CN1267456C - Process for preparing glucose-reducing sand sagebrush polysaccharide and its use - Google Patents

Abstract

The invention relates to a kind of Artemisia annua polysaccharide prepared by extracting, separating, enzymatically hydrolyzing and column chromatography using waste residue after removing oil from natural Artemisia annua seeds as raw material, and its preparation method and application, which is characterized in that it uses 50-80% Ethanol extraction, chloroform and n-butanol mixture for extraction, papain enzymatic hydrolysis, obtained through sephadex G-200 column chromatography, used to treat chemical diabetes caused by alloxan, and reduce the damage of diabetes to the liver and kidney , reduce blood aspartate aminotransferase, alanine aminotransferase, urea nitrogen content, and can inhibit weight loss caused by diabetes, simple production process, good hypoglycemic effect, no toxic side effects, easy to use, good effect for treating diabetes.

Description

A kind of preparation method and application of artemisia polysaccharide for hypoglycemic

technical field

The invention relates to a method for preparing Artemisia annua polysaccharide with hypoglycemic effect, which is prepared by extracting, separating, enzymatically hydrolyzing and column chromatography using the waste residue after removing oil from natural Artemisia annua seeds and its application.

Background technique

Diabetes mellitus is a metabolic disorder syndrome characterized by chronic hyperglycemia associated with genetic factors and various environmental factors. It is a common disease that seriously endangers human health worldwide. In recent years, the prevalence and mortality of diabetes in the world have shown a gradual upward trend, and it has become one of the key issues to be solved and overcome in the world's medical field today. Commonly used hypoglycemic drugs are mainly synthetic drugs, which have certain damage to the liver and kidney and are not easily accepted by patients.

Patent JP95/00560 applied for a new pyridine-type thiazolidine compound and its salt with hypoglycemic effect; patent CN86104075 (86104075) applied for the preparation method of hypoglycemic drugs-thiazolidinedione, and they are effective in treating hyperglycemic animals. Application methods and pharmaceutical formulations containing them; patent CN1252273 (99121819.1) applied for the preparation and application of a new generation of sulfonylurea hypoglycemic agents-selenomethionine chromium sulfonylurea hypoglycemic agents; patent CN1465342 (02112414.0) applied for a thiazole Alkyl salt derivative hypoglycemic drug; CN1113491 (95102055.2) prepares new hydroxylamine compound with hypoglycemic activity by selectively reducing suitable 1-(2-, 3- or 4-nitrobenzenesulfonyl) derivatives.

Patent US20030072822 applied for a method of ethanol extract of tarragon to treat diabetes; patent US20040151783 applied for a polysaccharide component of Gymnema sylvestris leaf with hypoglycemic effect; Among them, grapefruit oil has a hypoglycemic effect on diabetic patients. Patent US20030198697 applied for a fatty acid component extracted from coix, which has hypoglycemic effect. Patent CN1188651 (97100285.1) extracts a kind of high-efficiency pure natural hypoglycemic health care product or medicine containing hypoglycemic active ingredients extracted from wild stone lotus; A new technology for extracting blood sugar and nutrients from raw materials; patent CN1137411 (95105954.8) applied for a Chinese medicine "Rehabilitation Pancreatic Islet Hypoglycemic Pill" composed of 12 kinds of traditional Chinese medicines such as ginseng and gecko and its production method; patent CN1401318 ( 02138749.4) Extracting and separating the effective parts and active ingredients with hypoglycemic effect from the traditional Chinese medicine Guijianyu, including terpenoids, phenolic acids, etc., which have the effect of reducing blood sugar in diabetic mice, and at the same time affect malondialdehyde and superoxide dismutase , can reduce or inhibit the occurrence and development of diabetic complications; patent CN1456337 (03128089.7) applied for a bitter melon product containing multiple hypoglycemic active ingredients and its preparation method; patent CN1306752 (00101247.9) disclosed a Edible health biscuits—the formula and production process of food fiber potluck biscuits. The biscuit mainly contains flour, dietary fiber, active polysaccharide, yam, bitter gourd, etc., and is developed through scientific methods.

