JPS5910520A - Production of soybean saponin - Google Patents

Abstract

PURPOSE:Soysauce lees or its defatted product by treating with an organic solvent is subjected to extraction with water and/or a lower alcohol, subsequently to fractional extraction, adsorption and desorption, fractional precipitation or the like to separate soybean saponin having anti-inflammatory effect. CONSTITUTION:Soybean lees which are obtained as by-products, when a variety of soysauce or soysauce-like seasonings are produced, are used as starting materials to effect predefatting treatment with an organic solvent such as chloroform or ethyl ether to prepare defatted soysauce lees. The lees are combined with water and/or lower alcohol in an amount of 5-20 times the weight of the lees or defatted lees to effect extraction. The extract is subjected to fractional extraction with organic solvents such as methanol, acetone or ethyl ether, adsorption and desorption with nonionic adsorption resin, fractional precipitation, and chromatography to separate soybean saponin. The product is recrystallized from aqueous slcohol to give high-purity soybean saponin.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing soybean saponin using soda oil cake obtained as a by-product in the soy sauce production process as a starting material.

Generally, saponins are used as emulsifiers and dispersants in cosmetics and health drugs, as well as in the food industry. recently,
Saponin crude drugs have come to be reconsidered in the pharmaceutical field, and their anti-inflammatory, anti-ulcer, and anti-allergic effects have also come to be utilized.

Soybean saponin has long been known as a low-toxic saponin with the weakest hemolytic effect, which is a biological property of saponins, but the separation process from soybeans is complicated and economically disadvantageous, making it difficult to use in practical applications. It is considered a drawback.

The present inventors have discovered that this soybean saponin can be industrially advantageously produced using soy sauce lees, a by-product of the soy sauce production process, as a starting material.

The present invention involves extraction by adding water and/or lower alcohol to soy sauce lees or defatted oil lees obtained by degreasing the soy sauce lees with an organic solvent. characterized in that soybean saponin is separated by applying at least one type of separation means selected from adsorption/desorption using a chromatographic adsorption resin, fractional precipitation, and chromatography;
This is a method for producing soybean saponin.

The soy sauce lees used as a starting material in the present invention is a by-product in the process of producing cormorant oil, and the soy sauce lees that is a by-product in the production of various soy sauce and soy sauce-like seasonings can be used. Since soy sauce lees contains a large amount of salt, it is difficult to use or dispose of it, but according to the present invention, it can be turned into an industrial product with high utility value.

Since soy sauce lees usually contains lipids such as soy sauce oil, it is preferably used as defatted soy sauce lees obtained by defatting in advance using a conventional solvent. Examples of solvents for degreasing include chloroform and other halogenated hydrocarbons, ethers such as ethyl ether and isopropyl ether, alcohols such as methanol and ethanol, petroleum ether, hydrocarbons such as benzene, acetone and other ketones, Ethyl acetate, other esters, or mixtures of these solvents can be used.

According to the present invention, soy sauce lees or defatted soy sauce lees is extracted with water and/or lower alcohol. In this case, it is possible to extract with water alone, but it is more preferable to extract with aqueous lower alcohol or lower alcohol in terms of extraction efficiency.
. As the lower alcohol, for example, methanol, ethanol, n- and isopropanol, 1]-butanol, etc. can be used, but methanol or aqueous ethanol is particularly preferred from the viewpoint of the yield of soybean saponin.

Extraction is carried out using a conventional method, at a rate of 5% per weight of soy sauce lees or defatted soy sauce lees.
The extraction is carried out using a batch-type or continuous-type extraction device using ~20 times the amount of the above solvent at room temperature or under heating.

Next, one or a combination of two or more of the above-mentioned separation means for separating soybean saponin is applied to the thus obtained extract. Before this separation process, extract the extract by e.g.
Except when clarified by filtration, centrifugation or other means, and directly adsorbed and desorbed using a nonionic adsorption resin,
The solvent is usually recovered by concentration under reduced pressure.

To separate saponin from the extract obtained as a residue after the above-mentioned solvent recovery, first use an organic solvent at °C.
・The distribution sampling method can be applied. This method utilizes the difference in the solubility of thick q saponins and other substances in organic solvents, and includes a shaking extraction method, a one-step distribution method such as perforation that continuously performs this method, and a method using these methods. Examples include multi-stage distribution methods that combine the following.

Organic solvents used in this partition extraction method include, for example, alcohols such as methanol and ethanol, acetone and other ketones, ethers such as ethyl ether, ethyl acetate and other esters, chloroform and other halogens. Hydrocarbons, petroleum ether, and other hydrocarbons are selected as (1) and two or more mutually immiscible solvents (e).Water is also used. Example of a preferred solvent system: water and 11-butanol, an upper phase of a mixed system of chloroporum, methanol and water in various proportions that separate into two phases -
It is equivalent to F.

