JPH06206891A - Method for extracting saikosaponin - Google Patents

Abstract

PURPOSE:To extremely simply obtain an extracting method capable of collecting high-purity saikosaponin in a short time without using an expensive material and a large amount of solvent. CONSTITUTION:The method for collecting saikosaponin is characterized by extracting a plant of the genus Bupleurum with a water-containing lower alcohol, concentrating the extract and depositing saikosaponin.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention is directed to Bupleurum fa ( Umbelliferae ), which is important as a medicinal plant.
lcatum ) and other methods for extracting and purifying saikosaponin from plants belonging to the genus Lima .

[0002]

2. Description of the Prior Art Psychosaponins extracted from plants belonging to the genus Mishima are mainly Psychosaponins a, c and d.
It is divided into It is known that these saikosaponins and their derivatives have various pharmacological actions, and in particular, regarding saikosaponin d, recent studies have confirmed anti-inflammatory, anti-allergic, anti-tumor, etc. activities, and their usefulness. It is rising. The conventional method of collecting saikosaponin requires the use of partition extraction method and various column chromatography methods, which requires a lot of time and labor and inevitably requires the use of an expensive column or the like. Also, it is not industrially preferable because it requires a large amount of solvent. For example, a 60% (v / v: hereinafter, unless otherwise specified, "%" means "v / v") methanol extract (reflux for 2 hours / twice) extracted with Diaion (HP) There is a method in which it is adsorbed to -20), washed with 50% methanol, and then eluted with 90% methanol. However, since this method can obtain only a fraction containing about 30% of saikosaponin, further purification by column is required. In addition, a large amount of solvent is required, which is not an advantageous method. Therefore, a simple collection method is required, but at present, as such a method, for example, 60% of the plants of the genus Mishima
The method is limited to a method in which the extract is dried under reduced pressure after extraction with methanol. However, such a method has a problem that the content of saikosaponin in the obtained dried extract is not sufficient and the purity is low, so that further purification is required depending on the application.

[0003]

Therefore, a method for collecting saikosaponin, which does not require an expensive column or a large amount of solvent and can easily separate and purify high-purity saikosaponin in a short time, has not yet been found. It was not done, and its development was requested.

[0004]

Under such circumstances, as a result of various investigations by the present inventors, it is expected that the plant of the genus Mishima genus will be extracted with hydrous lower alcohol and the extract will be concentrated to recover the precipitate. It was found that highly pure saikosaponin can be collected extremely easily and in a short time without using expensive materials or a large amount of solvent, and based on this, the present invention was completed. That is, the present invention provides (1) a method for collecting saikosaponin, which comprises extracting a plant belonging to the genus Mishima genus with hydrous lower alcohol, concentrating the extract, and precipitating saikosaponin, (2) extracting under alkaline conditions The method for collecting according to item (1), (3) the method for collecting according to item (1) using leaves of the plant of the genus Lima, and (4) methanol having a concentration of 40% or more as a lower alcohol. (5) The method according to (1), wherein the extract is concentrated, and water at a temperature 10 ° C or more higher than the liquid temperature is added to precipitate the saikosaponin. Collection method described in section
(6) Extraction was performed with 40 to 70% (v / v) methanol in an amount of 1 to 10 times the weight of the plant of the genus Mishima, and the extract was concentrated to a methanol concentration of 5 to 30% (v / v). After that, 1 to 10 times the volume of the concentrated solution is added to water to precipitate the saikosaponin, and the sampling method according to item (1),

