CN112521524B - Composition for synergistically reducing blood sugar by using lycium barbarum polysaccharide and acaudina molpadioides functional peptide - Google Patents
Composition for synergistically reducing blood sugar by using lycium barbarum polysaccharide and acaudina molpadioides functional peptide Download PDFInfo
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Abstract
The invention provides a composition which takes lycium barbarum polysaccharide and acaudina molpadioides polypeptide as main active ingredients and has an auxiliary blood sugar reducing effect, and the composition has a protection and improvement effect on type II diabetic rats caused by streptozotocin. The composition prepared by the invention has a synergistic interaction effect and can be used for assisting in reducing blood sugar in foods, functional foods, foods with special medical application and medicines. The lycium barbarum polysaccharide with the blood sugar reducing function is prepared by taking lycium barbarum as a raw material, and is low in cost, green and environment-friendly; after the functional peptide of acaudina molpadioides is added, the hypoglycemic activity of the composition of the lycium barbarum polysaccharide and the functional peptide of acaudina molpadioides is obviously higher than that of the single lycium barbarum polysaccharide and the polypeptide of acaudina molpadioides, and the composition has wide medicinal value and application prospect.
Description
Technical Field
The invention belongs to the technical field of active substance extraction and application, and particularly relates to a composition for synergistically reducing blood sugar by using lycium barbarum polysaccharide and acaudina molpadioides functional peptide.
Background
With the development of society and the change of people's eating habits, diabetes has gradually developed into the third chronic common endocrine and metabolic diseases which endanger human health after cardiovascular diseases and tumors. The number of people with diabetes mellitus in China is the second in the world, and is a great burden affecting the physical health of the people. Because of the chronic nature of diabetes, long-term adjuvant therapy is very important in controlling the condition. Therefore, the current treatment scheme of diabetes is highly advocated for diet therapy in all countries, and the overall treatment scheme of combining exercise therapy and drug therapy on the basis of diet therapy is emphasized, wherein the diet therapy is the drive horse of the car and is most important for controlling diabetes. Therefore, the development of the health-care food for assisting in reducing blood sugar is an important way for reducing the harm of diabetes.
Lycium barbarum polysaccharides are the main active ingredients in lycium barbarum, and researches show that the lycium barbarum polysaccharides can promote the transformation, synthesis and storage of hepatic glycogen and relieve clinical symptoms of diabetes. The acaudina molpadioides contains rich proteins, polysaccharides, essential amino acids, trace elements and other active substances, is low in price and high in yield as low-value sea cucumbers, can be developed and utilized on a large scale, and has the activities of reducing blood sugar, reducing blood fat and the like. Most of domestic health-care foods with the auxiliary blood sugar reducing function take one or two blood sugar reducing functional factors as the functional components of the products. The development of a compound product which produces synergistic effect by two blood sugar reducing functional factors of lycium barbarum polysaccharide and acaudina molpadioides polypeptide has practical and important significance.
Disclosure of Invention
The invention aims to provide a composition which takes lycium barbarum polysaccharide and acaudina molpadioides polypeptide as main active ingredients and has the effect of assisting in reducing blood sugar. Animal experiments show that the composition has protection and improvement effects on type II diabetic rats caused by streptozotocin. The composition prepared by the invention has a synergistic interaction effect and can be used for assisting in reducing blood sugar in foods, functional foods, foods with special medical application and medicines.
The invention firstly provides a lycium barbarum polysaccharide with the efficacy of reducing blood sugar, and the preparation method comprises the following steps:
1) adding pure water into the medlar for shearing, and extracting the shearing liquid by using deionized water; centrifuging the extracting solution to obtain an extracting solution I;
2) passing the extracting solution I through a microfiltration membrane system with the molecular weight of 100 ten thousand, and collecting the extracting solution penetrating through the microfiltration membrane to obtain an extracting solution II;
3) concentrating the extracting solution II through an ultrafiltration membrane system with the molecular weight of 1000, and collecting the extracting solution intercepted by an ultrafiltration membrane to obtain an extracting solution III;
4) concentrating the extract III under reduced pressure at a temperature not higher than 60 ℃, adding ethanol, stirring, standing for precipitation, centrifuging, and drying the precipitate to obtain lycium barbarum polysaccharide;
the provided lycium barbarum polysaccharide is used for preparing a blood sugar reducing product;
in another aspect of the invention, the hypoglycemic composition is provided, wherein the lycium barbarum polysaccharide and the acaudina molpadioides functional peptide are proportioned according to the following parts by weight: 10-90 parts of wolfberry polysaccharide and 10-90 parts of acaudina molpadioides functional peptide.
