JPS6150921A - Purification of baicalin - Google Patents

Abstract

PURPOSE:To prepare baicalin useful as a remedy for allergic diseases, easily, in high yield and purity, by preparing crude baicalin from defatted OUGON (root of Scutellaria baicalensis) by conventional method, dissolving the crude baicalin in water, especially in boiling water, and leaving the solution for a whole day and night. CONSTITUTION:Defatted OUGON is extracted with water, and the filtrate is added with hydrochloric acid and left for a whole day and night. The produced residue is dissolved in methanol, purified by the gel-filtration with a polyamide, cellulose, etc., and concentrated under reduced pressure to obtain crude baicalin. The crude baicalin can be purified to the acicular crystal of highly pure baicalin by dissolving in water, especially in boiling water, and leaving for a whole day and night. Since the raw material is defatted in the pretreatment step, the purification can be facilitated and the yield can be improved. The repetition of the troublesome recrystallization processes is unnecessary because the crude baicalin is dissolved in water.

Description

[Detailed Description of the Invention] Purpose of the Invention Industrial Field of Application Baicalin or its aglycone pyryrhein has an antihistamine effect and is a pharmaceutical product (for internal or external use).
Also, its application to cosmetics is attracting attention. Baicalin or pyrite is considered to be used as a preventive and therapeutic agent for various 7-reel diseases, especially as an anti-itch agent for chronic dermatitis, eczema, and the accompanying itching. A herbal medicine used in Chinese herbal medicine.

It is known as one of the main components contained in Scutellariae.

<Prior Art> As a typical method for obtaining baicalin, the following method is known as a typical example.

(1) Extract the yellow pulp with water, add hydrochloric acid to the leaching liquid, precipitate it, repeat the leaching with methanol, collect the precipitate by filtration, recrystallize it with ethanol, yield 4
Get %.・・・・・・(Literature location: Inagaki Isaku Plant Chemistry> pages 194-195, Inae Yaku Publishing, 1972)
(Published on October 25th) (2) In an analytical study on flavonoid components such as suggonin contained in astragalus, an extract was obtained from astragalus by reflux extraction using methanol, and then the aqueous portion was passed through a polyamide column. After that, the adsorbate was eluted with methanol, and the methanol solution was concentrated.
The precipitated baicalin is removed and sugenin is obtained from the filtrate. (Reference location: Tsuyoshi Tomimori et al., Pharmaceutical Journal VOI, 102°Nα4388-391, 1982). In addition to this, the following methods are being considered.

(3) After water extraction, add hydrochloric acid to obtain a precipitate.

(4) A method of extraction using an organic solvent such as methanol or ethanol.

(5) A method of water-extracting the extract extracted by the method of (4) above.

(6) A method of purifying the composition obtained in (5) above by recrystallizing it with methanol.

(7) More recently, using the tissue culture method of yellow mustard,
The callus is obtained and research is being conducted on the separation of flavones in the yellow mustard.

<Problems to be Solved by the Invention> The present inventors have described above. No matter which method is adopted,
As a result, it was found that the final yield of high-purity baicalin was around 0.1 to 0.4%. For example, in (1) above, it is said that the yield is 4%, but if you analyze this.

It is still in the form of crude baicalin, with a purity of 70 to 8.
It is in the 0% range. In other words, it is well refined. In order to obtain baicalin, in the known methods shown in +11 to (6) above, baicalin of considerable purity can be obtained by repeating recrystallization many times, but in order to obtain baicalin,
While it requires a technique with considerable skill, it has the disadvantage that the yield decreases each time the purity is increased.

3. Structure of the Invention (Means for Solving the Problem) The present inventors have developed a technical method for obtaining high-purity baicalin at a high efficiency based on the known technology of fil to (7) described above. We have begun research to improve a simple purification method that does not require any skill.

As a result, by employing the method shown in the examples below, we succeeded in obtaining baicalin with a high purity of 98% to 100%. Moreover, the yield is around 1 to 4% when starting from yolk, and unlike conventional methods, it is necessary to repeat elution and recrystallization several times using organic solvents such as methanol and ethanol. There is no such thing.

