阅读说明：本技术 壳寡糖双胍衍生物在制备抑制肝损伤药物中的应用 (Application of chitosan oligosaccharide biguanide derivative in preparation of liver injury inhibiting medicine ) 是由 刘晓非 赵励彦 郑淇方 邹雅露 王园园 刘宇星 于 2019-08-21 设计创作，主要内容包括：本发明公开壳寡糖双胍衍生物在制备抑制肝损伤药物中的应用,将壳寡糖与盐酸在微波装置中搅拌反应,之后加入双氰胺水溶液,使用盐酸调节溶液pH,在微波装置中搅拌反应,得到壳寡糖双胍衍生物。本发明在体外实验中验证了衍生物对DPPH自由基的清除作用,证明了它具有抗氧化性；同时,建立2型糖尿病大鼠模型,壳寡糖双胍衍生物以灌胃的方式给药,经过衍生物治疗后,有效抑制了糖尿病大鼠肝脏的氧化应激损伤。(The invention discloses application of a chitosan oligosaccharide biguanide derivative in preparation of a medicament for inhibiting liver injury. The invention verifies the scavenging effect of the derivative on DPPH free radicals in vitro experiments, and proves that the derivative has oxidation resistance; meanwhile, a type 2 diabetes rat model is established, the chitosan oligosaccharide biguanide derivative is administrated in a gastric perfusion mode, and after treatment of the derivative, oxidative stress damage of the liver of a diabetes rat is effectively inhibited.)

1. The application of the chitosan oligosaccharide biguanide derivative in preparing the medicine for inhibiting liver injury is characterized in that the chitosan oligosaccharide biguanide derivative is prepared by taking chitosan oligosaccharide and dicyandiamide as raw materialsWith pendant chitosan oligosaccharide group NH₂And dicyandiamide react under the conditions of hydrochloric acid and microwave to form a biguanide group bonded with the chitosan oligosaccharide, wherein m and n are respectively the polymerization degrees of an acetylation unit and a deacetylation unit in the chitosan oligosaccharide.

2. Use of a chitosan oligosaccharide biguanide derivative according to claim 1 in the preparation of a medicament for inhibiting liver damage, wherein the chitosan oligosaccharide has a weight average molecular weight of 3000 or less, preferably 1500-3000 Da.

3. The use of a chitooligosaccharide biguanide derivative according to claim 1 in the preparation of a medicament for inhibiting liver damage, wherein the degree of deacetylation of the chitooligosaccharide is above 97%, preferably 97-9%.

4. Use of a chitosan oligosaccharide biguanide derivative according to claim 1 in the preparation of a medicament for inhibiting liver damage, wherein the degree of biguanide substitution in the chitosan oligosaccharide biguanide derivative is in the range of 40 to 60%, preferably 45 to 55%.

5. Use of a chitosan oligosaccharide biguanide derivative according to any one of claims 1 to 4 in the manufacture of a medicament for inhibiting liver damage, wherein the chitosan oligosaccharide biguanide derivative attenuates oxidative stress damage of liver tissue.

Technical Field

The invention belongs to the field of pharmacy, and particularly relates to a microwave synthesis preparation method of a chitosan oligosaccharide biguanide derivative and application of the chitosan oligosaccharide biguanide derivative in preparation of a medicament for inhibiting liver injury.

