JPH0959170A - Separation and purification of baicalin and baicalein - Google Patents

Abstract

PROBLEM TO BE SOLVED: To efficiently obtain baicalin and baicalein in a Chinese crude medicine such as Scutellaria root, etc., containing no admixture, by supercritical chromatography using a concentration gradient method with an alcohol and/or water as an auxiliary solvent. SOLUTION: An alcohol solution and/or an aqueous solution obtained by mixing and dissolving baicalin and baicalein is subjected to supercritical chromatography using a concentration gradient method with an alcohol and/or water as an auxiliary solvent. Ethanol is preferable as the alcohol and carbon dioxide is preferable as the supercritical fluid. Preferably, the amount of the auxiliary solvent is 5-20ml based on 1mg of baicalin and baicalein and that of carbon dioxide is 300-500ml in terms of economic efficiency. The temperature in the supercritical chromatography is 308.15-313.15K and the pressure is preferably 15-25MPa. The measuring flow velocity is adjusted to 5ml.minute<-1> , that of an eluting solution to 0.1-0.5ml.minute<-1> and the concentration gradient is prepared therefor.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for separating and purifying baicalin and baicalein in Chinese herbal medicines such as eugon using a supercritical fluid.

[0002]

2. Description of the Related Art Recently, physiologically active substances contained in plants and marine animals and plants have been attracting attention as raw materials for medicines. But,
Since these physiologically active substances have extremely similar physicochemical properties, separation and purification are not easy, and development of a separation and purification method for these substances on an industrial scale is desired. Conventionally, vacuum distillation method, extraction method, recrystallization method, etc. have been used for purification of these physiologically active substances, but these methods are sufficient fractionation for separation of substances with similar physicochemical properties. It was ineffective and purification of high concentration was difficult.

On the other hand, on the laboratory scale, purification of a physiologically active substance by high performance liquid chromatography (HPLC) has been studied, but since the eluent is used, the concentration of the target substance becomes extremely small and the target substance from the eluent is used. There has been a problem that a great amount of labor is required for the recovery and replacement of filling materials. Therefore, when liquid chromatography is used as an industrial separation means for physiologically active substances, the number of liquid chromatography is currently small.

On the other hand, extraction, separation, and
As a purification method, application of supercritical fluid extraction using a supercritical fluid that is easy to separate from solute as an eluent is considered promising (Japanese Patent Laid-Open No. 4-144655). In particular, carbon dioxide used as a supercritical fluid is generally non-toxic, and since its critical temperature is 304.2K, it can be operated at a low temperature of about 308.15K, and it is inexpensive, so many studies using it have been conducted. ing.

For example, as extraction from marine organisms, highly unsaturated fatty acids such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are separated from fish oil by supercritical carbon dioxide extraction using a temperature gradient method. Researches for purification (Japanese Patent Laid-Open No. 4-144655) have been conducted.

[0006] As the extraction of bio-related substances from plants, extraction of limonoids from citrus seeds by supercritical carbon dioxide extraction (Journal of Food Engineering, Vol. 39, No. 8,
(Pp. 684-689, 1992), Extraction of picifueric acid or its analogs from cypress family plants such as Shinobu Hiba and Sawara by supercritical carbon dioxide extraction (JP-A-62-270547), supercritical fluid extraction Leaves, stems, medicinal plants from South America by
It is known to extract oil containing γ-linoleic acid from roots in the presence of water and alcohol (JP-A-02-235996).

[0007] In the conventional supercritical carbon dioxide extraction, although the substance can be easily recovered, the separation of substances having similar physicochemical properties has a drawback that a sufficient fractionation ability cannot be obtained.

On the other hand, recently, studies have been conducted on fatty acid methyl esters by subjecting substances having similar physicochemical properties to separation and purification by supercritical chromatography combined with a temperature gradient method to recover the target substance. (J. Chromatography, Vol.605, 263-267, 1992).

On the other hand, there is a demand for extraction, separation and purification of active ingredients from Chinese herbs and their raw materials containing a large amount of various physiologically active substances, but effective separation and purification methods on an industrial scale have been established. That is not the case at present. For this reason, studies have also been conducted on the extraction, separation, and purification methods of baicalin and baicalein, which are contained in Aogon, which is a type of Chinese herbal medicine, and are widely used as antiallergic agents.

