Preparation method of radix ophiopogonis oligosaccharide

Abstract

The invention discloses a preparation method of radix ophiopogonis oligosaccharide, which comprises the following steps: taking a proper amount of radix ophiopogonis medicinal material, adding water to extract twice at the material-liquid ratio of 1: (6-10) and the extraction temperature of 90-100 DEG C for 1-2 hours each time, and concentrating the filtrate to prepare extract; adding 40% ethanol for alcohol precipitation, and concentrating filtrate to prepare extract; and adding 80% ethanol for alcohol precipitation, washing the precipitate with 95% ethanol, and drying the precipitate to obtain the product. According to the method, the molecular weight can be controlled to be 1000 or below, and the activity and safety of resisting type 2 diabetes mellitus are higher. According to the present invention, the expression of the insulin receptor and the substrate thereof can be improved, the sensitization effect on the insulin is significantly better than the metformin hydrochloride and the pyrrolitazone hydrochloride, and the blank of the diabetes caused by the insulin receptor and the substrate defect in the treatment drug can be filled. The preparation process is stable and controllable, the product yield is high, and good conditions are provided for quality control and industrial application of the radix ophiopogonis oligosaccharide.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of traditional Chinese medicine effective component extraction and pharmaceutical preparation, and particularly relates to a preparation method of ophiopogon japonicus oligosaccharide. BACKGROUND

[0002] Ophiopogon japonicus is the dried tuber of Ophiopogon japonicus of Liliaceae, rich in steroidal saponins, polysaccharides and high isoflavone components, and has the effects of nourishing yin, generating fluid, moistening the lungs and clearing the heart. Ophiopogon japonicus oligosaccharide is extracted from traditional Chinese medicine Ophiopogon japonicus, and the molecular weight is usually distributed between 1000-10000 Da. The molecular weight of Ophiopogon japonicus oligosaccharide is closely related to its biological activity, and the size of the molecular weight directly affects its solubility, absorption efficiency, target and activity intensity. The activities of oligosaccharides with different polymerization degrees are significantly different. For example, low molecular weight oligosaccharide (200-1000 Da) is easily absorbed and can quickly enter the blood or intestinal cells, and has strong effects of regulating blood sugar and antioxidant; medium and high molecular weight oligosaccharide (1000-3000 Da) has strong effects of immune regulation, prebiotic effect and anti-inflammatory effect. Therefore, the oligosaccharide combination with optimized molecular weight can be designed according to specific diseases (such as diabetes, intestinal inflammation).

[0003] The inventors have systematically verified that low molecular weight Ophiopogon japonicus oligosaccharide can improve T2DM glucose and lipid metabolism disorder through multi-target synergistic regulation, and its mechanism of action covers: improving insulin sensitivity, protecting and repairing islet beta cell function, regulating metabolic homeostasis and intestinal microecology, regulating liver glucose and lipid metabolism, inhibiting intestinal glucose absorption, activating energy metabolism and adipose tissue remodeling, etc., and has obviously better activity of resisting type 2 diabetes, and has broad clinical application prospects.

[0004] At present, the preparation method of low molecular weight Ophiopogon japonicus oligosaccharide mostly adopts water extraction and alcohol precipitation method, which is simple to operate, but has defects such as low yield, unstable molecular weight control, insufficient purity leading to product safety hazards, etc. However, for the activity of resisting type 2 diabetes, designing suitable oligosaccharide combination with appropriate molecular weight and successfully stably preparing and producing on a large scale are the basis for clinical application. Therefore, it is an urgent problem to optimize and improve the existing preparation method. SUMMARY

[0005] In order to overcome the above technical defects, the present application provides a preparation method of high-activity Ophiopogon japonicus oligosaccharide resisting type 2 diabetes. The method comprises the following steps: taking Ophiopogon japonicus medicinal materials, adding water to extract twice, the solid-liquid ratio is 1:6-10, the extraction temperature is 90-100℃, each time for 1-2 hours, and the filtrate is concentrated to prepare an extract; adding 40% ethanol for alcohol precipitation, concentrating the filtrate to prepare an extract; adding 80% ethanol for alcohol precipitation, filtering, washing the precipitate with 95% ethanol, and drying to obtain the product.

[0006] As a preferred solution, the feed liquid ratio is 1:10, the extraction temperature is 100 DEG C, and each time is 2 hours.

