CN114195906A - Screwtree root polysaccharide with sugar metabolism regulation activity and preparation method and application thereof - Google Patents
Screwtree root polysaccharide with sugar metabolism regulation activity and preparation method and application thereof Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
- A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
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Abstract
The invention discloses a screwtree root polysaccharide with sugar metabolism regulation activity and a preparation method and application thereof, and relates to the application of screwtree root polysaccharide in the field of improving sugar metabolism. Taking a certain amount of screwtree root powder, adding distilled water, soaking, performing ultrasonic-assisted extraction, combining extracting solutions, concentrating, centrifuging, removing protein from filtrate, precipitating with ethanol, redissolving, dialyzing, and freeze-drying to obtain screwtree root polysaccharide. Animal experiment results show that the screwtree root polysaccharide prepared by the invention has good sugar metabolism regulation activity, can regulate the level of intestinal flora, and has good development and application prospects.
Description
Technical Field
The invention relates to the technical field of polysaccharides, in particular to helichrysum sessiliflorum polysaccharide with sugar metabolism regulation activity and a preparation method and application thereof.
Background
Diabetes is a common disease and a frequently encountered disease, seriously harms human health, and has a remarkably rising trend in recent years. Diabetes patients need to take medicines for a long time and control diet, and no good medicine can control diabetic complications at present. In order to better control the occurrence of diabetes, people should pay attention to the prevention of diabetes and develop more health foods with good function of improving sugar metabolism.
Researches show that natural plants are an important treasury for developing hypoglycemic health-care foods, and polysaccharides contained in the natural plants have multiple activities of immunoregulation, sugar metabolism improvement and the like, and have low toxicity, multiple target points and certain prevention and control effects on complications, so that the natural plants gradually receive wide attention and attention of people.
Screwtree (L.) is a plant of the genus screwtree of the family Sterculiaceae, and its fruit is similar to sesame fruit. It is mainly distributed on hillsides and hilly lands in southeast Asia, belongs to shrubs, has a plant height of about 1m, and has rough and grey brown or dark brown root surface, slightly fragrant smell and bitter taste. The screwtree has rich chemical components and is rich in phenols, polysaccharides, linoleic acid, monoterpenes and the like. The screwtree root is a common Chinese herbal medicine, can be used as a medicine for treating cold, fever, dysentery, eczema, nonspecific colitis and the like, and has remarkable activity in the aspects of immunoregulation, enzyme inhibition and the like as a main chemical component polysaccharide. The inhibitory activity of helichrysum polysaccharose on alpha-glucosidase is researched by the banmulus trifoliatus. However, no report has been made on the study of polysaccharides from screwtree fruits to improve sugar metabolism.
Disclosure of Invention
Aiming at the defects of the prior art, the invention discloses helichrysum hypargyi polysaccharide with sugar metabolism regulation activity, a preparation method and application thereof.
The invention provides a preparation method of screwtree root polysaccharide with sugar metabolism regulation activity, which comprises the following steps:
firstly, soaking screwtree fruits by using a solvent, and then removing the solvent in the screwtree fruits, wherein the solvent is one or more of petroleum ether, alcohol and an alcohol aqueous solution: soaking screwtree fruits in petroleum ether at a material-liquid ratio of 1:25-40 (g/mL); soaking time: filtering for 5 h, and soaking screwtree with 90% ethanol at a material-liquid ratio of 1:25-40 (g/mL) for: soaking for 5 h, filtering, naturally drying filter residues, and grinding into coarse powder for later use;
the second step is that: soaking in distilled water at a ratio of 1:30-40 (g/mL) for 2-3 h at 80-90 deg.C for ultrasonic-assisted extraction, mixing extractive solutions, rotary evaporating, concentrating, centrifuging at 10000 rpm for 15 min, and mixing solutions;
the third step: removing protein from the solution obtained in the second step by adopting a Sevag method for 6-8 times, and performing rotary evaporation on the obtained aqueous solution for 5-10 min;
the fourth step: adding absolute ethyl alcohol into the solution subjected to rotary evaporation in the third step for alcohol precipitation, wherein the volume ratio of the solution to the absolute ethyl alcohol is 1:3, controlling the content of the ethyl alcohol in an alcohol precipitation system to be 75-85%, standing overnight at 4 ℃, precipitating, and centrifuging to obtain an alcohol precipitate;
the fifth step: adding distilled water into the alcohol precipitate obtained in the fourth step, and redissolving the alcohol precipitate by using the distilled water until the alcohol precipitate is completely dissolved;
and a sixth step: redissolving the alcohol precipitate with distilled water, dialyzing with a dialysis bag with molecular weight cutoff of 3500Da to obtain a trapped fluid, performing rotary evaporation and concentration on the trapped fluid at 70-80 ℃, and freeze-drying the concentrated solution to obtain helichrysum;
the seventh step: the content of polysaccharide is more than 90 percent when measured by a phenol-sulfuric acid method, and the characteristic peak of the polysaccharide of the screwtree fruits is detected by an infrared spectroscopy.
