Compound preparation containing agriophyllum squarrosum crude oligosaccharide and preparation and application thereof

Abstract

The invention relates to the technical field of Mongolian medicine biological preparations, in particular to a compound preparation containing agriophyllum squarrosum crude oligosaccharide and preparation and application thereof. The compound preparation containing the agriophyllum squarrosum crude oligosaccharide comprises the following raw materials in parts by mass: 4-8 parts of the agriophyllum squarrosum crude oligosaccharide, 1-2 parts of resveratrol, 5-10 parts of sodium carboxymethyl cellulose, 1-2 parts of cellulase, 1-4 parts of sodium alginate and 1-2 parts of lactoferrin peptide. The method is simple in process, low in cost and suitable for mass production; and the agriophyllum squarrosum crude oligosaccharide is loaded and compounded with lactoferrin and resveratrol, so that the stability of the product is improved, and meanwhile, the agriophyllum squarrosum crude oligosaccharide can be used for treating insulin resistance, and is small in dosage, high in drug effect, convenient to take and free of toxic and side effects.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of Mongolian medicine biological agents, and particularly relates to a compound preparation containing coarse oligosaccharides of Haloxylon ammodendron and preparation and application thereof. BACKGROUND

[0002] Haloxylon ammodendron is distributed in Northeast China, North China and Northwest China, and resources thereof are very rich; Haloxylon ammodendron likes to grow in semi-fixed sandy land with good moisture conditions and loose soil, has strong vitality, and has short breeding cycle, fast breeding speed, high photosynthetic efficiency and strong adaptability to harsh environments such as wind and sand.

[0003] Haloxylon ammodendron is a Mongolian medicine material in common use, and the whole grass thereof can be used as medicine, and resources thereof are particularly rich and curative effect thereof is reliable and reliable. In the whole grass of Haloxylon ammodendron, the water content is about 77%, the polysaccharide content is about 0.041%, the protein content is 4.4%, the crude fiber content is 3.7%, the fat content is about 0.4%, the ash content is about 5.1%, and the nitrogen-free extract is 8.7%.

[0004] At present, the chemical composition of Haloxylon ammodendron is less studied in the field of medicine, and according to the domestic and foreign literatures and work, it is found that Haloxylon ammodendron has good effects of reducing blood sugar and regulating blood fat in addition to the antioxidant effect, has the effects of delaying or inhibiting liver and kidney damage caused by diabetes. At present, it is found that the main effective component is coarse oligosaccharide (AOS), and oligosaccharide is also called oligosaccharide, and has certain research and development value.

[0005] Insulin resistance (IR) is the pathological basis of various metabolic diseases, is the key mechanism causing type 2 diabetes, and is the main feature of type 2 diabetes and the key factor for the progression of type 2 diabetes. Therefore, the research of drugs for improving IR is concerned by people. However, the current Haloxylon ammodendron preparation is unstable in blood sugar control, and the bioavailability is insufficient; and after long-term use, the sensitivity of patients to coarse oligosaccharides of Haloxylon ammodendron decreases, and the dosage needs to be continuously increased to maintain the curative effect, thereby increasing the metabolic burden of liver and kidney.

[0006] Diabetic osteoporosis (DOP) is a common complication of diabetes, and its mechanism involves inhibition of osteoblast activity and hyperactivity of osteoclasts. The existing Haloxylon ammodendron preparation has insufficient osteogenic stimulation, and the promotion effect of coarse oligosaccharides of Haloxylon ammodendron on osteogenic differentiation is weak; it is difficult to improve the abnormal bone mineralization of diabetes patients caused by calcium absorption disorder, and it is urgent to be solved. SUMMARY

[0007] The present application aims at solving the problems in the prior art, and provides a compound preparation containing coarse oligosaccharides of Haloxylon ammodendron and preparation and application thereof.

[0008] The compound preparation containing coarse oligosaccharides of Haloxylon ammodendron comprises, by mass fraction, Haloxylon ammodendron coarse oligosaccharides 4-8 parts, white resveratrol 1-2 parts, sodium carboxymethyl cellulose 5-10 parts, cellulase 1-2 parts, sodium alginate 1-4 parts and lactoferrin peptide 1-2 parts.

[0009] Preferably, the enzyme activity of the cellulase is 400-600 U / g.