Preferably, the weight part ratio of the lycium barbarum polysaccharide to the acaudina molpadioides functional peptide is 30: 70.
The preparation method of the acaudina molpadioides polypeptide comprises the following steps:
1) crushing a raw material acaudina molpadioides, and adding distilled water to obtain a suspension;
2) Adding compound protease into the suspension obtained in the step 1) for enzymolysis, adding ethanol for polysaccharide precipitation after enzymolysis is finished, centrifuging and taking supernatant to obtain total acaudina molpadioides polypeptide solution;
wherein the protease is a compound enzyme of trypsin and papain,
the enzymolysis condition is 1-5h, temperature is 50 ℃, pH value is 6-8;
the centrifugation conditions were as follows: 8000-12000rpm, centrifuging for 10-30 min;
3) separating the total acaudina molpadioides polypeptide solution to obtain an acaudina molpadioides active polypeptide solution; the separation conditions were as follows: firstly, membrane separation with molecular weight of 1000Da is utilized, and then membrane separation with molecular weight of 360Da is utilized to obtain polypeptide with the range of 360-1000 Da;
in order to facilitate taking, the blood sugar reducing composition is prepared into oral preparations such as capsules, tablets, granules and the like according to conventional pharmaceutical auxiliary materials and processes.
The invention adopts the medlar as the raw material to prepare the medlar polysaccharide with the function of reducing blood sugar, has low cost and is green and environment-friendly; after the functional peptide of acaudina molpadioides is added, the hypoglycemic activity of the composition of the lycium barbarum polysaccharide and the functional peptide of acaudina molpadioides is obviously higher than that of the single lycium barbarum polysaccharide and the polypeptide of acaudina molpadioides, and the composition has wide medicinal value and application prospect.
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FIG. 1: graph of the effect of different substances on oral glucose tolerance in type ii diabetic rats.
Detailed Description
The present invention will be described in detail with reference to examples.
Example 1 preparation of Lycium barbarum polysaccharides
In the research of the applicant, the lycium barbarum polysaccharide prepared by the existing method does not have obvious hypoglycemic activity, wherein the lycium barbarum polysaccharide prepared by the existing method comprises the following steps:
pulverizing 5kg fructus Lycii, adding 10 times of pure water, heating and boiling for 4 hr, filtering with gauze to obtain supernatant, adding 10 times of pure water into the residue, heating and boiling for 4 hr, filtering with gauze to obtain supernatant, mixing the two supernatants, drying under reduced pressure, adding 95% ethanol when the liquid concentration volume is 2.5 times of fructus Lycii material mass, stirring while adding until ethanol concentration reaches 75%, continuing stirring for 30min, standing for 8 hr, collecting precipitate, and air drying for 12 hr at 65 deg.C to obtain fructus Lycii polysaccharide.
The extract yield of the lycium barbarum polysaccharide prepared by the method is 23.2%, and the content of the lycium barbarum polysaccharide is 31.47% by performing content measurement by using a phenol-sulfuric acid method.