Patent PCT/KR98/00325 has applied for a kind of as hypoglycemic agent as the bioflavonoid of molecular formula (I) or the purposes that the plant extract that contains same bioflavonoid reduces the blood sugar level of mammal; Patent CN1348762 (00116167.9) uses bitter Preparation of medicaments for treating diabetes from ginseng flavonoids, and preparation of compound medicaments for treating diabetes by using at least one of the active ingredients of sophora flavonoids and astragalus polysaccharides, ginseng polysaccharides, ginsenosides, Ophiopogon japonicus polysaccharides, trichosanthin polysaccharides, and mulberry leaf total polysaccharides; patent CN1389146 (02138749.4) extract of Cyclocarya paliurus, together with flavoring agent and auxiliary substances, and add two or more substances in sodium alginate, carboxymethyl cellulose, agar, and xanthan gum as stabilizers to obtain Hypoglycemic, blood pressure, and blood fat-lowering health food; patent CN1245060 (98110398.7) combines (1) L-carnitine, (2) chitosan, (3) chromium-rich microbial polysaccharides in a high-efficiency weight-loss diet according to a certain formula And the preparation method of hypoglycemic medicine or health food; the patent CN1398630 (02136740.X) extracts the sugar complex with hypoglycemic activity from the Chinese medicinal material Atractylodes Rhizoma Atractylodes Rhizome, its production method and application. None of the patents at home and abroad involve the preparation method and application of the hypoglycemic natural product made of artemisia polysaccharide as raw material.

Contents of the invention

The purpose of the present invention is to use the waste residue after degreasing of Artemisia annua as raw material, and prepare a kind of natural can reduce blood sugar through extraction, separation, enzymolysis, column chromatography, etc., and can reduce the damage to liver and kidney caused by diabetes. The damage is to reduce the contents of aspartate aminotransferase, alanine aminotransferase and urea nitrogen in the blood, as well as the natural biological polysaccharide Artemisia polysaccharide that inhibits weight loss caused by diabetes, and its preparation method and application.

The steps of the present invention are as follows,

Step 1: After the Artemisia annua seeds are degreased by _CO2 supercritical extraction, fully dissolve in water, the ratio of raw materials to water is 1:30-50, add ethanol to the aqueous solution, and let the ethanol content reach 50-80%, then stand still and stay overnight , Centrifuge, collect the precipitate, and vacuum-dry to obtain substance A;

Step 2: Weigh a certain weight of substance A, dissolve it with 80-100 times the weight of water, add papain to the aqueous solution for enzymatic hydrolysis, then extract with a 1:2-4 mixture of chloroform and n-butanol, and collect the water layer The solution was concentrated under reduced pressure and dried to obtain substance B, the volume ratio of the aqueous solution of A and the extract was 4:1, and the amount of papain was 24.00U/2000ml of aqueous solution;

Step 3: Pass substance B through sephadex G-200 column chromatography, eluent: 0.05molNacl solution, flow rate: 8ml/h, collect 1.5ml of eluent after chromatography, concentrate under reduced pressure and release Put it into a dialysis bag for dialysis for 36-48 hours, add 2-3 times the amount of ethanol to the dialysate, let it stand overnight, centrifuge, collect the precipitate, and dry it in vacuum to obtain the natural biopolysaccharide of pure Artemisia annua seeds.

Molecular weight and chemical composition: the molecular weight Mn is about 1.42×10 ⁵ g/mol, and the second Veliy coefficient is 3.64×10 ^-5 cm ³ mol/g ² ; it consists of L-arabinose, D-xylose, and D-lysol Sugar, D-mannose, D-glucose, D-galactose and other six monosaccharides, the molar ratio is 0.14:0.09:1.51:0.17:0.07:1.45, white powder.

This natural biological polysaccharide is mainly used to treat chemical diabetes, and can reduce the damage of diabetes to liver and kidney, etc. Pharmacological experiments on rats showed that feeding the preparation for 30 days reduced the blood sugar of hyperglycemic animals by 13.98%, alanine aminotransferase ( GPT) decreased by 25.48%, aspartate transaminase (GOT) decreased by 28.7%, blood urea nitrogen content decreased by 51.27%, and the weight loss caused by diabetes was significantly slowed down.

The characteristics of the present invention: the present invention uses the waste residue after degreasing of Artemisia annua as raw material, and prepares a kind of natural blood sugar that can lower blood sugar and reduce the damage of diabetes to the liver and kidney through extraction, separation, enzymolysis, column chromatography, etc. Injuries are preparations that reduce the levels of aspartate aminotransferase, alanine aminotransferase, and urea nitrogen in the blood, and can inhibit weight loss caused by diabetes. Compared with other hypoglycemic drugs, it has the advantages of simple production process, good hypoglycemic effect, no toxic and side effects, convenient use, and good curative effect on type I and type II diabetes.

