JPS63122695A - Production of glycoside concentrate - Google Patents

Abstract

PURPOSE:To obtain the titled compound having oxygen resistance, antimutagenic and antitumor properties economically in an industrial scale, by fractioning an extract from a de-fatted sesame seed by a polar solvent with an organic solvent and fractioning the resultant glycoside-containing fraction with silica gel and ODS. CONSTITUTION:A sesame seed oil cake obtained by de-fatting a sesame seed is ground and stirred with a polar solvent (e.g. ethanol/water mixed solvent, etc.) added in an extracting vessel while maintaining the temperature at 50 deg.C for 3hr to carry out an extract treatment and filtered under a reduced pressure in order to exclude an insoluble residue to provide an extract, which is then fractioned with a organic solvent such as chloroform, etc., to afford a glycoside- containing fraction, which is treated with acetone and passed through a column of silica gel and ODS so as to fraction to afford a glycoside concentrate containing substances expressed by formulas I-III as an aglycon.

Description

DETAILED DESCRIPTION OF THE INVENTION ta+ Industrial Application Field The present invention relates to a method for fractionating and concentrating specific glycosides contained in sesame seeds.

(b) Conventional technology Sesame seeds are prepared using the following formula 1. Although it has long been known that glycosides having the substances shown in II and III as aglycones are included, details such as the chemical structure of the glycosides are not clear.

OCR.

OCH.

However, it has been suggested that the substances represented by formulas I, n, and II [ themselves have physiological effects such as antioxidant, antimutagenic, and antitumor properties, and we look forward to their effects and usefulness in the future. There is something big about this.

On the other hand, I, II, and III are fat-soluble, and the hydroxyl group, which is thought to be greatly involved in physiological activity, is in a free state, so they have difficulties in solubility, stability, and interaction with living organisms (cells). It is expected that.

For these reasons, it is expected that the use of I, II, and I in the form of glycosides will increase stability and hydrophilicity and improve interaction with living organisms.

However, the above glycosides present in sesame seeds are
Since it is only a few mg per gram and coexists with a large amount of glycosides with similar behavior, it has been a major obstacle in concentrating it.

In order to solve this problem, the present inventors first invented a method for concentrating the above-mentioned glycosides by solvent fractionation and silica gel treatment (Japanese Patent Application No. 61-82659). This method enabled effective concentration of glycosides.

(C) The problem that the invention seeks to solve However, such a method also provides the formula I as described above.

It is not possible to perform mutual fractionation of glycosides of If, I[[.

However, concentrating these I, I[, and III glycosides separately is an essential requirement to utilize their physiological activities more effectively, and the development of effective fractionation and concentration methods is strongly desired. ing.

Therefore, the object of the present invention is to obtain the formula without contamination with other glycosides. , n, and III as aglycones are mutually fractionated and each is separately concentrated to a high concentration.

(Means for solving the d1 problem) In order to achieve the above objective, the present inventors investigated the behavior of this glycoside in detail and found that by combining solvent fractionation, treatment with silica gel and ODS, We found that it can be effectively fractionated and concentrated.

The present invention was completed based on this knowledge, and after fractionating an extract of defatted sesame seeds with a polar solvent using an organic solvent to obtain a glycoside-containing fraction, silica gel and ODS
The following formula 1. n,
This is a method for producing a glycoside concentrate using the substance shown in DI as an aglycone.

CR5 0CH.

Each step will be explained in detail below.

A. Extraction from raw materials The raw material sesame seeds may be of any type or origin, but for later work, it is preferable that they be degreased in advance and crushed to 100 mesh or less. This is immersed in 5 to 20 times the amount of polar solvent, heated to 30 to 80°C and stirred for several hours to extract glycosides. Examples of polar solvents include alcohol, hydrous alcohol, tetrahydrofuran, ethyl acetate, and chloroform. Examples of alcohol include methanol, ethanol, propanol, and isobutanol; when mixed with water, the water content is 10 to 99%. Effective. After extraction, insoluble residues are removed by filtration, and most of the solvent is distilled off to obtain crude glycosides.

B. Solvent fractionation The crude glycoside is poured into about 10 times the amount of chloroform and after completely dissolved, the same amount of water is added and stirred vigorously. After standing still, transfer the chloroform phase to another container, add half the amount of water to the chloroform phase, and stir. After standing still, the chloroform phase is removed and the solvent is removed to obtain a chloroform-soluble component.

In this step, sugars, proteins and water-soluble glycosides are removed.

Next, a sufficient amount of acetone is added to the chloroform-soluble matter, and the mixture is vigorously stirred with a stirrer to bring it into sufficient contact with the acetone. After stopping stirring and leaving the mixture for several hours, the supernatant liquid was removed to obtain a sticky precipitate. The precipitate is heated to 30 to 50° C. under reduced pressure to remove residual acetone to obtain a powdery acetone-insoluble matter. In this step, oil-soluble substances such as ■, ■, ■ and sterols are removed.

C. Silica gel column fractionation As the silica gel fractionation, a method using a silica gel column will be described here, but the present invention is not limited thereto.