In order to ensure that the prepared lycium barbarum polysaccharide has obvious hypoglycemic activity, the applicant improves the preparation method and establishes the following preparation method:
Pulverizing 5kg fructus Lycii, adding 10L pure water, shearing with high speed shearing machine for 10min for 4 times to obtain fructus Lycii soaking solution, and the shearing rotation speed is 10000 rpm. Transferring the sheared wolfberry fruit soaking solution to an extraction tank, adding deionized water with the volume 7 times that of the wolfberry fruit soaking solution, and extracting for 2 hours at the extraction temperature of 60 ℃; 10 times of deionized water was added for the 2 nd extraction. Centrifuging with a tubular centrifuge at 16000rpm after extraction. Extracting for 2 times, and mixing to obtain extractive solution I. Passing the extractive solution I through a microfiltration membrane system with molecular weight of 100 ten thousand, and passing through the membrane at 50 deg.C and pressure of 2 bar. Collecting the extract penetrating through the microfiltration membrane to obtain an extract II. Concentrating the extractive solution II by ultrafiltration membrane system with molecular weight of 1000, filtering at 30 deg.C under 10 bar. Collecting the extract intercepted by the ultrafiltration membrane to obtain an extract III. And continuing to concentrate the extracting solution III under reduced pressure, wherein the temperature is lower than 60 ℃. When the concentration is 2.5 times of the wolfberry fruit, the wolfberry fruit is put into an alcohol precipitation tank. Adding ethanol while stirring to make ethanol concentration reach 75%, and stirring for 30 min. And standing for 8 h. Separating and recovering supernatant, and air drying the precipitate for 12h at 65 ℃. Obtaining brown lycium barbarum polysaccharide. The content of the lycium barbarum polysaccharide is 55.62% by measuring the content of the lycium barbarum polysaccharide by a phenol-sulfuric acid method. The product has obvious blood sugar reducing activity through animal experiment detection.
Example 3 preparation of functional peptide of acaudina molpadioides
Crushing 100g of dry acaudina molpadioides, and adding water to prepare a suspension, wherein the mass ratio of acaudina molpadioides to water is 1: 6; then adding 2% of compound enzyme (mass ratio is 1:1) of trypsin and papain for enzymolysis, wherein a magnetic stirrer is used in the enzymolysis process, the enzymolysis time is 4h, the temperature is 50 ℃, and the pH value is 7; after enzymolysis, adding ethanol to precipitate polysaccharide, centrifuging at 8000rpm for 20min, taking supernatant, filtering the supernatant through 1000Da and 360Da membranes, collecting 360-fold concentrated solution with molecular weight of 1000, decolorizing the concentrated solution with activated carbon, removing fishy smell, and spray drying to obtain 10.65g of acaudina molpadioides polypeptide. The product has obvious blood sugar reducing activity through animal experiment detection.
Example 4: preparation of hypoglycemic composition
The lycium barbarum polysaccharide prepared by the establishing method of the invention in the embodiment 1 and the acaudina molpadioides functional peptide are compounded according to the following proportions by weight: 10-90 parts of wolfberry polysaccharide and 10-90 parts of acaudina molpadioides functional peptide. Through activity screening analysis, the wolfberry polysaccharide: when the ratio of the functional peptide of the acaudina molpadioides to the functional peptide is 30:70, the effect of reducing blood sugar is best.
Example 5: preparation of capsules
The raw materials are proportioned (by weight percent): 80 parts of hypoglycemic composition (medlar polysaccharide: acaudina molpadioides functional peptide: 30:70) and 20 parts of microcrystalline cellulose. The preparation process comprises the following steps: mixing the blood sugar reducing composition and microcrystalline cellulose, granulating, and encapsulating. Each granule is 100 mg.
Example 6: preparation of tablets
The raw materials are proportioned (by weight percent): 80 parts of hypoglycemic composition (medlar polysaccharide: acaudina molpadioides functional peptide: 30:70) and 20 parts of microcrystalline cellulose. The preparation process comprises the following steps: mixing the blood sugar reducing composition and microcrystalline cellulose, granulating, mixing, granulating, and tabletting. Each granule is 100 mg.
Example 7: preparation of granules
The raw materials are proportioned (by weight percent): 80 parts of hypoglycemic composition (medlar polysaccharide: acaudina molpadioides functional peptide: 30:70) and 20 parts of microcrystalline cellulose. The preparation process comprises the following steps: mixing the blood sugar reducing composition and microcrystalline cellulose, granulating, mixing, granulating, and packaging. Each granule is 100 mg.
