounds are disclosed in Japanese Patent Publication No. 1986-2005 and Japanese Patent Publication No. 1972-2005.
No. 28687 and the techniques described in each publication are known.
That is, in Japanese Patent Publication No. 50-2005, a cobalt salt or an iron salt is added to a salt-saturated acetic acid solution of chlorophyllin, and a chlorophyllin-cobalt complex compound is precipitated by adjusting the pH (PH 5 to 7), and then an alkaline solution of thiamine is added. and then set the pH to 4 again.
The product was obtained by adjusting the temperature to ~6. The method disclosed in Japanese Patent Publication No. 51-28687 is based on carrying out the reaction with the pH level at the time of thiamine addition being made more alkaline. However, in any case, 1 equivalent of thiamine was coordinated to the cobaltichlorophyllin complex compound, that is, 1
A thiamine cobaltichlorophyllin complex compound having a thiol-type thiamine molecule as one ligand has not been confirmed so far, and no method for selectively producing it is known. Therefore, according to the present invention, there is provided a novel thiamine cobaltichlorophyllin complex compound in which one molecule of thiol-type thiamine is coordinated to one molecule of the cobaltichlorophyllin complex compound. The thiamine cobaltichlorophyllin complex compound of the present invention is produced by the following steps. The first step is to complex chlorophyllin in a water-free acetic acid solution, preferably an alcoholic, for example, methanolic acetic acid solution, with a divalent cobalt salt such as cobalt acetate in the presence of oxygen. A chlorophyllin complex compound is prepared, and this product is distilled to remove the dissolution, particularly under reduced pressure, to obtain a highly pure crystalline cobaltichlorophyllin complex compound. In the second step, the high-purity cobaltichlorophyllin complex compound obtained in the first step is dissolved in methanol, and thiamine, which has been made into an alkaline methanol solution in advance, is mixed and reacted to form a thiamine cobaltichlorophyllin complex compound, and then the reaction takes place. Remove as much methanol as possible from the mixture. Instead of removing the solvent by distillation from the cobaltichlorophyllin complex compound-containing mixture obtained by the complexation reaction in the first step, the reaction is carried out by adding a solvent in which the reaction product is insolubilized to the reaction mixture. The product may be precipitated and the precipitate collected. The crude thiamine cobalt chlorophyllin complex compound obtained as described above is finally purified by dissolving it in, for example, hydrous alcohol and subjecting it to column chromatography and gel permeation chromatography of hydrophobic porous polymers. Attach. The present invention will be explained in more detail below. The chlorophyllin used as a raw material in the present invention is derived from plant chloroplasts, such as higher plant leaves such as mulberry leaves, alpha alpha leaves, timothy grass leaves, algae such as chlorella and scenedesmus, or plant chloroplasts. It can be obtained from highly concentrated grains such as silkworm feces through solvent extraction, saponification and acidification, and purification operations such as solvent fractional extraction, fractional crystallization and chromatographic separation. This product mainly consists of chlorin e ₆ , but a portion of chlorin p ₆ , purpurin 7, etc. may be mixed in without any problem. When converting the raw material chlorophyllin into an acetic acid solution, it is preferable to coexist with alcohol, and methanol, ethanol, isopropanol, etc. can be used, but methanol is preferred. In this case, the acetic acid content is between 5 and 80%, preferably between 10 and 80%.
It is 30%. As the divalent cobalt salt, cobalt chloride, cobalt sulfate, cobalt acetate, etc. can be used, but cobalt acetate is preferable. This divalent cobalt salt is subjected to the reaction, for example, as a methanol solution. The reaction temperature ranges from room temperature to 50-60°C, preferably 40-50°C. At this time, it is important to avoid the presence of water as much as possible in terms of product quality and yield. In the present invention, the above reaction is carried out in the presence of oxygen. As a result of various studies, the inventor discovered that the addition of hydrogen peroxide minimizes the effect on the unstable chlorin ring, and as a result, a highly pure product can be obtained. That is, by adding hydrogen peroxide in advance to the cobalt salt solution to be introduced into the reaction system, a reaction that normally requires several hours can be completed within one hour. It is also possible to carry out the reaction with air or oxygen blowing.