The operational or process characteristics of the present invention are as follows: 1. It was discovered that in the final step, that is, after obtaining crude baicalin, when it is dissolved in boiling water, a large amount of acicular crystals of baicalin precipitates over time. This triggered the completion of the present invention.

The greatest feature of the method for purifying baicalin according to the present invention is that it eliminates the need for repeated and troublesome recrystallizations, and as a result, it can be expected to be further utilized in medicines, etc.

"Example 1" The ends of yellow scallions or scraps are immersed in benzene, black bean sanitation, ether, or n-hexane, preferably ether or n-hexane, or heated under reflux to dissolve the phase contained in the scallions. Remove dissolved components. If this operation is performed in advance, the final yield of baicalin will be 1
~30% increase. Furthermore, the following extraction or purification operability is improved.

For the yellow pulp that has been defatted in the above process, the organic solvent has been completely removed and 100 parts of water is added to 1 part of the dried product.
After digestion under heating at ~100 C0, either σ8 cloth or filter paper may be used to obtain the solution.

In the conventional method, a method is known in which the yellow pulp is extracted without degreasing it.

In this case, the number of battles mentioned above will increase.

Since the oil is degreased in advance, filtration is very smooth and the filtrate is clean. If the filtrate is not clear, repeat II.
It is necessary to add an extra layer. Add hydrochloric acid to the clear filtrate. As for the amount of hydrochloric acid to be used, it is best to add around 1% of the σ filtrate. After adding hydrochloric acid, mix well and let it stand overnight, then filter with a filter paper and remove the residue on the filter paper. , using methanol to dissolve this solution, polyamide or cellulose, or Sephadex L
H-20 (manufactured by Pharmasha, Fine Chemicals.

Alternatively, when the resulting solution is passed through either nylon 66 or nylon 12 and concentrated under reduced pressure using an evaporator, crude baicalin gradually precipitates around the inside of the container and becomes resistant to adhesion. The precipitated crude baicalin is taken out and dissolved by adding it to ice, cold water, hot water, or boiling water, preferably boiling water. After dissolving QC at room temperature.

If you leave it for a day or night, yellow needle-like crystals will precipitate.
Separate this. This product has a purity of 98% or more
% of high-purity baicalin, and the yield depends on the yolk used, but the lowest is baicalin! You can get 10-401 from illusion.

In the above-mentioned process, the more impurities other than baicalin are present in the step number ζ where it is dissolved in methanol.

It tends to dissolve easily in methanol, so it is necessary to take this point into consideration during operation.
As an alternative to methanol, it may be dissolved in ethanol, but the final yield of baicalin is better with methanol.

In addition, in the final step, ice, cold water, hot water, and boiling water were used to precipitate high-purity baicalin.

In addition, high-purity baicalin could be precipitated and fractionated even when dissolved in the primary mixed solution in combination with water.

Water and ethanol, water and methanol, water and acetone, water and ether, water and ethyl acetate, water and 0-hexane, water and tetrahydrofuran, water and beef sanitation, water and benzene, water and dioxane, water and dichloromethane, water and chloroform, water and carbon tetrachloride, water and carbon disulfide.

"Example 2" After degreasing the ends of yellow pulp or cracks in the same manner as in Example 1, 1 part of dried yellow pulp was added with 10 parts of ethanol, and heated (78σ Filter the extracted liquid, concentrate the M liquid under reduced pressure to remove ethanol, and then add water and dissolve it to obtain an aqueous solution (hereinafter referred to as liquid A for convenience), or perform a degreasing process. After that, add 1 part of saturated sodium bicarbonate aqueous solution, other weakly basic aqueous solution, or strongly basic diluted solution to 1 part of well-dried yellow pulp.
Add 0 parts, stir at 0 to 40C', and after a day and a night of concentration,
I+a filter, IIa filter residue.

A solution obtained by bath dissolving in ethanol (hereinafter referred to as solution B for convenience) is obtained.

Or 1 part number ζ of well-dried degreased yellow mustard, 2 parts water
00 parts, perform extraction at room temperature to 100c' to obtain the filtrate, add hydrochloric acid to the filtrate, and calculate the concentration of hydrochloric acid in the filtrate! Make it about χ.