[0007] The relative density of the extract in the concentration step is greater than or equal to 1.25.

[0008] The preferred drying method of the precipitate is hot air drying at 55 DEG C.

[0009] The present application is particularly designed for the preparation method of the molecular weight oligosaccharide combination for resisting type 2 diabetes activity, and the molecular weight of the ophiopogon oligosaccharide can be stably controlled to be below 1000, and the oligosaccharide combination is stable. The ophiopogon oligosaccharide obtained by the method has the effect of resisting diabetes at multiple targets, and the activity is higher. The activity of resisting type 2 diabetes of the high-dose group is basically equivalent to that of metformin hydrochloride or pioglitazone hydrochloride, and in some aspects, it is even better. For example, in the aspect of promoting the expression of insulin receptor (InsR), insulin receptor substrate (IRS-1 / IRS-2) of islet beta cells, liver and fat, the effect is obviously stronger than that of metformin hydrochloride and pioglitazone hydrochloride. At present, there is no drug with the same mechanism of action on the market, which can fill the gap in the treatment of diabetes caused by defects of insulin receptor (InsR) and insulin receptor substrate (IRS-1 / IRS-2).

[0010] Toxicological studies show that the ophiopogon oligosaccharide obtained by the preparation method has a wide safety window (the maximum tolerance of mice is 12.8 g / kg, and no toxic reaction is found when the long-term administration dose of dogs reaches 5.94 g / kg), and the safety is high.

[0011] The preparation process of the present application is stable and controllable, the product yield is high, the obtained product oligosaccharide combination is stable, has obvious light and heat stability and biological safety, and provides good conditions for quality control and industrial application of ophiopogon oligosaccharide. BRIEF DESCRIPTION OF DRAWINGS

[0012] Figure 1 Figure 1 is a MALDI-TOF-MASS diagram of 20% alcohol precipitation impurity removal of ophiopogon oligosaccharide extract of the embodiment;

[0013] Figure 2 Figure 2 is a MALDI-TOF-MASS diagram of 30% alcohol precipitation impurity removal of ophiopogon oligosaccharide extract of the embodiment;

[0014] Figure 3 Figure 3 is a MALDI-TOF-MASS diagram of 40% alcohol precipitation impurity removal of ophiopogon oligosaccharide extract of the embodiment. DETAILED DESCRIPTION

[0015] The present application will be further described below in combination with specific embodiments.

[0016] Study on extraction, separation and purification process of olygosaccharide from ophiopogon japonicus

[0017] 1. Selection of extraction conditions of olygosaccharide from ophiopogon japonicus

[0018] Take ophiopogon japonicus, and carry out orthogonal experiment according to table 1. Each group of test is 20g ophiopogon japonicus. The total volume of water extract of each group of test is determined, and the total sugar content is determined by anthrone-sulfuric acid method.

[0019] Total sugar amount = total volume of water extract x total sugar content

[0020] Extraction rate = (total sugar amount / weight of medicinal material) x 100%

[0021] Table 1 Factor level table of ophiopogon japonicus water decoction extraction test

[0022] horizontal A extraction temperature / °C B extraction time / h C extraction times D water amount / multiple 1 80 0.5 2 10 2 90 1 2 8 3 100 (boiling) 2 3 6

[0023] Table 2 Orthogonal experiment table of ophiopogon japonicus water decoction extraction test

[0024]

[0025] The size of range R reflects the influence degree of each factor on the determination index. According to the value of range R, the primary and secondary order of factors affecting the extraction of total sugar in ophiopogon japonicus in the investigation range is B > A > D > C, that is, extraction time > extraction temperature > water amount > extraction times, and the optimal scheme is B3A3D1C3. Because the influence of extraction twice and extraction three times on the extraction rate is not significant, from the perspective of energy saving, extraction twice can be considered.

[0026] Take another four ophiopogon japonicus, each 500g, and extract according to the optimal conditions obtained by orthogonal experiment. The water amount of the first group is designed to be 10 times the amount of water for the first time, and 6 times the amount of water for the second time, and other extraction conditions are the same. The total volume of water extract of each group of test is determined, and the total sugar content is determined by anthrone-sulfuric acid method. The results are shown in table 3.