30 to 2 for a cylinder with an inner diameter to height ratio of 1/10 to 1/30
A silica gel column is prepared by filling the cylinder with 0.00 mesh silica gel to approximately 80% of the height of the cylinder. Any silica gel may be used as long as it has a pore size of 30 to 100 angstroms, and is used without activation treatment. At this time, the silica gel is preliminarily mixed with a sufficient amount of chloroform/
It is immersed in a mixed solution of acetate 7 = 10/2 to 2/1, and after the liquid phase has sufficiently soaked into the silica gel, it flows from the top of the cylinder. Remove excess liquid until the silica gel has finished settling and the liquid level is approximately equal to the silica gel surface.

Next, the acetone-insoluble portion is dissolved using as little as possible of the chloroform/acetone mixture used for column preparation, and the solution is allowed to flow gently from the top of the column. At this time, the acetone-insoluble portion to be subjected to the column is 5% of the weight of the silica gel column used.
% or less, and if it exceeds this, the loss of the target substance will gradually increase.

After lowering the liquid level, the chloroform/acetone mixture used for column preparation is flowed at a volume of 0.85 to 1.5 times the volume of the cylinder. Subsequently, acetone/methanol = 20/1 to 5/1 is poured into the cylinder 0.8 to 1.5 times the volume of the cylinder, and the eluate is collected. The optimal composition of the eluent varies depending on the activity of the silica gel, the shape of the cylinder, the amount added, etc., but usually acetone/methanol =
10/1 is appropriate. This fraction is desolventized to obtain a powdery crude concentrate.

D, ODS column fraction ODS refers to silica gel to which octadecyl groups are chemically bonded.

As the ODS fractionation, a method using ODS column fractionation will be described here, but the present invention is not limited thereto.

60 to 2 for a cylinder with an inner diameter to height ratio of 1710 to 1/20
An ODS column is prepared by packing 00 mesh of ODS to approximately 80% of the height of the cylinder.

ODS is immersed in advance in a sufficient amount of water/alcohol (methanol is good) = 2/1 to 1/1 mixed solution, and after heating to 60 to 70°C so that the liquid phase is sufficiently infiltrated into ODS, Degas by exposing to vacuum. This is packed into a cylindrical column, flowing from the top to form a column, and water/methanol = 2/1 is flowed through to completely replace the column.

The crude concentrate obtained in step C above is suspended (or dissolved) in water/methanol=271 and applied to a column.

At this time, the crude concentrate to be fed to the ODS column is desirably 1.5% or less of the weight of the ODS column packing material, and if it exceeds this, the loss of the target substance will gradually increase.

After lowering the liquid level, a 2/1 mixed solution of water/methanol is poured into the cylinder to remove highly polar substances.

The optimal composition of the eluent varies depending on the shape of the cylinder, the amount of load, etc., but in general, methanol is suitable, and 1.
Flow 5 to 2 times. This category contains the target glycoside, but by performing this treatment, mixed fatty acid esters, etc. can be removed.

Furthermore, the concentrate obtained by removing the solvent from the methanol elution section is suspended again in water/methanol=2/1, and the suspension is applied to a cylinder filled with ODS. Again, the concentrate is preferably 1.5% or less of the weight of the ODS column. After lowering the liquid level, water/
Methanol = 271 to 0/10 to 1.5 to 2 of the cylinder volume
．． Run 0x and collect the eluate. The optimal composition of the eluent varies depending on the shape of the cylinder, the amount of load, etc., but usually a mixture of four types: water/methanol = 2/l, 6/4. It is appropriate to flow 5 to 2.0 times as much. The solvent is removed from each of these sections to obtain the desired concentrate in powder form.

At this time, water/methanol=6/4 and 4/6 each contain different glycosides, and 515 contains a mixture of both. These are removed from the solvent to obtain the desired glycoside concentrate.

As a method for evaluating the concentrate obtained by the present invention, there is no known method to date that directly measures the content of the target substance, which is a glycoside. Therefore, the present inventors decomposed the target product using a methanol solution of an inorganic acid, and the generated I, n, lll0
We decided to evaluate the degree of enrichment based on the amount. Examples of inorganic acids include HCl and HNOi.

There are H3P0a, HsBOa, etc., especially H,3PO.

Good. In addition, I, II, which exists in a free state
Regarding I[I, the amounts of 1, n, and m derived from glycosides were determined by the difference in the amounts of these substances before and after decomposition.

Next, to describe the analytical method for I, n, n[, sample 50
Accurately measure ~200 mg into a small test tube and dissolve or disperse by adding 5% phosphoric acid and 5 mj2 of methanol.

The mixture is tightly stoppered and heated in a 150°C water bath for 3 to 6 hours, then cooled, 1 ml of ethyl acetate and 5 mA of saturated saline are added, the mixture is stoppered and shaken vigorously. After standing, the upper layer is subjected to a gas chromatograph equipped with a flame ionization detector. Inject a solution of a certain concentration of cholesterol as a standard substance,
From the peak area ratio, 1. Calculate the total content of n and III. At the same time, a blank test was performed using methanol instead of 5% methanol phosphate, and I, n, which is not derived from glycosides, was
, m are determined, and the difference is taken as the amount derived from the glycoside.