Background

Oxidative stress is an important factor in the development and progression of type 2 diabetes. It refers to the imbalance between the rate of production of Reactive Nitrogen (RNS) and Reactive Oxygen Species (ROS) and the clearance of its antioxidant defense system, resulting in excess RNS and ROS in the body, and damage to biological macromolecules such as proteins and nucleic acids in organs, tissues and cells of the body (Reddy VP, Zhu X, Perry G, et al]Alzheimer's Diseases,2009,16(4): 763-. Hyperglycemia is a major cause of oxidative stress, and hyperglycemia increases the ROS and RNS content in the body by means of mitochondrial electron transport chains, glucose autooxidation, and polyol pathways. Among these ROS-generating pathways, the mitochondrial electron transport chain is predominant. The mitochondrial electron transport chain is mainly involved by several substances: enzyme complexes I to IV, cytochrome c and coenzyme Q, in which little superoxide product including hydroxyl radical (. OH), superoxide anion (O) is produced uninterruptedly for a long period^2-) And hydrogen peroxide (H)₂O₂) While superoxide dismutase (SOD), Catalase (CAT) and glutathione peroxidase (GSH-Px) will catalyze the conversion of superoxide products to oxygen and water.

In the diabetic state, prolonged hyperglycemia can lead to abnormalities in the oxidative stress system of the liver, resulting in specific immune responses and ultimately liver damage. The liver is an organ very sensitive to oxidative stress and free radicals can cause direct damage to the liver (Ito K, Ozasa H, Noda Y, et al. effects of free radial scavenger on acid liver induced by d-galactosamine and lipopolysaccharides in rats [ J ]. Hepatology Research the Official Journal of the Japan Society of Hepatology,2010,38(2):194 and 201). Diabetic patients can develop glucose metabolism disorder in a hyperglycemic state, and liver injury is promoted. In addition, high sugars, in addition to inducing oxidative stress, can also disrupt the normal clearance of tissue cells (Matough F A, Budin S B, Hamid Z A, et al. the role of oxidative stress and antioxidants in biological compounds [ J ]. Sultan Qaboos University Medical Journal,2012,12(1): 5-18). For example, glycosylation or oxidation of antioxidant enzymes can lead to the reduction of activities of superoxide dismutase (SOD), Catalase (CAT) and other antioxidant enzymes, the reduction of antioxidant levels such as vitamin C, vitamin E, Glutathione (GSH) and the like, and the weakening of the ability of the organism to remove free radicals, thereby promoting the development of liver injury. Thus, during the course of liver damage, on the one hand, the production of free radicals increases and, on the other hand, the antioxidant capacity decreases, so that both coexist and lead to the development of oxidative stress (Leclercq I A, Silva MoraisA D, Schroyen B, et al. insulin resistance in adipocytes and systemic cells: Mechanisms and sequences [ J ]. Journal of Hepatology,2007,47(1): 142-. Therefore, oxidative stress is a key factor for causing the diabetic liver injury, and inhibition of oxidative stress can cause obvious delay in the occurrence and development of the diabetic liver injury, which has extremely important significance for effectively treating type 2 diabetes.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides the application of the chitosan oligosaccharide biguanide derivative in preparing the medicine for inhibiting liver injury.

The chitosan oligosaccharide biguanide derivative used in the invention adopts the technical scheme of chitosan oligosaccharide biguanide derivative and microwave synthesis method thereof (application No. 2017108673840, application date 2017, 9 and 22 days), the microwave synthesis method is used for introducing the biguanide structure of the classic diabetes treatment medicament metformin into chitosan oligosaccharide, and the product has the effect of treating the liver injury of diabetes with guanidino, has the naturalness, safety and unique biological activity of chitosan oligosaccharide, and can relieve the stimulation side effect of biguanide medicament on gastrointestinal tract.

The chitosan oligosaccharide biguanide derivative is prepared by taking chitosan oligosaccharide and dicyandiamide as raw materials and taking chitosan oligosaccharide side group NH₂And dicyandiamide under hydrochloric acid and microwave conditions and form a biguanide group bonded to the chitosan oligosaccharide, as shown in the following chemical formula:

the chitosan oligosaccharide is the only basic amino oligosaccharide with positive charges in nature, has small molecular weight, good solubility and easy absorption and utilization by organisms, the weight average molecular weight is below 3000, preferably 1500-3000 Da, and the deacetylation degree is above 97%, preferably 97-99%.

In the chitosan oligosaccharide biguanide derivative, the substitution degree of biguanide is 40-60%, preferably 45-55%.