Example 8: animal experiments
Referring to the health food function evaluation file of the State food and drug administration: national food and drug administration [2012] No. 107 annex 3 auxiliary hypoglycemic function evaluation method.
60 Wistar male rats (180-220 g) are kept at room temperature of 25 +/-0.5 ℃ and relative humidity of 50 +/-5%, and a light/dark cycle of 12h/12h is maintained. All mice were given the same amount of basal diet and provided adequate drinking water acclimatization for one week. The normal group was given ordinary diet, and the diabetic group was given high-fat diet (high-fat diet formulation: 66.5% basal diet, 2.5% cholesterol, 20% sucrose, 10% lard, 1% bile salt) for 4 consecutive weeks, with free diet of water for each group of experimental rats. After 4 weeks, the rats in the experimental group were injected with 28mg/kg bw STZ (dissolved in citrate buffer, pH 4.4) and the rats in the normal control group were injected with the same dose of physiological saline. And (4) collecting blood from the tail vein to measure the fasting blood glucose value, and successfully modeling by taking the blood glucose value of more than or equal to 11.1mmol/L as a model II type diabetes rat model. Gavage was continued for 4 weeks according to experimental groups.
Twelve of the groups were randomly selected as normal control groups, and the remainder were used as diabetic groups. The experimental components are a model control group, a composition treatment group (the sum of the polypeptide and polysaccharide contents of the acaudina molpadioides polypeptide and lycium barbarum polysaccharide composition used by each kilogram of rats is 0.05 g/kg-bw for low dose, 0.1 g/kg-bw for medium dose, 0.2 g/kg-bw for high dose, 0.1 g/kg-bw for acaudina molpadioides polypeptide treatment group and 0.1 g/kg-bw for lycium barbarum polysaccharide treatment group). 34d is continued until the end of the experiment, and the blank control group and the model group are perfused with the same volume of solvent.
(1) Determination of fasting plasma glucose (FGB)
After the last administration, fasting is not forbidden for 5h, 20 μ L of blood from rat tail vein is added into 0.4ml protein precipitant, mixed, stood for 7min at room temperature, centrifuged for 15min at 7000r/min, 250 μ L of supernatant is taken to determine fasting blood glucose content according to the method of glucose determination kit specification.
Compared with the blank control group, the fasting blood glucose value of the model group is obviously reduced, and the statistical difference is generated (P is less than 0.01) (Table 1). Compared with a model group, the blood sugar level of the rats with type II diabetes can be reduced by the composition in the low, medium and high dose groups, wherein the blood sugar level of the rats in the composition in the high dose group is obviously reduced (15.5 +/-3.3 mmol/L) (P < 0.01). When the lycium barbarum polysaccharide and the acaudina molpadioides polypeptide are used independently, the effect is not good as that of the composition, and the synergistic effect of the lycium barbarum polysaccharide and the acaudina molpadioides polypeptide in the composition enhances the blood sugar reducing effect.
Table 1: fasting blood glucose level
Note: p < 0.05, P < 0.01, compared to the blank control group; comparison with model control#P＜0.05，##P＜0.01。
(2) Oral glucose tolerance assay
After the last administration, the patient is fasted for 5 hours without water prohibition, and the gastric lavage glucose solution is 2g/(kg bw). The blood sugar content was measured at 0, 0.5, 1 and 2h after gastric lavage, and the area under the curve (AUC) was calculated according to the formula: AUC [ mmol/(h.L) ] -0.5A + B + C +0.5D
As can be seen from fig. 1, from 0min, the blood glucose of rats in each group is increased and then decreased, compared with the rats in the type ii diabetes model group, the blood glucose levels of the rats in the high, medium and low dose composition groups are decreased from 60min, and the glucose AUC is significantly decreased compared with the model group (table 2), which indicates that the high, medium and low dose hypoglycemic compositions can inhibit the blood glucose increase induced by glucose, thereby alleviating the decrease of glucose tolerance of the diabetic rats, and the hypoglycemic effect is more obvious compared with the effect of using acaudina molpadioides polypeptide and lycium barbarum polysaccharide alone, indicating that the synergistic effect of the two in the composition enhances the hypoglycemic effect.
