JPS6393794A - Extracting and purifying method of beetsaponin - Google Patents

Abstract

PURPOSE:To readily obtain the titled material useful for treatment of hyperlipidemia, etc., from sugar extract residue of a beet sugar etc., in good yield, by bringing a extract of ground beet sugar, etc., by an aqueous alkali solution into contact with a synthetic adsorbing resin and eluting the adsorbed beetsaponin. CONSTITUTION:A ground beet sugar is subjected to extraction treatment with an aqueous alkali solution, for example, NaOH aqueous solution having pH 8-12. Then, the extract is brought into contact with a synthetic adsorbing resin to extract a beetsaponin, which is eluted with a solvent such as ethanol, etc. When 3-7% alkali metal salt is contained in above-mentioned extract, the adsorption of the beetsaponin is preferably improved. An insoluble, porous and non-polar polymer obtained by polymerizing styrene with divinyl benzene and exhibiting a macro network structure is preferably used as the synthetic adsorbing resin.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to a method for extracting and purifying beet saponin from sugar beet.

[Prior Art] and [Problems to be Solved by the Invention] Sugar beet is a plant belonging to the Chenopodiaceae family and is commonly known as beet, and is cultivated in large quantities to produce sugar from its leaves and roots. Sugar beet contains beet saponin, which has a strong foaming property, which reduces production efficiency in the sugar manufacturing process. For this reason, although it has been a long time since Vander Harr confirmed that beet saponin is a glycoside of oleanolic acid in 1927, its effective use has not been studied. There are no reports of this.

In view of the above-mentioned circumstances, the present inventors aimed to utilize beet saponin effectively (a) medicinal efficacy of beet saponin and (b)
We have conducted repeated research on the content of beet saponin in sugar beet.

As for the results (a), we confirmed the effectiveness of beet saponin against hyperlipidemia and ulcers, and as for (b);
Beet saponin was confirmed in roots used for sugar production, leaves not used for sugar production, and even sugar extraction residue produced as waste during the sugar production process. In addition,
Table 1 shows the results of measuring the content of beet saponin in sugar beets.

Table I Based on the above results, the present inventor came to the knowledge that extraction and purification of beet saponin from sugar beet is extremely appropriate, and conducted research on a method for extracting and purifying beet saponin from sugar beet.

First, the following experiment was conducted to examine a method for extracting beet saponin from sugar beet using a solvent.

1. Method of directly extracting beet saponin from sugar beet with methanol (method A).

2. Method of treating sugar beet with water to remove water-soluble components and then extracting beet saponin with methanol (Method B)
.

3. A method in which beet saponin from sugar beet is extracted with an aqueous alkaline solution, the alkaline extract is made acidic, and the resulting precipitate is extracted with methanol (method C).

The specific method described above is as follows.

Method A-1: 100 g of crushed sugar beet roots and 300 ml of methanol were stirred at room temperature for 1 hour and then filtered, the residue was washed twice with a small amount of methanol, and the methanol in the filtrate and washing liquid was distilled off under reduced pressure. 5.0 g of extract was obtained.

Method A-2: 100 g of crushed sugar beet roots and 30 Qmf of methanol were refluxed for 1 hour, filtered, and then operated in the same manner as Method A-1 to obtain 5.2 g of an extract.

Method B-1: 100g of crushed sugar beet root and 300m of water
1 was stirred at room temperature for 30 minutes and then filtered. The residue was refluxed with 300 ml of methanol for 1 hour and filtered. The residue was washed twice with a small amount of methanol, and the methanol in the filtrate and washing liquid was distilled off under reduced pressure. 0.7 g of extract was obtained.

Method B-2: 100g of crushed sugar beet root and 30g of pH 1 water Qmj! The mixture was stirred at room temperature for 30 minutes, filtered, and then operated in the same manner as Method B-1 to obtain an extract of 0.88.

Method C-1: 100 g of crushed sugar beet roots and 200 mL of pH 10 water were stirred at room temperature for 2 hours, filtered, the residue was washed twice with a small amount of pH 10 water, and the filtrate and washings were combined. This was adjusted to pH 2 with hydrochloric acid and the precipitate was collected by filtration.

The precipitate and 10 ml of methanol were refluxed for 1 hour and filtered, the residue was washed twice with a small amount of methanol, and the methanol in the filtrate and washing liquid was distilled off under reduced pressure to obtain 0.3 g of an extract.

Method C-2: 100 g of crushed sugar beet roots and 200 ml of pH 10 water are stirred at room temperature for 2 hours and then filtered, the residue is washed twice with a small amount of pH 10 water, the filtrate and the washing liquid are combined, This was adjusted to pH 5 with hydrochloric acid, the precipitate was collected by filtration, and the following procedure was performed in the same manner as in Method C-1 to obtain 0.2 g of an extract.

Table 2 shows the results of measuring the content of built saponin and oleanolic acid in the extract obtained by the above method. In either method, the recovery rate of beet saponin was low, and the extract was contaminated with oleanolic acid resulting from the decomposition of beet saponin.

From the experiments shown in Table 2 above, it was found that beet saponin decomposes into oleanolic acid in acidic conditions, but is stable in weakly alkaline conditions.Therefore, we conducted further research on extraction and purification methods for beet saponin and arrived at the present invention.

(Means for solving the problem) and [action] That is, the present invention extracts ground sugar beet with an aqueous alkaline solution, and then brings the obtained extract into contact with a synthetic adsorption resin to adsorb beet saponin. , provides a method for extracting and purifying beet saponin, which is characterized in that beet saponin is eluted with a solvent from a synthetic adsorption resin on which beet saponin has been adsorbed.

Here, the sugar beet leaves and sugar extraction residue may be crushed and subjected to the treatment of the present invention as a crushed sugar beet product. However, after crushing the root, water or warm water is added, stirring and filtering is performed to remove water-soluble substances such as carbohydrates, and this is desirably used as a crushed sugar beet product to be subjected to the treatment of the present invention. In addition, add crushed sugar beet roots to 3 times the amount of warm water (60℃).
) for 1 hour to remove water-soluble substances, the loss of beet saponin is less than 2% of the original saponin content and has almost no effect.

However, as mentioned earlier, in sugar factories, sugar beet roots become sugar extraction residue after water-soluble substances such as carbohydrates are removed during the sugar manufacturing process. When used as a crushed sugar beet product, there is no need to separately perform the above-mentioned removal operation for water-soluble substances.

Beet saponin is extracted by stirring the ground sugar beet and an aqueous alkaline solution at room temperature for 1 to 2 hours. As the alkaline aqueous solution, for example, an aqueous solution of sodium hydroxide, potassium hydroxide, sodium carbonate, sodium bicarbonate, ammonia, etc. having a pH of 8 to 12 can be used.

Next, the extract is brought into contact with a synthetic adsorption resin to adsorb beet saponin. The synthetic adsorption resin referred to here is an insoluble, porous, non-polar polymer with a giant network structure manufactured by polymerizing styrene and divinylbenzene.
For example, it is sold as Amberlight XAD (Organo Co., Ltd.) or Diaion HP (Mitsubishi Chemical Industries, Ltd.). The amount of synthetic adsorption resin varies depending on the type, but it is usually 30Qm per gram of beet saponin contained.
1 or more preferably 400-500 m 1! It is.

The saponin concentration of the extract brought into contact with the synthetic adsorption resin is 0.
．． 03 to 0.5% by weight. The liquid during adsorption is preferably slightly alkaline, particularly preferably around pH 8. When the extract contains 3 to 7% of alkali metal salt, adsorption properties are improved. For example, when an extract with a pH of 8 is passed through at 5 V = 2, the recovery rate of beet saponin is in the 40% range, but the extract contains sodium chloride, potassium chloride, sodium sulfate,
When sodium nitrate or the like is contained at 5%, the recovery rate increases to the 90% range.

The solvent used to elute the beet saponin adsorbed on the synthetic adsorption resin is, for example, a lower alcohol such as methanol or ethanol. By distilling off the solvent of the eluent, a purified product of beet saponin is obtained.

Note that if the used synthetic adsorption resin is washed with IN sodium hydroxide solution, then washed with water until it becomes neutral, and then washed with methanol to activate it, it can be used again for extraction and purification of beet saponin.

[Examples, etc.]

The present invention will be explained in more detail with reference to Examples below.

Example 1 Sugar beet sugar extraction residue was finely ground, and 1 kg (
Water 21 was added to beet saponin (containing 3.923 g), adjusted to pHIO with a 10% aqueous sodium hydroxide solution, stirred at room temperature for 1 hour, and then filtered. The residue was washed twice with a small amount of water at pH 10, and the filtrate and washings were combined and purified with 35% hydrochloric acid.
The solution was brought to H8 and sodium chloride was added to make a 5% solution. This liquid was mixed with synthetic adsorption resin (Diaion HP-20) 1.5
The beet saponin was adsorbed by flowing it through a column packed with x at 5V=2, and then water II! was washed at 5 V = 4, beet saponin was eluted by flowing 3.51 methanol at 5 V = 4, and the methanol in the eluent was distilled off under reduced pressure to obtain 14.1 g of yellow purified beet saponin (beet saponin). (Content: 3.687 g, purity: 26%) was obtained. The recovery rate of beet saponin according to this example was 94%.

In addition, when the above operation was performed in the same manner as above without adding sodium chloride to the extract, the recovery rate of beet saponin was 41%.

Example 2 Sugar beet root was finely ground, 1 kg of this (beet saponin content: 1.266 g) was added with water 21, and the mixture was heated at 60°C.
After stirring for 30 minutes, filter and add water 11 to the residue to give a 10%
The pH was adjusted to 10 with an aqueous sodium hydroxide solution, stirred at room temperature for 1 hour, and then filtered. The residue was washed twice with a small amount of water at pH 10, and the filtrate and washings were combined and adjusted to pH 8 with 35% hydrochloric acid.
Sodium chloride was added to make a 5% solution. This liquid was passed through a column packed with 500 ml of synthetic adsorption resin (Diaion HP-20) at 5 V = 2 to adsorb beet saponin, and then washed by flowing 500 ml of water at 5 V = 4.
Beet saponin was eluted by flowing 1.5 n of methanol at 5V=4, and the methanol in the eluent was distilled off under reduced pressure to obtain 5.5 g of yellow purified beet saponin (contains 1.152 g of beet saponin, purity 21%). ) was obtained. The recovery rate of beet saponin according to this example was 91%.

Pharmacological test of beet saponin The results of the test on beet saponin (purity 26%) extracted and purified by the method of the present invention are as follows.

1. Effect on hyperlipidemia (1) Fat emulsion-induced hyperlipidemia model After fasting for about half a day, male ddY mice weighing about 23 g were injected with a fat emulsion (olive oil 32.7%, cholesterol 2.0%). 5
%, sodium cholate 1.8%, sucrose 41.9%, water 21.1%) 25.5 ml/kg was administered orally, and immediately after and 7 hours later, the test drug was administered by a total of 2 inlet openings. . Seventeen hours after the second administration, blood was collected from the abdominal aorta under ether anesthesia, and serum total cholesterol was measured at C0.
D-p-chlorophenol coloring method (cholesterol C■
Measured using Test Wako (Wako Pure Chemical Industries, Ltd.). Nicomol was used as a comparative drug. The test drug was suspended in 5% gum arabic powder.

As shown in Table 2, beet saponin (26%) significantly improved serum total cholesterol at a dose of 500 μ/kg.

Table Ⅲ *Significant difference from control group (treatment) (P <0.05) Each value represents the mean ± S, IE.

2. Effects on various ulcers (11 Hydrochloric acid-ethanol ulcer model The test drug was orally administered to Wistar male rats weighing approximately 200 g after fasting for 24 hours, and 1 hour after drug administration, hydrochloric acid-ethanol (60% ethanol + 150 mM hydrochloric acid)
The rats were orally administered at a rate of 1 ml/rat, and killed with ether 1 hour after the administration of hydrochloric acid-ethanol.The total length of damage occurring in the blue gland area was taken as the damage factor.

As shown in Table 2, administration of beet saponin (26%) had a significant dose-dependent effect on reducing the damage coefficient, and the inhibition rate was 100% at a dosage of 300 µ/kg.

Table ■ * Significant difference from control group (treatment) (P < 0.05) ** Significant difference from control group (treatment) (P < 0.01) (2) Indomethacin ulcer model Wistar male rats weighing approximately 170 g After fasting for 24 hours, the test drug was orally administered, and 1 hour after drug administration, indomethacin was injected subcutaneously at a rate of 200 trg/kg. Seven hours after indomethacin administration, the animals were killed with ether, and the damage factor was determined in the same manner as in the hydrochloric acid-ethanol ulcer model. Ta.

As shown in Table 7, administration of beet saponin (26%) had a significant effect of lowering the damage coefficient in a dose-dependent manner, and the inhibition rate was also quite high.

Table V** Significant difference from control group (treatment) (P <0.01) (3)
Restraint water immersion stress ulcer model ddY male mice weighing approximately 20 g were fasted for 24 hours, then the test drug was orally administered, and 10 minutes after drug administration, they were subjected to restraint water immersion (water temperature approximately 22°C) for 5 hours, resulting in analogous dislocation. The animals were sacrificed and their stomachs removed and fixed with Polmarin. Then, an incision was made along the major groove, and the ulcer was observed and graded into the following four stages.

O: Asymptomatic 1: Mild bleeding 2: Bleeding and Elodion 3: Severe bleeding and Elodion As shown in Table ■, administration of beet saponin (26%) had a significant dose-dependent effect on reducing the damage factor. This was observed, and the inhibition rate was 100% at a dose of 300 μ/kg.

Table ■ ** Significant difference from control group (treatment) (P < 0.01) [Effects of the invention] According to the present invention, sugar extraction residue from sugar beets, etc., which are discarded in large quantities at sugar factories, are used. As a result, beet saponin, which is effective against hyperlipidemia and ulcers, can be extracted and purified relatively easily and with a good recovery rate.

Claims (2)

[Claims] (1) After extracting the crushed sugar beet with an alkaline aqueous solution, the extract obtained here is brought into contact with a synthetic adsorption resin to adsorb beet saponin, and the beet saponin is extracted from the synthetic adsorption resin with a solvent. A method for extracting and purifying beet saponin, characterized by elution. (2) The method for extracting and purifying beet saponin according to claim 1, wherein an alkali metal salt is added to the extract.