In the chemical formula, m and n are respectively the polymerization degrees of acetylation units and deacetylation units in the chitosan oligosaccharide.

The preparation method of the chitosan oligosaccharide biguanide derivative comprises the following steps: dispersing chitosan oligosaccharide in hydrochloric acid and stirring under the microwave condition of 160W-560W to perform the first step of microwave reaction; adding dicyandiamide aqueous solution, adjusting the pH value of a reaction system to 1-2 by using hydrochloric acid, continuously stirring under the microwave condition of 160-560W, and carrying out a second-step microwave reaction to react chitosan oligosaccharide and dicyandiamide to prepare the chitosan oligosaccharide biguanide derivative, wherein the specific formula is shown as follows:

in the first step of microwave reaction, the microwave power is 300-400 w, the reaction time is 3-7 min, preferably 5-7 min, and the stirring speed is 100-200 rpm.

In the second step of microwave reaction, the microwave power is 300-400 w, the reaction time is 10-20 min, preferably 15-20 min, and the stirring speed is 100-200 rpm.

And after the reaction is finished, cooling the reaction liquid to room temperature, carrying out alcohol precipitation on the mixed solution, drying and grinding to obtain chitosan oligosaccharide biguanide derivative powder.

The molar ratio of dicyandiamide to amino groups in the chitosan oligosaccharide is (0.5-3): 1, preferably (1-3): 1, and more preferably 1: 1.

Hydrochloric acid is 0.2-0.8 mol/L HCl aqueous solution, and the pH value of the reaction system is adjusted to 1 by using the hydrochloric acid.

The invention has the advantages of high production efficiency, simple operation process, clean and environment-friendly reaction process and good product performance. Animal experiments prove that after a type 2 diabetes rat model is established, the chitosan oligosaccharide biguanide derivative is administrated by gastric perfusion, ALT and AST enzyme activity is reduced, the contents of TP, TBIL and FFA in serum are reduced, related enzymes are prevented from leaking from liver cells to extracellular circulation, the degree of damage of free radicals to livers is reduced, on the other hand, the MDA activity value is reduced, the activities of SOD, CAT and GSH-Px are increased, the oxidative stress damage of liver tissues is weakened, the treatment effect on the oxidative stress damage of the livers is finally exerted, and compared with the single use of the traditional diabetes treatment medicament metformin, the chitosan oligosaccharide biguanide derivative has a better treatment effect, namely the application of the chitosan oligosaccharide biguanide derivative in the preparation of the medicament for inhibiting the hepatic damage.

Drawings

FIG. 1 is a comparison spectrum diagram of infrared spectra of raw materials Chitosan Oligosaccharide (COS) and chitosan oligosaccharide biguanide hydrochloride (COSG) in the invention.

FIG. 2 is a nuclear magnetic carbon spectrum contrast line chart of Chitosan Oligosaccharide (COS) and chitosan oligosaccharide biguanide hydrochloride (COSG) as raw materials in the invention.

FIG. 3 is a graph showing the in vitro antioxidant activity of chitooligosaccharide biguanide hydrochloride (COSG) of the present invention, wherein (a) is the UV absorption spectrum of the COSG and Vc solution, (b) is the scanning spectrum of the DPPH-ethanol solution and DPPH free radical system after adding the COSG and Vc solution, (c) is the absorbance value change curve of DPPH solutions with different concentrations (0.05mM, 0.10mM, 0.15mM, 0.20mM) after cooling at room temperature for 0,10,20,30min, and (d) is the test curve of the scavenging ability of COSG and Vc to DPPH free radicals.

FIG. 4 is a graph of data showing the variation of weight (a), fasting glucose (b) and urine glucose (c) of STZ-induced diabetic rats improved by COSG according to the present invention, wherein the data are mean. + -. standard deviation, n is 6,^#P<0.05,^##P<0.01,^###P<0.001 vs. blank control group,. P<0.05,**P<0.01,***P<0.001 compared to the model group.