A polysaccharide of Helicterus sibiricus L with sugar metabolism regulating activity is prepared by the above preparation method, and has an average molecular weight of 150 kDa.
The invention also provides application of the screwtree root polysaccharide with the sugar metabolism regulation activity in preparing products with the function of improving sugar metabolism.
Further, in the first step, the alcohol is ethanol, and the aqueous solution of the alcohol is 90% ethanol.
Further, the screwtree root powder ground into coarse powder in the first step has a particle size of 60-80 meshes.
Furthermore, in the second step, the ultrasonic power is 600- & lt1000 & gt W, the ultrasonic extraction time is 20-40 min, and the extraction times are 3-5.
Further, the centrifugation speed in the fourth step is 8000-12000 rpm.
Further, the feed-liquid ratio of the alcohol precipitate to the distilled water in the fifth step is 1:30-60g/mL until the alcohol precipitate is completely dissolved.
Further, the dialysis time in the sixth step is 3 to 5 days.
Has the advantages that:
compared with the application and the prior art, the screwtree root polysaccharide with the sugar metabolism regulation activity and the preparation method thereof provided by the invention have the following advantages:
1. the preparation method has the advantages of mild process, simple operation, high yield and contribution to industrial production;
2. the screwtree polysaccharide has stable structure, high activity and glycolipid metabolism regulation activity;
3. provides the application of the screwtree root in the preparation of the product with the sugar metabolism function;
4. the content of polysaccharide is over 90% by phenol-sulfuric acid method. Detecting a characteristic peak of the screwtree root polysaccharide by an infrared spectroscopy;
5. the screwtree root polysaccharide can obviously reduce the fasting blood sugar value;
6. the polysaccharide of screwtree root has obvious influence on sugar tolerance.
Drawings
Fig. 1 is an infrared spectrum of the screwtree root polysaccharide provided in example 1 of the present invention.
FIG. 2 is a graph showing the change in body weight of mice according to example 4 of the present invention.
Fig. 3 is a graph of experimental FBG of screwtree polysaccharide animal provided in example 4 of the present invention.
FIG. 4 is an OGTT chart of an animal experiment of the screwtree root polysaccharide provided in example 4 of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
The preparation process of the present invention is formulated in view of the following considerations: the preparation method comprises the steps of preparing high-purity silicon nitride powder by using rice hulls as raw materials, using silicon nitride as a raw material, using walnut shells as a pore-forming agent, adding a sintering aid, and changing the porosity and mechanical properties of a sample by changing the doping amount of the walnut shells and the sintering aid; the porosity is controlled by changing the particle size of the silicon nitride powder and the walnut shell powder.
The features and properties of the present invention will be described in further detail below with reference to the accompanying drawings and examples.
