JPH02247196A - Recovery of purified saponin from asparagus - Google Patents

Abstract

PURPOSE:To obtain the subject compound having high purity by immersing crushed asparagus in an alcohol, defatting the obtained concentrated extract, extracting with a solvent, treating by column chromatography to separate foreign materials and repeating the above procedures. CONSTITUTION:A lower alcohol such as methanol is added to the edible part of preferably a white asparagus and to the root and stalk of the asparagus and crushed with a mixer, etc. The crushed asparagus is immersed at room temperature in a state left at rest and the solvent is distilled off from the resultant extract to obtain a concentrated extract. A solvent such as benzene, ether, etc., is separately added to the extract and the defatting treatment is repeated once or more. The treated liquid is added with e.g. a 1:1 mixture of n-butane and water to extract the objective compound to the n-butane layer and the extract is passed through the column of a silica gel column chromatography to separate the foreign materials. The objective compound is produced by treating the product by LH-20 column chromatography.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention relates to a method for recovering saponin from asparagus in a purified form, and particularly relates to recovering high-grade saponin components with few impurities. .

[Conventional technology]

It has been well known that saponins are contained in plants. In general, saponin is a glycoside whose aglycone is a complex alicyclic compound, and is a general term for a group of plant components whose aqueous solutions exhibit oxidative properties, fish toxicity, and hemolytic properties. Additionally, animal saponins are known, such as sea cucumber and starfish saponins.

Subsequent research revealed the properties of saponins themselves, and they were broadly classified into steroid saponins and triterpenoid saponins.

A solvent extraction method is known as a method for recovering the above-mentioned saponin components from these plants and animals.

An extraction method is also known as a means of recovering saponin from white asparagus, an edible plant;
Since this method uses the edible part of asparagus as the raw material, a large amount of edible white asparagus must be used.
There was a cost problem.

As an example of a means for solving such a problem, the present inventors
Previously, in Japanese Patent Application No. 63-279349 (method for recovering saponin from asparagus waste), asparagus stems and roots, which were conventionally considered waste, were used as raw materials to produce saponin at low cost and in large quantities. A method for recovering the components has been disclosed.

However, subsequent research revealed that the saponin obtained by this method contained some impurities (impurities) that were inconvenient for practical use, and there was a desire to develop a means to remove these impurities.

[Problem that the invention seeks to solve]

As mentioned above, there is no conventional saponin recovery method to obtain purified, high-quality saponin in large quantities at low cost, and commercially available saponin is therefore expensive and contains many impurities. Ta.

[Means for solving problems]

The present inventors have conducted extensive research to solve the above-mentioned problems, and have used asparagus, an edible plant (white asparagus or green asparagus), which is relatively inexpensive and available in large quantities, as a raw material, and in the extraction process. We succeeded in developing a saponin recovery method that can obtain purified saponin with high purity by combining the purification process using column chromatography.

That is, the present invention is a method for purifying and recovering saponin components in asparagus, which comprises: a first step of pulverizing asparagus as a starting material and immersing it in alcohol to obtain a concentrated extract; A second step of defatting the concentrated extract by treating it with one or more solvents or alcohols; After adding a solvent to the degreasing process liquid obtained in the second step to extract saponin components into the organic layer, A method for recovering purified saponin from asparagus is provided, which comprises a fourth step of passing the crude purified liquid obtained in the third column chromatography step through column chromatography again to separate impurities.

[For production]

The raw materials used in the present invention are the edible parts of white asparagus and green asparagus, as well as the roots and stems, but white asparagus contains more saponin components than green asparagus. It has been confirmed that it contains.

The outline of the method of the present invention will be explained below according to the flow sheet shown in FIG.

As the alcohol used in the first step of the present invention, lower alcohols such as methanol and ethanol can be used, but in the examples described below, an example of obtaining a concentrated extract by immersing in 99% methanol is shown.

Conventional extraction methods require steps such as heating to several tens of degrees or stirring in order to increase extraction efficiency, but in the method of the present invention, the process is simply immersed at room temperature and in a stationary state. That's fine.

In the second step, the extracted extract is defatted, and this can be done at least once by separately adding at least one of organic solvents such as benzene, ether, and chloroform, or by using the same or different solvents. te2
This can be achieved by removing lipids and impurities in the extracted extract by treating it multiple times.