As another method for separating Nobonin, adsorption and desorption using a nonionic adsorption resin can also be used. As the nonionic adsorption resin, for example, styrene-based adsorption resins such as Serverchrome XAD-2 (manufactured by Server Co., Ltd.), Amberlite XAD-2 (manufactured by Rohm and Haas), etc. are used. In the case of an extract with water or an aqueous lower alcohol of generally less than 60% V/V, soybean saponin can be selectively adsorbed on the adsorption resin as it is. When extracting with a higher concentration, aqueous lower alcohol, or pure lower alcohol, the lower alcohol concentration in the extract is generally reduced to 60% V/V by concentrating under reduced pressure to recover the lower alcohol or adding water. Adsorption is performed after reducing the amount to less than Next, the soybean saponin adsorbed on the adsorption resin is treated with lower alcohol or a highly concentrated aqueous lower alcohol at °C.
・Can be shaken and removed.

Note that if a relatively large amount of lipids other than soybean saponin coexists in the extract or its concentrate in a suspended state, the selective adsorption and desorption of soybean saponin by the adsorption resin will be hindered, so static separation must be performed in advance. Or use a super-assistant such as diatomaceous earth.
If you remove this, you will get better results.

Similar to the above-mentioned partition extraction method, the fractional precipitation method can also be advantageously applied to the separation of soybean saponin by taking advantage of the difference in solubility in organic solvents between soybean saponin and other substances. That is, raw materials containing %'l-saponin and other substances are dissolved in a solvent in which they can be dissolved, and this solution is gradually added to a solvent in which one of them is more soluble. The final fraction containing the precipitated soybean saponin is collected and the fraction containing other substances is removed.

This operation can be repeated with the same solvent system, but it is also possible to select different solvent systems and combine them to make the separation of soybean saponins more effective.

The solvent system for this method is preferably a mutually miscible solvent system consisting of two or more solvents selected from the solvents listed in the above-mentioned partition extraction method. Examples include combinations of methanol and ethyl acid, methanol or highly concentrated aqueous ethanol and acetone.

Chromatography is also an effective means of separating soybean saponins.゛The treated starting materials, in this case a mixture of soybean saponin and other substances, are treated in organic solvents or mixed solvents in which they are soluble, such as methanol alone, methanol and water, chloroform, methanol, The soybean saponin is dissolved in a mixed solvent of soybean saponin and water, and passed through a column pre-packed with a packing capable of adsorbing or distributing soybean saponin and other substances, such as silica gel, chemically bonded silica gel, granular activated carbon, alumina, etc. or adsorb other substances.

When soybean saponin is adsorbed, the soybean saponin is further eluted by developing with the same series of organic solvents with different mixing ratios or with a different series of organic solvents that easily elute soybean saponin. If substances other than soybean saponin are adsorbed,
Soybean saponin is obtained as a flowthrough. The soybean saponin in these solutions can be collected by recovering the organic solvent by concentration under reduced pressure. In addition, reverse phase type chemically bonded silica gel, such as Micro Honda Pack C8 or 0
18 (manufactured by Waters) and similar fillers are:
It is suitable for purifying the target large-9 saponin.

Furthermore, by appropriately combining the above separation means,
Good results can be obtained.

51. The separated soybean saponin may be used as a product as it is, but if necessary, it can be further recrystallized using an aqueous lower alcohol such as aqueous methanol, aqueous ethanol, etc. to obtain highly purified soybean saponin. can.

The present invention has extremely high industrial value in that it provides useful saponin at low cost from soy sauce lees, which has low utility value.

Example 1 Koikuchi brewed soy sauce lees was crushed into powder, and 40
0 g was placed in a 21 volume round bottom flask, ethyl acetate 1-e was added thereto, and the mixture was heated under reflux for 5 hours. Next, the ethyl acetate extract is removed by r-filtration, and the remaining residue is extracted with ethyl acetate in the same manner five times in total.

The defatted soy sauce lees thus obtained was placed in a 21-volume round-bottomed flask, and one portion of methanol was added to it. and heated under reflux for 5 hours. Next, the methanol extract is collected by filtration, and the same methanol extraction is performed a total of 6 times to check for remaining residue. These methanol extracts are combined and the solvent is distilled off under reduced pressure to obtain 65 g of methanol extract.

Add 65g of this methanol extract to 60g of n-butanol.
After partitioning between 0me and 300ml of water, and distilling off the solvent in the fraction transferred to n-butanol, an extract of 1917 is obtained.

19 g of this extract was dissolved in 30 ml of methanol, poured into 1.5 p of ethyl acetate, and the resulting precipitate was collected by centrifugation to obtain 6 g of precipitate, which was filtered through A1.

Dissolve 6 g of this precipitate in 300 rd methanol 1-,
, 20.9 g of activated carbon was added and stirred for 12 hours.