Specific examples of the plant belonging to the genus Mishima used in the present invention include Bupleurum falcat.
um ), Kyushu Psycho ( Bupleurum falcatum va
r. kiusianum ), Manshumi Shima Psycho ( Bupleurum )
chinese ), Hosoba Mishima Psycho ( Bupleurum scorzone
raefolium ), Daphlia Psycho ( Bupleurum euphorbioid )
es ), tofu ( Bupleurum jeholense ) and the like are used, and preferred among them are, for example, Mishima-Psycho and Kyushu-Psycho, and more preferred are, for example, Mishima-Psycho. For example, when using a plant belonging to the genus Mishima genus cultivated by a normal cultivation method, it is preferable to use a plant of the first to fourth year grade, preferably the first to second year grade. For example, the hydroponic method described in JP-A-01-160440 and the like ( In the following, when using a plant of the genus Mishima cultivated by the "hydroponics method", 2 to 6 months,
Preferably, the one cultivated for 3 to 4 months may be used.
In the extraction of the present invention, the whole plant can be used, but preferably leaves or roots can be used. Since the leaves contain more saikosaponin d than the roots, it is preferable to use the leaves when it is desired to mainly recover the saikosaponin d, and to use the roots otherwise. Washing plants of the genus Lynx can be used for extraction without any special treatment if they are washed (for example, sufficiently washed with water) according to a conventional method, but after washing, the dried powder is used. Is preferred. Examples of the lower alcohol of the hydrous lower alcohol used for extraction include methyl alcohol, ethyl alcohol, propyl alcohol, isopropyl alcohol, n-butyl alcohol,
Alcohols having 1 to 6 carbon atoms such as i-butyl alcohol, s-butyl alcohol and t-butyl alcohol are used, preferably carbon having a boiling point of less than 100 ° C. such as methyl alcohol, ethyl alcohol, propyl alcohol and isopropyl alcohol. Alcohol of number 1 to 4,
More preferably, for example, methyl alcohol, ethyl alcohol and the like are used, and especially methyl alcohol and the like are frequently used. The concentration of alcohol used for extraction varies depending on the alcohol used, but is usually 25% or more, preferably 35 to 90%, more preferably 50 to 75%. Specifically, for example, the concentration of methanol used is usually 40% or higher, preferably 50 to 70%, more preferably 55 to 65%. The extraction is preferably carried out under alkaline conditions in order to prevent the saikosaponin from being denatured by the organic acids emerging from the plant. Extraction usually takes place at a pH of 8.0 to 13. Although it depends on the weight ratio of the plant to the hydrous lower alcohol and the concentration of the hydrous lower alcohol.
0, preferably for example 10.0 to 12.0, more preferably pH 10.5 to 11.5. In reality, for example, 25
% Ammonia water 0.5 to 3%, preferably 1.5 to 2
%, For example, 0.5 to 3%, preferably 1.5 to 2% of an alkali metal hydroxide such as potassium hydroxide or sodium hydroxide.
%, Preferably in the extraction solvent. The ratio of the water-containing lower alcohol used varies depending on the kind of the water-containing lower alcohol, but usually 0.1 to 1 dry weight of the plant is used.
To 100 times (weight), preferably 0.5 to 50
Double amount is used, and more preferably 1 to 25 times amount is used. Specifically, for example, in the case of methanol, 1 to 50 times the dry weight of the plant can be used,
The amount used is preferably 3 to 20 times, more preferably 5 to 10 times, and particularly preferably about 7 times. As the extraction method, known means generally used in the extraction of active ingredients in plants is applied. Specifically, the extraction may be carried out by leaving it in a cool dark place, but it may be carried out under reflux or under ultrasonic waves, preferably under reflux. Further, in order to avoid denaturation by light, it is preferable to perform extraction in a dark place. The temperature at the time of extraction varies depending on the type and proportion of alcohol in the extract, but it may be a temperature above the temperature at which the extract does not freeze and below 150 ° C, preferably a temperature above the temperature at which the extract does not freeze and below 100 ° C. Is performed, more preferably at a temperature of 0 ° C to 90 ° C, particularly preferably at a temperature of 4 ° C to 70 ° C. However, depending on the kind of saikosaponin, there are some that are easily decomposed at high temperature (for example, saikosaponin d etc.), so if you want to extract a large amount of such saikosaponin, you should extract at a temperature not exceeding 70 ° C. preferable. The obtained extract is preferably filtered to remove the plant body used for extraction before depositing saikosaponin.
As the filtration method, for example, centrifugal filtration, suction filtration, etc. are used, for example, centrifugal filtration is used when a large amount is desired to be filtered, and suction filtration is used when a small amount is simply to be filtered. As a concentration method for subsequently concentrating the thus obtained extract, for example, a known concentration method such as a vacuum concentration method is used, and preferably a rotary vacuum evaporator is used, for example. The temperature at the time of concentration varies depending on the type and proportion of alcohol in the concentrate, but it may be a temperature above the temperature at which the concentrate does not freeze and below 150 ° C, preferably a temperature above the temperature at which the concentrate does not freeze and below 100 ° C. And more preferably at a temperature of 0 ° C or higher and 90 ° C or lower,
Particularly preferably, it is performed at a temperature of 4 ° C. or higher and 70 ° C. or lower.
However, depending on the type of saikosaponin, there are some that are easily decomposed at high temperature (for example, saikosaponin d etc.), so if you want to extract a large amount of such saikosaponin, concentrate at a temperature not exceeding 70 ° C. preferable. The concentration is carried out until the methanol concentration of the extract becomes usually 2 to 50%, preferably 3 to 30%. Psychosaponin may precipitate during concentration, but it is preferable to leave it after concentration in order to completely precipitate it. Furthermore, it is more convenient to add water to the concentrate to precipitate the saikosaponin. The volume ratio of the concentrate to water when water is added to the concentrate to cause precipitation is 1: 0.1 to 1:20, preferably 1: 0.5 to 1:10, and more preferably Is performed from 1: 1 to 1: 5. The water temperature used for precipitation may be the same as the temperature of the concentrated liquid, but it is preferable to use a water temperature higher than the liquid temperature of the concentrated liquid by 10 ° C or more, more preferably 10 ° C or higher than the liquid temperature of the concentrated liquid. 30 ~
The temperature is in the range of 70 ° C., and particularly preferred is water having a temperature in the range of 45 to 55 ° C. which is higher than the liquid temperature of the concentrated liquid by 10 ° C. or more. This is because when water at the same temperature as the concentrate is used, the precipitate will be suspended in the whole liquid, and the whole liquid must be filtered or centrifuged to recover the precipitate, When water with a temperature higher than 10 ° C is used, most of the precipitates rise to the liquid surface, so collecting the rising precipitates is simpler and the recovery labor can be greatly reduced. is there. Further, if most of the precipitates can be collected in advance in this manner, the filter paper is less likely to be clogged when the remaining precipitates are collected by filtration, so that efficient collection is possible.