[0010] Preferably, the polysaccharide of the Phragmites australis is prepared by the following method: steaming, drying, crushing, adding 90-95% ethanol aqueous solution to reflux extract 2-3 times, filtering to obtain the first extract; adding 70-75% ethanol aqueous solution to the residue to reflux extract 2-4 times, filtering to obtain the second extract; combining the first extract and the second extract, recovering ethanol under reduced pressure, concentrating, diluting with water, standing for 20-40 h, passing through an adsorption resin column, eluting, collecting the eluate, decolorizing, concentrating under reduced pressure to extract, and drying under reduced pressure.

[0011] More preferably, the mass ratio of the Phragmites australis to the ethanol aqueous solution is 1:5-10, and the mass ratio of the residue to the ethanol aqueous solution is 1:5-10.

[0012] More preferably, the adsorption resin column is a D101 macroporous adsorption resin column.

[0013] The above method for preparing the compound preparation containing the polysaccharide of the Phragmites australis comprises the following steps: S1. Adding sodium carboxymethylcellulose to water, stirring at 60-70℃ for 1-2 h, cooling to 5-15℃, adding cellulase and sodium alginate to the mixture and stirring for 10-20 min, adding dropwise to a calcium chloride solution and stirring for 10-20 min, standing for 1-2 h, filtering, washing, and freeze-drying to obtain a pre-preparation; S2. Adding the pre-preparation to water, adjusting the pH of the system to 5-5.5, stirring at 45-55℃ for 10-15 h, filtering after inactivation, washing, and freeze-drying; adding the product and the polysaccharide of the Phragmites australis to water, ultrasonic treatment for 10-20 min, adding lactoferrin peptide and resveratrol, stirring for 1-2 h, and freeze-drying.

[0014] Preferably, in S1, the particle size of the pre-preparation is 50-80 μm.

[0015] Preferably, in S1, the concentration of the calcium chloride solution is 0.5-1 mol / L.

[0016] Preferably, in S2, the ultrasonic frequency is 60-70 kHz.

[0017] The polysaccharide of the Phragmites australis or the compound preparation containing the polysaccharide of the Phragmites australis is used for preparing a medicament for treating insulin resistance or osteoporosis. Beneficial effects

[0018] The sodium carboxymethyl cellulose used in the application has good compatibility with sodium alginate, and the microspheres obtained after the two are compounded by hydrogen bonds and then solidified have high stability, the sodium carboxymethyl cellulose is hydrolyzed by cellulase to form a hydrolysis product which penetrates from the inside to the outside of the microspheres, the internal void structure of the product is uniform, which is beneficial to the stable loading of the psammophila crude oligosaccharide, and the lactoferrin peptide and the resveratrol form a complex through hydrogen bond interaction, so that the psammophila crude oligosaccharide is not easy to overflow, and the loading stability is high.

[0019] The application realizes the slow release and intestinal target delivery of the psammophila crude oligosaccharide, avoids gastric acid degradation, and the hydrogen bond complex formed by the lactoferrin peptide and the resveratrol further reduces the risk of burst release of the psammophila crude oligosaccharide and prolongs the half-life thereof. The psammophila crude oligosaccharide, in combination with the lactoferrin peptide and the resveratrol, together improves insulin resistance and cooperatively corrects the bone resorption-formation imbalance of diabetic osteoporosis. The inventors found in the research process that the psammophila crude oligosaccharide, after being stably loaded and compounded with the lactoferrin peptide and the resveratrol, can produce obvious synergistic effect, is used for the prevention and treatment of diabetes, has rapid onset, and can reduce the incidence of gastrointestinal reactions during use.

[0020] The compound preparation of the application reduces the dosage of the psammophila crude oligosaccharide, avoids receptor desensitization, reduces the metabolic load of liver and kidney, and at the same time can reduce the direct stimulation of the psammophila crude oligosaccharide on the gastrointestinal tract, and the natural ingredient combination avoids the hepatotoxicity risk of chemically synthesized drugs.