FIG. 5 is a photograph showing morphological observation of liver tissue (HE staining X100 times) in the example of the present invention.

Detailed Description

The following is a further description of the invention and is not intended to limit the scope of the invention. After the chitosan oligosaccharide biguanide derivative is prepared, the subject group successively tests and evaluates the medicinal value of the chitosan oligosaccharide biguanide derivative, such as the application of the previous Chinese patent application of ' application of the chitosan oligosaccharide biguanide derivative in preparing a medicament for treating diabetic nephropathy ' (application No. 2017108663660, application No. 2017, 9 and 22 days), ' application of the chitosan oligosaccharide biguanide derivative in preparing a medicament for treating lipid metabolism disorder ' (application No. 2017108917900, application No. 2017, 9 and 27 days), ' application of the chitosan oligosaccharide biguanide derivative in preparing a medicament for treating pancreatic islet deficiency ' (application No. 2018111171793, application No. 2018, 9 and 25 days), ' application of the chitosan oligosaccharide biguanide derivative in preparing a medicament for inhibiting apoptosis (application No. 2018111202043, application No. 2018, 9 and 25 days), ' application of the chitosan oligosaccharide biguanide derivative in preparing a medicament for treating skeletal muscle insulin resistance ' (application No. 2018111927902, application date 2018, 10 month 13). In the present invention, this subject group continues the test of medicinal value for the chitosan oligosaccharide biguanide derivatives, and first prepares and characterizes the chitosan oligosaccharide biguanide derivatives, and studies on liver damage.

Example 11500 Da preparation of Chitosan oligosaccharide biguanide derivatives

The raw material ratio is 1:1, weighing 1.44g of dicyandiamide in a conical flask, adding 15mL of distilled water, and shaking and dissolving dicyandiamide in a constant-temperature shaking incubator at 50 ℃. After the chitosan oligosaccharide (1500Da) and the dicyandiamide are completely dissolved, pouring the dicyandiamide aqueous solution into a four-mouth bottle containing the chitosan oligosaccharide hydrochloric acid solution, and adjusting the pH value of the solution to 1 by using 1mol/L hydrochloric acid. The reaction was carried out under conditions of microwave power of 400W and microwave time of 15 min. After the reaction is finished, carrying out alcohol precipitation, suction filtration, drying and grinding on the mixed solution to obtain chitosan oligosaccharide biguanide derivative powder COSG with the degree of substitution of 55%.

Example 23000 Da preparation of Chitosan oligosaccharide biguanide derivatives

The raw material ratio is 1:1 weighing 2.88g of dicyandiamide in a conical flask, adding 15mL of distilled water, and shaking and dissolving dicyandiamide in a constant-temperature shaking incubator at 50 ℃. After the chitosan oligosaccharide (3000Da) and the dicyandiamide are completely dissolved, pouring the dicyandiamide aqueous solution into a four-mouth bottle containing the chitosan oligosaccharide hydrochloric acid solution, and adjusting the pH value of the solution to 1 by using 1mol/L hydrochloric acid. The reaction was carried out under conditions of microwave power of 400W and microwave time of 15 min. After the reaction is finished, carrying out alcohol precipitation, suction filtration, drying and grinding on the mixed solution to obtain chitosan oligosaccharide biguanide derivative powder COSG with the degree of substitution of 55%.

The chitosan oligosaccharide biguanide derivative and the raw material chitosan oligosaccharide obtained in the embodiment 1 of the invention are identified by infrared spectroscopy, nuclear magnetic resonance spectroscopy and the like, as shown in attached figures 1 and 2.

