CN105859901B - Alisma orientale polysaccharide with function of improving insulin resistance and extraction method and application thereof - Google Patents
Alisma orientale polysaccharide with function of improving insulin resistance and extraction method and application thereof Download PDFInfo
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Abstract
The invention relates to the technical field of traditional Chinese medicine preparation, and in particular relates to alisma polysaccharide with an insulin resistance improving effect, and an extraction method and application thereof. The method for extracting the alisma polysaccharide with the effect of improving insulin resistance comprises the following steps: selecting rhizoma Alismatis powder with particle size of 40-100 mesh, extracting with cellulase at enzyme concentration of 0.4%, enzymolysis temperature of 35-40 deg.C for 120-; heating and inactivating to obtain Alismatis rhizoma polysaccharide extractive solution. The invention has the beneficial effects that: the method for extracting the alisma orientale polysaccharide with the effect of improving the insulin resistance adopts an enzyme extraction method to extract the alisma orientale polysaccharide, the process is simple, the yield of the alisma orientale polysaccharide is improved, and the extraction rate of the alisma orientale polysaccharide can reach 18.89%. The alisma orientale polysaccharide obtained by the extraction method has an obvious function of reducing blood sugar, and a new way is developed for the fine and deep processing of alisma orientale.
Description
Technical Field
The invention relates to the technical field of traditional Chinese medicine preparation, and in particular relates to alisma polysaccharide with an insulin resistance improving effect, and an extraction method and application thereof.
Background
Insulin Resistance (IR) refers to a decrease in the sensitivity of the target organ of insulin action to insulin action, manifested by impaired glucose utilization by peripheral tissues, mainly muscle and adipose tissues. IR is not only a key link and a major feature of the development of type 2 diabetes (T2DM), but also has close correlation with the development of other complications such as hypertension, atherosclerosis, metabolic syndrome, ovarian cysts, alzheimer's disease, tumors. At present, the drugs for improving and treating insulin resistance, such as insulin sensitizer thiazolidinediones, such as pioglitazone and rosiglitazone, are clinically applied, and side effects caused by the drugs comprise edema and weight gain; metformin causes the accumulation of lactic acid in the blood. Meanwhile, the 2 kinds of drugs have high hepatotoxicity. Therefore, the research on the prevention and treatment of insulin resistance is very important.
Alismatis rhizoma is dried tuber of Alisma orientale (Samuels) Juzep. Recent research shows that the alisma orientale essential oil mainly contains polysaccharide, triterpene, sesquiterpene and other components, and pharmacodynamic research shows that the alisma orientale has the effects of reducing blood sugar, promoting urination, resisting fatty liver, reducing blood fat, reducing blood pressure, resisting allergy, regulating immunity and the like. The polysaccharide is one of the main active ingredients of the Alisma orientale. The polysaccharide is a natural product, has wide sources, has the advantages of small toxic and side effects, no residue, no drug resistance and the like, and is thus valued by researchers. At present, with the research on polysaccharide, the polysaccharide is found to have the efficacy of reducing blood sugar, and has wide sources and no cytotoxicity, so the polysaccharide has great significance for the research on the traditional Chinese medicine polysaccharide, and becomes a hot research field in the medical field.
The extraction method of the polysaccharide mainly comprises a heating extraction method, an enzymolysis method, an ultrasonic extraction method and the like, the extraction methods have own advantages and disadvantages, the existing alisma polysaccharide is quite complex in composition, the separation and purification of various substances are difficult, the separation and purification of the polysaccharide are difficult, and the single saccharide for improving the insulin resistance is difficult to define.
Disclosure of Invention
The technical problem to be solved by the invention is as follows: rhizoma Alismatis polysaccharide with effect of improving insulin resistance, and its extraction method and application are provided.
In order to solve the technical problems, the invention adopts the technical scheme that: provides an extraction method of alisma orientale polysaccharide with the function of improving insulin resistance, which comprises the following steps: selecting crushed rhizoma alismatis powder with the particle size of 40-100 meshes, and extracting by adopting cellulase, wherein the mass volume concentration of the enzyme is 0.4-0.6%, the enzymolysis temperature is 35-40 ℃, the enzymolysis time is 120-180min, the pH is 4-4.5, and the mass-volume ratio of the material liquid is 1:20-1: 30; heating and inactivating to obtain Alismatis rhizoma polysaccharide extractive solution.