That is, in this case, degreasing may be performed at least once using only benzene as the organic solvent, but in order to perform complete degreasing, ether or chloroform may be used.
It was confirmed that even better results could be obtained if each sample was degreased at least once.

In the third step, an organic solvent is added to the degreasing process liquid obtained in the second step, and further processing is performed for purification. That is, in this step, for example, n-butanol and water (1:
Using the mixed solvent of 1), extract the saponin component into the n-butanol layer, and then extract the saponin component into the n-butanol layer (Pra.l).
The saponin fraction (Pra, Ii) and impurities (Pra, 1.2
) and separate.

In the fourth step, in order to further remove impurities in the saponin, a saponin mixture (Fra.

1.1.1) and impurities (Pra, 11.2) to recover purified saponin with almost no impurities.

Hereinafter, the present invention will be explained in detail with reference to Examples.

〔Example〕

As a raw material, we weighed 70.0g of dried raw white asparagus (consisting of edible parts, stems, and roots),
To this was added 300ml of 99% methanol.

These were ground using a food mixer (National MXM3) to obtain a mixture.

Next, this mixture was put into an Erlenmeyer flask and heated for 1 hour at room temperature.
After allowing the mixture to stand for 0 hours to extract saponin, the extract was collected into a suction filtration bottle by suction filtration.

Add 300ml of 99% methanol to the extraction residue again.
After stirring, the mixture was allowed to stand at room temperature for 10 hours for extraction. This extraction operation was repeated a total of 5 times to obtain 1.2 g of extracted product.
This was concentrated using a rotary evaporator and the solvent was distilled off to obtain about 35 g of a brown concentrated extract (first step).

Next, add 50 ml of water to the concentrated extract and shake well to suspend, then add 50 ml of benzene and suspend.
A milky white solution was obtained. This solution is centrifuged for 1
Centrifuge at 10,000 rpm for 30 minutes, remove the lipid components in the extract by tilting the centrifuge tube and discarding the upper layer of benzene (or you can remove only the upper layer by sucking it up with a pipette). I did it. Add another 50ml of benzene to the remaining water layer and repeat the same operation as above to further degrease. Next, repeat the same procedure as above except that 50 ml of ether was added instead of benzene to perform degreasing with ether.After repeating this twice, 50 ml of chloroform was added instead of ether.
In addition, the same process was repeated, and degreasing with chloroform was performed twice in total (second step).

These operations may be performed once for each extraction solvent, or
Although it is sufficient to perform the process twice with benzene, in order to recover saponin with fewer impurities, in this example, as described above,
Degreasing was carried out twice each using benzene, ether and chloroform.

Next, water was distilled off from the obtained aqueous layer using a rotary evaporator, and the water was concentrated and dried.
Approximately 100 ml of a mixed solvent of butanol and water (1:1, l) and the aqueous layer (Pra,
2) was obtained.

The obtained butanol layer (Pra, 1) was then concentrated to dryness using a rotary evaporator to obtain 2.4 g of crude saponin. This was dissolved in a solvent of methanol:water (10:1) and applied to a silica gel column equilibrated with a solvent of the same composition. By elution with a solvent of the same composition, 1.4 g of saponin fraction (Pra, 1.1) was obtained, and impurities (Pra, 1.2) were removed (third step).

The results of TLC analysis of the saponin fraction (Pra, 1.1) are shown in FIG. 3, and 1m-2 of the impurities Im-1 and lm-2 shown in FIG. It turns out there wasn't.

The saponin fractions obtained in the third step were then collected, concentrated using a rotary evaporator, and passed through an LH-20 column chromatograph to remove impurities (Pral, 1.2) and form a pure saponin mixture (Pral, 1.2).

1.1.1) was obtained (4th step).

The results of TLC analysis of the saponin mixture (Fra, 1.1.l) are shown in FIG. From this figure, it can be seen that 1 m -2 of impurities that could not be removed in the third step were removed.

As described above, 0.24 g of the saponin mixture was finally obtained, and the proportions of the saponin mixture in each step were as shown in Table 1.