In the infrared spectrum of COS, 3414cm^-1The strong absorption peak in the vicinity is a multiple absorption peak widened by partially overlapping O-H and N-H stretching vibration absorption peaks associated with hydrogen bonds. Because a large number of intermolecular and intramolecular hydrogen bonds exist in COS molecules, and the lengths and the strengths of the hydrogen bonds are different, the stretching vibration peak of the COS molecules appears in a wider frequency range. 2918cm^-1The left and right sides are C-H stretching vibration absorption peaks. 1626cm^-1The vicinity is the N-H stretching vibration peak of secondary amine. Furthermore, 1060cm of the fingerprint area^-1An N-H in-plane bending vibration absorption peak of secondary amine is close to the point, because COS deacetylation is not complete and a small amount of-NH-CO-structure is contained in the molecule, an absorption peak is formed at the point; 602cm^-1The vicinity corresponds to the out-of-plane rocking vibration absorption band of N-H. Compared with COS, COSG 1626cm^-1At 1060cm^-1The absorption peak near the molecule is obviously enhanced, and both the peaks are characteristic peaks of secondary amine groups, which indicates that secondary amine groups are increased in the molecule, and indicates that the guanidination reaction is performed on COS amino groups. In addition, 3414cm^-1The nearby absorption peak is a multiple absorption peak widened by partial overlapping of O-H and N-H stretching vibration absorption peak parts associated with hydrogen bonds, because a large number of intermolecular and intramolecular hydrogen bonds exist in COS molecules, and the length and the strength of the hydrogen bonds are different, the stretching vibration peak appears in a wider frequency range, and the peak is obviously enhanced relative to COS, which also shows that the content of N-H in the molecule is increased, thereby indicating that the guanidination reaction occurs on COS amino.

The chemical shift of each C on the chitosan ring is: c1: 97.03, C4: 76.03, C5: 75.01, C3: 71.49, C6: 60.23, C2: 56.23. the chitosan oligosaccharide is obtained by degrading chitosan, so that the chemical environment and chemical shift of each C on a sugar ring of the chitosan oligosaccharide are consistent with those of chitosan. Further determining the structural change of the modified chitosan oligosaccharide by using a 13CNMR analysis technology, wherein the chemical shifts of the chitosan oligosaccharide guanidine salt are respectively as follows: c7: 165.21, C8: 155.13, C1: 98.91, C4: 78.17, C5: 77.03, C3: 69.13, C6: 60.17, C2: 56.43. wherein C7: 165.21 and C8: 155.13 the chemical shift of the newly emerging carbon determines the presence of the guanidine group.

After the reaction of chitosan oligosaccharide and dicyandiamide to generate guanidine groups is determined, the product is titrated by using NaOH standard solution, and the guanidine substitution degree DS of the product is measured and taken as a judgment standard. NH in NaOH and Chitosan oligosaccharide guanidine₂ ⁺Cl^-Reaction, titration, equivalence point, pH mutation, NaOH mole equal to NH in product₂ ⁺Cl^-The number of moles. The DS of chitosan oligosaccharide guanidine can be calculated by the following formula:

in the formula:

n₁-moles of building blocks substituted with guanidino groups;

n₂-moles of building blocks not substituted with guanidino groups;

Δ v — the volume of NaOH consumed to reach the breakthrough point (pH 9.1)/ml;

c_NaOHconcentration/mol. L of NaOH Standard solution^-1；

G-product mass/G;

318-relative molar Mass of units substituted by guanidino groups/g.mol^-1；

161-relative molar masses of structural units which are not substituted by guanidino groups/g.mol^-1；

DS- (bis) guanidine degree of substitution/%.

Example 3 the chitosan oligosaccharide biguanide derivative powder COSG prepared in example 1 was selected for in vitro antioxidant performance studies.

1) DPPH free radical solution preparation

Weighing a certain amount of DPPH, dissolving in absolute ethyl alcohol, fixing the volume in a bottle with the capacity of 250mL to prepare DPPH-ethanol solution with the concentration of 0.20mM, and storing at low temperature in a dark place.