Preferably, in the above method for extracting alisma orientale polysaccharide with insulin resistance improving effect, the temperature rise is specifically: treating at 95-100 deg.C for 10 min.
Preferably, the method for extracting alisma orientale polysaccharide with insulin resistance improving effect further comprises the following steps: concentrating, precipitating and drying the obtained Alisma orientale polysaccharide extract to obtain Alisma orientale polysaccharide powder for improving insulin resistance.
Preferably, in the above method for extracting alisma orientale polysaccharide with insulin resistance improving effect, the concentration specifically comprises: filtering the obtained rhizoma Alismatis polysaccharide extractive solution, collecting filtrate, centrifuging at 4000r/min for 15min, collecting supernatant, and concentrating the supernatant to 1/3 of original volume to obtain concentrated solution.
Preferably, in the above method for extracting alisma orientale polysaccharide with insulin resistance improving effect, the "precipitation and drying to obtain alisma orientale polysaccharide powder with insulin resistance improving effect" is specifically: adding 4 times of ethanol into the concentrated solution, collecting the lower layer precipitate, centrifuging at 4000r/min for 15min, collecting precipitate, and vacuum drying to obtain Alismatis rhizoma polysaccharide powder with insulin resistance improving effect.
Preferably, the method for extracting alisma orientale polysaccharide with insulin resistance improving effect specifically comprises the following steps: taking rhizoma alismatis powder of 40 meshes, and mixing the raw materials in a mass-volume ratio of 1:20, adding a citric acid buffer solution with the mass volume concentration of 0.4% of cellulase and the pH value of 4.5 to obtain a mixed solution, heating and carrying out enzymolysis on the mixed solution in a constant-temperature water bath kettle at the temperature of 40 ℃ for 2 hours, and then inactivating the mixed solution in boiling water for 10min to obtain the alisma orientale polysaccharide extracting solution.
The invention provides another technical scheme for providing the alisma orientale polysaccharide prepared by the extraction method of the alisma orientale polysaccharide with the effect of improving insulin resistance.
The invention provides another technical scheme as follows: provides the function of the alisma orientale polysaccharide in preparing the medicine for improving the insulin resistance.
The invention provides another technical scheme as follows: provides a pharmaceutical composition, which comprises a pharmaceutically acceptable carrier and the alisma polysaccharide with a therapeutically effective amount.
Preferably, in the above pharmaceutical composition, the pharmaceutical composition is a granule, powder, tablet, capsule or oral liquid.
The invention has the beneficial effects that: the method for extracting the alisma orientale polysaccharide with the effect of improving the insulin resistance adopts an enzyme extraction method to extract the alisma orientale polysaccharide, the process is simple, the yield of the alisma orientale polysaccharide is improved, and the extraction rate of the alisma orientale polysaccharide can reach 18.89%. The alisma orientale polysaccharide obtained by the extraction method has an obvious function of reducing blood sugar, and a new way is developed for the fine and deep processing of alisma orientale.
Drawings
FIG. 1 is a graph of normal and model glucose tolerance over 8 weeks of molding in example 2 of the present invention;
FIG. 2 is a graph showing the results of glucose tolerance test conducted at the end of 2 weeks by intervention of Alisma orientale polysaccharides;
FIG. 3 is a graph showing the results of the experiment of intervention of Alisma orientale polysaccharide in pyruvate tolerance at the end of 4 weeks.
Detailed Description
In order to explain technical contents, achieved objects, and effects of the present invention in detail, the following description is made with reference to the accompanying drawings in combination with the embodiments.
The most key concept of the invention is as follows: the method for extracting alisma orientale polysaccharide with the effect of improving insulin resistance adopts an enzyme extraction method to extract alisma orientale polysaccharide, has a simple process, and improves the yield of alisma orientale polysaccharide.
