JPS6212777A - Separation and purification of berberine alkaloid - Google Patents

Abstract

PURPOSE:To deposit and separate berberine alkaloid as an ionic salt, by treating a tissue piece or cell of a plant containing the berberine alkaloid with sulfuric acid and adding a salt exchange agent containing chlorine ion, sulfuric acid ion, etc., to the resultant extract solution. CONSTITUTION:A tissue piece or cell of a plant containing berberine alkaloid, e.g. goldthread or Amur cork, is dipped in a 0.2-0.5N aqueous solution of sulfuric acid for >=1hr to give an extract solution. A salt exchange agent, preferably an aqueous solution of hydrochloric acid or nitric acid, containing chlorine ion and/or nitric acid ion is then added to the extract solution to convert the berberine alkaloid into a slightly soluble ionic salt at 10-30 deg.C deposit and separate the berberine alkaloid. EFFECT:The aimed substance is obtained in high purity and recovery ratio by the simple method in a few steps. USE:A medicine for intestinal disorder, remedy for bacterial intestinal diseases and yellowish dyes.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to a method for separating and purifying berberine alkaloids from materials containing berberine alkaloids. Berberine alkaloids are compounds that are in demand as intestinal regulators, bacterial intestinal disease treatments, and yellow dyes.

[Conventional technology]

Regarding conventional methods for separating berberine alkaloids from substances containing berberine alkaloids, berberine alkaloids can be obtained by tissue culture of plants that produce berberine alkaloids, such as plants such as Oriental cypress belonging to the Rutaceae family and Oriental oleracea belonging to the Ranunculaceae family. Several methods have been reported for separating and purifying berberine from callus.

For example, Japanese Patent Application Laid-open No. 47-30897 discloses that callus obtained by tissue culture is crushed, extracted with methanol, concentrated, and then acidified by adding hydrochloric acid, followed by ether extraction. The ether-insoluble part was made alkaline with ammonia, extracted with chloroform, and the chloroform-soluble part was concentrated and subjected to thin layer chromatography (
T, L, C,) is disclosed.

Also, JP-A-50-13519 and JP-A-51-
Publication No. 12993 discloses that alumina callus obtained by tissue culture was crushed, extracted with hot water or methanol, concentrated under reduced pressure, acidified with hydrochloric acid, and used methanol as an eluent. A method is disclosed in which impurities are removed by column chromatography, hot water is added to the obtained methanol solution containing berberine to make it homogeneous, and then berberine chloride is precipitated and separated by cooling.

In addition, JP-A-55-155056 discloses that 95% ethanol is added to crushed yellowfin tuna to perform heating extraction, and then, after concentrating this extract, water and talc are added to it for heating extraction. A method for removing impurities by filtration to obtain a pigment extract containing berberine is disclosed. This method is a method for obtaining a berberine extract, and the publication does not disclose anything about a method for isolating berberine.

Further, JP-A-57-144992 discloses a method for separating berberine alkaloids such as jatrolidine and palmatine from a culture obtained by tissue culture of a plant of the family Tiliaceae. According to this method, after the culture is filtered into cultured cells and culture solution, the air-dried cultured cells are extracted with ethanol, while the culture solution is made alkaline and then extracted with chloroform, and then each extract is extracted with chloroform. After concentrating, this is developed using thin layer chromatography (T, L, C,) to fractionate spots containing the target component, which is made into an aqueous solution and then potassium iodide is added to precipitate it as an iodized salt. A method for separating the desired berberine alkaloid is shown.

All of the conventional methods for separating and purifying berberine alkaloids in water are complicated and have many operational steps, and the recovery efficiency of the berberine alkaloids to be obtained is low.
In addition to low efficiency, various fatty acid esters, which are impurities, are accompanied during extraction, which requires degreasing using ether treatment later.

As a new separation method different from the above methods, the applicant has
According to Japanese Patent Application No. 59-82951, chloride ions or nitric acid were added to a culture mixture consisting of cells and a culture medium obtained by tissue culture of Japanese larch, or to a culture medium containing berberine alkaloid released from cultured cells into the culture medium. We demonstrated a method for separating and purifying berberine alkaloids by adding ions to precipitate berberine alkaloids as poorly soluble yellow crystals, isolating these, and recrystallizing if necessary.