[0027] Total sugar amount = total volume of water extract x total sugar content

[0028] Extraction rate = (total sugar amount / weight of medicinal material) x 100%

[0029] Table 3 Verification test of ophiopogon japonicus water decoction extraction

[0030] Group Extraction condition Total sugar amount (g) Extraction rate (%) 1 Extraction 2 times (10, 10 times water) 331.5 66.3 2 Extraction 2 times (10, 10 times water) 341.8 68.4 3 Extraction 2 times (10, 10 times water) 335.2 67 4 Extraction 2 times (10, 6 times water) 327.4 65.5

[0031] According to the above experimental results, considering the extraction efficiency and economic benefit in experimental production, the optimal scheme is selected as follows: take appropriate amount of ophiopogon japonicus, extract twice with water, the solid-liquid ratio is 1:6-10, the extraction temperature is 100℃, and each time is 2 hours.

[0032] 2. Separation of olygosaccharide from ophiopogon japonicus

[0033] Ophiopogon japonicus oligosaccharides were obtained by fractional alcohol precipitation. Two portions of Ophiopogon japonicus, each weighing 1 kg, were extracted twice with water (10 times and 6 times the concentration), each time for 2 hours. The extracts were filtered, and the filtrates were collected and concentrated under reduced pressure to an extract with a relative density of 1.25 or higher. 2 L of 20%, 30%, and 40% ethanol were added to each extract, and the mixture was stirred and allowed to stand for 12 hours. The extracts were then filtered, and the filtrates were concentrated under reduced pressure to an extract with a relative density of 1.25 or higher. 1 L of 80% ethanol was added to each extract, and the mixture was stirred and allowed to stand for 12 hours. The precipitates were washed with 95% ethanol and dried with hot air at 55°C to obtain Ophiopogon japonicus oligosaccharides.

[0034] The comparison results of Ophiopogon japonicus oligosaccharide extract at three alcohol concentrations are shown in the figure. Figure 1 , Figure 2 , Figure 3 .Depend on Figure 1 , Figure 2 , Figure 3 It is evident that 40% alcohol precipitation yields better results in removing impurities from samples, significantly altering the molecular weight range and concentrating the molecular weights below 1000. Furthermore, filtration is difficult and cumbersome when using 20% ​​or 30% alcohol precipitation for impurity removal. Therefore, 40% alcohol precipitation is employed for impurity removal.

[0035] The optimal process was initially determined as follows: 1 kg of Ophiopogon japonicus was extracted twice with water (10 times and 6 times the water volume), at 100℃ for 2 hours each time. The extract was filtered, and the filtrate was collected and concentrated under reduced pressure to an extract with a relative density greater than 1.25. 2 L of 40% ethanol was added, stirred, and allowed to stand for 12 hours. The extract was then filtered, and the filtrate was concentrated under reduced pressure to an extract with a relative density greater than 1.25. 1 L of 80% ethanol was added, stirred, and allowed to stand for 12 hours. The extract was then filtered, and the precipitate was washed with 95% ethanol and dried with hot air at 55℃ to obtain the Ophiopogon japonicus oligosaccharide extract. The prepared Ophiopogon japonicus oligosaccharide was used in the following experiments.

[0036] 3. Pilot-scale amplification test of Ophiopogon japonicus oligosaccharide extraction process

[0037] Three parallel portions of Ophiopogon japonicus, each weighing 5 kg, were extracted twice with water (10x and 6x dilution), each time for 2 hours. The extracts were filtered, and the filtrates were collected and concentrated under reduced pressure to a relative density greater than 1.25. 2 L of 40% ethanol was added, the mixture was stirred, and allowed to stand for 12 hours. The extracts were then filtered, and the filtrates were concentrated under reduced pressure to a relative density greater than 1.25. 1 L of 80% ethanol was added, the mixture was stirred, and allowed to stand for 12 hours. The precipitate was then washed with 95% ethanol and dried with hot air at 55°C to obtain the Ophiopogon japonicus oligosaccharide extract. The results are shown in Table 4. As can be seen from Table 4, the scale-up experiment was stable and feasible.