Mix well and leave for a day and night, then filter to obtain the residue. This residue is dissolved in a mixed solution of ethanol and amethane to obtain a solution (hereinafter referred to as solution C for convenience).

semi-treated, polyamide, cellulose, Sephadex-L
H20, nylon 12. The crude baicalin that precipitates around the inside of the container is separated out by concentrating each liquid under reduced pressure. After drying, the crude baicalin is dissolved in water, and the precipitated yellowish baicalin is collected. Crystalline baicalin can be obtained.

The purity of baicalin obtained here is at least 98%
That's all for now.

Regarding the purification process for the above-mentioned liquids A, B, and C that have passed through polyamide, etc.

Additionally, in some cases it may be a good idea to perform the following operations.

First, in the case of liquid A, crude baicalin can be better precipitated by adding water from the same boat to the liquid passed through polyamide and then concentrating it under reduced pressure.

On the other hand, in the case of B liquid, the liquid passed through polyamide etc.
After concentrating to a certain degree, add twice the amount of water to the concentrate and shake well.

If left for a while, crude baicalin will precipitate, so take a fraction of it, recrystallize it using methanol, take one fraction, and dry it thoroughly.This dried recrystallization Highly purified baicalin in the form of yellow needle-like crystals can also be obtained by adding and dissolving it in #1llll+! water and leaving it overnight at room temperature from OCo. In this case, the purity is at least 98%. It is.

Regarding liquid C, the liquid passed through polyamide etc.

After concentrating to a certain extent, apply 2
Add about twice as much ice water and shake well.The precipitate will be separated, add it to a mixture of ethanol and acetone, recrystallize it, take it out, dry it thoroughly, and then , dissolved in a small amount of ethanol, and then added 10% to this solution.
Add up to 100 times as much water and leave it for about a day or night to obtain high purity baicalin in the form of yellow needle-like crystals.

Effects of the Invention The present invention, as shown in the Examples, obtains highly pure baicalin by obtaining crude baicalin and dissolving it in ice, cold water, hot water, or boiling water in the final step. Unlike the conventional recrystallization method, in which crude baicalin is repeatedly used in various organic solvents, this method does not require any special skill and is easy to operate.

If this method is adopted, high-purity baicalin can be produced, for example, from callus obtained from astragalus and its tissue culture method, or from crude baicalin obtained by a synthetic method expected in the future. It is useful for purification. In other words, in the method of the present invention, even if crude baicalin is obtained by any conventional method, once it is dissolved in water and the precipitated needle-like crystals are collected, baicalin with a purity of 98% or more can be obtained. .

In addition, as a condition for obtaining high-purity baicalin from yellow pulp, as shown in the example, a degreasing step is adopted in the pretreatment, but in the conventional method, various solvents are suddenly used without degreasing. In some cases, it may be extracted, but in such cases, adding a degreasing operation using xane or ether will make the subsequent purification process much smoother and will also improve the yield.

In Example 1 or 2 according to the present invention, when obtaining crude baicalin, there is an operation of filtering the extract and dissolving the residue in various solvents. However, it is best to use methanol as the solvent for this residue. In other words, it becomes easier to obtain a product of high purity in terms of yield. Furthermore, when the residue is dissolved in methanol, the worse the solubility, the more baicalin is contained in the residue.

Therefore, the amount of methanol used to dissolve the residue is large (if this is the case, the yield of high-purity baicalin will also increase in proportion to this amount), so unlike the conventional method, there is no guideline. Unlike methods that require repeated recrystallization, it is more rational in that the yield can be determined in advance during the production process.

On the other hand, if the residue dissolves well in methanol when obtained, it is still at the stage of extracting from the yellow mustard, and the baicalin content is extremely low, so there are Management becomes easier.

Next, the high-purity baicalin obtained according to the present invention is also optimal as a raw material for producing pyrykrein. That is,
Since the purity is high (in order to obtain pyrite, high purity pyrite is easily obtained by following Reference Examples 1 to 3 shown below).

"Reference Example" To the high-purity Picalici obtained in Examples 1 and 2, (l
Add 12N sulfuric acid 2e or add Lt (2+ 2.5%
Add 2e of hydrochloric acid or 2e of +312N alcoholic sulfuric acid, and heat and reflux for about 2 hours in case (1) and about 3 hours in case 2-50P (3) in case (2).