(e) Examples Example 1 1 kg of sesame oil cake obtained by defatting Chinese sesame seeds is crushed to 100 mesh or less and placed in a 2 (l) extraction tank. Ethanol/water =
Add 85/15 to 15/15 and stir for 3 hours while maintaining the temperature at 50°C. After cooling, insoluble residues were removed by vacuum filtration to obtain 12.5A of extract. The entire amount of the extract was subjected to a rotary evaporator, most of the solvent was distilled off, and 45 g of crude glycosides were obtained.
I got it.

The crude glycoside was dissolved in 453 mA of chloroform, transferred to a separating funnel, 450 ml of water was added, and the mixture was shaken vigorously. After standing for 1 hour, the lower layer was transferred to another container, 250 mj2 of diluted chloroform was added, and the mixture was shaken vigorously again. The lower layer is combined with the previous lower layer and the solvent is distilled off to obtain 11 g of chloroform soluble portion.

Next, 200mj of acetone! and stir vigorously with an electric stirrer to completely disperse the chloroform-soluble matter. After standing for 3 hours, the supernatant was removed and the precipitate was incubated under reduced pressure for 30 minutes.
The mixture was kept at ~35° C. for 2 hours, and residual acetone was removed to obtain 5.3 g of an acetone-insoluble portion.

Next, the inner diameter is 3. Silica gel (Wako Gel C-100 manufactured by Wako Pure Chemical Industries, Ltd.) in a glass column with a height of 45 cm.
A silica gel column is prepared by injecting 100 g using chloroform/acetone=4/1. Same liquid 2Qm
The entire amount of acetone-insoluble matter is dissolved using 1.1 liter of acetone, and the acetone-insoluble matter is dissolved in the solution from the top, and the solution is adsorbed onto the silica gel. 120ml of the same liquid
After flushing, acetone/methanol = 9/2 120m
7! The eluate was collected and the solvent was removed to obtain 0.41 g of a powdery crude concentrate.

After that, 0DS (manufactured by Yamamura Kagaku ■005 60/200 mesh) '40 was added to a glass column of the same size.
g using aqueous methanol to prepare an ODS column. Using 20ml of the same solution, dissolve 1g of the powdery concentrate of the acetone/methanol-9/2 elution category,
Injected from the top. After the liquid level drops, 40 Qrrl
of water-containing methanol, then methanol alone
The eluate was collected and the solvent was removed to obtain 0.68 g of a powdery concentrate.

Further, 180 g of ODS (same as above) is injected into a glass column of the same size using a water-containing methanol (30% (v/v) methanol) solution to prepare an ODS column. Using the same liquid I Qmff, the above powdered concentrate 0.
68g was dissolved and injected from the top. After the liquid level has fallen, add 509 ml of water-containing methanol (30% Cv/v in order)
, 40% (v/v) , 50% (v/v)
, 70% (v/v)) and desolventized each section to obtain powdered concentrates of 0.04 g and 0.0 g, respectively.
3g.

0.20 g, 0.22 g were obtained. In addition, I, I derived from the target substance contained in the concentrated glycoside in this example
The glycosides of I and I were as shown in Table 1.

Table-1 In addition, the composition ratio of this concentrate is 29% of glycosides.

18% of glycosides (■), 58% of glycosides (■), and I, II, and I[[, which are not derived from glycosides, were not detected.

Example 2 1 kg of sesame oil cake was crushed to 100 mesh or less, and 201
Transfer to extraction tank. Add chloroform 151 and continue extraction overnight with constant stirring. Insoluble residues were removed by filtration under slight pressure, and the extract 12. H! get.

After concentrating to about 0.51 using a rotary evaporator, add 500 ml of water and shake vigorously.

Thereafter, the same procedure as in Example 1 was carried out to reduce the acetone insoluble area to 4.8
I got g. 1.0 g of the product was applied to a silica gel column and an ODS column under the same conditions as in Example 1 to obtain 0.09 g of the desired concentrate. The content of the present glycoside concentrate is shown in Table-2.

Table 2 The composition ratio of glycosides was almost the same as in Example 1. None of I, n, and III, which are not derived from glycosides, was detected.

(f) Effects of the Invention According to the present invention, a target glycoside exists in only a trace amount in sesame seeds and is difficult to concentrate because it coexists with a large amount of other glycosides with similar behavior. can be mutually fractionated and each can be concentrated to a high concentration.

These are expected to have useful physiological effects such as antioxidant, antimutagenic, and antitumor properties.

Claims (1)

[Claims] (1) A polar solvent extract of defatted sesame seeds is fractionated with an organic solvent to obtain a glycoside-containing fraction, and then fractionated with silica gel and ODS, using the following formula.
A method for producing a glycoside concentrate using the substances shown in I, II, and III as aglycones. I ▲There are mathematical formulas, chemical formulas, tables, etc.▼ II▲There are mathematical formulas, chemical formulas, tables, etc.▼ III▲There are mathematical formulas, chemical formulas, tables, etc.▼