[Problem that the invention seeks to solve]

The present inventors studied the conventional methods for separating and purifying berberine alkaloids, and found that
It was acknowledged that all the other methods except for No. 51 have various drawbacks, such as having many operational steps, being complicated, and having low recovery efficiency, as mentioned above. On the other hand, the method disclosed in Japanese Patent Application No. 59-82951 uses an aqueous solution containing berberine alkaloid released from cultured cells into a liquid medium and dissolved therein during culture, or crushing the cultured cells to extract berberine alkaloid to the outside of the cells. This method uses an aqueous solution containing berberine alkaloid as a raw material and adds chlorine ions or nitrate ions to it to precipitate berberine alkaloid.
Further improvements are desired. For example, a method that does not necessarily require mechanical disruption of cultured cells and that can increase the purity of the separated berberine alkaloid is desired.

Against this background, the present inventors separated and purified berberine alkaloids from berberine alkaloid-containing substances by using a simple method with a small number of steps and a high recovery rate, unlike conventional methods. We earnestly considered ways to do this.

[Means and operations for solving the problems] As a result, the inventors discovered that the above object could be achieved by employing the following method, and completed the present invention.

That is, according to the method of the present invention, a salt exchanger containing chloride ions and/or nitrate ions is added to an extract obtained by treating tissue pieces or cells of plants containing berberine alkaloids with sulfuric acid, thereby producing berberine alkaloids. Provided is a method for separating and purifying berberine alkaloids, which comprises precipitating and separating berberine alkaloids as salts of the ions.

Specific examples of plant tissue pieces or cells containing berberine alkaloids used as raw materials in the berberine alkaloid separation and purification method of the present invention include plants of the family Ranunculaceae such as Oriental japonica and Japanese larch, plants of the Rutaceae family such as yellowfin, plants of the family Rutaceae, etc. A tissue piece of a berberine alkaloid-producing plant or cultured cells (including callus) obtained by tissue culture of the plant tissue piece. Since cultured cells are usually a mixture of cultured cells and a medium, for example, when a liquid medium is used as the medium, the culture mixture is divided into cultured cells and culture medium as necessary. Cultured cells containing berberine alkaloids can be dried in a wet state or by air-drying, and used as a raw material for the next extraction operation of the present invention, which is treated with sulfuric acid, which will be described later. On the other hand, some berberine alkaloids may be released from the cultured cells to the outside of the culture solution, but in this case, the cultured cells and the liquid medium are separated and the cultured cells are treated with sulfuric acid (described later). The salt exchange agent described below may be added to the culture mixture together with the culture medium, or the entire culture mixture may be treated with sulfuric acid to extract the berberine alkaloids present in the cells into an extracellular aqueous solution. Next, a method may be adopted in which a salt exchanger described below is added to the berberine-containing aqueous solution after cell residues in the culture mixture treated with sulfuric acid are removed by filtration or the like.

Specifically, the berberine alkaloid separated and purified by the method of the present invention is berberine represented by formula (I) (R,, R2=-OCII20-1R3, R4=-OC
H3), palmatine (R,, R2, R3, R4=-O
CH3), cobuticin (R1, R2=-OCII2
0-, R3, Ra=-OCH20-), Jatrolidine (R1=-011, R2, R3, R4=-OCH
3), columbamine (Rt, R2, R3, R4=-O
CH3), Salif engine (R,, R2=-OCH20
-, R3=-OCH3, R4=-011) and the like. Among these, berberine is preferred in the present invention.

In the present invention, the above-described berberine alkaloid-containing material is treated with sulfuric acid to obtain an extract containing berberine alkaloid. The sulfuric acid used as the extractant in this case is usually a sulfuric acid aqueous solution, and the sulfuric acid concentration at this time is usually in the range of 0.1 to 3 normal (N), preferably 0.2 to 0.5 normal. . In the present invention, the amount of the extractant used is usually 1 to 5 parts by weight of an aqueous sulfuric acid solution having the concentration range described above per 1 part by weight of the berberine-containing material;
Preferably, 3 to 4 parts by weight is used, and extraction is carried out by immersion at a temperature of usually 10 to 50° C. for about 1 hour or more, usually with stirring. After the extraction treatment, the solid raffinate is removed by filtration or the like in a conventional manner to obtain an extract containing berberine alkaloids.

In the present invention, the berberine alkaloid-containing extract obtained by the above method has chloride ions or/and
By adding a salt exchanger containing nitrate ions, the berberine alkaloid is precipitated as a salt of the ions.