[0006]

Since the precipitate obtained by the method of the present invention contains a high concentration of saikosaponins, it can be used as it is as a raw material for medicines, cosmetics, soaps, etc. It may be further purified before use. Psychosaponin is an important raw material as an active ingredient of medicines, cosmetics, soaps and the like, and is used, for example, as a raw material of medicines according to a method known per se or a method analogous thereto (for example, Japanese Patent Publication No. 6690/1987). 60-25933, JP-A-60
166623, etc.). In particular, since the saikosaponin obtained by the method of the present invention contains a large amount of saikosaponin d, it is preferably used for anti-inflammatory, anti-allergic, anti-tumor, etc. applications known to be effective (for example, JP-A-1-160440). ). The saikosaponin obtained by the method of the present invention is a known pharmaceutically acceptable excipient (eg starch, gum arabic, carboxymethyl cellulose, crystalline cellulose etc.), lubricant (eg magnesium stearate, talc etc.), Oral dosage forms such as capsules, powders, fine granules, granules, tablets, etc. by mixing with disintegrants (eg carboxymethyl calcium, talc etc.) and parenteral administration of ointments, poultices etc. The dosage form can be The dose is, for example, 2 to 400 mg per day for an adult as a psychosaponin d when orally administered as an anti-inflammatory agent, and 1 to 20 when parenterally administered.
It is 0 mg.

