KR0155231B1 - Proteoglycan extracted from artemisza swa yomogi and their preparation method - Google Patents

Abstract

Protein polysaccharide obtained by separating from Artemisia swayomogi according to the present invention is excellent in the treatment and prevention of liver disease, glucose 71.2%, xylose 25.4%, mannose 2.8%, rhamno It contains 0.6% (mol%) of rhamnose, 1.4% of nitrogen, and the protein consists of the following amino acids.

Description

Mugwort Protein Polysaccharide and Extract Purification Method thereof

FIG. 1 is an ultraviolet-visible spectral spectrum of the final fraction (KP) of phospholipid protein polysaccharides

FIG. 2 is an infrared spectral spectrum of the final fraction (KP) of phospholipid protein polysaccharide.

FIG. 3 is an amino acid analysis chromatogram of the final fraction (KP) of phospholipid protein polysaccharide.

[Purpose of invention]

The present invention relates to the extraction and purification of the erythroid protein polysaccharide excellent in hepatocyte regeneration effect and hepatitis treatment effect.

Injin mugwort (Artemisia iwayomogi) is also called injin, injinho, heat keeper or evergreen mugwort, and is a very rich plant resource native to all parts of Korea.

Injin mugwort is not only rich in resources, but is also a plant of the Asteraceae, which is frequently used for various liver diseases such as hepatitis, liver cancer, and cirrhosis in folk medicine. In Korea, since there are many drinking populations and various liver-related diseases have emerged as a national problem, it is an object of the present invention to separate and purify protein polysaccharide fractions having excellent liver function improvement effect as a jinjin mugwort.

[Prior Art and Problems]

The conventional technique of using jinjin mugwort in liver disease is mainly to extract the active ingredients such as capillarisin, scoparon, etc. using an organic solvent to produce a crude state (JP 90142781).

However, when organic solvents are used, a small amount of organic solvents may remain in the final product, which may be harmful to the human body, and may cause serious environmental pollution even in the disposal of organic waste liquids.

On the other hand, there are no patents related to the purification of Injin mugwort protein polysaccharides with Sephadex by extracting Injin mugwort with water.

[Configuration, Action and Effect of the Invention]

The present invention was purified by extracting Injin mugwort with water to determine the constituents of the extract to determine that it is a protein polysaccharide, this protein polysaccharide has excellent hepatoprotective function. When explaining the present invention.

Finely chopped Injin mugwort and extract 2-3 times with water of 40-100 ° C. and pH 3-8 for 2-3 hours, followed by filtration.

The filtrate was concentrated under reduced pressure, and excess water-soluble alcohol (meaning C1-C5 aliphatic alcohol such as methanol, ethanol, butanol) was added thereto, and the mixture was left to stand in a 0-10 ° C refrigerator for 24 hours.

At this time, a large amount of precipitate is formed. Filtering this precipitate and freeze-drying can yield the Injin mugwort protein polysaccharide.

The purification method for determining the fraction of the phospholipid protein polysaccharide having excellent activity in improving liver function is as follows.

Phosphorus wormwood protein polysaccharide is dissolved in water and passed through a column filled with Sephadex, Rose, Sephacryl, etc., or passed through an ultrafiltration membrane to receive a fraction.

Each fraction is obtained by ultraviolet-visible spectrum at a wavelength of 200-800 nm, and fractions having high absorbance are selected and taken as final fractions (KP).

Lyophilize this KP.

This KP has a very good ability to rejuvenate hepatocytes and to cure hepatitis. Next, the present invention will be described in more detail as Examples and Experimental Examples.

Example 1

Finely grind 200 gr of phosphorus mugwort and extract twice with 9 hours with an aqueous solution of 45-55 ° C. adjusted to pH 4.

After filtration, the filtrate was concentrated under reduced pressure and lyophilized to obtain 50.2 gr of X.

After dissolving the extract in 120 ml of water, 20 gr of ammonium sulfate was added and 500 ml of ethanol was added and left in a refrigerator at 5 ° C. for 24 hours to obtain 5.0 gr of phosphorus polysaccharide, a precipitate.

After filtering the phospholipid protein polysaccharide, 5 ml fractions were collected from the column packed with Sephadex G 200, and the ultraviolet-visible spectrum was obtained at a wavelength of 400 nm, respectively.

The main fraction was lyophilized for 20 hours at -40 ° C, 10 hours at -30 ° C, 5 hours at -20 ° C, 3 hours at -10 ° C, and 1 hour at 0 ° C to obtain the final fraction, KP.