Example 1
Taking 40-mesh rhizoma alismatis powder, adding a citric acid buffer solution with pH of 4.5 and cellulose mass concentration of 0.6% according to the mass-volume ratio of the material liquid of 1:20 to obtain a mixed solution, heating the mixed solution in a 40-DEG C constant-temperature water bath kettle for 2 hours, and then inactivating the mixed solution in boiling water for 10min to obtain rhizoma alismatis polysaccharide extracting solution.
Taking 100-mesh rhizoma alismatis powder, adding a citric acid buffer solution with the mass volume concentration of 0.5% of cellulase and the pH value of 4 into the rhizoma alismatis powder according to the mass volume ratio of feed liquid of 1:25 to obtain a mixed solution, heating the mixed solution in a constant-temperature water bath kettle at 40 ℃ for 2 hours, and then inactivating the mixed solution in boiling water for 10min to obtain the rhizoma alismatis polysaccharide extracting solution. Filtering the obtained rhizoma Alismatis polysaccharide extractive solution, collecting filtrate, centrifuging at 4000r/min for 15min, collecting supernatant, and concentrating the supernatant to 1/3 of original volume to obtain concentrated solution. Adding 4 times of ethanol into the concentrated solution, collecting the lower layer precipitate, centrifuging at 4000r/min for 15min, collecting precipitate, and vacuum drying to obtain Alismatis rhizoma polysaccharide powder with insulin resistance improving effect.
Accurately weighing 2.000g of rhizoma alismatis powder of 80 meshes, and adding citric acid buffer solution with pH of 4.5 and cellulase concentration of 0.4% according to the feed-liquid ratio of 1:20 (g/mL); heating the above mixed materials in a 40 deg.C constant temperature water bath kettle for 2 hr, and inactivating in boiling water for 10min to obtain extractive solution. Filtering the extractive solution with gauze, collecting filtrate, centrifuging at 4000r/min for 15min, collecting supernatant, vacuum concentrating to 1/3 of original volume, adding 4 times of anhydrous ethanol into the concentrated solution, mixing with ethanol to obtain ethanol with volume fraction of 80%, sealing, standing in 4 deg.C refrigerator overnight, centrifuging at 4000r/min for 15min the next day to obtain Alismatis rhizoma polysaccharide, and drying the precipitate in freeze drier to obtain Alismatis rhizoma polysaccharide powder.
Example 2 extraction analysis of Alisma orientale polysaccharide with insulin resistance improving effect
The invention relates to alisma polysaccharide and an extraction method thereof
1. The particle size (<40, 40-80, 40-100, 100-150, 150-180, >180 meshes), the material-liquid ratio (1:10, 1:20-1:30, 1:40g/mL), the pH (4, 4.5, 5, 5.5, 6, 6.5), the cellulase concentration (0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6%), the enzymolysis temperature (35, 40, 45, 50, 55, 60 degrees), and the influence of the enzymolysis time (30, 60, 90, 120, 150, 180min) on the extraction rate of the alisma polysaccharides are respectively examined. Obtaining the optimal process, and then optimizing the extraction process by using a response surface method to obtain the optimal process conditions.
2. Accurately weighing 2.000g of rhizoma Alismatis powder of 40-100 meshes, adding 0.4% citric acid buffer solution of cellulase with pH of 4.5 at a material-to-liquid ratio of 1:20 (g/mL); heating the above mixed materials in a 40 deg.C constant temperature water bath kettle for 2 hr, and inactivating in boiling water for 10min to obtain extractive solution. Heating the above mixed materials in a 40 deg.C constant temperature water bath kettle for 2 hr, and inactivating in boiling water for 10min to obtain extractive solution. Filtering the extractive solution with gauze, collecting filtrate, centrifuging at 4000r/min for 15min, collecting supernatant, vacuum concentrating to 1/3 of original volume, adding 4 times of anhydrous ethanol into the concentrated solution, mixing with ethanol to obtain ethanol with volume fraction of 80%, sealing, standing in 4 deg.C refrigerator overnight, centrifuging at 4000r/min for 15min the next day to obtain Alismatis rhizoma polysaccharide, and drying the precipitate in freeze drier to obtain Alismatis rhizoma polysaccharide powder.
3. Response surface experimental design: the BOX-Behnken test Design principle in software Design-expert.V8.0.6 is utilized, the feed-liquid ratio, the enzyme concentration and the pH value are used as independent variables, and the extraction rate of polysaccharide is used as a response value to Design a test.