For example, a method of purifying baicalin by dissolving crude baicalin obtained from defatted sardine in water and boiling water for a whole day and night (Japanese Patent Publication No. 62-32173), sorghum which is a medicinal plant After adding water to Kizami, it is pretreated under a specific condition for a specific time to change baicalin into baicalein, and a method for extracting an extract containing a large amount of baicalein useful for medicines (Japanese Patent Publication No. 04-70286). ) Etc. have been reported.

[0011]

However, there are few reports of separating and purifying substances having similar physicochemical properties using the above-mentioned supercritical fluid from crude drug materials with high pharmaceutical demand, particularly baicalin and baicalein. There has been no report that the and were effectively separated and purified.

Therefore, an object of the present invention is to provide a method for industrially separating and purifying baicalin and baicalein, which are the medicinal components contained in a kind of Chinese herbal medicine such as augon, using a supercritical fluid. It is in.

[0013]

[Means for Solving the Problems] The inventors of the present invention have diligently studied the use of a supercritical fluid in order to solve the above problems. In particular, carbon dioxide, which is harmless to biological substances as a supercritical fluid, is used. As a result of diligent studies on separation and purification of baicalin and baicalein by supercritical chromatography using alcohol such as ethanol and water as cosolvents, a method capable of solving the above problems was found, and the present invention was completed.

That is, the method for separating and purifying baicalin and baicalein of the present invention is carried out by supercritical chromatography using an alcohol solution and / or an aqueous solution in which baicalin and baicalein are mixed and dissolved together with a concentration gradient using alcohol and / or water as a cosolvent. It is characterized by using a graph.

The alcohol as the cosolvent in the present invention is ethanol, 1-propanol or methanol, preferably ethanol. Carbon dioxide is suitable as the supercritical fluid.

[0016]

BEST MODE FOR CARRYING OUT THE INVENTION In the present invention, the amount of alcohol and / or water used is preferably 5 to 20 ml with respect to 1 mg of baicalin and baicalein, from the viewpoint of economic efficiency. In addition, the amount of carbon dioxide used is 300-500 ml for 1 mg baicalin and baicalein.
Is preferable from the viewpoint of economic efficiency.

In the present invention, the temperature for supercritical chromatographic separation is 305.15 to 323.15 K, especially 308.1.
5 to 313.15 K is preferable in terms of efficiently performing supercritical chromatography. Furthermore, the pressure for supercritical chromatographic separation is 7.2 to 30 MPa, especially 15 to 25 MPa.
It is preferable that the supercritical chromatograph can be efficiently performed.

Further, in the present invention, the eluent to be provided with a concentration gradient is a carbon dioxide measuring flow rate of 0.05 to 10 ml.
Adjust the flow rate to min ^-1 , preferably 5 mlmin ^-1.
0.01 to 1.0 ml ・ min ^-1 , preferably 0.1 to 0.5 ml ・ min ^-1
And adjust the concentration gradient. To establish such a concentration gradient, for example, the cosolvent flow rate is 0.5 mlmin ^-1 for the first 10 minutes.
At a flow rate of 20%, and 1 minute later, 0.5 ml · min ⁻¹ of 10
Set to flow at 0% flow rate.

The supercritical chromatographic apparatus used in the present invention will be specifically described below with reference to the drawings. As shown in FIG. 1, the supercritical chromatographic apparatus includes a pressure increasing section in which a path from the cylinder 1 to the stop valve V4 is formed, a column separating section provided downstream thereof, and a detecting section.

First, the booster unit will be specifically described.
The booster unit has two booster pumps 4A and 4B for carbon dioxide as a supercritical fluid and for an eluent. Further, the booster unit is equipped with a cylinder 1 for supplying a pump 4A for boosting liquid carbon dioxide. As the cylinder 1, a siphon-type liquid carbon dioxide cylinder can be used.

A drying pipe 2 filled with a desiccant is provided between the cylinder 1 and the pressurizing pump 4A, and liquid carbon dioxide from the cylinder 1 passes through the drying pipe 2,
Water in liquid carbon dioxide is removed. Such drying tube 2
As a carrier gas drying tube manufactured by GL Science Co., Ltd. (Material SUS316, maximum working pressure 20MPa, inner diameter 35.5mm, length
310 mm) can be used. Also, as a desiccant, GL Science Co., Ltd. molecular sieve 5A
(1/16 inch pellets) can be used.

Further, the booster unit is a cooling unit (cooler).
Equipped with 6. As such a cooling unit 6, a product name BL-22 manufactured by Yamato Scientific Co., Ltd. can be used. The cooling unit 6 is filled with ethylene glycol so that the ethylene glycol is cooled to about -261.15K. The liquid carbon dioxide from which water has been removed by the desiccant is cooled by this ethylene glycol and supplied to the pressurizing pump 4A.