After hydrolysis of KP, constituent sugars were measured by HPLC, and the results were 71.2% glucose, 25.4% xylose, 2.8% mannose, and 0.6% rhamnose. .

Elemental analysis of KP contains 1.4% nitrogen, suggesting that KP is a protein polysaccharide with amino acids.

Experimental Example 1

[Measurement of GOP and GPT]

The following animal experiments were conducted to determine KP's hepatocyte regeneration ability.

As experimental animals, Sprague-Dawley rats with a body weight of about 200 gr were acclimated for at least one week in an animal breeding room maintained at constant temperature and humidity.

The experimental method is as follows.

CCl ₄ and corn oil mixed solution (1: 9v / v%) was intraperitoneally administered 0.2ml per 100gr body weight, and then 48 hours later, under anesthesia with ether, the body was circulated in the abdominal aorta and centrifuged. , GPT activity was measured.

Each dose of the drug treatment group was administered 0.5% CMC solution, and suspended in a mixture of PVP and tween 80 CCl ₄ CCl ₄ and administered 24 hours before administration After 2 hours and 24 hours after each of 3 single oral.

The experimental results are as follows.

Under normal conditions, GOT and GPT levels in rats were 60 ± 6U / L and 25 ± 2U / L, respectively, but the GOT value was 1207 ± 139U / L in the CCl-treated control group and the GPT value was increased to 731 ± 133U / L, respectively. It was.

However, in the group treated with KP three times, the GOT value was found to be greatly reduced to 206 ± 97U / L.

Therefore, KP, a fraction of the Artemisia erythrocyte protein polysaccharide, purified by Sephadex, is considered to have strong hepatocyte regeneration ability.

Experimental Example 2

[Measurement of AST and ALT]

As experimental animals, Sprague-Dawley rats with a body weight of about 200 gr were acclimated for at least one week in an animal breeding room maintained at constant temperature and humidity.

The experimental method is as follows.

Galactosamine (sigma chemical co., USA) was intraperitoneally administered 0.2ml (800mg / kg) per 100gr body weight in the rat, and 48 hours later, it was opened under ether anesthesia, collected in the abdominal aorta, and centrifuged. ALT activity was measured.

Each drug in the treatment group was suspended orally in a 0.5% CMC solution containing PVP and tween 80 once a day from 3 days before galactosamine administration.

The experimental results are as follows.

Under normal conditions, AST and ALT levels in rats were 60 ± 6U / L and 25 ± 2U / L, respectively, but AST was 2655.8 ± 144.7U / L for galactosamine-treated control group, and ALT was 1935.1 ± 128.6U / L, respectively. Increased to.

However, in the group treated with KP three times, the AST value was significantly decreased to 1137.6 ± 192.1U / L and ALT to 742.8 ± 97.4U / L.

Therefore, KP, a protein polysaccharide fraction of Injin mugwort purified with Sephadex, seems to have a strong acute hepatitis treatment effect.

Experimental Example 3

The ultraviolet-visible spectral spectrum of KP is shown in FIG. 1, and the infrared spectral spectrum is shown in FIG.

Amino acid qualitative and quantitative analysis of KP was carried out using an amino acid autoanalyzer.

The analysis results are shown in the following table.

Amino acid analysis chromatogram is shown in FIG.

In addition, elemental analysis of KP shows that carbon: hydrogen: nitrogen is composed of 34.2: 3.7: 1.4 ratio, respectively.

As confirmed in the above experimental results, it can be seen that the erythroid polysaccharide of the present invention has an excellent hepatoprotective function.

Until now, it is known that capillarisin and scoparone, which are low molecular materials, are extracted from organic solvents when the substances that improve liver function in jinjin mugwort have been extracted with organic solvents. Phosphorus mugwort protein polysaccharide, a high-molecular substance, was found to improve liver function.

Experimental Example 4

[Acute Toxicity Test]

Injin mugwort has been commercially available for a long time, and 2 g of protein polysaccharide per Kg was orally administered to Sprague-Dawley rats weighing 200 gr. Therefore, it is considered that Injin mugwort polysaccharide is not toxic.

Claims (2)

Contains 71.2% glucose, 25.4% xylose, 2.8% mannose and 0.6% (mol%) rhamnose, contains 1.4% nitrogen and the following amino acids Injin mugwort polysaccharide excellent in the prevention and treatment of liver disease with a constitution. Extraction of Artemisia swayomogi with water of pH 3-8, and filtration. The filtrate is concentrated, precipitated with ethanol and purified to extract Injin mugwort protein polysaccharide with excellent liver disease prevention and treatment.