After the completion of the reaction, a highly purified cobaltichlorophyllin complex compound was obtained by simply distilling the reaction solution or by adding thereto a solvent that insolubilizes the reaction product and separating the resulting precipitate. Examples of the solvent in which the reaction product is insolubilized include water, ether, isopropyl ether, and chloroform. Another method is to add an alkali to adjust the pH and cause precipitation and crystallization, but this is not so preferred as it causes not only industrial problems but also affects quality. The cobaltichlorophyllin complex compound thus obtained is of high purity and can be provided as it is to the next step. A methanol solution of a cobaltichlorophyllin complex compound and a hydrochloride, nitrate, or free base of thiamine (vitamin B ₁ ) is adjusted to pH 7 to 11, and the raw material thiamine is mixed and reacted in the thiol form. Less than 2 moles of thiamine should be used per mole of cobaltichlorophyllin complex compound. Preferably, substantially equimolar amounts are used. At this time, an ammonia buffer can be used to adjust the pH if necessary. The reaction is desirably carried out at room temperature to 50° C. while blocking oxygen, preferably under light-shielding conditions. The reaction solution is concentrated under reduced pressure to obtain a crude thiamine cobaltichlorophyllin complex compound. As a purification method, crude thiamine cobalt chlorophyllinacetic acid compound may be dissolved in 30 to 70% aqueous alcohol to adjust the pH to 3 to 7, preferably 4 to 6, and column chromatographic fractionation of the hydrophobic porous polymer may be performed. can. The larger the unit surface area of the resin is, the more suitable the porous polymer is.
It is 100 m ² /g or more, preferably 400 m ² /g or more. The pore diameter of the porous synthetic resin is usually preferably 5.0 Å or more, and the larger the average pore diameter, the better the result. Examples of these porous polymers include styrene-divinylbenzene copolymers [Amberlite XAD-2 (manufactured by Rohm and Haas), Diaion HP (manufactured by Mitsubishi Chemical Corporation)]. By passing the purified thiamine cobaltichlorophyllin complex compound fraction through a gel permeation chromatography column, a thiamine cobaltichlorophyllin complex compound of constant quality is obtained. As a gel perchromatography carrier, Sephadex LH-
Dextran gel such as 20 or Toyo Pearl HW
Synthetic gelling agents such as -40 are preferred. Suitable developing solvents include methanol, ethanol, acetone, and the like, with methanol being preferred. Confirmation of the fractionation of the thiamine cobaltichlorophyllin complex compound is carried out by visible absorption spectroscopy and thin layer chromatography. From the thus obtained thiamine cobaltichlorophyllin complex compound fraction, a thiamine cobaltichlorophyllin complex compound in the form of a black-green glossy fine powder is obtained by concentration to dryness, spray drying, or the like. This product has extremely high purity and can be provided as a pharmaceutical raw material as is. The thiamine cobaltichlorophyllin complex compound thus obtained has the following estimated structural formula. or The elemental analysis results for C ₄₆ H ₅₅ CoN ₈ O ₁₀ S (molecular weight 971.08) are as follows, and this actual value is 1 molecule of chlorophyllin (chlorin e ₆ ), 1 atom of cobalt and 1 molecule of vitamin B ₁ (thiol). This agrees well with the theoretical value, which assumes that one molecule (type thiamine) is bound. C (%) H (%) N (%) Theoretical value: 56.89 5.72 11.54 Actual value: 57.63 5.33 11.76 O (%) S (%) Co (%) Theoretical value: 6.48 3.30 6.07 Actual value: 17.07 3.23 5.99 Here The obtained thiamine cobaltichlorophyllin complex compound further has the following physical properties. 1. The thiamine cobaltichlorophyllin complex compound of the present invention is a dark green amorphous or shiny fine lump with a weak specific odor. This substance is extremely soluble in methanol and ethanol, slightly soluble in water, and almost insoluble in ether, chloroform, benzene, and ethyl acetate. 2. Accurately weigh approximately 100 mg of the thiamine cobaltichlorophyllin complex compound of the present invention, and dissolve in methanol to make exactly 50 ml. 1ml of the liquid
and add methanol to make exactly 50ml. Furthermore, take 2 ml of this liquid and add 3 ml of methanol.
ml to make the sample solution. When the visible absorption spectrum is measured using the Japanese Pharmacopoeia General Test Method "Absorbance Measurement Method" using methanol as the control solution for the sample solution, it has maximum absorption at wavelengths of 428 to 432 nm and 640 to 667 nm, and the absorption strength is at 428 to 432 nm. ＞640～
The order is 667nm. 3 Analysis by infrared absorption spectrum Thiamine cobaltichlorophyllin compound according to the present invention, cobaltichlorophyllin (cobaltichlorophyllin e ₆ ), and chlorophyllin (chlorin e ₆ )
The infrared absorption spectrum (KBr tablet method) was measured. The infrared spectra are first and second, respectively.