Table 1 Weight (g) % Material 70 100Pra,
1 2.4 3.4Fra, 1
．． 1 1.4 2.0 Pra, 1
．． 1. 1 O, 240, 34 A comparison of the TLC analysis results for each of these fractions is shown in FIG.

From these results, the saponin mixture finally obtained is S
It can be seen that it is a saponin of -2 to S-5. It is also understood that the above method results in a saponin mixture that is substantially free of impurities.

[Comparison example]

The following test was conducted to compare whether there are differences in the purification degree and recovery efficiency of saponin depending on the type of asparagus used as a raw material.

Raw white asparagus and raw green asparagus (consisting of edible parts, stems, and roots) as raw materials were each dried and weighed 70.0 g, and a concentrated extract was obtained in the same manner as in Example 1 ( After defatting these extracts (second step), n-butanol and water (1:
]) was added to dissolve the extract, and the mixture was allowed to stand overnight in a separatory funnel to extract saponin components into the butanol layer.

The butanol fraction was added to a silica gel plate such as a thin layer chromatography plate, developed with a mixed solvent of chloroform:methanol:water (70:30:5), and then anisaldehyde-sulfuric acid solution was sprayed as a coloring agent. After, 70
The results obtained after heating at .degree. C. for several minutes are shown in FIG. 2(I).

Further, the results obtained by spraying the Ehrlich reagent as a coloring agent and heating it for several minutes at 70° C. are shown in FIG. 2 (If).

Furthermore, after spraying a 10% sulfuric acid solution as a coloring agent,
The results obtained by heating at C for several minutes are shown in FIG. 2(m).

]　2 The above test results revealed the following.

That is, in an example using white asparagus as a raw material, when anisaldehyde reagent is used, S-1 to S- are the main saponins as shown on the left side of Figure 2 (I).
In addition to 5 types of saponins, trace amounts of 38 other saponins were also recognized.

In addition, when Ehrlich reagent is used, Fig. 2 (I
As shown on the left side of I), 10 types of saponins believed to be furostanol type were observed.

On the other hand, in an example using green asparagus as a raw material, when anisaldehyde reagent is used, the main saponins are S-2 and S-4, as shown on the right side of Figure 2 (I).
, S-5, and Ehrlic
In the case of using reagent h, as shown on the right side of FIG. 2 (II), only one type of saponin, which appeared to be furostanol type, was observed.

In addition, as shown on the left and right sides of Fig. 2(m), F
ra, W-1 (white asparagus) and Pra
, G-1 (green asparagus) were confirmed to contain major impurities 1 m-1 and 1 m-2 and some other small amounts of impurities.

From the above, it was confirmed that white asparagus is more suitable than green asparagus as a raw material for saponin extraction.

〔Effect of the invention〕

As described above, the saponin component obtained by the purified saponin recovery method of the present invention has extremely high purity compared to conventional products. Furthermore, since it can be purified by simple means, it can be said to be a preferable method from an industrial standpoint.

[Brief explanation of drawings]

FIG. 1 is a flow sheet of the method of the present invention. Figure 2 (1) (n) (m) shows the analysis results of the butanol fraction by TLC method when white asparagus was used as the raw material (left side) and when green asparagus was used as the raw material (right side). FIG. FIG. 3 is an analysis diagram showing the analysis results of the silica gel chromatography fraction by TLC method. Figure 4 shows TLC
FIG. 2 is an analysis diagram showing the analysis results of LH-20 Lamb chromatography fractions according to the method. Figure 5 shows F by TLC method.
ra, 1. Fra, 1.1. Pra, 1.1
．． FIG. 1 is an analysis diagram showing a comparison of the analysis results of No. 1 and Pra, 1.1.2.

Claims (1)

[Claims] The following first to fourth steps: (a) A first step of grinding asparagus as a starting material and then soaking it in alcohol to obtain a concentrated extract; (b) A first step of obtaining a concentrated extract by crushing asparagus as a starting material; A second step in which the concentrated extract is treated with one or more solvents or alcohols to perform at least one degreasing process; (c) A solvent is added to the degreasing liquid obtained in the second step to add saponin to the organic layer. After extracting the components, a third step of passing through a column chromatography to separate impurities; and a fourth step of passing the crude liquid obtained in the third step through a column chromatography again to separate impurities; A method for recovering purified saponin from asparagus, comprising: