KR930001078B1 - Extraction from croton tiglium for hepatitis - Google Patents

Abstract

A liver protecting agent is prepd. by (a) defatting croton tiglium L. (I) with solvent, (b) extracting the defatted (I) with cold D.W. or 0.5M NaOH soln., and filtering to obtain the filtrate, (c) dissolving the obtd. filtrate with D.W., (d) ultra- filtrating the obtd. soln. to obtain the extract of (I) and (e) mixing the extract of (I) with other liver protecting agent, e.g. alpha tocopherol etc., to obtain the final product. The M.W. of the extract is 300,000 or more.

Description

Hepatoprotective composition containing degreased green onion extract

The present invention relates to a hepatoprotective composition containing an extract extracted from degreased green beans.

Polysaccharides and glycoproteins contained in plant components have been found to be particularly effective in protecting the liver. Representative herbal medicines containing such polysaccharides and glycoproteins are known as yellow white, frankincense, ginseng, and sapling. Polysaccharides extracted from such plants Glycoprotein has been found to have an effect of significantly reducing hepatotoxicity caused by carbon tetrachloride. In addition, Japanese Laid-Open Patent Publication No. 55-76818 discloses experimental results that polysaccharides extracted from cloud mushroom (Coriolus versicolor) improve liver abnormalities, and glucan obtained from the cell wall of Saccharomyces cerevisiae. Is a macrophage stimulant that has been shown to improve the viability and phagocytic dysfunction of mice and have effects on viral hepatitis in mice. Science, Vol., 208, pp 67-69 (1980). .

The inventors pay attention to the fact that the polysaccharides or glycoproteins contained in plant constituents have an effect of remarkably improving the hepatic function. Therefore, the present inventors extracted and separated polysaccharides and glycoproteins from many herbal plants and studied the effects on the hepatic functions of the substances. Among them, especially found in a large amount of polysaccharides and glycoproteins to significantly improve the liver function in Padu and completed the present invention through the pharmacological activity test and toxicity test.

Padu is a seed of Croton tiglium L. Its fat component is pardon oil, which is used for the treatment of constipation, indigestion and amoeba dysentery in Chinese medicine. However, since soybean oil contains povol esters, known as tumor promoting agents, and is treated as a toxic substance, soybean extract used in the liver protection composition of the present invention is essential to remove fat components. In other words, the fat component is degreased from the soybean curd, and then the desired polysaccharide or glycoprotein is extracted and purified from the residue. As a result of various pharmacological comparison experiments, the extracts of green beans contain large amounts of polysaccharides and glycoproteins. Among them, those with molecular weight of 300,000 or less are not suitable because of low activity and protein has minimal hepatoprotective effect. More than 10,000 polysaccharides and glycoproteins were identified.

Therefore, in the present invention, the use of total extract (CT) degreasing fat from the soybean, or polysaccharides and glycoproteins and protein mixtures (HT) having a high molecular weight of 300,000 or more (high molecular weight) among them, in particular, 300,000 molecular weight Polysaccharide and glycoprotein mixture (HP) were separated and used as a hepatoprotective composition.

The process of degreasing from soybean, the method of extracting and separating polysaccharide glycoprotein and protein from degreased soybean can all be easily carried out using conventional methods of degreasing, extracting, separating and purifying from herbal plants.

Brief description of the degreasing, extraction, separation, and purification methods used in the present invention are as follows.

● Degreasing process:

Using the petroleum ether as a solvent and pulverized in a low-temperature room at about 40 ℃ using a Asahina continuous extraction device for about 24 hours continuous extraction to obtain the oil residue completely removed.

● Obtaining Total Extract:

The degreased padu residue is filtered by cooling or warming with constant or distilled water, and the extract filtrate is lyophilized to obtain a total extract of padu.

Separation of high molecular weight fraction (HT) and low molecular weight fraction (LT):

The degreased parduzan residue was precipitated overnight in cold distilled water or 0.5M NaOH aqueous solution, and the precipitate was filtered off. Then, three times of cold 95% ethanol was added to the filtrate, and the resultant was left overnight in a low-temperature chamber. Using an ultrafiltration membrane capable of fractionating more than 300,000, the material remaining on the membrane without filtration is high molecular weight fraction (HT), and the filtrate passing through the membrane is a low molecular weight fraction (LT) and a low molecular weight fraction (LT).

Separation of high molecular weight polysaccharides and glycoproteins (HP) and low molecular weight polysaccharides and glycoproteins (LP):

Dissolve 95% ethanol precipitate in cold distilled water, add cold 15% trichloroacetic acid (TCA), leave for 1 hour in ice bath, centrifuge for 1 hour at about 12,000Xg, collect supernatant and add 4 times cold 95% ethanol overnight. Neglect produces a precipitate. The precipitate was dissolved in 2% sodium acetate solution and neutralized. 2 times cold ethanol was added and left overnight at about 4 ° C., and the supernatant was centrifuged at 12,000 × g for 15 minutes to obtain a precipitate. The precipitate was dissolved in distilled water to obtain an ultrafiltration membrane. To obtain a high molecular weight polysaccharide and glycoprotein having a molecular weight of 300,000 or more and a polysaccharide and a glycoprotein having a molecular weight of less than 300,000.

The total extraction (CT) or high molecular weight polysaccharides and glycoproteins (HP) of green onions obtained in the present invention were treated with carbon tetrachloride to cause acute hepatic insufficiency in cystine SD rats. .

Therefore, human oral or parenteral administration of total extract (CT) or high molecular weight extract (HT) or high molecular weight polysaccharide and glycoprotein (HP) of soybeans alone or in combination with a pharmaceutically acceptable carrier Liver damage can be prevented or treated. Soybean extract or fractions thereof of the present invention may be used in combination with other agents having a hepatoprotective effect. Hereinafter, the present invention will be described in more detail as examples.

Example 1

Isolation of Total Extract from CT

In a low-temperature chamber at 4 ° C., the amount of papdu was finely pulverized in the larynx, and extracted continuously with an Asahina continuous extraction device using petroleum ether as a degreasing solvent for 24 hours to completely remove oil components and then dried. The defatted parduits were chilled or warmed overnight with constant or distilled water, filtered to remove the residue and lyophilized to obtain total extract (CT) of the pardues.

Example 2

Separation of Low Molecular Weight Padu Extract (LM)

In Example 1, 1 g of defatted soybeans were leached overnight with 5 ml of cold distilled water or 0.5 M-NaOH aqueous solution, filtered, and the filtrate was added to 3 times cold 95% ethanol, left overnight in a low temperature chamber, and filtered. Ethanol was removed, neutralized and freeze-dried to obtain low molecular weight soybean extract (LM) dissolved in water and alcohol.

Example 3

Separation of High Molecular Weight (HT) and Low Molecular Weight (LT) from Polysaccharides, Glycoproteins and Proteins

In Example 2, the precipitate obtained by standing in 95% ethanol was dissolved in distilled water to remove ethanol, and then pretreated by repeating the procedure of immersing the ultrafiltration membrane which can fractionate more than 300,000 molecular weight in distilled water and leaving it for 1 hour. The sample was charged into an ultrafiltration cell in a low temperature chamber at 4 ° C., filtered under pressure at about 55 psi and 3.7 atm on a magnetic stirrer, and washed several times. At this time, the filtrate passed through the ultrafiltration membrane is low molecular weight polysaccharides, glycoproteins and proteins (LT), and the remaining on the membrane is high molecular weight proteins, glycoproteins and polysaccharides (HT).

Example 4

Separation of high molecular weight polysaccharide and glycoprotein (HP) and low molecular weight polysaccharide and glycoprotein (LP)

The supernatant obtained by dissolving the 95% ethanol precipitate obtained in Example 2 in cold distilled water and adding cold 15% trichloroacetic acid (TCA) for 1 hour in an ice bath and centrifuging for 1 hour at 12,000Xg was about 4 times cold. It was left overnight in a low temperature room with 95% ethanol. At this time, the resultant precipitate was separated and obtained by centrifugation. The precipitate was neutralized with 2% sodium acetate solution and left overnight in 2x cold ethanol, and the supernatant was centrifuged at 12,000Xg for 15 minutes to obtain a precipitate, and then the precipitate was distilled water. Ultrafiltration was performed in the same manner as in Example 3 to obtain a high molecular weight polysaccharide and glycoprotein mixture (HP) having a molecular weight of 300,000 or more, and a low molecular weight polysaccharide and glycoprotein mixture (LP) having a molecular weight of less than 30 mg.

The following animal tests were performed to compare and measure the hepatoprotective effects using CT, LM, HT, LT, HP and LP obtained above.

Experiment 1

Effects of Total Green Bean Extract (CT) on Acute Hepatic Injury Induced by Carbon Tetrachloride

Thirty female SD rats were divided into five groups, each of which was divided into the following five groups, and the total amount of carbon tetrachloride or green onion extract was administered at different doses. The carbon tetrachloride was mixed with corn oil orally, and the total green onion extract was suspended in 0.9% saline solution. The administration period was 4 days, and the administration schedule is shown in Table 1 below.