2) Preparation of sample solution

Chitosan oligosaccharide guanidine hydrochloride solution: accurately weighing 0.020g of chitosan oligosaccharide guanidine hydrochloride product, placing the product in a 25mL conical flask, adding 10mL of distilled water into the conical flask to oscillate and dissolve the product to obtain a chitosan oligosaccharide guanidine hydrochloride solution, diluting the solution to a solution with a certain concentration gradient, taking 1mL of DPPH solution, adding 2mL of chitosan oligosaccharide guanidine hydrochloride solution into the solution, starting adding the solution from the lowest concentration during adding the solution, observing the fading condition of the solution, recording the concentration of the sample solution, which is just fading the color of the solution, wherein the concentration is the maximum concentration of the sample solution, and diluting the sample to the solution with the certain concentration gradient for later use on the basis of the concentration.

Ascorbic acid solution: 0.020g of ascorbic acid was accurately weighed out in a 25mL Erlenmeyer flask, and 10mL of distilled water was added thereto to dissolve it with shaking, to obtain an ascorbic acid solution.

3) Ultraviolet scanning of sample solutions

3mL of chitosan oligosaccharide guanidine hydrochloride solution and ascorbic acid solution are taken, and an ultraviolet visible spectrophotometer is used for respectively scanning at the wavelength of 800nm and 250-.

4) Ultraviolet scanning of DPPH free radical systems

Respectively adding 2mL of chitosan oligosaccharide guanidine hydrochloride solution, 2mL of ascorbic acid solution and 2mL of absolute ethyl alcohol into 1mL of DPH-ethanol solution, oscillating to uniformly mix the solutions, standing for 10min in a dark place, and continuously scanning the solutions at the wavelength of 400-800nm by using an ultraviolet visible spectrophotometer to obtain the full-wavelength absorption spectrum of the solutions, and finding out the wavelength lambda max with the maximum light absorption value of the DPPH-ethanol solution.

5) Stability analysis of DPPH solutions

0.20mM MDPPH-ethanol solution is diluted to ethanol solution with concentration of 0.05mM, 0.10mM, 0.15mM, 0.20mM respectively. Storing the diluted DPPH-ethanol solution in a shade place, and measuring absorbance photometric values of the DPPH-ethanol solution with different concentrations at the lambda max position determined in 4) after 0min, 10min, 20min and 30min by using an ultraviolet-visible spectrophotometer.

6) Determination of the scavenging Rate of Chitosan oligosaccharide guanidine for DPPH free radical

Chitosan oligosaccharide guanidine hydrochloride was formulated as a 0.05mg/mL sample solution. Mixing 2mL sample solution and 0.05mg/mL ascorbic acid solution with 1mL LDPPH-ethanol solution, standing in shade for 30min, and measuring absorbance at λ max (A) obtained in 4) with UV-visible spectrophotometer_sample)。

Mixing 2mL of anhydrous ethanol and 1mL of DPPH-ethanol solution, standing in shade for 30min, and measuring the absorbance (A) at λ max_DPPH)。

2mL of the sample solution was thoroughly mixed with 1mL of absolute ethanol, and the absorbance (A) was measured at the maximum wavelength_blank). The contents of the components of the test solution prepared above are shown in table 1.

The DPPH radical clearance calculation formula is as follows:

TABLE 1DPPH radical scavenging Rate methods the amounts of reagents added

2. Study conditions (in vitro antioxidant Activity of COSG)

FIG. 3a shows the UV absorption spectrum of the COSG and Vc solutions, the sample solution has no significant absorption at 400-800nm wavelength, and thus it can be determined that the effect of COSG on DPPH clearance is almost not affected, and FIG. 3b shows the scanning spectra of DPPH-ethanol solution and DPPH radical system after the addition of COSG and Vc solutions. It can be seen that the DPPH-ethanol solution has a distinct absorption peak at 516nm, which is the maximum absorption wavelength. In addition, after the mixed solution is mixed with the solution of COSG and Vc, the absorption peak of DPPH-ethanol is greatly weakened, and the COSG has a certain degree of scavenging effect on DPPH free radicals.