The eluent is a sample bottle with a stopper (entrainer)
7 and install it upstream of the booster pump 4B. The eluent pressurized by the pressure boosting pump 4B passes through the stop valve V2, is mixed with the pressurized carbon dioxide in front of the stop valve V4, and is supplied to the column separation unit. A filter is installed at the sample inlet to prevent impurities such as dust from entering. As such a filter, GL Science
FT type replacement filter (FC-10 pore size 10 μm)
Can be used. Furthermore, for discharging the eluent,
Install stop valve V3. Such stop valve V
2. As V3, it is possible to use WHITEY bonnet-integrated flow control stop valve SS-0KS2BKB (product name).

The booster pumps 4A and 4B are high pressure single plunger pumps manufactured by GL Science Co., Ltd., trade name APS-5L (maximum pressure 58.8 MPa, normal pressure 49.0 MPa).
, With a flow rate of 0.5 to 5.2 ml · min ⁻¹ ) can be used. A cooler (not shown) is attached to the head portion of the boosting pump 4A to prevent vaporization of liquid carbon dioxide. Further, a filter 3A is provided between the drying pipe 2 and the cooling unit 6, and impurities such as dust are removed by the filter 3A to prevent the impurities from being mixed into the booster pump 4A. As such a filter 3A, a filter having an average pore size of about 10 μm (FT Science Model FT4-10 (trade name)) can be used.

Further, a pressure control valve is provided in the carbon dioxide pressure increasing section.
V1 is provided, and the pressure in the system of the pressure increasing section and the column separating section is set to an arbitrary pressure by this pressure control valve V1. The pressure control valve V1 is a product name of TESCOM
26-17 21-24 can be used. This pressure control valve V1 can control the pressure in the system with an accuracy within ± 0.1MPa, and the maximum working pressure is 41.5MPa.

A pressure gauge 5A is provided in the pressure increasing section, and the pressure in the system is measured by the pressure gauge 5A. An upper limit contact output terminal is attached to the pressure gauge 5A, and the pressure pump 4A is set to be turned off at a specified pressure. As the pressure gauge 5A, a Bourdon-type pressure gauge 5A manufactured by GL Science Co., Ltd. under the product name LCG-350 (maximum operating pressure 34.3 MPa) can be used. As the pressure gauge 5A, an economy pressure gauge manufactured by Jiken Co., Ltd., which is certified by PE-33-A (trade name, strain gauge type, accuracy ± 0.3%), can be used.

A stop valve V4 is arranged between the pressurizing section and the column separation section, and the outflow of the fluid in the column separation section can be controlled by the stop valve V4. As such a stop valve V4, 2 manufactured by GL Science Co., Ltd.
Directional valve, product name 02-0120 (maximum operating pressure 98.0 MPa)
Can be used.

Further, a safety valve 8A is provided between the booster section and the column separation section in order to ensure safety. As the safety valve 8A, a spring type valve made by NUPRO can be used. In this case, the pressure in the system is, for example,
Adjust and calibrate to operate at 34.3MPa. In the booster, except for the section from the cylinder 1 to the filter 3A, 1/16 inch stainless steel pipe (SUS316, outer diameter)
1.588mm, inner diameter 0.8mm), all other parts are 1/8
Inch stainless steel tube (SUS316, outer diameter 3.175mm, inner diameter 2.1)
7 mm) can be used.

Next, the column separation section will be described. The column separation unit is an air temperature chamber in the supercritical chromatograph.
Installed within 14. The internal volume of the constant temperature bath is 20 liters, and the temperature can be controlled with an accuracy of ± 0.1 K or less by, for example, the temperature controller DB1000 (trade name) of Chinault. The temperature measuring part is a platinum resistance temperature measuring device 1TPF48 made by Chino.
3 (trade name) can be used. The liquid carbon dioxide and the eluent supplied from the pressurizing section are sent to the preheating column 9. The preheating column 9 is for preheating a solvent (carbon dioxide) to an equilibrium temperature and converting it into a supercritical fluid.
1/8 inch stainless steel tube (SUS316, outer diameter 3.175 mm, inner diameter 2.
17mm, length about 4m) is transformed into a spiral shape with a diameter of 55mm and a length of 140mm and installed in a constant temperature bath.