[0007]

EXAMPLES The present invention will be described in more detail with reference to Examples and Comparative Examples, but the present invention is not limited to the following Examples unless it exceeds the gist. The symbols used in Examples and Comparative Examples have the following meanings. S. a: Saikosaponin a, S. c: Saikosaponin c,
Sd: saikosaponin d, kg: kilogram, mg: milligram, ml: milliliter, Example 1 5 kg of dried crude powder of Mishima Psycho leaf subjected to hydroponics, 1.5% of ammonia water, 60% of 35 liters Extraction was performed by leaving it to stand in a cool dark place with methanol. The extraction temperature was 4 ° C and the number of extraction days was 14 days. It was obtained by filtering 87.
5 ml of the extract was concentrated using a rotary vacuum evaporator (manufactured by Tokyo Rika Kikai Co., Ltd., NE-1R type) until the concentration of methanol reached 30% at 60 ° C. for about 15 minutes, and 450 ml of 18 ° C. After adding water and mixing well, the precipitated insoluble matter was collected on a filter paper (Advantech No. 1, manufactured by Advantech) with a Buchner funnel.
100% methanol was poured into the filter paper after filtration to separate the filtrate, which was then filtered through a glass filter (17G3, manufactured by Iwaki Glass Co., Ltd.). This filtrate is concentrated to dryness, and Mishima Psycho Extract 1
01 mg was obtained. The concentration of saikosaponin in this extract (Sa, Sc, Sd in the total weight of the extract and wt% of the total amount thereof) and recovery rate (Sa, S. in the extraction material).
[Table 1] shows Sa, Sc, Sd and the total amount thereof in the obtained extract when the contents of c and Sd are 100%. The content of saikosaponin was measured by HPLC (High Performance Liquid Chromatography (High P
performance liquid chromatography), hereinafter abbreviated as HPLC).

[Table 1] Concentration and recovery rate of saikosaponin in the extract ───────────────────────────────── S.a S. c S.d Total ──────────────────────────────── Saponin concentration 2.3 0 73.3 75.5 Recovery Rate 20.6 0 84.5 73.8 ──────────────────────────────────

Examples 2 to 6 87.5 ml of the extract obtained in the same manner as in Example 1 was used as a rotary vacuum evaporator (NE-1R type manufactured by Tokyo Rika Kikai Co., Ltd.) until the methanol concentration reached 10%.
Was concentrated at 60 ° C. for about 20 minutes, and 1, 3, 6 and 9 times the amount of water at 18 ° C. were added, and the mixture was stirred well. The precipitate was recovered as an extract in the same manner as in Example 1. The content of saikosaponin in these extracts was measured using HPLC in the same manner as in Example 1. The concentration and recovery rate are shown in [Table 2].

[Table 2] Concentration and recovery rate of psychosaponin in the fraction ────────────────────────────────── Example Water amount Weight Psychosaponin concentration (%) Recovery rate ────────────────────── (No.) (times) (mg) S.a.S.c.S. d Total (%) ────────────────────────────────── 2 0 56 1.1 0.0 71.2 72.3 61.9 3 1 59 1.3 0.0 73.9 75.2 66.9 4 3 61 2.1 0.0 73.7 75.8 70.2 5 6 60 2.0 0.0 80.8 82.8 71.1 6 9 60 1.2 0.0 78.3 79.5 70.1 ───────────────────── ─────────────

Example 7 87.5 ml of the extract obtained in the same manner as in Example 1 was used as a rotary vacuum evaporator (NE-1R type manufactured by Tokyo Rika Kikai Co., Ltd.) until the methanol concentration reached 10%.
Concentrate for about 20 minutes at 60 ° C using
Cooled to ° C. 50 ml of this concentrated liquid is added to water at 6 ° C or 50
Each 150 ml of water at ℃ was added and well stirred, and the floating state of the precipitate was observed. A liquid containing this precipitate was used in Example 1.
The mixture was filtered in the same manner as in (1) and concentrated to dryness to obtain a Mishima psycho extract (first extract). Further, the filtrate was allowed to stand in a cool dark place at 4 ° C. for 4 hours, and the precipitates recognized again were similarly concentrated to dryness to obtain an extract (second extract). The content of saikosaponin in these extracts was adjusted by HP in the same manner as in Example 1.
It was measured using LC. The results are shown in [Table 3].