The activated carbon was then removed by IF' filtration, and the r liquid was concentrated under reduced pressure to obtain crude soybean saponin fraction 2I. Dissolve the coarse vzabonin fraction 2I in 500 ml of methanol (1-2.1 volume round bottom flask). Add 5 parts and 4 parts of caustic horn IJ-methanol solution'lrJme to the mixture, heat under reflux for 1 hour, and then neutralize the strongly acidic ion exchange resin. The solvent was distilled off under reduced pressure.The remaining 1.8 g of the 1!j r) gel was then transferred to a reverse phase silica gel column chromatograph [carrier: 100 g of Bondervac Cl8 (manufactured by Waters Co., Ltd.], solvent: methanol-water ( 1:2 to 1:1) yields 1.5 p of large-i saponin.

This Ovzabonin was analyzed by thin layer chromatography using silica gel [precoated silica gel 60 F254, layer thickness 0.25 m+n (manufactured by Merck & Co., Ltd.), developing solvent: chloroform-methanol-water (lower layer of 65:35:10 ~
6:4:1)), sprayed with 50% sulfuric acid solution, and then heated to 90-95°C for 5 minutes, marked spots were detected and Rf values were 0.26, 0.50, 0. 3
4 and 0°67, reddish-purple spots characteristic of soybean saponin are obtained. This is, for example, Journal Fist of the American Oil φ Chemistry Society 47
This result agrees well with the results of thin layer chromatography of soybean saponin described in Vol. 3, pp. 86-9o (1970), and it is confirmed that the saponin obtained as described above is soybean saponin.

Example 2 Koikuchi brewed soy sauce lees was crushed into flakes, 2kIP was put into a 20n enamel tank,
Add 12 ffl of 8% V7/V aqueous ethanol to this, close the lid, and immerse and extract overnight at room temperature. This extract is collected by a conventional decanting method, and the residue is poured into a similar aqueous ethanol extraction twice. Concentrate the aqueous ethanol extract by combining 1. Place the concentrate in a liquid chromatograph with a diameter of 60 cm and a height of 50 mm, and set it up overnight. At this time, the concentrated liquid is separated into two layers, so the lower layer is taken out from the top of the column and passed through 1T1 using diatomaceous earth as an aid to remove some remaining lipids, resulting in a clear liquid of 1p. The solvent is distilled off from this clear liquid using a crash evaporator under reduced pressure to obtain 230 g of extract.

, 230g of extract and 1 part of methanol! Add about 70
° (:) to dissolve as much of the extract as possible, cool and leave to stand. The formed precipitate was removed by r-filtration, and the II solution was similarly concentrated to dryness under reduced pressure to obtain 160 g of methanol-soluble extract. It will be done.

, J) Extract 160 gwon-7'ta/-) Ishi 400
ml and 600 m of water, and the solvent in the fraction transferred to r]-butanol is distilled off, yielding 42.9 g of extract.

Dissolve 42g of this extract in 60me of methanol, pour it into 1.21ml of acetone, and pour the resulting precipitate into
Separate and collect (yield 24 g). This precipitate 24j9 was dissolved in 1.2 methanol, 40 g of activated carbon was added thereto, and the mixture was stirred for 12 hours.

This solution is removed by filtration with activated carbon, and the solvent is distilled off from liquid f-1 to obtain 10 g of a fraction of paddy soybean zabonin.

When 10 g of this crude soybean saponin fraction was further purified in the same manner as in Example 1, the desired soybean saponin was 7.2! j
can get.

Example 6 Useful brewed soy sauce lees 2! I was treated by the method of Example 2-(
The obtained lower layer 1 of the concentrated separated solution of the aqueous ethanol extract
! Add 200 mJ of water to make the alcohol concentration 30%.
After adjusting to V/V, suspended matter is removed by filtration using diatomaceous earth to obtain a clear liquid 1.2-e.

This clear liquid12. e to styrene-based adsorption resin A, / Burlite
After washing with 400me of 30% V/V aqueous ethanol at a flow rate of V=1, the adsorption part was washed with methanol 1e.
Then, the solvent of the eluate was distilled off under reduced pressure to obtain 40 g of extract.

Dissolve 40g of this extract in 60ml of methanol, pour it into 1.2ml of acetone, separate the resulting precipitate and collect it, and you'll get 20g of precipitate! Get 1. From now on
Activated carbon treatment was carried out in the same manner as in Example 2. /i1'! i1~
Further, the same purification treatment as in Example 1 was carried out to obtain 14 g of the desired ovusabonin.

Applicant Gizkoman Co., Ltd. Agent: Patent Attorney Masao Kobayashi 137-

Claims (1)

[Claims] Water and/or lower alcohol is added to the soybean oil lees or the defatted soy sauce lees obtained by degreasing this with an organic solvent for extraction, and the resulting extraction part is subjected to partition extraction with an organic solvent and non-ionic adsorption resin. A method selected from adsorption-desorption, fractional precipitation, and chromatography;
-/, method for producing zaponin.