[0021] The application provides an extraction process of the psammophila crude oligosaccharide, which is simple in process, low in cost, suitable for mass production, improves the product stability after the psammophila crude oligosaccharide is loaded and compounded with the lactoferrin peptide and the resveratrol, and can be used for treating insulin resistance, has small dosage, high drug efficacy, is convenient to take, and has no toxic side effects. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 The blood glucose comparison chart of the blank control group, the model group, the AOS group, the example 5 group and the comparative example group of mice.

[0023] Figure 2 The serum inflammatory factor (TNF-α, IL-1β) comparison chart of the blank control group, the model group, the AOS group, the example 5 group and the comparative example group of mice.

[0024] Figure 3 The left tibia HE staining comparison chart of the blank control group, the model group, the AOS group, the example 5 group and the comparative example group of mice.

[0025] Figure 4 The bone volume fraction (Tb.BV / TV) and cortical bone thickness (Ct.Th) comparison chart of the blank control group, the model group, the AOS group, the example 5 group and the comparative example group of mice.

[0026] Figure 5 The bone structure model index (SMI) and the trabecular bone number (Tb.N) of the mice in the blank control group, the model group, the AOS group, the Example 5 group and the Comparative Example group are compared. DETAILED DESCRIPTION

[0027] The application will be further described below with reference to specific examples. Example 1

[0028] A compound preparation containing a crude oligosaccharide of Suaeda salsa, which raw materials include: 40 g of the crude oligosaccharide of Suaeda salsa, 10 g of resveratrol, 50 g of sodium carboxymethyl cellulose, 10 g of cellulase with an enzyme activity of 400 U / g, 10 g of sodium alginate and 10 g of lactoferrin peptide.

[0029] The crude oligosaccharide of Suaeda salsa is prepared by the following operation: Suaeda salsa is steamed for 5 h, dried in the air, freeze-dried, ground and passed through a 100-mesh sieve, 90% ethanol aqueous solution (the mass ratio of the ethanol aqueous solution to the Suaeda salsa is 5:1) is added for reflux extraction twice, the filtrate is combined after filtration, the first extract is obtained; 70% ethanol aqueous solution (the mass ratio of the ethanol aqueous solution to the residue is 5:1) is added to the residue for reflux extraction twice, the filtrate is combined after filtration, the second extract is obtained; the first extract and the second extract are combined, ethanol is recovered under reduced pressure, concentrated to no alcohol taste, diluted with an equal volume of deionized water, placed for 20 h, the filtrate is filtered, the D101 macroporous adsorption resin column is used for elution with deionized water, the water eluate is collected, activated carbon is used for decolorization, concentrated under reduced pressure to extract, dried under reduced pressure and ground.

[0030] The preparation method of the compound preparation containing the crude oligosaccharide of Suaeda salsa includes the following steps: S1, sodium carboxymethyl cellulose is added to 400 g of deionized water, stirred at a temperature of 60℃ for 1 h, cooled to 5℃, cellulase and sodium alginate are added thereto, stirred for 10 min, added dropwise to a calcium chloride solution with a concentration of 0.5 mol / L, stirred at a speed of 1000 r / min for 10 min, placed for 1 h, filtered, washed with deionized water and freeze-dried to obtain preformed material with a particle size of 80 μm; S2, the preformed material is added to 1000 g of deionized water, the pH value of the system is adjusted to 5-5.5, stirred at a temperature of 45℃ for 1 h at a stirring speed of 50 r / min, filtered after inactivation, washed and freeze-dried; the product and the crude oligosaccharide of Suaeda salsa are added to 1000 g of deionized water for ultrasonic treatment for 10 min at an ultrasonic frequency of 60 kHz, lactoferrin peptide and resveratrol are added and stirred for 1 h, and freeze-dried. Example 2

[0031] A composite preparation containing a crude oligosaccharide of halodule japonica okamura, raw materials of which include: a crude oligosaccharide of halodule japonica okamura 80g, resveratrol 20g, sodium carboxymethyl cellulose 100g, cellulase with enzyme activity of 600U / g 20g, sodium alginate 40g, lactoferrin peptide 20g.