However, in the case of refluxing using the above (3), next:
After adding about 20 liters of ice water, a precipitated pyrite is obtained. As a result, the precipitated pyrite is removed. If necessary, polyamide, cellulose, Sephadex LH
-20, nylon 66, nylon 12, etc., or recrystallized with methanol, benzene, etc. to obtain pyrite.

"Reference Example 2j It
1% potassium hydroxide aqueous solution l-% hydroxide l-I
Add 200 J of Jum aqueous solution and heat under reflux for 10 minutes. Next, either hydrochloric acid or sulfuric acid is added to make it acidic to obtain the precipitated pyrein, or the precipitated baicalin is separated and dissolved in a solvent such as ethyl acetate or ether.
Concentrate under reduced pressure to obtain pyrite.

On the other hand, instead of (4) above, (5) ethanol 10
After heating and refluxing for 5 minutes, neutralize by adding hydrochloric acid or sulfuric acid, filtrate, distill off the solvent in the filtrate, add water to the residue, dissolve it, and add hydrochloric acid or sulfuric acid. In addition, it is acidic. This results in a precipitated pi-force rain. 1
The pyrite obtained using 4) or 5) may be passed through polyamide or the like or recrystallized using methanol or the like, if necessary, according to Reference Example 1.

"Reference Example 3" It is also possible to add glucurondase to the high-purity pyrite obtained in Examples 1 and 2 and hydrolyze it to obtain pyrite, but in that case, refer to the following literature. It is a good idea to use this as a reference when performing enzyme treatment.

(Reference location) (1) Chemical and Pharmaceutical Pretin Volume 8, page 238 (1% 0 years): (ChlM
, pki+m, 3++l+,, ) (2) Same journal, vol. 12, page 664 (1% 4 years) (3) Journal of Biological Chemistry, vol. 239, page 59 (1% 4 years); (J, 3+・' + Ch
``'+) Above, baicalin obtained in Example 1 or 2 or Reference Examples 1 to
The high-performance liquid chromatography charts of baicalin obtained in step 3 are as shown in Figures 1 and 2, and in terms of purity, the standard products of baicalin and pyriterein (manufactured by Maru Falcos, (manufactured by Wako Pure Chemical Industries, Ltd.) to prepare a calibration curve, and when determined from this, the purity is 98% to 100%.

(High performance liquid chromatography conditions) Column: ODS system power 5 μL S-410 (4,6
Eluent: Baicalin (Figure 1) Acetonitrile: Water ±32: 680,005 M tetra-n-aluminum ammonium iodite (PH = 4.0, phosphoric acid) (Figure 2)〉 Acetonitrile:water:o, 6% phosphoric acid aqueous solution M 43:2
7:30 Eluent flow fA' 1.0 m11/m+n Column temperature: 40C' Detector wavelength: 280nm Detector sensitivity: 0.16AUFS

[Brief explanation of the drawing]

FIG. 1 shows a chart of high purity icarin obtained in Example 1 or 2 by high performance liquid chromatography. FIG. 2 is a high-performance liquid chromatography chart of pyrites obtained in Reference Examples 1 to 3.

Claims (2)

[Claims] (1) After digesting defatted Scutellaria scutellariae with water, separate the filtrate, add hydrochloric acid to the filtrate to a concentration of about 1%, and leave it for a day and night. After that, the residue was separated, and a solution of the residue dissolved in methanol was dissolved in polyamide, cellulose, or Sephadex LH-2.
0 (manufactured by Pharmacia Fine Chemicals), and when this solution is concentrated under reduced pressure, the crude baicalin is gradually deposited around the inner surface of the solid crude baicalinga container. Separate. Next, after dissolving the crude baicalin in water, cold water, hot water, or boiling water, preferably boiling water, high purity baicalin in the form of needle-shaped crystals is precipitated by leaving it for about a day or night. A method for purifying baicalin, which is characterized by fractionating it. (2) In the final step, after dissolving the crude baicalin in ice, cold water, hot water, or boiling water, preferably boiling water, high-purity baicalin in the form of needle-shaped crystals is precipitated by leaving it for about a day and night. A method for purifying baicalin, which is characterized by separating the baicalin.