As shown in FIG. 3c, after cooling at room temperature for 0,10,20, and 30min, no significant change in absorbance values of DPPH solution was observed, and DPPH radicals had good stability at room temperature. FIG. 3d shows that the Vc scavenging ability of DPPH free radicals is very excellent and does not vary with concentration, whereas COSG has a significant dose-dependent effect on DPPH free radical scavenging ability. As the concentration increases, it tends to stabilize and eventually has similar ability to Vc. Therefore, COSG is effective in scavenging free radicals, has antioxidant activity, can improve oxidative stress of diabetic rats, and is used for treating T2DM rats.

Example 4 the chitosan oligosaccharide biguanide derivative powder COSG prepared in examples 1, 2 was selected for animal pharmacodynamic experiments. The effect of the present invention will be further described by the following animal experiments.

The experimental animals selected SD adult male rats 70, 10 in each group, 7 in total, clean grade, body weight of about 500 g, provided by animal experiment center in Tianjin. The animals are raised in cages, and 4-5 animals are raised in each cage. The animal feeding laboratory conforms to the national standard clean level and has a space of 30m²The indoor illumination is controlled at the light-dark period rhythm of 12h/12 h. The indoor average temperature is 23 ℃ and the relative humidity is 50-60%.

70 rats were divided into the following 7 groups: control group (labeled C, blank), model group (labeled M, diabetes-inducing and water-filling), metformin group (labeled Met, diabetes-inducing and metformin-administering), chitosan oligosaccharide 1 group (labeled COS1, diabetes-inducing and chitosan oligosaccharide-administering, 1500Da), chitosan oligosaccharide 2 group (labeled COS2, diabetes-inducing and chitosan oligosaccharide-administering, 3000Da), chitosan oligosaccharide guanidine 1 group (labeled COSG1, diabetes-inducing and chitosan oligosaccharide biguanide derivative COSG prepared in example 1), chitosan oligosaccharide guanidine 2 group (labeled COSG2, diabetes-inducing and chitosan oligosaccharide biguanide derivative COSG prepared in example 2). The control group was fed a normal diet and the other groups were fed a high fat and high sugar diet for 8 weeks, followed by fasting for 12 hours, followed by injection of STZ at a dose of 25mg/kg into the tail vein, to construct a type 2 diabetic rat model. When the random blood sugar is larger than 16.7mmol/L, the molding is considered to be successful. In the next 8 weeks, the normal control group was fed with basal diet, the drug-dry group was added with the corresponding drug on the basis of high-fat high-sugar diet, and the drug was gavaged at a dose of 500mg/kg (solvent: distilled water, pH 7), the blank group was gavaged with the corresponding dose of distilled water, and each group of rats was gavaged once a day in the afternoon at the same time.

2. Specimen collection

According to the indexes to be detected in animal experiments, the serum and the tissues of experimental rats are treated as follows for the following detection. 1) Collecting a blood sample: at the end of the experimental period, the rats were fasted for 12 hours and then sacrificed by bleeding under ether anesthesia by femoral artery puncture and blood samples were collected. Standing for 1h, centrifuging at 2500r/min for 20min, and collecting supernatant to obtain serum. Serum samples were stored in a-20 ℃ refrigerator for further analysis.

2) Weight: the weight of the rats was measured at the same time chosen weekly in units: g.

3) liver specimen: after the experiment, each group of rats was sacrificed by exsanguination, livers were immediately taken, a part of liver tissues was fixed with 10% formaldehyde solution after weighing, and were cut into blocks by conventional paraffin, and HE-stained, and observed under a light microscope.

3. Study conditions

1) Effects on the general condition of diabetic rats:

diabetic rats develop typical symptoms of weight loss. All diabetic rats had significant weight loss (P <0.001) compared to normal rats. The body weight of all other groups of mice gradually went normal during the course of the experiment as compared to the diabetic rats, as shown in figure 4 a.