The carbon dioxide made into a supercritical fluid by the preheating column 9 together with the eluent, the check valve 10 for preventing the backflow of the fluid.
(For example, Nupro SS-CHS4-10: Maximum working pressure 41.2MPa
) And is introduced into the column 13 for separating the sample to be extracted. In addition, an injector 11 is installed between the preheating column 9 and the separation column 13 to drive the sample. The injector 11 has a syringe loading sample injector (maximum operating pressure 48.1
MPa) can be used. The separation column 13 is, for example, a column of Superpak Crest C18T-5 (TP) manufactured by Nihon Bunko Kogyo Co., Ltd., and the packing has a total porosity that suppresses metal impurities with an average particle size of 5 μm as much as possible. A monomeric type filler obtained by chemically bonding an octadecyl group to the hydrophilic silica gel and then treating the residual silanol group can be used. The column has an inner diameter of 4.6 mm and a length of 15
A 0 mm one can be used.

The separation column 13 is installed in the hexagonal valve 12, and except when the separation operation is performed by the separation column 13.
The pipe can be cleaned by switching the hexagonal valve 12. In addition, stop valves V5 and V6 are provided to discharge the pressure in the column. Such a hexagonal valve
As 12, a high pressure switching valve manufactured by GL Sciences Co., Ltd. with a product name HPV-6 (maximum working pressure 34.3 MPa) can be used. In addition, as the stop valves V5 and V6, Whity's bonnet integrated flow control stop valve
You can use SS-0KS2BKB (trade name).

The pressure after passing through the column is measured by a pressure gauge 5B and adjusted to a set pressure by a flow rate control valve V7. The pressure gauge is of the Bourdon type, manufactured by GL Science Co., Ltd.
LCG-350 (maximum working pressure 34.3MPa) can be used, and the pressure gauge is certified by Jiken Co., Ltd. economy pressure gauge, trade name PE-33-A (strain gauge type, accuracy ± 0.3 % FS, F
S: kgf · cm ^-2 ) can be used.

Further, a safety valve 8B is installed on the upstream side of the column 13 for the purpose of preventing an explosion due to a pressure increase in the column. Safety valve 8B is made by Nupro (spring type, 177-R3AK
IG (trade name)) can be used, and the pressure in the system is
For example, adjust and calibrate so that it operates at 34.3 MPa.

Supercritical fluid (carbon dioxide) in which the sample is dissolved
Is discharged to the outside of the system using the flow rate control valve V7 (expansion valve). The flow control valve V7 is equipped with a back pressure regulator (880-81) manufactured by JASCO Corporation.
Type automatic pressure control valve, working pressure range 0 ~ 49.0MPa, pressure adjustment accuracy ± 2%) can be used. This valve is used to adjust the flow rate of the supercritical fluid and perform decompression operation. Further, in order to prevent clogging of the pipe due to condensation of the sample, the high-pressure filter 3B is installed on the upstream side of the flow rate control valve. The filter 3B has a product name of 2TF-7 manufactured by Nupro (average pore size of about 7 μ
m) can be used.

A heater is provided in the flow rate control valve V7 in order to prevent condensation of the sample due to depressurization and generation of dry ice due to the supercritical fluid (carbon dioxide). Further, by vibrating the outlet of the pipe, it is possible to prevent the pipe from being blocked by the precipitate.

After depressurizing with the flow control valve V7, the deposited sample is collected with the trap 16. In addition, the flow rate of supercritical fluid is
Measure from 17. As the flowmeter, an integrating wet gas meter manufactured by Shinagawa Seiki, with the product name W-NK-0.5B (measurement accuracy 0.1 ml) can be used. Since the target gas is carbon dioxide, the water in the flowmeter should be saturated with carbon dioxide before measurement.

Finally, the detecting section will be specifically described. The detector is installed upstream of the flow rate control valve V7, the sample in the supercritical fluid that has passed through the column is guided to the UV detector 15, and the absorption wavelength is measured. Then, it is discharged to the outside of the system by using the flow rate control valve V7 (expansion valve).
As the UV detector, a UV-970 type intelligent UV / VIS detector (trade name) manufactured by JASCO Corporation can be used. As the recorder, 807-IT type integrator (trade name) manufactured by JASCO Corporation can be used.

[0038]

[Examples] Using a supercritical chromatograph device (Nippon Bunko Kogyo Co., Ltd., SUPER-200 (trade name)) configured as described above, baicalin and baicalein are separated from an alcohol mixed solution of baicalin and baicalein, The case of purification will be specifically described based on Examples.