In the present invention, when a salt exchanger is added to the above-mentioned extract, the extract may be concentrated to a solid-free solution having an appropriate concentration, if necessary.

The salt exchange agent used in the present invention is a water-soluble compound containing chloride ions and/or nitrate ions and is r! KR8;
:C 1. Okay, 11wi39. Sea urchin: Examples include sodium chloride, potassium chloride, nitric acid, lithium nitrate, sodium nitrate, and potassium nitrate. In the present invention, it is preferable to use hydrogen chloride, hydrochloric acid, and nitric acid, and in this case, hydrochloric acid is used.

In the present invention, when adding a salt exchanger to the extract, the amount of chlorine ions and/or nitrate ions is usually 10 to 40 times the molar part of berberine alkaloid in the extract. , preferably 20 to 3
It is 0 times molar part. In the present invention, by selecting the amount of the salt exchanger added in this way, it is possible to efficiently convert the berberine alkaloid in the extract into hydrochloride or nitrate of berberine alkaloid, which is sparingly soluble in the extract, and precipitate it. . In this case, the temperature during precipitation is usually 0 to 50°C, preferably 10 to 30°C. In the present invention, after converting the berberine alkaloid in the solution into a sparingly soluble salt and precipitating it, the precipitate is separated from the solution by a conventional method such as filtration.

In the present invention, the berberine alkaloid hydrochloride or/nitrate separated from the solution by the above method is washed with a detergent such as water, aqueous methanol, aqueous ethanol/methanol, ethanol, acetone, or the like. After washing, the berberine alkaloid is obtained as a highly pure hydrochloride or nitrate of the alkaloid by performing purification operations such as recrystallization an appropriate number of times using a recrystallization solvent such as a lower aliphatic alcohol such as methanol or ethanol. I can do it.

〔Effect of the invention〕

According to the method of the present invention, berberine alkaloid can be separated and purified from a berberine alkaloid-containing substance with high purity and high recovery rate by a simple method with fewer steps than conventional methods.

〔Example〕

Hereinafter, the method of the present invention will be specifically explained using examples.

Example 1 Into a 251 extraction tank equipped with a stirrer, 6.7 kg of oren callus obtained by tissue culture method (berberine content: 2
60%), add 14.7 kg of 0.3N sulfuric acid and heat at 25°C.
After stirring for hr, it was filtered. 550 g of NaCIt was added to this filtrate while stirring at room temperature, and stirring was continued for 3 hours. The resulting precipitate of crude berberine chloride was filtered off, washed with water, and dried. 110g of this crude chlorinated berberine was added to 99g of methanol.
00 g, and the insoluble matter was filtered while hot. When the filtrate was gradually cooled to room temperature while stirring, yellow purified berberine chloride was precipitated. This is filtered, washed with 90g of methanol, and then dried to obtain highly purified purified chlorinated berberine.
00g (recovery rate 75%/berberine standard containing raw callus,
A purity of 99.5% was obtained.

Example 2 Example 1 except that when extracting berberine from chemical callus, the slurry of chemical callus made of 0.3N sulfuric acid was simply immersed at 25° C. for 4 hours without stirring.
When treated in the same manner as in Example 1, the same results as in Example 1 were obtained.

Example 3 The same procedure as in Example 1 was carried out except that 950 g of concentrated hydrochloric acid was added instead of NaCl in the salt exchange reaction of the extract. As a result, 101 g of purified chlorinated berberine (recovery rate 76%, purity 99%) was obtained. .5%) was obtained.

Example 4 The same procedure as in Example 1 was repeated except that 1200 g of ethanol was used instead of the methanol used to recrystallize crude berberine chloride in Example 1, and the resulting purified crystals were washed with 120 g of ethanol. , 99 g (recovery rate 94%, purity 99.0%) of purified chlorinated berberine was obtained.

Example 5 The same procedure as in Example 1 was carried out except that 940 g of 13.4N sulfuric acid was used instead of adding NaCl to the berberine-containing filtrate, and high purity berberine nitrate was obtained with a recovery rate of 69%. Ta.

Claims (1)

[Claims] (1) A salt exchanger containing chloride ions and/or nitrate ions is added to the extract obtained by treating plant tissue pieces or cells containing berberine alkaloids with sulfuric acid to precipitate berberine alkaloids as salts of the ions. 1. A method for separating and purifying berberine alkaloids, which is characterized by separating berberine alkaloids.