4. The response surface results are shown in tables 1 and 2.
TABLE 1 Box-Behnken test design protocol and results
| Experiment number | A | B | C | Y |
| 1 | 5.0 | 0.4 | 30 | 9.26 |
| 2 | 4.5 | 0.3 | 10 | 11.63 |
| 3 | 4.0 | 0.4 | 30 | 13.93 |
| 4 | 4.0 | 0.4 | 10 | 9.12 |
| 5 | 4.0 | 0.5 | 20 | 13.52 |
| 6 | 4.5 | 0.4 | 20 | 18.12 |
| 7 | 5.0 | 0.4 | 10 | 4.77 |
| 8 | 4.5 | 0.4 | 20 | 17.72 |
| 9 | 5.0 | 0.5 | 20 | 10.07 |
| 10 | 4.0 | 0.3 | 20 | 12.43 |
| 11 | 4.5 | 0.4 | 20 | 17.65 |
| 12 | 4.5 | 0.4 | 20 | 18.51 |
| 13 | 4.5 | 0.4 | 20 | 18.12 |
| 14 | 4.5 | 0.5 | 30 | 16.07 |
| 15 | 4.5 | 0.5 | 10 | 11.78 |
| 16 | 4.5 | 0.3 | 30 | 16.46 |
| 17 | 5.0 | 0.3 | 20 | 8.13 |
TABLE 2 model regression coefficient significance test results
In conclusion, on the basis of single factor, a regression equation is established through Box-Behnken analysis, and the optimal combination conditions obtained through response surface regression analysis are as follows: the ratio of the feed to the liquid is 1:20, the enzyme concentration is 0.4%, the enzymolysis temperature is 40 ℃, the enzymolysis time is 120min, and the pH value is 4.5. The extraction rate of the alisma polysaccharide extracted by the cellulase is up to 18.89%.
(II) the efficacy test of the alisma polysaccharide
1. Test conditions
1.1 sample: the alisma orientale polysaccharide prepared by the invention.
1.2 test animals: c57BL/6J mouse is suitable, the weight is 18-22 g, SPF grade, purchased from Beijing Huafukang Biotechnology GmbH; license number: SCXK (Jing) 2009-.
1.3 Molding, grouping and handling
1) Molding die
Mice were randomly assigned to a normal group and a high-fat group by weight, and the normal group was given a low-fat diet and the high-fat group was given a high-fat diet. Freely drinking water, feeding for 10 weeks, and judging the success of molding according to the weight difference and the sugar tolerance test result.
Sugar tolerance test: 5 mice were randomly selected for each group and fasted for 12 h. After fasting blood glucose was measured, glucose solution (2g/kg body weight) was injected intraperitoneally. Accurately timing, taking blood from tail vein at 0, 15, 30, 60, 120min to measure blood sugar, drawing blood sugar-time curve, and comparing blood sugar values at each time point.
A pyruvic acid tolerance test;
2) grouping
After the molding is successful, except for the normal group of mice, the high-fat group of mice is randomly divided into 5 groups, namely a model group, a positive medicine rosiglitazone group, an alisma polysaccharide low-dose group and an alisma polysaccharide high-dose group. To prevent self-healing of the model, normal groups of mice continued to be fed low-fat diet during dosing, and the remaining groups continued to be fed high-fat diet.
Blank control group: normal group mice were administered daily
Model group: equivalent distilled water for daily intragastric administration of insulin resistance model mice
Administration to the test groups: the low and high dose groups are 0.15 g/kg-1·d-1，0.3g·kg-1·d-1Alisma orientale polysaccharide gastric administration insulin resistance model mouse.
Positive drug group: the concentration of rosiglitazone was 1 mg/mL. The dosage is 0.01g kg-1·d-1。
1.4 data analysis
Data analysis Using SPSS
2. Test results
The modeling results and the effect of alisma polysaccharide on the glucose tolerance of the tested animals are shown in figure 1, figure 2 and figure 3.
FIG. 1: modeling of normal glucose tolerance and model group glucose tolerance at 8 weeks end
Note a: comparison with the normal group: p < 0.05, P < 0.01.