It was as shown in Figures 2 and 3. The absorption of 3400 cm ^- ' derived from NH and water of the raw material chlorophyllin (Figure 3) shifts to 3430 cm - ' in the cobaltichlorophyllin complex compound, and at the same time, the absorption of 3400 cm ^- ' derived from NH and water of the cobaltichlorophyllin complex compound (Figure 2) and In thiamincobaltichlorophyllin, absorption of 1080 cm ^- '(νCo-N) occurs due to cobaltium complexation (first
figure). From this, it is inferred that the cobalt metal in the thiamine-cobaltichlorophyllin and cobaltichlorophyllin complex compounds has a complex bond with the four N atoms of the pyrrole nucleus of the raw material chlorophyllin. In addition, 1625 cm ^- ′(NH) of the complex compound of the present invention
and thiamine (bimin B ₁ ) at 1040 cm ^- '(C-O)
_Absorption thought to be _derived from
) is not present in cobaltichlorophyllin or chlorophyllin. From the above results, it is estimated that cobalt in the complex compound of the present invention is coordinately bonded to four N atoms of the pyrrole nucleus of chlorophyllin, and that thiamine is bound in the complex compound of the present invention. Ta. 4 Analysis by visible region absorption spectrum The thiamin cobaltichlorophyllin compound of the present invention, its precursor cobaltichlorophyllin complex compound, and the raw material chlorophyllin were analyzed using methanol as a solvent and a layer length of 10 mm at 340~
Visible absorption measurements at 700 nm were performed. The visible absorption spectrum is shown in Figure 4, and the results are summarized in the table below.

[Table] In the visible absorption spectra of the complex compound of the present invention and the cobaltichlorophyllin complex compound, the characteristic absorption band of 500 to 530 nm of chlorophyllin almost disappears, and the red band shifts to a slightly shorter wavelength region, and The Soret's band has moved to longer wavelengths, and the ε value has decreased. These changes are considered to indicate a complex bond between chlorophyllin and metal cobalt in the complex compound and cobaltichlorophyllin complex compound of the present invention. The toxicity of the thiamine cobaltichlorophyllin compound obtained by the present invention is as follows.

[Table] The pharmacological effects of thiamine cobaltichlorophyllin on experimental gastric ulcers induced by the stress method and the restraint cortisone method are shown below. The animal is a male Wistar rat (body weight
200-300g) and 10 animals in each group. All drugs to be administered were dissolved in physiological saline. The dose was 100 mg/Kg body weight per day, and 1 ml of physiological saline was administered to the control saline group. In an acute ulcer prevention test using the stress method, the drug was orally administered continuously for 7 days before stress loading. In addition, in a treatment trial for chronic ulcers using the restraint cortisone method, 7 days after the restraint was removed.
It was orally administered continuously for 1 day. Experimental ulcer generation by stress method is described in "Chem.Pharm.Bull." Volume 12, No. 465
(1964), and the generation of experimental ulcers by the restraint cortisone method was published in ``Journal of the Japanese Society of Gastroenterology,'' No. 62.
(12), page 1533 (1965). The results are shown in the table below.

[Table] Example 1 5.0 g of chlorophyllin (chlorin e ₆ ) is dissolved in a mixed solvent of 500 ml of methanol and 100 ml of glacial acetic acid. Separately, add 10.0 g of cobalt acetate tetrahydrate to methanol.
Add 30% hydrogen peroxide to the solution dissolved in 50ml
Add 0.75 ml and drop the resulting solution to the previously prepared chlorophyllin solution at 50°C while stirring. After further stirring at the same temperature for 1 hour, the mixture was cooled to room temperature while continuing to stir. After separating the insoluble matter from the reaction solution, the solvent was distilled off under reduced pressure, and the resulting dry solid was approx.
Suspend in 100ml of water, filter through a glass filter, and wash by pouring the same amount of water over the glass filter. The residue was then dried to obtain 4.26 g (yield 73.7%) of cobaltichlorophyllin. This cobaltichlorophyllin is dissolved in 300 ml of methanol. Separately, 3.20 g of thiamine hydrochloride was dissolved in 190 ml of methanol, and the pH was further adjusted to 10.5 with a methanol solution containing about 5% sodium hydroxide. This solution is added dropwise to the above methanol solution of cobaltichlorophyllin at 40° C. while stirring. Continue stirring for an additional 3 hours after addition. Insoluble materials generated during the reaction were separated, and the liquid was concentrated to dryness under reduced pressure to obtain crude thiamin cobaltichlorophyllin.
5.15g (yield 63.3%) was obtained. Next, add diamond ions to 40% methanol in advance.