Example 1
The embodiment provides a preparation method of screwtree root polysaccharide with sugar metabolism regulation activity, which comprises the following specific steps:
the first step is as follows: adding petroleum ether with a certain volume into screwtree root, soaking for 5 h at a material-liquid ratio of 1:25 (g/mL), filtering, soaking for 5 h with 90% ethanol at a material-liquid ratio of 1:25 (g/mL), removing pigment, polyphenol and other impurities, soaking for 5 h, filtering, naturally drying filter residues, and grinding into coarse powder;
the second step is that: extracting polysaccharides from radix Helicteris with distilled water at 80-90 deg.C at a material-to-liquid ratio of 1:30 (g/mL), soaking for 2-3 h, ultrasonic-assisted extracting at an ultrasonic power of 600W for 20 min with microwave extraction time of 3 times, mixing extractive solutions, centrifuging at 10000 rpm for 15 min, and mixing solutions;
the third step: removing protein by Sevag method for 6 times, and rotary evaporating the obtained solution for 6-8 min;
the fourth step: adding absolute ethyl alcohol into the solution subjected to rotary evaporation in the third step for alcohol precipitation, controlling the concentration of the ethyl alcohol in an alcohol precipitation system to be 75%, standing overnight at 4 ℃, and centrifuging to obtain an alcohol precipitate;
the fifth step: adding distilled water into the alcohol precipitate, and redissolving. Dialyzing with 3500Da molecular weight cutoff dialysis bag for 5 days to obtain the cutoff liquid. Rotary evaporating and concentrating the trapped solution at 70-80 deg.C, and freeze drying the concentrated solution to obtain polysaccharides from radix Helicteris.
Example 2
The embodiment provides a preparation method of screwtree root polysaccharide with sugar metabolism regulation activity, which comprises the following specific steps:
the first step is as follows: adding petroleum ether with a certain volume into sesame, and soaking for 5 h, wherein the material-liquid ratio is 1:30 (g/mL). Filtering, soaking in 90% ethanol at a material-to-liquid ratio of 1:30 (g/mL) for 5 h, removing pigment, polyphenol and other impurities, air drying, and grinding into coarse powder;
the second step is that: extracting polysaccharides from radix Helicteris with distilled water at 80-90 deg.C at a material-liquid ratio of 1:30 (g/mL), ultrasonic power of 800W, microwave extraction time of 30 min for 4 times, mixing extractive solutions, concentrating, and centrifuging;
the third step: removing protein by Sevag method for 7 times, and rotary evaporating the obtained solution for 5-10 min;
the fourth step: adding absolute ethyl alcohol into the water solution for alcohol precipitation, controlling the concentration of the ethyl alcohol in an alcohol precipitation system to be 80%, standing overnight at 4 ℃, and centrifuging to obtain an alcohol precipitate;
the fifth step: adding distilled water into the alcohol precipitate, and redissolving. Dialyzing with 3500Da dialysis bag for 4 days to obtain retentate. Rotary evaporating and concentrating the trapped solution at 70-80 deg.C, and freeze drying the concentrated solution to obtain polysaccharides from radix Helicteris.
Example 3
The embodiment provides a preparation method of screwtree root polysaccharide with sugar metabolism regulation activity, which comprises the following specific steps:
the first step is as follows: adding petroleum ether with a certain volume into the screwtree root, and soaking for 5 hours, wherein the material-liquid ratio is 1:40 (g/mL). Filtering, soaking in 90% ethanol at a material-to-liquid ratio of 1:40 (g/mL) for 5 h, removing pigment, polyphenol and other impurities, air drying, and grinding into coarse powder;
the second step is that: extracting polysaccharides from radix Helicteris with distilled water at 80-90 deg.C at material-liquid ratio of 1:40 (g/mL), ultrasonic power of 1000W, microwave extraction time of 40 min, extraction times of 5 times, mixing extractive solutions, concentrating, and centrifuging;
the third step: removing protein by Sevag method for 8 times, and rotary evaporating the obtained solution for 5-10 min;
the fourth step: adding absolute ethyl alcohol into the water solution for alcohol precipitation, controlling the concentration of the ethyl alcohol in an alcohol precipitation system to be 85%, standing overnight at 4 ℃, and centrifuging to obtain an alcohol precipitate;
the fifth step: adding distilled water into the alcohol precipitate, and redissolving. Dialyzing with 3500Da molecular weight cutoff dialysis bag for 3 days to obtain the cutoff fluid. Rotary evaporating and concentrating the trapped solution at 70-80 deg.C, and freeze drying the concentrated solution to obtain polysaccharides from radix Helicteris.