TABLE 2 Effect on oral glucose tolerance in type II diabetic rats
Note: p < 0.05, P < 0.01, compared to the blank control group; comparison with model control #P＜0.05，##P＜0.01。
(3) Serum insulin assay:
after the last administration, the rats were anesthetized with ether without fasting for 10h, blood was taken from the abdominal aorta, and serum was isolated. Serum insulin levels were determined with reference to kit instructions.
As shown in Table 3, compared with the normal control group, the serum insulin content of the model control group is reduced by 29.81 percent (P < 0.05), and the serum insulin level of the diabetic rats in the high-dose group can be increased to the level of normal rats (P < 0.05) after the dry prognosis of the composition.
Table 3: effect on serum insulin of type II diabetic rats
Note: p < 0.05, P < 0.01, compared to the blank control group; comparison with model control#P＜0.05，##P＜0.01。
In conclusion, the blood sugar reducing composition provided by the invention can improve the serum insulin level of diabetic rats and has an effect of improving insulin resistance of type II diabetic rats. The effect of the acaudina molpadioides polypeptide and the lycium barbarum polysaccharide which are used independently is obviously lower than that of the composition, and the synergistic effect of the acaudina molpadioides polypeptide and the lycium barbarum polysaccharide is shown.
Claims (7)
1. The blood sugar reducing composition is characterized in that the blood sugar reducing composition comprises the following wolfberry polysaccharide and acaudina molpadioides functional peptide in parts by weight: 10-90 parts of wolfberry polysaccharide and 10-90 parts of acaudina molpadioides functional peptide with the blood sugar reducing effect;
The preparation method of the lycium barbarum polysaccharide comprises the following steps:
1) adding pure water into the medlar for shearing, and extracting the shearing liquid by using deionized water; centrifuging the extracting solution to obtain an extracting solution I;
2) passing the extracting solution I through a microfiltration membrane system with the molecular weight of 100 ten thousand, and collecting the extracting solution penetrating through the microfiltration membrane to obtain an extracting solution II;
3) concentrating the extracting solution II through an ultrafiltration membrane system with the molecular weight of 1000, and collecting the extracting solution intercepted by an ultrafiltration membrane to obtain an extracting solution III;
4) concentrating the extractive solution III at 60 deg.C or below under reduced pressure, adding ethanol, and stirring
Standing for precipitation, centrifuging, and drying the precipitate to obtain fructus Lycii polysaccharide.
2. The hypoglycemic composition of claim 1, wherein the ratio of the lycium barbarum polysaccharides to the acaudina molpadioides functional peptides in the composition is 30:70 parts by weight.
3. The hypoglycemic composition according to claim 1 or 2, wherein the functional peptide of acaudina molpadioides is prepared by the following steps:
1) crushing a raw material acaudina molpadioides, and adding distilled water to obtain a suspension;
2) adding compound protease into the suspension obtained in the step 1) for enzymolysis, adding ethanol for polysaccharide precipitation after enzymolysis is finished, centrifuging and taking supernatant to obtain total acaudina molpadioides polypeptide solution; the protease is a compound enzyme of trypsin and papain;
3) Separating the total acaudina molpadioides polypeptide solution to obtain an acaudina molpadioides functional peptide solution; the separation conditions were as follows: firstly, the membrane with the molecular weight of 1000Da is used for separation, and then the membrane with the molecular weight of 360Da is used for separation, so that the polypeptide with the range of 360-1000Da is obtained.
4. The hypoglycemic composition of claim 3, wherein the enzymatic hydrolysis conditions are 1-5 hours, 50 ℃ and 6-8 pH.
5. The hypoglycemic composition of claim 3, wherein the centrifugation conditions in 2) are as follows: 8000-12000rpm, and centrifuging for 10-30 min.
6. The hypoglycemic composition of claim 1, further comprising pharmaceutically acceptable conventional excipients.
7. The hypoglycemic composition of claim 6, which is in the form of capsule, tablet or granule.
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