[Table 3] ────────────────────────────────── Water used Weight temperature of saikosaponin concentration (%) ──────────────────── (℃) (mg) S.a S.c S.d Total ─────────────── ──────────────────── 6 (1st extract) 64 0.0 0.0 63.4 63.4 (2nd extract) 31 0.0 0.0 56.1 56.1 ───────── ───────────────────────── 50 (1st extract) 82 1.1 0.0 63.4 64.5 (2nd extract) 16 0.0 0.0 19.9 19.9 ──── ────────────────────────────── [Table 3] As a result, after the extract was concentrated, the temperature was higher than the temperature of the extract by 10%. In the method of the present invention in which water having a temperature higher than ℃ (that is, water having a temperature of 50 ℃) is added, the temperature of the extract and It can be seen that the saikosaponin can be efficiently obtained by one precipitation as compared with the case of adding water at the same temperature (that is, water at 10 ° C.).

Example 8 200 g of dried powder of Mishima psycho or 200 g of dried powder of root were placed in 2.1 liter of 60% methanol containing 1.5% of ammonia water in a cool dark place at 4 ° C. for 6 days or 2
It was refluxed for a time (Examples 10 and 12) and extracted. These were rotary vacuum evaporators (manufactured by Tokyo Rika Kikai Co., Ltd., NE-1R) until the methanol concentration reached 10%.
Type) for about 80 minutes at 60 ° C.
The mixture was cooled to 4 ° C, and water at about 50 ° C, which was 3 times the amount of the concentrated solution, was added and stirred. The suspended precipitate was collected by filtration,
The saikosaponin concentration was adjusted to HP by the same method as in Example 1.
It was measured using LC.

[Table 4] ─────────────────────────────── Psychosaponin concentration (%) ────────── ────────── Extraction site Extraction method S.a S.c S.d Total ─────────────────────────── ───── Leaves standing 4.42 0.0 78.15 82.59 Reflux 4.09 0.0 80.69 84.78 Root standing 14.45 3.35 37.38 55.18 Reflux 9.39 2.12 31.27 42.78 ───────────────────── ────────── [Table 4] As a result, when the leaves were extracted, a precipitate containing about 80% of S.d was obtained in both static and reflux extractions, and the roots were statically extracted. At times, a precipitate containing about 50% of Sa, Sc, and Sd was obtained.
It can be seen that Sd is efficiently recovered.

[0011]

INDUSTRIAL APPLICABILITY The method of the present invention does not require an expensive column, a large amount of solvent, or the like, is easy to separate the saikosaponin, and can easily obtain a high-concentration saikosaponin in a short time. Provide a collection method.

Claims (6)

[Claims] 1. A method for collecting saikosaponin, which comprises extracting a plant belonging to the genus Mishima genus with hydrous lower alcohol, concentrating the extract, and precipitating saikosaponin. 2. The collection method according to claim 1, wherein the extraction is performed under alkaline conditions. 3. The collecting method according to claim 1, wherein leaves of a plant belonging to the genus Lynx are used. 4. A concentration of 40% (v / v) as lower alcohol
The method of claim 1, wherein the methanol is used. 5. The collecting method according to claim 1, wherein after concentration of the extract, water at a temperature higher than the liquid temperature by 10 ° C. or more is added to precipitate the saikosaponin. 6. The weight of the plant of the genus Mishima is 1 to 1
After extracting with 0-fold volume of 40-70% (v / v) methanol and concentrating until the methanol concentration becomes 5-30% (v / v), the volume of the concentrate is 1-10 times volume. The extraction method according to claim 1, wherein the saikosaponin is precipitated by adding water.