[0038] Table 4. Scale-up Experiment of Oligosaccharide Extraction Process from Ophiopogon japonicus

[0039] Group Radix ophiopogonis oligosaccharide extract (kg) Sugar content (%) 1 2.815 76.3 2 2.738 78.2 3 2.770 72.4

[0040] 4. Scale-up test of Ophiopogon japonicus oligosaccharide extraction process (outsourced to GMP-compliant medicinal material production)

[0041] 285 kg of Ophiopogon japonicus was used as raw material. It was extracted twice with water (10x and 6x dilution), each time for 2 hours. The extract was filtered, and the filtrate was collected and concentrated under reduced pressure to a relative density greater than 1.25. 2 L of 40% ethanol was added, stirred, and allowed to stand for 12 hours. The extract was then filtered, and the filtrate was concentrated under reduced pressure to a relative density greater than 1.25. 1 L of 80% ethanol was added, stirred, and allowed to stand for 12 hours. The precipitate was washed with 95% ethanol and dried with hot air at 55℃ to obtain the Ophiopogon japonicus oligosaccharide extract. The results are shown in Table 5, indicating that the extraction process is stable and feasible.

[0042] Table 5. Scale-up Experiment of Ophiopogon japonicus Oligosaccharide Extraction Process

[0043] Batch number Radix ophiopogonis oligosaccharide extract (kg) Sugar content (%) 20190625 167.2 78.67

[0044] Example 2: Quality Study of Ophiopogon japonicus Oligosaccharides

[0045] Ophiopogon japonicus oligosaccharide is a pale yellow to pale brown powder, odorless, slightly sweet, and readily soluble in water, with a maximum absorption peak at 625±2 nm. Ophiopogon japonicus oligosaccharide is dried under reduced pressure at room temperature to constant weight using phosphorus pentoxide as a desiccant; the weight loss should not exceed 6.0%, the residue on ignition should not exceed 0.6%, and the heavy metal content should not exceed 20 mg / kg. Based on anhydrous glucose, it should not be less than 70%.

[0046] Example 3: Stability Study of Ophiopogon japonicus Oligosaccharides

[0047] Under high temperature (60℃) and strong light (4500±500Lx) conditions, the content of its active ingredient showed no significant change, indicating that the substance has good photothermal stability. However, in the high humidity test, under the test conditions of relative humidity of 90%±5% and 75%±5%, the water absorption exceeded 5%, indicating that this product should not be stored in a high humidity environment. Accelerated stability test showed that the sample color deepened significantly at 40℃, while the properties were stable at 30℃. Therefore, this product should be sealed in double-layer vacuum aluminum foil packaging and stored in a cool, dry place.

[0048] Example 4: Study on the hypoglycemic effect and mechanism of this Ophiopogon japonicus oligosaccharide on spontaneously diabetic type II OLETF rats

[0049] 1. Materials: Ophiopogon japonicus oligosaccharides prepared by the method of this invention.

[0050] Spontaneous type 2 diabetic OLETF rats: purchased from the Tokushima Research Institute of Otsuka Pharmaceutical Co., Ltd., Japan.

[0051] Pioglitazone hydrochloride, metformin hydrochloride, glucagon-like peptide-1 (GLP-1) assay kit, glucagon assay kit, muscle glycogen assay kit, liver glycogen assay kit, glucokinase assay kit, phosphoenolpyruvate carboxykinase assay kit, blood glucose assay kit, streptozotocin, blood glucose meter, etc.

[0052] 2. Methods: After 2 weeks of acclimatization feeding, rats were assessed for their basal condition and randomly divided into four groups: a normal control group (LETO group), a model group (OLETF group), a positive control group (metformin hydrochloride group), a positive control group (pioglitazone hydrochloride group), and low, medium, and high dose groups of Ophiopogon japonicus oligosaccharide. Animals in each group were administered the drug via gavage from 12 weeks of age until 40 weeks of continuous administration. Administered the drug once daily, and the animals were weighed daily to adjust the gavage dosage as needed.

[0053] Blood glucose measurement: After the last administration, the animals were fasted for 12 hours but allowed to drink water. Blood was collected from the tail and measured using a blood glucose meter.

[0054] Urine protein and urine glucose determination: During the third and final administration of the drug to the animals, the rats were placed in a metabolic cage, and urine was collected over 24 hours. After recording the urine volume, 5 mL of urine was taken, centrifuged at 3000 rpm for 10 min, and the supernatant was collected to determine urine glucose and urine protein according to the kit method.

[0055] Plasma glycated hemoglobin (HbA1c) determination: After the last administration, the animals were fasted for 12 hours but allowed free access to water. Blood was collected and analyzed using a Bayer DCA2000 glycated hemoglobin analyzer. Reagents were also provided by Bayer.