Infrared spectrum analysis: FIG. 1 shows the infrared spectrum of screwtree root polysaccharide. 3425.91 cm-1A strong absorption peak appears at the position, which is the stretching vibration of the O-H functional group; 1628.58 cm-1The absorption peak at (a) is a vibration absorption peak of C = O; 1419.59 cm-1A bending vibration absorption peak at C-H; 1033.37 cm-1Pyran type sugar rings exist nearby, and the absorption peaks are characteristic absorption peaks of the polysaccharide.
Example 4
The embodiment provides an application of screwtree polysaccharide with sugar metabolism regulation activity:
1. experimental animals:
male mice, purchased from the center of wu's animal, fuzhou, four weeks old. All mice were acclimated to free diet water for one week following the strict schedule.
2. Type 2 diabetes modeling:
randomly selected 10 mice were fed with mouse food as Normal group, and the remaining 30 mice were fed with high-sugar high-fat diet (15% lard, 15% sucrose, 1% cholesterol, 10% egg yolk, 0.2% sodium deoxycholate and 58.8% Normal diet) to induce type 2 diabetes.
After four weeks, 30 mice were injected intraperitoneally with 45 mg/kg STZ solution (dissolved in 0.1mol/L sodium citrate buffer, pH 4.5). Normal group mice were injected intraperitoneally with sodium citrate buffer solution once every other day for three times. Fasting blood glucose values were measured 48 h after the last injection. If the fasting blood glucose value is higher than 11.1mmol/L, the molding is considered to be successful.
3. The grouping and processing method comprises the following steps:
type 2 diabetic mice were randomly divided into 3 groups of 10 mice each. The Model, the screwtree polysaccharide group (300 mg/kg. d), and the metformin hydrochloride group (100 mg/kg. d) were separately subjected to intragastric administration for four weeks. The Normal and Model groups were gazed with purified water (100 mg/kg. d). After the experiment is finished, the mice are killed according to ethical criteria, and all animal experiments meet the ethical guidelines for nursing and using experimental animals of Fujian agriculture and forestry university
4. Observation index
The body weights (g) of the mice were weighed before and after the experiment, respectively, on an electronic scale, and the results are shown in fig. 2:
after 2 weeks of continuous administration, the body weight of mice in both the normal group and the administered group was increased compared to that in the model group; after continuous administration for 4 weeks, the weight of the mice in the model group is obviously reduced, the requirements of a diabetes model are met, and the weight of the mice in the administration group can still ensure gradual increase, which shows that the glucose metabolism function of the mice is improved.
5. Fasting Blood Glucose (FBG)
The mice were gavaged for 0, 2, 4 weeks, the mice were fasted for 12 h before night, 8 o' clock in the morning the next day, and the tail of the mice was sampled and fasting blood glucose values were measured with a glucometer (ohlong, japan).
As shown in FIG. 3, the change of fasting blood glucose FBG after 4 weeks of gavage is shown. As can be seen from the figure, the fasting blood glucose value was significantly reduced in the administration group as compared with the model group, and was comparable to the control group. The application proves that the screwtree polysaccharide has the activity of remarkably improving the sugar metabolism.
6. Sugar tolerance (OGTT)
And in the 4 th week of gastric lavage, fasting before night without water prohibition, measuring fasting blood glucose value as blood glucose value of 0 h at 8 o' clock in the next morning, gavaging 2 g/kg glucose solution in the stomach of the mouse after 5min, and collecting blood at the tail part of 0.5 h, 1 h and 2 h after administration to measure blood glucose value.
As shown in FIG. 4, the blood glucose values of the administered group were all lower than those of the model group from 0 h, 0.5 h, 1 h and 2 h, indicating that the polysaccharides from screwtree have the function of regulating carbohydrate metabolism.
In summary, the screwtree root polysaccharide disclosed by the embodiment of the invention has a stable active structure, can obviously improve sugar metabolism, and can be applied to preparing functional products for regulating sugar metabolism. The preparation method is simple, mild in process conditions, easy to operate, green and environment-friendly in preparation process, and suitable for large-scale production.