Detailed ways

Example 1:

Preparation method and application of hypoglycemic natural product from Artemisia annua seeds

Step 1: The part of Artemisia annua seeds degreased by _CO2 supercritical extraction is fully dissolved in water (the ratio of raw material to water is 1:30), and ethanol is added to the aqueous solution (the ethanol content reaches 50%) to stand still, overnight, and centrifuged , collect the precipitate and dry it in vacuum to obtain substance A;

Step 2: take a certain weight of material A and dissolve it in water with 80 times its weight, add papain to the water solution for enzymolysis, then extract with chloroform and n-butanol (1:2) mixture, collect the aqueous layer solution to reduce Concentrate under pressure and dry to obtain substance B, the volume ratio of A aqueous solution and extract is 4:1, and the amount of papain is 24.00U/2000ml aqueous solution;

Step 3: Pass substance B through sephadex G-200 column chromatography (eluent: 0.05molNacl solution, flow rate: 8ml/h, collect 1.5ml of eluent after chromatography), and concentrate the eluent under reduced pressure Put it into a dialysis bag for dialysis for 36 hours, add 2 times the amount of ethanol to the dialysate, let it stand overnight, centrifuge, collect the precipitate and dry it in vacuum to obtain a pure hypoglycemic natural product.

Hypoglycemic effect: Alloxan-induced chemical diabetic rats took this product and the blood sugar, alanine aminotransferase, aspartate aminotransferase, and urea nitrogen all decreased. The results are shown in the following table:

control group High blood sugar group     Hyperglycemia group + this product Blood sugar (m mol/L) 3.77 5.71 4.48 13.98% reduction Alanine aminotransferase (Iμ/L) 27.0375 77.025 51.5136 Decrease by 25.48% Aspartate aminotransferase (Iμ/L) 146.375 230 165 28.7% reduction Burea nitrogen (m mol/L) 7.0225 26.7425 13.0136 51.27% reduction

Body weight change: the average weight of each group is shown in (Table 1). The weight of the control group increased, and the body weight of the hyperglycemia group decreased significantly, with a reduction rate of 7.69%. The rate is 1.56%, indicating that it has a certain inhibitory effect on the weight loss of individuals with high blood sugar.

Table 1 Body weight of rats in each experimental group (g/rat) number of days 1 30 days growth trend Hyperglycemia group 260.1 240.1 -7.69% High blood sugar + artemisinin 269.2 265.5 -1.56% control group 266.2 289 +8.56%

The molecular weight and chemical composition of pure hypoglycemic natural products were characterized by dynamic and static light scattering, gas chromatography, infrared spectroscopy, and nuclear magnetic resonance.

Dynamic and static light scattering instrument and gas chromatography confirmed that the weight-average molecular weight Mn of the pure hypoglycemic natural product is about 1.42×10 ⁵ g/mol, and the second Veliy coefficient is 3.64×10 ^-5 cm ³ mol/g ² ; Composed of six monosaccharides including arabinose, D-xylose, D-lyxose, D-mannose, D-glucose, and D-galactose, the molar ratio is 0.14:0.09:1.51:0.17:0.07:1.45, white powder.

Glucose (Glu) was determined by glucose oxidase method, alanine aminotransferase (GPT) and aspartate aminotransferase (GOT) were determined by continuous detection method, and urea nitrogen (BuN) was determined by endpoint method. The instrument uses a fully automatic biochemical analyzer, OLYcupus AU400 (Japan). Alloxan was purchased from sigma company.

After taking this product, the blood sugar, alanine aminotransferase, aspartate aminotransferase, and urea nitrogen content of alloxan-induced chemical diabetic animals were all reduced, indicating that the hypoglycemic effect is better.