Suspend 60ml of HP-20 and fill the column to prepare a hydrophobic porous polymer column. 40% of the crude thiamine cobaltichlorophyllin
After adjusting the pH to 5.0 with a methanol solution containing about 5% sodium hydroxide, the resulting precipitate was removed by filtration.
A column (diameter 2.5 cm, polymer part length 15 cm) filled with this liquid was filled with the hydrophobic porous polymer Diaion HP-20 (manufactured by Mitsubishi Chemical Industries, Ltd.).
50ml/hour. Then 40% of about 300ml
Methanol is passed through at 50 ml/h. Further about
200 ml of pure methanol is passed through the column at 100 ml/hour, and a green eluate fraction (approximately 100 ml) is collected. Methanol was distilled off from the fractionated eluate under reduced pressure, and dark green thiamin cobaltichlorophyllin was obtained.
Obtain 942 mg (yield 18.3%). Next, a chromatography column was prepared by suspending 50 g of Cephadex LH-20 in methanol and filling a column with a diameter of 3 cm and a length of 30 cm. 42 mg of the previously obtained thiamine cobaltichlorophyllin dissolved in about 10 mg of methanol is loaded onto this column, and then developed and eluted with methanol. Approximately 100 ml of thiamine cobaltichlorophyllin was collected by range chromatography and visible absorption spectrum absorption ratio of the eluate, and methanol was distilled off under reduced pressure to obtain 460 mg of dark green thiamine cobaltichlorophyllin powder ( Yield: 48.8%). When this thiamine cobaltichlorophyllin powder was dissolved in methanol and used as a sample solution to measure the visible absorption spectrum, maximum absorption was found at wavelengths of 430 nm and 646 nm. Chlorophyllin (chlorin)
The total yield from _e6 ) is 5.65%. Example 2 4.88 g of crude thiamine cobaltichlorophyllin obtained in the same manner as in Example 1 was dissolved in about 70% methanol.
Dissolve in 100 ml and adjust the pH to 4.0 with a methanol solution containing about 5% sodium hydroxide, and separate the resulting precipitate. Next, add diamond ions to 70% methanol in advance.
Suspend 300 ml of HP-20 (manufactured by Mitsubishi Chemical Industries, Ltd.) and fill the column to prepare a hydrophobic porous polymer column. The liquid is passed through a column (diameter 4.0 cm, polymer part length 25 cm) packed with a hydrophobic porous polymer at a rate of 50 ml/hour. Approximately 200 ml of 70% methanol are then passed through at 70 ml/hour. Add approximately 300ml of pure methanol to 100ml.
ml/hour and exhibits a green color (approx.
200ml). Methanol is distilled off from the fractionated eluate under reduced pressure to obtain 752 mg (yield 15.4%) of dark green thiamincobaltichlorophyllin. Next, suspend 50 g of Cephadex LH-20 (manufactured by Pharmacia) in methanol, and
Fill a cm column and prepare it as a chromatography column. 752 mg of thiamine cobaltichlorophyllin previously obtained was dissolved in about 10 methanol and loaded onto this column, and then developed and eluted with methanol. Approximately 80 ml of thiamine cobaltichlorophyllin was collected by thin layer chromatography and visible absorption spectrum absorption ratio of the eluate, and methanol was distilled off under reduced pressure to obtain 389 mg of dark green thiamine cobaltichlorophyllin powder ( A yield of 51.7% was obtained. When this thiamine cobaltichlorophyllin powder was dissolved in methanol and used as a sample solution, the visible absorption spectrum was measured, and maximum absorption was found at wavelengths of 430 nm and 646 nm. Example 3 4.92 g of crude thiamine cobaltichlorophyllin obtained in the same manner as in Example 1 was dissolved in about 30% methanol.
After dissolving in 100 ml and adjusting the pH to 6.0 with a methanol solution containing about 5% sodium hydroxide, the resulting precipitate was separated. Next, add diamond ions to 30% methanol in advance.
Suspend 60 ml of HP-20 (manufactured by Mitsubishi Chemical Industries, Ltd.) and fill the column to prepare a hydrophobic porous polymer column. The liquid is passed through a column packed with a hydrophobic porous polymer (diameter 2.5 cm, polymer section length 10 cm) at a rate of 50 ml/hour. Approximately 400 ml of 30% methanol are then passed through at 100 ml/hour. Add approximately 200ml of 80% methanol.
The elution section (approx.