[0056] Muscle glycogen and liver glycogen assays: After blood collection, the animal was euthanized by dislocation. Hind limb muscle and liver were harvested, rinsed with physiological saline, immediately frozen in liquid nitrogen, and stored at -80°C. Muscle glycogen and liver glycogen were measured according to the kit instructions.

[0057] Oral glucose tolerance test: After the last administration, fasting was allowed for 12 hours, but water was not. Except for the blank control group, all other groups were given glucose at 2.0 g / kg BW. Blood samples were collected from the tail at 0 min, 30 min, 60 min, 90 min and 120 min after glucose administration and measured with a blood glucose meter.

[0058] Pancreatic islet tissue pathology sections: After blood collection, the abdominal cavity is opened and the pancreas is quickly removed. The pancreas is then washed with physiological saline, the floating blood is aspirated, and the tissue is fixed in 10% formalin solution. After being embedded in paraffin, the sections are prepared, stained with hematoxylin and eosin (HE), and finally, the changes in tissue structure are observed under a microscope.

[0059] Measurement of InsR and IRS-1 / IRS-2 in pancreatic islet cells: After blood collection, the abdominal cavity was opened and the pancreas was quickly removed. The pancreas was washed with physiological saline, and the blood was aspirated. The pancreas was then fixed in 10% formalin solution, embedded in paraffin, and sectioned. The expression of InsR and IRS-1 / IRS-2 was measured using standard immunohistochemistry according to the SABC kit. Six fields containing islets were selected from each group. Image-Proplus 6.0 pathological image analysis system was used to analyze the positive staining of InsR and IRS-1 / IRS-2 in each islet. The integrated optical density and area were recorded, and the ratio of integrated optical density to area was used as the average optical density for statistical analysis.

[0060] Determination of InsR and IRS-1 / IRS-2 mRNA expression in rat liver, skeletal muscle, and adipose tissue: Total RNA was extracted from each tissue according to the Trizol manufacturer's instructions. After determining the RNA concentration, 2 μg of RNA was reverse transcribed into cDNA using M-MLV reverse transcriptase, followed by PCR amplification. The PCR products were subjected to 1.2% agarose gel electrophoresis, and gel images were taken using a gel imaging system. The gel images were analyzed using Smart View to obtain the brightness scan ratio of the target gene and the internal reference gene.

[0061] Determination of InsR and IRS-1 / IRS-2 protein expression in rat liver, skeletal muscle, and fat: Referring to the literature, tissue lysis buffer was used to obtain total tissue protein. A small amount was taken and the protein concentration was determined using the Coomassie Brilliant Blue method. The expression levels of each protein were determined using the conventional Western blot method. Finally, gel imaging was performed, and the scan density values ​​were obtained by analyzing the colorimetric images using Smart View.

[0062] 3. Results

[0063] 3.1 Effects of Ophiopogon japonicus oligosaccharides on blood glucose levels and glucose tolerance in spontaneously diabetic type II rats. The results are shown in Table 6.

[0064] As shown in Table 6, compared with the blood glucose level of the normal control group, the blood glucose level and area under the blood glucose curve of the model group increased significantly, indicating that the model was successful.

[0065] After administration, the blood glucose levels in the low, medium, and high dose groups of Ophiopogon japonicus oligosaccharide were significantly lower than those in the model group, and a dose-response relationship was observed; the higher the dose, the greater the decrease in blood glucose levels, indicating that Ophiopogon japonicus oligosaccharide has a significant hypoglycemic effect. Furthermore, the area under the blood glucose curve in the low, medium, and high dose groups of Ophiopogon japonicus oligosaccharide was significantly lower than that in the model group (P<0.05), and a dose-response relationship was observed. This suggests that Ophiopogon japonicus oligosaccharide can significantly improve glucose tolerance in type II diabetic rats and is more resistant to hyperglycemia induced by exogenous sugars.

[0066] Table 6. Effects of Ophiopogon japonicus oligosaccharides on blood glucose levels and glucose tolerance in spontaneously diabetic rats.

[0067]

[0068] Note: Compared with the normal control group: **P<0.01; Compared with the model group: #P<0.05; ##P<0.01.

[0069] 3.2 Effects of Ophiopogon japonicus oligosaccharides on urinary protein, urinary glucose, liver glycogen, and muscle glycogen in spontaneously diabetic type II rats. The results are shown in Tables 7 and 8.