[0032] The crude oligosaccharide of halodule japonica okamura is prepared by the following operation: halodule japonica okamura is steamed for 10h, dried in air, freeze-dried, crushed to pass through a 100-mesh sieve, 95% ethanol aqueous solution (mass ratio of ethanol aqueous solution to halodule japonica okamura is 10:1) is added for reflux extraction for 3 times, the filtrate is combined after filtration, to obtain a first extract; 75% ethanol aqueous solution (mass ratio of ethanol aqueous solution to the residue is 5:1) is added to the residue for reflux extraction for 4 times, the filtrate is combined after filtration, to obtain a second extract; the first extract and the second extract are combined, ethanol is recovered under reduced pressure, concentrated to no alcohol taste, diluted with an equal volume of deionized water, placed for 40h, the filtrate is filtered, and then passed through a D101 macroporous adsorption resin column, eluted with deionized water, the water eluate is collected, decolorized with activated carbon, concentrated under reduced pressure to extract, dried under reduced pressure, and crushed.

[0033] The preparation method of the composite preparation containing the crude oligosaccharide of halodule japonica okamura includes the following steps: S1, sodium carboxymethyl cellulose is added to 600g deionized water, stirred at a temperature of 70℃ for 2h, cooled to 15℃, cellulase and sodium alginate are added thereto, stirred for 20min, added dropwise to a calcium chloride solution with a concentration of 1mol / L, stirred at a speed of 2000r / min for 20min, placed for 2h, filtered, washed with deionized water, and freeze-dried to obtain a preform with a particle size of 50μm; S2, the preform is added to 2000g deionized water, the pH value of the system is adjusted to 5-5.5, stirred at a temperature of 55℃ for 2h at a stirring speed of 100r / min, filtered after inactivation, washed, and freeze-dried; the product and the crude oligosaccharide of halodule japonica okamura are added to 2000g deionized water for ultrasonic treatment for 20min at an ultrasonic frequency of 70kHz, lactoferrin peptide and resveratrol are added and stirred for 2h, and freeze-dried. Example 3

[0034] A composite preparation containing a crude oligosaccharide of halodule japonica okamura, raw materials of which include: a crude oligosaccharide of halodule japonica okamura 80g, resveratrol 20g, sodium carboxymethyl cellulose 100g, cellulase with enzyme activity of 600U / g 20g, sodium alginate 40g, lactoferrin peptide 20g.

[0035] The following operations are used to prepare the coarse oligosaccharides of Spartina: Spartina is steamed for 9 hours, dried in the air, and then freeze-dried. The Spartina is ground and passed through a 100-mesh sieve. A 95% ethanol aqueous solution (the mass ratio of the ethanol aqueous solution to the Spartina is 7:1) is added to the Spartina for reflux extraction three times. The filtrate is obtained by filtering the Spartina. A 75% ethanol aqueous solution (the mass ratio of the ethanol aqueous solution to the Spartina is 7:1) is added to the residue for reflux extraction four times. The filtrate is obtained by filtering the residue. The first filtrate and the second filtrate are combined. The ethanol is recovered under reduced pressure. The filtrate is diluted with an equal volume of deionized water. The solution is allowed to stand for 35 hours. The filtrate is obtained by filtering the solution. The filtrate is passed through a D101 macroporous adsorption resin column. The filtrate is eluted with deionized water. The water eluate is collected. The water eluate is decolorized with activated carbon. The eluate is concentrated under reduced pressure. The eluate is dried under reduced pressure. The dried eluate is ground.

[0036] The above method for preparing the composite preparation containing the coarse oligosaccharides of Spartina includes the following steps: S1. Sodium carboxymethyl cellulose is added to 450 g of deionized water. The solution is stirred at a temperature of 68°C for 80 minutes. The solution is cooled to a temperature of 12°C. Cellulase and sodium alginate are added to the solution. The solution is stirred for 12 minutes. A 0.8 mol / L calcium chloride solution is added dropwise to the solution. The solution is stirred at a speed of 1200 r / min for 17 minutes. The solution is allowed to stand for 80 minutes. The solution is filtered. The filter cake is washed with deionized water. The filter cake is freeze-dried to obtain a preformed material with a particle size of 70 μm. S2. The preformed material is added to 1800 g of deionized water. The pH value of the system is adjusted to 5-5.5. The solution is stirred at a temperature of 42°C for 100 minutes. The stirring speed is 90 r / min. The solution is filtered after inactivation. The filter cake is washed. The filter cake is freeze-dried. The product is added to 1200 g of deionized water. The solution is ultrasonically treated for 18 minutes. The ultrasonic frequency is 62 kHz. Lactoferricin and resveratrol are added to the solution. The solution is stirred for 100 minutes. The solution is freeze-dried. Example 4

[0037] A composite preparation containing coarse oligosaccharides of Spartina includes the following raw materials: coarse oligosaccharides of Spartina 70 g, resveratrol 12 g, sodium carboxymethyl cellulose 90 g, cellulase with an enzyme activity of 450 U / g 18 g, sodium alginate 20 g, and lactoferricin 18 g.

