JPS6232173B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION A. OBJECTS OF THE INVENTION The present invention relates to a simple method for highly efficient purification of 98% or higher purity baicalin into needle-like crystals.

<Industrial Application Fields> Baicalin or its aglycone baicalein has an antihistamine effect, and is attracting attention for its application to pharmaceuticals (internal or external use) and cosmetics. Baicalin or baicalein is considered to be used for the prevention and treatment of various allergic diseases, especially as an anti-itch agent for chronic dermatitis, eczema, and the accompanying itching. used in prescription,
It is known as one of the main ingredients contained in Scutellariae.

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

(1) Extract yellow with water, add hydrochloric acid to the leaching solution, precipitate it, repeat the leaching with methanol, collect the precipitate by filtration, and recrystallize with ethanol to obtain a yield of 4%. obtain. ...(Reference location: Isao Inagaki <Phytochemistry> pages 194-195,
(Ishiyaku Publishing, October 25, 1972) (2) In an analytical study on flavonoid components such as suggonin contained in Huang, an extract was obtained from Huang through reflux extraction using methanol. Thereafter, the aqueous portion is passed through a polyamide column, the adsorbed material is eluted with methanol, and the methanol solution is concentrated to remove precipitated baicalin and obtain sugonin from the filtrate. ...(Reference location: Takeshi Tomimori et al. Pharmaceutical journal
VOl・102, No. 4 388-391 1982). In addition to this, the following methods are being considered.

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

(4) Extraction method using organic solvents such as methanol and ethanol.

(5) A method in which the extract extracted by the method (4) above is extracted with water.

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

(7) More recently, Huang et al.'s tissue culture method has shown that
The callus is obtained and research is being conducted on the separation of flavones in the callus.

<Problems to be Solved by the Invention> The present inventors have found that no matter which method is adopted as described above, the yield of high-purity baicalin ultimately obtained is within the range of 0.1 to 0.4%. It turns out that it is. For example, in (1) above, the yield is 4
%, but if you analyze it, it is still in the state of crude baicalin, and the purity is 70-80%.
It's on the stand. In other words, in order to obtain well-purified baicalin, baicalin of considerable purity can be obtained by repeating recrystallization many times using the known methods shown in (1) to (6) above. However, while this requires a highly skilled technique, it also has the disadvantage that the yield decreases each time the purity is increased.

B. Structure of the Invention <Means for Solving the Problem> The present inventors have developed a technique for obtaining highly pure baicalin at a high efficiency based on the publicly known techniques (1) to (7) described above. We have begun research to improve a simple purification method that does not require technical 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 1-4% when starting from yellow, 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 include the discovery that in the final step, that is, after obtaining crude baicalin and dissolving it in boiling water, a large amount of needle-like crystals of baicalin precipitates over time. This was the trigger that led us to complete the present invention.

The most important 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 used in medicines and the like.

"Example 1" Soaking or heating reflux powder or scraps of yellow radish in benzene, cyclohexane, ether, or n-hexane, preferably ether or n-hexane, to remove the fat contained in yellow turmeric. Remove dissolved components. If this operation is performed in advance, the final yield of baicalin will increase by 10 to 30%. Furthermore, the following extraction or purification operability is improved.

The yellow leaves defatted in the above process are completely free of organic solvents, and 100 parts of water is added to 1 part of the dried product, digested under heating at 80 to 100°C, and then the resulting liquid is prepared. In obtaining this, either filter cloth or filter paper may be used. In the conventional method, it is known to extract yellow lily without degreasing, but in this case, the number of filtration steps mentioned above increases, but in this case, degreasing is performed in advance. Therefore, filtration is very smooth and the filtrate is clean. If the filtrate is not clear,
At this point, it is necessary to add filtration again. Add hydrochloric acid to the clear filtrate. As for the amount of hydrochloric acid to be used, it is best to add around 1% to the filtrate.After adding hydrochloric acid, mix well and leave it for a day and night.Next, perform filter paper filtration and remove the residue on the filter paper. Dissolve this solution using methanol,
Polyamide or cellulose or Cephadex
LH-20 (manufactured by Pharmacia Huain Chemical,
Alternatively, after passing through either nylon 66 or nylon 12, the resulting solution is concentrated under reduced pressure using an evaporator, and crude baicalin gradually precipitates and adheres to the surroundings of the container. 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 dissolution, from 0℃ to room temperature,
If it is left to stand for about a day or night, yellow needle-like crystals will precipitate, and these will be collected. This stuff is 98% pure
It is more than 100% high purity baicalin, and the yield is
Depending on the yellow used, the minimum weight is 1 kg to 10 ~
40g obtained.