A: Normal control

B: carbon tetrachloride administration control group

C: carbon tetrachloride and CT 1mg / Kg / day administration group

D: carbon tetrachloride and CT 20mg / Kg / day administration group

E: group of carbon tetrachloride and CT 100 mg / Kg / day

TABLE 1

Remarks: ○ Saline: 0.9%

○ Carbon tetrachloride: 2.5ml / Kg / day

○ Numeric Unit of CT: mg / kg / day

○ Carbon tetrachloride is mixed orally with corn oil

○ CT is suspended orally in 0.9% saline solution.

After administering the drug as described above, the blood was collected from the heart under anesthesia after 12 hours of fasting in advance, and the activity of serum enzymes was measured as shown in Table 2 below.

TABLE 2

From the above experiment, it can be seen that the hepatoprotective effect is remarkably improved as the total dose of green bean extract is increased. In addition, the liver was extracted from rats of the A, B, and D experimental groups, and the amount of lipid peroxide, which is another indicator of liver damage, was determined by the TBA method. It was confirmed that the decrease significantly.

TABLE 3

Experiment 2

Comparison of Protective Effects of Different Fractions of Green Bean Extract

As an experimental animal, 48 mice were divided into eight groups, such as total head extract (CT), each fraction (LM, HT, HP, LT, LP), carbon tetrachloride administration group, and control group, and the drugs were administered in the same manner as in Example 1. . At this time, the extracts were suspended in 0.9% physiological saline to use a dose of 20ml / Kg / day, carbon tetrachloride was administered orally by mixing 2.5ml / Kg / day with corn oil as in Experiment 1. From the serum obtained on the 5th day, the activity values of S-ALT (GOT) and S-AST (GPT) were measured as shown in Table 4 below.

TABLE 4

From the above experimental results, it was confirmed that the low molecular weight soybean extract (LM, LT, PL) having a molecular weight of less than 300,000 showed little hepatoprotective effect, and the high molecular weight extract showed a particularly high hepatoprotective effect, especially in the HP-administered group.

Experiment 3

Comparison of Hepatoprotective Effects of HP and Alpha Tocopherols

To compare the hepatoprotective effect of alpha-tocopherol known to have hepatoprotective effect and HP of the present invention, experiments were performed using mice as in Experiment 3, and blood of each experimental group was collected to compare S-AST and A-ALT. The results are shown in Table 5 below. However, HP was used as a dose of 20mg / Kg / day, tocopherol was used a dose of 350mg / Kg day, the other experimental group was administered the same dose as in Experiment 3.

TABLE 5

From the above experiments, it can be seen that HP administration shows better hepatoprotective effect than tocopherol.

Experiment 4

Material composition analysis of each fraction of green onion extract

The anthrone test and Lowry analysis were used to determine the sugar and protein content in each fraction of the green bean extract. However, because the content is different depending on the measurement sample, it is shown in the range.

TABLE 6

In addition, the following experiments were carried out to identify the sugar type of the HP with the highest sugar content.

end. reagent

The developing solvent was used by mixing ethyl acetate, pyridine and distilled water in a ratio of 120: 50: 40. The fixation reagent (Location reagent) uses aniline phosphate and its manufacturing method is as follows. 20 ml aniline was mixed with 200 ml distilled water and 180 ml glacial acetic acid and 10 ml of limited phosphoric acid, and then stored at 4 ° C. Immediately before use, the mixture was mixed with acetone 2: 3.

Standard monosaccharides were spotted by dissolving glucose, galactose, arabinose and xylose in 10% (V / V) isopropanol demand.

I. Test contents and results

10 ml of 4N trifluoroacetic acid was added to 50 mg of HP fraction and hydrolyzed in a reflux apparatus. After complete hydrolysis, trifluoroacetic acid was evaporated with a rotary distillation apparatus, and then dissolved in 1 ml of distilled water. Samples and standard monosaccharides were contacted on a HPTLC plate (5 × 10 cm in size pre-coated with silica gel) and developed as a developing solvent. The developed plate was dried and then sprayed with aniline phosphate reagent and heated at 100 ° C. for 2-5 minutes to compare with the spots of standard monosaccharides. As a result, the HP fraction was composed mainly of glucose, arabinose and xylose. It was confirmed that it is composed.

Claims (6)

Hepatoprotective composition comprising a high-molecular weight of soybean extract (HT) that is a polysaccharide, glycoprotein or protein mixture. The hepatoprotective composition according to claim 1, wherein the extract is a mixture of polysaccharides and glycoproteins having high molecular weight (HP). The composition according to claim 1 or 2, wherein the high molecular weight means a molecular weight of 300,000 or more. A hepatoprotective composition comprising a mixture of degreased high molecular weight soybean extract and other hepatoprotective agents. The composition of claim 4, wherein the other hepatoprotective agent is alphatocopherol. The composition of claim 4 or 5, wherein the extract is a polysaccharide and a glycoprotein mixture (HP).