150ml). Methanol is distilled off from the fractionated eluate under reduced pressure to obtain 964 mg (yield 19.6%) of dark green thiamincobaltichlorophyllin. Next, suspend 50 g of Cephadex LH-20 (manufactured by Pharmacia) in methanol, and
Fill a cm column and prepare it as a chromatography column. 946 mg of thiamine cobaltichlorophyllin obtained earlier was dissolved in about 10 mg of methanol and placed on this column, and then developed and eluted with methanol. Approximately 80 ml of thiamine cobaltichlorophyllin was collected by thin layer chromatography and visible absorption spectrum absorption ratio of the eluate, and methanol was distilled off under reduced pressure to obtain 457 mg of dark green thiamine cobaltichlorophyllin powder ( Yield: 47.4%). When this thiamine cobaltichlorophyllin powder was dissolved in methanol and used as a sample solution, the visible absorption spectrum was measured, and maximum absorption was found at wavelengths of 430 nm and 646 nm. Example 4 2.00 g of chlorophyllin (chlorin e ₆ ) and 1.00 g of cobalt acetate tetrahydrate were dissolved in a mixed solvent of 20 ml of methanol and 6 ml of glacial acetic acid, and stirred at 50 to 55°C for 2 hours while aerating at 150 ml/hour. . After concentrating the reaction product to 5.3 g under reduced pressure, 30 ml of isopropyl ether was added, stirred, and filtered to obtain cobaltichlorophyllin. Dissolve this cobaltichlorophyllin and thiamine hydrochloride 1.30g in 30ml of methanol, and add to this solution.
Add 10% caustic soda/methanol solution to PH7.30
Stirred at room temperature for 1.5 hours at ~7.20. Next, the pH was adjusted to 5.10 with 3N hydrochloric acid and methanol, and the mixture was concentrated to dryness under reduced pressure to obtain 3.10 g of crude thiamine cobaltichlorophyllin. This crude thiamine cobaltichlorophyllin was purified in the same manner as in Example 1 to obtain 0.65 g of dark green thiamine cobaltichlorophyllin powder. Chlorophyllin (chlorin)
The total yield from _e6 ) is 19.9%. When this thiamine cobaltichlorophyllin powder was dissolved in methanol and used as a sample solution, visible absorption spectrum measurement was performed, and maximum absorption was found at wavelengths of 431 nm and 648 nm. Example 5 2.00 g of chlorophyllin (chlorin e ₆ ) and 1.00 g of cobalt acetate tetrahydrate were dissolved in a mixed solvent of 20 ml of methanol and 6 ml of glacial acetic acid and stirred at about 55° C. for 2 hours. After concentrating the reaction product to 5.3 g under reduced pressure,
30 ml of isopropyl ether was added, stirred, and filtered to obtain cobaltichlorophyllin. Dissolve 1.30 g of cobaltichlorophyllin and thiamine hydrochloride obtained above in 30 ml of methanol, and add 10%
A caustic soda/methanol solution was added, and the mixture was stirred at room temperature for 3 hours at around pH 7. Next, the pH was adjusted to 5.10 with 3N hydrochloric acid and methanol, and the mixture was concentrated to dryness to obtain 3.05 g of crude thiamine cobaltichlorophyllin. Thereafter, the product was purified in the same manner as in Example 1 to obtain 0.56 g of thiamine cobaltichlorophyllin. The total yield from chlorophyllin (chlorin e ₆ ) is 17.2%.

[Brief explanation of drawings]

In the accompanying drawings, FIGS. 1, 2, and 3 are infrared absorption spectrum curves of the thiamine cobaltichlorophyllin complex compound of the present invention, cobaltichlorophyllin as an intermediate, and chlorophyllin as a raw material, respectively; The figure shows curves showing the visible absorption of the three substances.

Claims (1)

[Claims] 1. Reacting chlorophyllin (chlorin e ₆ ) with a divalent cobalt salt in the presence of oxygen in a water-free acetic acid solution to form a cobalt chlorophyllin complex, and then distilling the resulting reaction mixture or reacting thereto. recovering the cobaltichlorophyllin complex compound by addition of a solvent in which the product becomes insolubilized;
Thiamine, which was made into an alcohol solution, was added in an amount less than 2 moles per 1 mole of the cobaltichlorophyllin complex compound to a solution in which it was dissolved in alcohol.
7 to 11, remove alcohol from the reaction mixture as much as possible, and reduce the almost solid residue to 30 to 70%.
One molecule of thiol-type thiamine per molecule of the cobaltichlorophyllin complex compound is dissolved in aqueous alcohol and the resulting solution is subjected to chromatography and gel filtration treatment using a hydrophobic porous polymer. A method for producing a novel coordinated thiamine cobaltichlorophyllin complex.