[0038] The following operations are used to prepare the coarse oligosaccharides of Spartina: Spartina is steamed for 7 hours, dried in the air, and then freeze-dried. The Spartina is ground and passed through a 100-mesh sieve. A 95% ethanol aqueous solution (the mass ratio of the ethanol aqueous solution to the Spartina is 9:1) is added to the Spartina for reflux extraction three times. The filtrate is obtained by filtering the Spartina. A 75% ethanol aqueous solution (the mass ratio of the ethanol aqueous solution to the Spartina is 9:1) is added to the residue for reflux extraction four times. The filtrate is obtained by filtering the residue. The first filtrate and the second filtrate are combined. The ethanol is recovered under reduced pressure. The filtrate is diluted with an equal volume of deionized water. The solution is allowed to stand for 25 hours. The filtrate is obtained by filtering the solution. The filtrate is passed through a D101 macroporous adsorption resin column. The filtrate is eluted with deionized water. The water eluate is collected. The water eluate is decolorized with activated carbon. The eluate is concentrated under reduced pressure. The eluate is dried under reduced pressure. The dried eluate is ground.

[0039] The above method for preparing the composite preparation containing the coarse oligosaccharides of Spartina includes the following steps: S1. Sodium carboxymethyl cellulose is added to 550 g of deionized water. The solution is stirred at 62°C for 100 minutes. The solution is cooled to 8°C. Cellulase and sodium alginate are added to the solution. The solution is stirred for 18 minutes. A 0.6 mol / L calcium chloride solution is added dropwise to the solution. The solution is stirred at a speed of 1800 r / min for 13 minutes. The solution is allowed to stand for 100 minutes. The solution is filtered. The filter cake is washed with deionized water. The filter cake is freeze-dried to obtain a preformed material with a particle size of 70 μm. S2. The preformed material is added to 1200 g of deionized water. The pH value of the system is adjusted to 5-5.5. The solution is stirred at 48°C for 80 minutes at a stirring speed of 70 r / min. The solution is filtered after inactivation. The filter cake is washed and freeze-dried. The product and the coarse oligosaccharides of Spartina are added to 1800 g of deionized water. The solution is ultrasonically treated for 12 minutes at an ultrasonic frequency of 68 kHz. Lactoferricin and resveratrol are added to the solution. The solution is stirred for 80 minutes. The solution is freeze-dried. Example 5

[0040] A composite preparation containing coarse oligosaccharides of Spartina includes the following raw materials: coarse oligosaccharides of Spartina 60 g, resveratrol 15 g, sodium carboxymethyl cellulose 80 g, cellulase with an enzyme activity of 500 U / g 15 g, sodium alginate 25 g, and lactoferricin 15 g.

[0041] The crude oligosaccharide of *Polygonum cuspidatum* was prepared by the following operation: *Polygonum cuspidatum* was steamed in a water bath for 8 hours, air-dried, freeze-dried, pulverized and passed through a 100-mesh sieve, and extracted three times by reflux with a 95% ethanol aqueous solution (ethanol aqueous solution to *Polygonum cuspidatum* mass ratio of 8:1). After filtration, the filtrates were combined to obtain the first extract. A 75% ethanol aqueous solution (ethanol aqueous solution to filter residue mass ratio of 8:1) was added to the filter residue and extracted four times by reflux. After filtration, the filtrates were combined to obtain the second extract. The first and second extracts were combined, the ethanol was recovered under reduced pressure, concentrated until no alcohol odor was detected, diluted with an equal volume of deionized water, allowed to stand for 30 hours, filtered, and then passed through a D101 macroporous adsorption resin column. Deionized water was used for elution, the water eluent was collected, decolorized with activated carbon, concentrated under reduced pressure to an extract, dried under reduced pressure, and pulverized.