In the above-mentioned process, in the step of dissolving in methanol, the more impurities other than baicalin, the easier it is to dissolve in methanol, so this point must be fully taken into consideration during operation. Furthermore, as an alternative to methanol, it may be dissolved in ethanol, but methanol is superior in terms of the final yield of baicalin.

In addition, in the final step, ice, cold water, hot water, and boiling water were used to precipitate high-purity baicalin, but high-purity baicalin can also be dissolved in the following mixed liquid in combination with water. Let it precipitate,
I was able to separate it.

Water and ethanol, water and methanol, water and acetone, water and ether, water and ethyl acetate, water and n-hexane, water and tetrahydrofuran, water and cyclohexane, 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 or cracks of yellow leaves in the same manner as in Example 1, the yellow leaves were thoroughly dried.
Add 10 parts of ethanol to 1 part and heat (78°C).
Filter the extracted solution (near the area), concentrate the filtrate under reduced pressure to remove ethanol, then add water and dissolve it to obtain an aqueous solution (hereinafter referred to as liquid A for convenience), or perform a degreasing treatment. After washing, add 1 part of well-dried yellow powder to a saturated aqueous solution of sodium bicarbonate or
Add 10 parts of another weakly basic aqueous solution or a strongly basic diluted solution, stir at 0 to 40°C, let stand overnight, filter, and remove the filtration residue from a solution dissolved in ethanol ( (hereinafter referred to as liquid B for convenience) is obtained.

Alternatively, extract the filtrate from room temperature to 100°C using 1 part of well-dried defatted yellow with 200 parts of water, and add hydrochloric acid to the filtrate to extract the Make sure the concentration of hydrochloric acid is around 1%, mix well, leave it for a day and night, then filter it.
Obtain the residue. This residue was dissolved in a mixture of ethanol and acetone (hereinafter, for convenience,
(referred to as liquid C).

The A liquid, B liquid, and C liquid obtained in the above steps are as follows:
The following operations are basically the same as in Example 1 shown above.
The crude baicalin that gradually precipitates around the inside of the container is collected by passing it through polyamide, cellulose, Cephadex-LH20, or nylon 12, and then concentrating each solution under reduced pressure. After drying, yellow crystalline baicalin can be obtained which precipitates after being dissolved in water. The purity of baicalin obtained here is at least 98% or higher.

Regarding the purification process for the above-mentioned liquids A, B, and C that have passed through polyamide, etc., it may be better to perform the following operation in some cases.

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

On the other hand, in the case of liquid B, after concentrating the liquid passed through polyamide etc. to a certain extent, add twice the amount of water to the concentrated liquid and shake well.
If left to stand for a while, crude baicalin will precipitate, so it is collected and recrystallized using methanol, then collected and thoroughly dried. High-purity baicalin in the form of yellow needle-like crystals can also be obtained by adding this dried recrystallized product to boiling water, dissolving it, and then leaving it for a day and night at 0°C to room temperature. The purity in this case is at least 98% or higher.

Regarding liquid C, the liquid passed through polyamide etc. is concentrated to a certain extent, then about twice the amount of ice water is added to this concentrated liquid, shaken well, and left for a while. Crude baicalin is precipitated, so take it out, add it to a mixture of ethanol and acetone, recrystallize it, take it out, dry it well, and dissolve it in a small amount of ethanol. Add 10 to 100 times more water to this solution and leave it for about a day or night to obtain high purity baicalin in the form of yellow needle-like crystals.

<C> Effects of the invention As shown in the examples, the present invention provides the following advantages: After obtaining crude baicalin, in the final step, ice, cold water,
By dissolving the crude baicalin in hot or boiling water to obtain highly pure baicalin, as in the past, crude baicalin was repeatedly recrystallized using various organic solvents. Unlike the chemical method, it does not require special skill and is easy to operate.

If this method is adopted, for example, high-purity baicalin can be produced based on callus obtained from yellow baicalin and its tissue culture method, or crude baicalin obtained through synthetic methods, etc. It is useful for purifying baicalin. 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 medium yellow, a degreasing step is adopted in the pretreatment as shown in the example, but in the conventional method,
In some cases, extraction is carried out immediately using various solvents without degreasing, but in such cases, the n ―
Adding a degreasing operation using hexane or ether makes the subsequent purification process very smooth and improves 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, if the amount of methanol used to dissolve the residue increases, the yield of high-purity baicalin will also increase in proportion to this amount, so there is no guideline as with conventional methods. Leave it to me,
Unlike previous methods that required multiple recrystallizations, this method is more rational in that the yield can be determined in advance during the production process.