FIG. 2: and (b) interfering the glucose tolerance test result at 2 weeks, and comparing the result with the model group, wherein P is less than 0.05 and P is less than 0.01.
FIG. 3: and (b) the experimental result of pyruvic acid tolerance of the alisma polysaccharide intervention 4 weeks later, wherein a is compared with the model group, and P is less than 0.05 and less than 0.01.
At the 8 th week of the model building, the results of the glucose tolerance data show that the blood sugar values of the high-fat group at the time points of 30min, 60min and 120min after the intraperitoneal injection of glucose are obviously different from those of the normal group. The molding is successful.
When the medicine is administered for 2 weeks, the results of glucose tolerance data show that the blood sugar values of the rosiglitazone positive medicine group at the time points of 15min, 30min, 60min and 120min after the glucose is injected into the abdominal cavity are obviously different from those of the model group, and the positive medicine rosiglitazone can play the role of reducing the blood sugar of the high-fat feed induced mouse insulin resistance model. The low-dose and high-dose groups of alisma polysaccharide have obvious difference with the model group at 15min, 30min, 60min and 120min, and the pyruvic acid tolerance data result shows that the polysaccharide high-dose group has obvious difference with the model group at 15min, 30min, 60min and 120min after the intraperitoneal injection of pyruvic acid, which indicates that alisma polysaccharide has certain insulin resistance improving effect and dose correlation.
In summary, 0.15 g/kg of alisma polysaccharide-1·d-1、0.3g·kg-1·d-1The fasting blood glucose level of the insulin resistance model mouse can be obviously reduced in the dose group and the model control group.
The above description is only an embodiment of the present invention, and not intended to limit the scope of the present invention, and all equivalent changes made by using the contents of the present specification and the drawings, or applied directly or indirectly to the related technical fields, are included in the scope of the present invention.
Claims (3)
1. The extraction method of alisma orientale polysaccharide with the function of improving insulin resistance is characterized by comprising the following steps: selecting crushed rhizoma alismatis powder with the particle size of 40-100 meshes, and extracting by adopting cellulase, wherein the mass volume concentration of the enzyme is 0.4-0.6%, the enzymolysis temperature is 35-40 ℃, the enzymolysis time is 120-180min, the pH is 4-4.5, and the mass volume ratio g/mL of feed liquid is 1:20-1: 30; heating and inactivating to obtain Alismatis rhizoma polysaccharide extractive solution; filtering the obtained Alisma orientale polysaccharide extract, collecting filtrate, centrifuging at 4000r/min for 15min, collecting supernatant, and concentrating the supernatant to 1/3 of the original volume to obtain concentrated solution; adding 4 times of ethanol into the concentrated solution, collecting the lower layer precipitate, centrifuging at 4000r/min for 15min, collecting precipitate, and vacuum drying to obtain Alismatis rhizoma polysaccharide powder with insulin resistance improving effect.
2. The method for extracting alisma orientale polysaccharide with insulin resistance improving effect according to claim 1, wherein the temperature raising is specifically as follows: treating at 95-100 deg.C for 10 min.
3. The method for extracting alisma orientale polysaccharide with insulin resistance improving effect according to claim 1, comprising the following steps: taking rhizoma alismatis powder of 40 meshes, and mixing the raw materials in a mass-volume ratio of 1:20, adding a mixed solution of a citric acid buffer solution with the mass volume concentration of 0.4% of cellulase and the pH value of 4.5, putting the mixed solution into a constant-temperature water bath kettle at 40 ℃ for heating and enzymolysis for 2 hours, and then inactivating in boiling water for 10min to obtain an alisma polysaccharide extracting solution; filtering the obtained Alisma orientale polysaccharide extract, collecting filtrate, centrifuging at 4000r/min for 15min, collecting supernatant, and concentrating the supernatant to 1/3 of the original volume to obtain concentrated solution; adding 4 times of ethanol into the concentrated solution, collecting the lower layer precipitate, centrifuging at 4000r/min for 15min, collecting precipitate, and vacuum drying to obtain Alismatis rhizoma polysaccharide powder with insulin resistance improving effect.
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