[0042] The preparation method of the above-mentioned compound formulation containing crude oligosaccharides from *Polygonum cuspidatum* includes the following steps: S1. Sodium carboxymethyl cellulose was added to 500g of deionized water and stirred at 65℃ for 90min. The temperature was then lowered to 10℃, and cellulase and sodium alginate were added. The mixture was stirred for 15min and then added dropwise to a 0.7mol / L calcium chloride solution. The mixture was stirred at 1500r / min for 15min and allowed to stand for 90min. The mixture was then filtered, washed with deionized water, and freeze-dried to obtain a pre-formed material with a particle size of 60μm. S2. Add the pre-prepared material to 1500g of deionized water, adjust the pH of the system to 5-5.5, stir at 50℃ for 90min at a stirring speed of 80r / min, filter after inactivation, wash, and freeze dry; add the product and crude oligosaccharide of *Polygonum cuspidatum* to 1500g of deionized water and sonicate for 15min at a sonic frequency of 65kHz, add lactoferrin peptide and resveratrol and stir for 90min, then freeze dry.

[0043] Comparative Example A compound preparation containing crude oligosaccharide of *Polygonum cuspidatum*, the raw materials of which include: 90g crude oligosaccharide of *Polygonum cuspidatum*, 80g sodium carboxymethyl cellulose, 15g cellulase with an enzyme activity of 500U / g, and 25g sodium alginate.

[0044] The following operations are used to prepare the coarse oligosaccharides of Spartina: the Spartina is steamed for 8 hours, dried in the air, and then freeze-dried, crushed and passed through a 100-mesh sieve, 95% ethanol aqueous solution (the mass ratio of the ethanol aqueous solution to the Spartina is 8:1) is added to reflux extract the Spartina for 3 times, the filtrate is combined after filtration, and the first extract is obtained; 75% ethanol aqueous solution (the mass ratio of the ethanol aqueous solution to the residue is 8:1) is added to the residue to reflux extract the residue for 4 times, the filtrate is combined after filtration, and the second extract is obtained; the first extract and the second extract are combined, ethanol is recovered under reduced pressure, concentrated to be alcohol-free, diluted with an equal volume of deionized water, and then left to stand for 30 hours, the filtrate is obtained after filtration, and then the filtrate is passed through a D101 macroporous adsorption resin column, eluted with deionized water, the water eluate is collected, decolorized with activated carbon, concentrated under reduced pressure to a extract, dried under reduced pressure, and crushed.

[0045] The above preparation method of the compound preparation containing the coarse oligosaccharides of Spartina includes the following steps: S1, sodium carboxymethyl cellulose is added to 500 g of deionized water, stirred at 65℃ for 90 min, cooled to 10℃, cellulase and sodium alginate are added thereto, stirred for 15 min, and then added dropwise to a 0.7 mol / L calcium chloride solution, stirred at a speed of 1500 r / min for 15 min, left to stand for 90 min, filtered, washed with deionized water, and freeze-dried to obtain a preformed material with a particle size of 60 μm; S2, the preformed material is added to 1500 g of deionized water, the pH value of the system is adjusted to 5-5.5, stirred at 50℃ for 90 min, and the stirring speed is 80 r / min, after inactivation, filtered, washed, and freeze-dried; the product and the coarse oligosaccharides of Spartina are added to 1500 g of deionized water, ultrasonic treatment is performed for 15 min, the ultrasonic frequency is 65 kHz, and then freeze-dried.

[0046] Since the probability of osteoporosis in postmenopausal women is greatly increased, male db / db mice are used as an animal model of type 2 diabetes complicated with osteoporosis to exclude the influence of the change of sex hormones on osteoporosis and to simply observe the effect of the drug on bone. After 8-week-old male db / db mice are adaptively fed for 1 week, db / db diabetic mice that meet the conditions (random blood glucose ≥ 16.7 mmol / L is considered to have formed type 2 diabetes) are screened; and male homologous and same-age healthy db / m mice are used as a blank control group.

[0047] The applicant randomly divides the db / db mice that meet the conditions into 5 groups, and each group is administered once a day (at 8:00 am), as shown in Table 1, and the test is continuously performed for 8 weeks.