Diabetic rats developed hyperglycemia within 8 weeks and the abdominal blood glucose levels were significantly elevated at the end of the experiment. As can be seen in FIG. 4b, fasting blood glucose levels were significantly increased in group M and decreased after treatment with metformin, COS and COSG (P <0.01) compared to group C. Among them, the COSG2 group with 3000Da performed better in lowering fasting glucose.

The effect of metformin, COS and COSG on urine glucose levels is shown in figure 4 c. Urine glucose content in group M was about 1.25 times that in group C (P <0.001), and the drug group exhibited significantly reduced urine glucose levels in diabetic rats (P <0.01) compared to group M. Additionally, the COSG1 group performed best.

2) Analysis of liver pathology

FIG. 5 shows the liver tissue of the HE-stained diabetic rat observed with a high power microscope, the liver lobules of the rats in group C are clear in structure, the hepatocytes are regularly arranged in a radial shape, the nuclei are located in the center of the cells, the morphology of the cells is normal and regular, the tissue structure is complete and clear, and no obvious pathological changes occur. Compared with the group C, a large number of liver cells in the group M have edema, degeneration, structural disorder of liver tissues and irregular arrangement. Treatment with metformin, COS and COSG reduced pathological changes in rat liver to a different extent compared to group M. Wherein, the repairing effect of the COSG2 on the liver cells is better, the shapes, the arrangement and the tissue structures of the liver cells are basically close to normal, and no obvious cell swelling and degeneration exist. While the difference between the different molecular weights is not significant.

3) Effect on rat liver injury serum protein and enzyme

In order to explore the hepatoprotective potential of COSG, biochemical analysis of blood was performed. As shown in table 2, ALT, AST, TP, TBIL and FFA activities were significantly higher in group M than in group C, but were inhibited by metformin, COS, COSG treatment. Among them, the effect of COSG is most significant (p <0.05), and the effect of metformin is slightly inferior to that of COSG. In addition, COS and COSG significantly increased the concentrations of SOD, CAT and GSH-Px, and reduced the level of MDA. All data show that the therapeutic effect of COSG is superior to COS.

Table 2 also shows that the insulin content in group M is significantly lower than in group C (p < 0.01). Mean FSI levels were 26.25% higher in Met group rats than in M group (p < 0.01). In addition, FSI levels were 30.04% higher for the COSG2 group than for the M group (p < 0.01). The HOMA-IR was significantly increased in the M group rats compared to the C group. However, after 8 weeks of drug treatment, the HOMA-IR decreased to varying degrees, with a COSG of 3000Da having the best therapeutic effect.

The experiments and analysis show that the chitosan oligosaccharide biguanide derivative disclosed by the invention has certain antioxidant activity, can inhibit weight reduction and blood sugar and urine sugar increase, improve insulin level, reduce ALT and AST enzyme activity, reduce TP, TBIL and FFA content in serum, prevent related enzymes from leaking from liver cells into extracellular circulation, reduce the degree of damage of free radicals to liver, reduce MDA, increase SOD, CAT and GSH-Px activity values and weaken oxidative stress damage of liver tissues. The chitosan oligosaccharide biguanide derivative has better treatment effect compared with the traditional diabetes treatment medicament metformin which is used alone, namely the application of the chitosan oligosaccharide biguanide derivative in the preparation of the medicament for inhibiting liver injury.

TABLE 2

Table 2 shows the effect of COSG on liver function indices associated with STZ-induced liver injury in diabetic rats. Data are mean ± standard deviation, n is 6,^#P<0.05,^##P<0.01,^###P<0.001 vs. blank control group,. P<0.05,**P<0.01,***P<0.001 compared to the model group.

The invention has been described in an illustrative manner, and it is to be understood that any simple variations, modifications or other equivalent changes which can be made by one skilled in the art without departing from the spirit of the invention fall within the scope of the invention.