As alcohol as a cosolvent, ethanol and a special grade reagent (purity 99.7% or more) manufactured by Wako Pure Chemical Industries, Ltd. were used. Carbon dioxide (purity 99.5% or more) manufactured by Fukuoka Oxygen Co., Ltd. was used as the supercritical fluid.

The concentrations of baicalin and baicalein in ethanol were 1.046 mg / ml and 1.002 mg / ml, respectively, and the concentrations of baicalin and baicalein in the mixed solution were 0.523 mg / ml and 0.501 mg / ml, respectively. .
The operation method is shown below.

First, the column 13 shown in FIG. 1 was set at a predetermined position in the constant temperature bath 14. Next, with the valve V4 closed, carbon dioxide was supplied from the cylinder 1 to the pressure rising section, and the upper limit pressure of carbon dioxide was adjusted to 19.7 MPa by the pressure control valve V1.
At the same time, the pressurization of ethanol was started. Furthermore, the constant temperature bath
The temperature inside 14 was controlled to 313.15 ± 0.2 K by the temperature controller.

Next, with the stop valves V3, V5 and V6 closed, the valves V2 and V4 were opened and ethanol and carbon dioxide gas were sent to the column separation section. Then, the entire system was pressure-adjusted to the operating pressure (19.7 MPa), and after the pressure became constant, the detection wavelength of the detector was set to 275 nm.

Next, after allowing the apparatus to stand for 10 minutes to stabilize, 20 μl of a sample was injected from the injector 11 with a microsyringe to start separation of baicalin and baicalein. At this time, the flow rate control valve V7 was used to adjust the measurement flow rate of carbon dioxide to 5.0 ml · min ⁻¹ .

On the other hand, the cosolvent flow rate is 0.5 ml.min for 10 minutes.
^-1 at a flow rate of 20%, and 1 minute later 0.5 ml ・ min ^-1
The flow rate was adjusted to 100% to give a concentration gradient. The UV detector confirmed that the peaks of baicalin and baicalein were divided into two by applying a concentration gradient. Further, in the trap 16, a sample (baicalin, baicalein, alcohol and water) deposited from carbon dioxide having lost the dissolving power was collected.

The conditions for the supercritical chromatographic separation operation in the examples are summarized in Table 1 below.

[Table 1]

FIG. 2 shows the relationship between the retention time of baicalin and baicalein separated by the above supercritical chromatographic separation operation and the peak height of the UV detector. Further, FIG. 3 shows the results of supercritical chromatographic separation under the same conditions as described above except that the eluent was not provided with a concentration gradient.

From FIG. 2, when the concentration gradient is applied to the eluent, the retention times of baicalin and baicalein are significantly different (baicalin: 13.425 minutes, baicalein: 2.275 minutes). Therefore, supercritical chromatography was used. It was shown that baicalin and baicalein can be separated and purified. The recovery rates of baicalin and baicalein in this operation were both 95% or more.

On the other hand, from (a) and (a) of FIG. 3, the retention times of baicalin and baicalein are close to each other (baicalin: 1.433 minutes, baicalein: 0.992 minutes) when the concentration gradient is not applied to the eluent. ),
It was shown that it is difficult to separate and purify baicalin and baicalein.

[0049]

As described above, in the method for separating and purifying baicalin and baicalein of the present invention, impurities are contained by using a supercritical chromatography with a concentration gradient in the eluent. Without
Baicalin and baicalein can be efficiently separated and purified.

[Brief description of drawings]

FIG. 1 is a schematic configuration diagram of a supercritical chromatography device used for separating baicalin and baicalein according to the present invention.

FIG. 2 is a graph showing the relationship between the retention time of baicalin and baicalein and the peak height when a concentration gradient is applied to the eluent.

FIG. 3 is a graph showing the relationship between the retention time of baicalin and baicalein and the peak height when the eluent has no concentration gradient.

[Explanation of symbols]

 1 cylinder 2 drying pipe 3A, 3B filter 4A, 4B boosting pump 5A, 5B pressure gauge 6 cooling unit (cooler) 7 sample bottle (entrainer) 8A, 8B safety valve 9 preheating column 10 check valve 11 injector 12 6-way valve 13 Separation column 14 Constant temperature bath 15 UV detector 16 Trap 17 Flow meter V1 Pressure control valve V2-V6 Stop valve V7 Flow control valve

Claims (1)

[Claims] 1. Separation of baicalin and baicalein from an alcoholic solution and / or an aqueous solution in which baicalin and baicalein are mixed and dissolved, using supercritical chromatography in which a concentration gradient is used in combination with alcohol and / or water as a cosolvent. , Purification method.