[0048] Table 1 Grouping and administration Group Dose Control group db / m mice were administered with the same volume of distilled water by gavage Model group db / db mice were administered with the same volume of distilled water by gavage AOS group db / db mice were administered with 750 mg / kg (equivalent to the clinical human dose) of the crude oligosaccharides of Agropyron medium by gavage Example 5 group db / db mice were administered with the compound preparation obtained in Example 5, in which the dose of the crude oligosaccharides of Agropyron medium was 375 mg / kg Comparative example group db / db mice were administered with the compound preparation obtained in the comparative example, in which the dose of the crude oligosaccharides of Agropyron medium was 375 mg / kg After the test is continuously performed for 8 weeks, the following indexes are detected: (1) Blood glucose determination: measure blood glucose, measure the mouse fasting 8h, then take blood from the mouse tail vein, the mouse blood glucose value is determined by blood glucose meter and displayed. If the mouse blood glucose value is higher than the maximum value of the blood glucose meter, it is recorded as 33.3mmol / L.

[0049] (2) Serum inflammatory factor detection: ELISA kit was used to determine the serum TNF-α, IL-1β of each group of mice; (3) HE staining analysis: the left tibia of the mouse was taken for pathological examination, fixed in 4% paraformaldehyde for 48h, then decalcified, gradient ethanol dehydrated, paraffin embedded, section thickness was 5μm, then HE staining analysis was carried out, observed under ordinary optical microscope and photographed.

[0050] (4) Micro-CT scanning of left femur of mice: the attached tissue of left femur of each group of mice was removed clean and washed with normal saline. The tibial tissue of each group of mice was subjected to Micro-CT scanning. The unified parameters were set, and the bone volume fraction (Tb.BV / TV), cortical bone thickness (Ct.Th), trabecular bone number (Tb.N) and bone structure model index (SMI) of the tissue were calculated by software.

[0051] As shown in Figure 1 , the blood glucose of the model group was significantly higher than that of the blank control group (P<0.05), while the blood glucose of the AOS group, the example 5 group and the comparative example group was significantly lower than that of the model group (P<0.05), and the blood glucose of the example 5 group was significantly lower than that of the AOS group and the comparative example group (P<0.05).

[0052] As shown in Figure 2 , the serum inflammatory factor of the model group was significantly higher than that of the blank control group (P<0.05), while the serum inflammatory factor of the AOS group, the example 5 group and the comparative example group was significantly lower than that of the model group (P<0.05), and the serum inflammatory factor of the example 5 group was significantly lower than that of the AOS group and the comparative example group (P<0.05).

[0053] As shown in Figure 3 , the AOS group, the example 5 group and the comparative example group respectively used the coarse oligosaccharide of sand rush, the composite preparation obtained in example 5 and the composite preparation obtained in comparative example, which all had protective effect on the formation of diabetic osteoporosis.

[0054] As shown in Figure 4 and Figure 5As shown, Tb.BV / TV, Ct.Th and Tb.N of the model group were significantly lower than those of the blank control group (P<0.05), and Tb.BV / TV, Ct.Th and Tb.N of the AOS group, the example 5 group and the comparative example group were significantly higher than those of the model group (P<0.05), wherein Tb.BV / TV, Ct.Th and Tb.N of the example 5 group were the highest (P<0.05); SMI of the model group was significantly higher than that of the blank control group (P<0.05), and SMI of the AOS group, the example 5 group and the comparative example group was significantly lower than that of the model group (P<0.05), and SMI of the example 5 group was significantly lower than that of the AOS group and the comparative example group (P<0.05).

[0055] The reason for the above results is that the sodium carboxymethyl cellulose used in the application has good compatibility with sodium alginate, and after the two are compounded by hydrogen bonds and then solidified, the obtained microspheres have high stability, the hydrolysis product of the sodium carboxymethyl cellulose hydrolyzed by cellulase penetrates from the inside to the outside of the microspheres, the internal void structure of the product is uniform, which is beneficial to the stable loading of the psammochloa crude oligosaccharide, and the hydrogen bond complex of the lactoferrin peptide and the resveratrol makes the psammochloa crude oligosaccharide not easy to overflow, and the loading stability is high. The application realizes the slow release and intestinal targeted delivery of the psammochloa crude oligosaccharide, avoids the degradation of gastric acid, and the hydrogen bond complex of the lactoferrin peptide and the resveratrol further reduces the risk of burst release of the psammochloa crude oligosaccharide and prolongs its half-life. The psammochloa crude oligosaccharide cooperates with the lactoferrin peptide and the resveratrol to improve insulin resistance (IR) and correct the bone resorption-formation imbalance of diabetic osteoporosis (DOP).