The embodiments described above are some, but not all embodiments of the invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the invention as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Claims (9)
1. A preparation method of screwtree root polysaccharide with sugar metabolism regulation activity is characterized by comprising the following steps:
firstly, soaking screwtree fruits by using a solvent, and then removing the solvent in the screwtree fruits, wherein the solvent is one or more of petroleum ether, alcohol and an alcohol aqueous solution: soaking screwtree fruits in petroleum ether at a material-liquid ratio of 1:25-40 (g/mL); soaking time: filtering for 5 h, and soaking screwtree with 90% ethanol at a material-liquid ratio of 1:25-40 (g/mL) for: soaking for 5 h, filtering, naturally drying filter residues, and grinding into coarse powder for later use;
the second step is that: soaking in distilled water at a ratio of 1:30-40 (g/mL) for 2-3 h at 80-90 deg.C for ultrasonic-assisted extraction, mixing extractive solutions, rotary evaporating, concentrating, centrifuging at 10000 rpm for 15 min, and mixing solutions;
the third step: removing protein from the solution obtained in the second step by adopting a Sevag method for 6-8 times, and performing rotary evaporation on the obtained aqueous solution for 5-10 min;
the fourth step: adding absolute ethyl alcohol into the solution subjected to rotary evaporation in the third step for alcohol precipitation, wherein the volume ratio of the solution to the absolute ethyl alcohol is 1:3, controlling the content of the ethyl alcohol in an alcohol precipitation system to be 75-85%, standing overnight at 4 ℃, precipitating, and centrifuging to obtain an alcohol precipitate;
the fifth step: adding distilled water into the alcohol precipitate obtained in the fourth step, and redissolving the alcohol precipitate by using the distilled water until the alcohol precipitate is completely dissolved;
and a sixth step: redissolving the alcohol precipitate with distilled water, dialyzing with a dialysis bag with molecular weight cutoff of 3500Da to obtain a trapped fluid, performing rotary evaporation and concentration on the trapped fluid at 70-80 ℃, and freeze-drying the concentrated solution to obtain helichrysum;
the seventh step: the content of polysaccharide is more than 90 percent when measured by a phenol-sulfuric acid method, and the characteristic peak of the polysaccharide of the screwtree fruits is detected by an infrared spectroscopy.
2. A polysaccharide of Helicterus communis having sugar metabolism-regulating activity, characterized in that: the screwtree polysaccharide with the sugar metabolism regulating activity is prepared by the preparation method, and the average molecular weight of the screwtree polysaccharide with the sugar metabolism regulating activity is 150 kDa.
3. Use of the polysaccharide of screwtree root having sugar metabolism-regulating activity according to claim 2 for the preparation of a product having a function of improving sugar metabolism.
4. The method for producing polysaccharides from screwtree fruits having sugar metabolism-controlling activity according to claim 1, wherein the method comprises the steps of: in the first step, the alcohol is ethanol, and the aqueous solution of the alcohol is 90% ethanol.
5. The method for producing polysaccharides from screwtree fruits having sugar metabolism-controlling activity according to claim 1, wherein the method comprises the steps of: the screwtree root powder ground into coarse powder in the first step has a particle size of 60-80 meshes.
6. The method for producing polysaccharides from screwtree fruits having sugar metabolism-controlling activity according to claim 1, wherein the method comprises the steps of: in the second step, the ultrasonic power is 600- & lt1000 & gt W, the ultrasonic extraction time is 20-40 min, and the extraction times are 3-5.
7. The method for producing polysaccharides from screwtree fruits having sugar metabolism-controlling activity according to claim 1, wherein the method comprises the steps of: the centrifugation rate in the fourth step is 8000-12000 rpm.
8. The method for producing polysaccharides from screwtree fruits having sugar metabolism-controlling activity according to claim 1, wherein the method comprises the steps of: and in the fifth step, the feed-liquid ratio of the alcohol precipitate to the distilled water is 1:30-60g/mL until the alcohol precipitate is completely dissolved.
9. The method for producing polysaccharides from screwtree fruits having sugar metabolism-controlling activity according to claim 1, wherein the method comprises the steps of: the dialysis time in the sixth step is 3 to 5 days.
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