Example 2:

Preparation method and application of hypoglycemic natural product from Artemisia annua seeds

Step 1: Part of Artemisia annua seeds degreased by _CO2 supercritical extraction is fully dissolved in water (the ratio of raw material to water is 1:50), and ethanol is added to the aqueous solution (the ethanol content reaches 80%) to stand still, overnight, and centrifuged , collect the precipitate and dry it in vacuum to obtain substance A;

Step 2: take a certain weight of material A and dissolve it in 100 times the weight of water, add papain to the water solution for enzymolysis, then extract with chloroform and n-butanol (1:4) mixture, collect the aqueous layer solution to reduce Concentrate under pressure and dry to obtain substance B, the ratio of A aqueous solution and extract is 4:1 by volume, and the amount of papain is 24.00U/2000ml aqueous solution;

Step 3: Pass substance B through sephadex G-200 column chromatography (eluent: 0.05molNacl solution, flow rate: 8ml/h, collect 1.5ml of eluent after chromatography), and concentrate the eluent under reduced pressure Put it into a dialysis bag for dialysis for 48 hours, add 3 times the amount of ethanol to the dialysate, let it stand overnight, centrifuge, collect the precipitate and dry it in vacuum to obtain the pure hypoglycemic natural product.

The results of the rat test are the same as in Example 1, and the molecular weight and chemical composition of the pure hypoglycemic natural product are the same as in Example 1.

Example 3:

Preparation method and application of hypoglycemic natural product from Artemisia annua seeds

Step 1: Part of Artemisia annua seeds degreased by CO _{supercritical} extraction is fully dissolved in water (the ratio of raw material to water is 1:40), and ethanol is added to the aqueous solution (the ethanol content reaches 70%) to stand still, overnight, and centrifuged , collect the precipitate and dry it in vacuum to obtain substance A;

Step 2: take a certain weight of material A and dissolve it in water with 90 times its weight, add papain in the water solution to carry out enzymolysis, then extract with chloroform and n-butanol (1:3) mixed solution, collect the aqueous layer solution to reduce Concentrate under pressure and dry to obtain substance B, the ratio of A aqueous solution and extract is 4:1 by volume, and the amount of papain is 24.00U/2000ml aqueous solution;

Step 3: Pass substance B through sephadex G-200 column chromatography (eluent: 0.05molNacl solution, flow rate: 8ml/h, collect 1.5ml of eluent after chromatography), and concentrate the eluent under reduced pressure Put it into a dialysis bag for dialysis for 36-48 hours, add 2.5 times the amount of ethanol to the dialysate, let it stand overnight, centrifuge, collect the precipitate and dry it in vacuum to obtain the pure hypoglycemic natural product.

The results of the rat test are the same as in Example 1, and the molecular weight and chemical composition of the pure hypoglycemic natural product are the same as in Example 1.

Claims (3)

1. the artemisia desertorum polysaccharide preparation method of a hypoglycemic is characterized in that: the waste residue that utilizes the natural biological sand sagebrush (Artemisia filifolia) to slough behind the oil obtains through the following step for raw material:

Step 1: the sand sagebrush (Artemisia filifolia) seed is through CO ₂After supercritical extraction was sloughed grease, water fully dissolved, and the ratio of raw material and water is 1: 30-50, in the aqueous solution, add ethanol, and that ethanol content reaches is static behind the 50-80%, spend the night, centrifugal, collecting precipitation, vacuum-drying gets substance A;

Step 2: the substance A that takes by weighing certainweight, water dissolution with 80-100 times of weight, in water dissolution, add papoid and carry out enzymolysis, use 1 again: 2-4 chloroform and propyl carbinol mixed solution extract, collect that the water layer solution decompression concentrates, dry substance B, the A aqueous solution and extraction liquid ratio by volume are that 4: 1, papoid consumption are the 24.00U/2000ml aqueous solution;

Step 3: substance B is passed through sephadex G-200 column chromatography, elutriant is: 0.05molNacl solution, flow velocity: 8ml/h, every part of 1.5ml of elutriant behind the collection chromatography, put into the dialysis tubing 36-48h that dialyses behind the elutriant concentrating under reduced pressure, dialyzate add 2-3 doubly measure ethanol static, spend the night, centrifugal, collecting precipitation, vacuum-drying gets pure sand sagebrush (Artemisia filifolia) natural seed biological polyoses.

2. the artemisia desertorum polysaccharide of the preparation method of artemisia desertorum polysaccharide according to claim 1 preparation, it is characterized in that: it is made up of L-arabinose, D-wood sugar, D-lyxose, D-seminose, D-glucose, six kinds of monose of D-semi-lactosi, mol ratio was followed successively by 0.14: 0.09: 1.51: 0.17: 0.07: 1.45, weight-average molecular weight Mn was 1.42 * 10 ⁵G/mol.

3. the application of artemisia desertorum polysaccharide according to claim 2 is characterized in that: be used for the treatment of the chemotype diabetes that tetraoxypyrimidine causes.