[0056] The above is only a preferred specific embodiment of the application, but the protection scope of the application is not limited thereto, and any person skilled in the art can make equivalent replacements or changes to the technical solution and the inventive concept of the application within the technical range disclosed by the application, which should be covered within the protection scope of the application.

Claims

1. A compound preparation containing crude oligosaccharides from *Polygonum cuspidatum*, characterized in that, The raw materials, by weight, include: 4-8 parts of crude oligosaccharide from *Polygonum aviculare*, 1-2 parts of resveratrol, 5-10 parts of sodium carboxymethyl cellulose, 1-2 parts of cellulase, 1-4 parts of sodium alginate, and 1-2 parts of lactoferrin peptide.

2. The compound preparation containing *Potentilla anserina* crude oligosaccharides according to claim 1, characterized in that, The enzyme activity of cellulase is 400-600 U / g.

3. The compound preparation containing *Potentilla anserina* crude oligosaccharides according to claim 1, characterized in that, Crude oligosaccharides from *Solanum tuberosum* were prepared as follows: *Solanum tuberosum* was steamed, dried, and pulverized. It was then extracted 2-3 times by reflux with a 90-95% ethanol aqueous solution. After filtration, the filtrates were combined to obtain the first extract. A 70-75% ethanol aqueous solution was added to the residue and extracted 2-4 times by reflux. After filtration, the filtrates were combined to obtain the second extract. The first and second extracts were combined, the ethanol was recovered under reduced pressure, the extract was concentrated, diluted with water, allowed to stand for 20-40 hours, passed through an adsorption resin column, eluted, and the eluent was collected. The eluent was decolorized, concentrated under reduced pressure to a paste, dried under reduced pressure, and pulverized.

4. The compound preparation containing *Potentilla anserina* crude oligosaccharides according to claim 3, characterized in that, The mass ratio of *Potentilla anserina* to ethanol aqueous solution is 1:5-10, and the mass ratio of filter residue to ethanol aqueous solution is 1:5-10.

5. The compound preparation containing *Potentilla anserina* crude oligosaccharide according to claim 3, characterized in that, The adsorption resin column is a D101 type macroporous adsorption resin column.

6. A method for preparing a compound formulation containing crude oligosaccharides of *Polygonum cuspidatum* as described in any one of claims 1-5, characterized in that, Includes the following steps: S1. Add sodium carboxymethyl cellulose to water and stir at 60-70℃ for 1-2 hours. Cool down to 5-15℃, add cellulase and sodium alginate and stir for 10-20 minutes. Add dropwise to calcium chloride solution and stir for 10-20 minutes. Let stand for 1-2 hours, filter, wash and freeze dry to obtain the pre-made material. S2. Add the pre-made material to water, adjust the pH of the system to 5-5.5, stir at 45-55℃ for 1-2 hours, filter after inactivation, wash, and freeze dry; add the product and sand pine crude oligosaccharide to water and sonicate for 10-20 minutes, add lactoferrin peptide and resveratrol and stir for 1-2 hours, then freeze dry.

7. The method for preparing the compound formulation containing *Potentilla anserina* crude oligosaccharide according to claim 6, characterized in that, In S1, the particle size of the preform is 50-80μm.

8. The method for preparing the compound formulation containing *Potentilla anserina* crude oligosaccharide according to claim 6, characterized in that, In S1, the concentration of calcium chloride solution is 0.5-1 mol / L.

9. The method for preparing the compound formulation containing *Potentilla anserina* crude oligosaccharide according to claim 6, characterized in that, In S2, the ultrasonic frequency is 60-70kHz.

10. The use of *Prunus armeniaca* crude oligosaccharide or a compound preparation containing *Prunus armeniaca* crude oligosaccharide as described in any one of claims 1-5 in the preparation of a drug for treating insulin resistance or osteoporosis.

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