On the other hand, when the residue is dissolved in methanol, if it dissolves well, it is still at the stage of extract from yellow baicalin, and the content of baicalin is
Since the amount is extremely small, process management becomes easy.

Next, the highly purified baicalin obtained by the present invention is also optimal as a raw material for producing baicalein. That is, since the purity is high, high purity baicalein can be easily obtained by following Reference Examples 1 to 3 shown below.

"Reference Example" 1 g of high purity baicalin obtained in Examples 1 and 2
(1) Add 2 parts of 2N sulfuric acid, or (2) Add 2 parts of 2.5% hydrochloric acid, or (3) Add 2 parts of 2N alcoholic sulfuric acid.
2 hours in case (1) and 2 hours in case (2) by adding
For 2.5 hours, heat under reflux for about 3 hours in case of (3). However, in the case of refluxing using (3) above,
Next, after adding about 20 minutes of ice water, baicalein is precipitated. The precipitated baicalein is treated with polyamide, cellulose,
Sefadex LH-20, nylon 66, nylon
Baicalein is obtained by purification using either 12 or by recrystallization using methanol, benzene, etc.

"Reference Example 2" 1 g of high purity baicalin obtained in Examples 1 and 2
To this, add 200 ml of (4) 1% aqueous potassium hydroxide solution or 1% aqueous sodium hydroxide solution, and heat under reflux for 10 minutes. Next, add hydrochloric acid or sulfuric acid to make it acidic,
Precipitated baicalein is obtained, or the precipitated baicalin is separated, dissolved in a solvent such as ethyl acetate or ether, and then concentrated under reduced pressure to obtain baicalein.

On the other hand, instead of (4) above, (5) ethanol 100
ml, heated under reflux for 5 minutes, neutralized by adding hydrochloric acid or sulfuric acid, filtered, distilled off the solvent in the filtrate, added water to dissolve the residue, added hydrochloric acid or sulfuric acid, Acidic and eggplant. This gives precipitated baicalein. Baicalein obtained using (4) or (5) may be passed through polyamide or the like or recrystallized using methanol or the like according to Reference Example 1, if necessary.

"Reference Example 3" Baicalein can also be obtained by adding glucurondase to the high purity baicalein obtained in Examples 1 and 2 and hydrolyzing it. It is better to process it.

(Reference location) (1) Chemical and pharmaceutical
Bulletin Volume 8, Page 238 (1960):
(Chem.Pharm.Bull.,) (2) Journal of Biological Chemistry, Volume 12, Page 664 (1964) (3) Journal of Biological Chemistry, Volume 239, Page 59 (1964): (J.
Biol, Chem.) The charts of baicalin obtained in Example 1 or 2 or Reference Examples 1 to 3 by high performance liquid chromatography are shown in Figures 1 and 2. In terms of purity, when a standard curve of baicalin and baicalein (manufactured by Ichimaru Fulcos, Wako Pure Chemical Industries, Ltd.) is used to prepare a calibration curve, the purity is determined from 98% to 100%. %.

(High performance liquid chromatography conditions) Column: ODS column LS-410 (4.6φ x 250m/
m) Eluent: <Baicalin (Fig. 1)> Acetonitrile: Water = 32:68 0.005M tetra-n-aluminum ammonium iodide (PH = 4.0, phosphoric acid) <Baicalein (Fig. 2)> Acetonitrile: Water: 0.6% phosphoric acid aqueous solution = 43:
27:30 Eluent flow rate: 1.0ml/min Column temperature: 40℃ Detector wavelength: 280nm Detector sensitivity: 0.16AUFS

[Brief explanation of the drawing]

FIG. 1 shows high-performance liquid chromatography of high-purity baicalin obtained in Example 1 or 2.
Chart. FIG. 2 shows the results obtained in Reference Examples 1 to 3.
Chart of baicalein by high performance liquid chromatography.

Claims (1)

[Claims] 1. After dissolving crude baicalin in ice, cold water, hot water, or boiling water, high-purity baicalin in the form of needle-shaped crystals is precipitated by allowing it to stand for about a day and night, and this is fractionated. A method for purifying baicalin, characterized by: 2. After digesting defatted scutellariae with water, separate the filtrate, add hydrochloric acid to the filtrate so that the concentration is about 1%, and leave it for a day and night. The residue was separated, and a solution of the residue dissolved in methanol was passed through polyamide, cellulose, or Sephadex LH-20 to obtain a solution, and this solution was concentrated under reduced pressure. 2. The method for purifying baicalin according to claim 1, which uses crude baicalin.