[0070] As shown in Tables 7 and 8, compared with the normal control group, the model group had significantly higher levels of urinary protein and urinary glucose, and significantly lower levels of liver glycogen and muscle glycogen, indicating that the model was successful.

[0071] After administration, compared with the model group, the low, medium, and high dose groups of Ophiopogon japonicus oligosaccharide showed significant decreases in urinary protein and glucose, and significant increases in liver glycogen and muscle glycogen, suggesting that Ophiopogon japonicus oligosaccharide has significant hypoglycemic and renal damage-inhibiting effects. Furthermore, the high dose group of Ophiopogon japonicus oligosaccharide showed better improvement in urinary protein, glucose, and liver glycogen than the positive control drugs metformin hydrochloride and pioglitazone hydrochloride groups. This indicates that Ophiopogon japonicus oligosaccharide can significantly improve blood glucose and inhibit renal damage in type II diabetic rats, significantly increase liver and muscle glycogen content, inhibit liver glycogen loss and breakdown, and increase muscle glycogen reserves.

[0072] Table 7 Effects of Ophiopogon japonicus oligosaccharides on urinary protein and urinary glucose in type II diabetic rats

[0073] Group Dose (mg / kg) Urine sugar (mmol / L) Urine protein (mg / L) Normal control group - 13.2±2.5 68.46±4.36 Model group - 38.3±5.2** 246.48±16.32** Metformin hydrochloride group 250 31.4±2.5# 216.39±35.69 Pioglitazone hydrochloride group 5.5 30.5±5.6# 196.39±29.87# Radix ophiopogonis oligosaccharide low-dose group 112 32.9±1.4# 185.44±26.59## Radix ophiopogonis oligosaccharide medium-dose group 225 26.5±2.6## 178.90±21.46## Radix ophiopogonis oligosaccharide high-dose group 450 22.3±2.4##△※ 150.17±16.52##△※

[0074] Note: Compared with the normal control group: ***P<0.01; Compared with the model group: #P<0.05; ##P<0.01; Compared with the metformin hydrochloride group: △P<0.05; △△P<0.01; Compared with the pioglitazone hydrochloride group: ※P<0.05, ※※P<0.01.

[0075] Table 8. Effects of Ophiopogon japonicus oligosaccharides on muscle and liver glycogen in type II diabetic rats.

[0076] Group Dose (mg / kg) Muscle glycogen (g / mg) Liver glycogen (g / mg) Normal control group - 2.06±0.25 12.65±1.53 Model group - 1.35±0.10** 5.64±0.99** Metformin hydrochloride group 250 1.65±0.14## 8.96±0.89## Pioglitazone hydrochloride group 5.5 1.84±0.06## 8.66±1.25## Radix ophiopogonis oligosaccharide low-dose group 112 1.59±0.09# 6.92±0.86# Radix ophiopogonis oligosaccharide medium-dose group 225 1.76±0.21## 8.21±1.68## Radix ophiopogonis oligosaccharide high-dose group 450 1.79±0.15## 10.64±1.51##△※

[0077] Note: Compared with the normal control group: **P<0.01; Compared with the model group: #P<0.05; ##P<0.01; Compared with the metformin hydrochloride group: △P<0.05; △△P<0.01; Compared with the pioglitazone hydrochloride group: ※P<0.05, ※※P<0.01.

[0078] 3.3 Effects of Ophiopogon japonicus oligosaccharides on plasma HbA1c in spontaneously diabetic rats

[0079] Table 9 shows that plasma HbA1c in the model group was significantly higher than that in the normal control group, indicating successful model establishment. After administration, compared with the model group, plasma HbA1c in the low, medium, and high dose groups of Ophiopogon japonicus oligosaccharide was significantly lower, suggesting that Ophiopogon japonicus oligosaccharide has a significant and good effect on controlling blood glucose. Moreover, the high dose group of Ophiopogon japonicus oligosaccharide showed better improvement in plasma HbA1c than the positive control drug metformin hydrochloride group, indicating that Ophiopogon japonicus oligosaccharide can significantly inhibit the increase of plasma HbA1c and has a significant effect on controlling blood glucose.

[0080] Table 9. Effects of Ophiopogon japonicus oligosaccharides on plasma HbA1c in type II diabetic rats.

[0081] Group Dose (mg / kg) HbA1c (mmol / L) Normal control group - 2.48±0.32 Model group - 11.25±2.13** Metformin hydrochloride group 250 8.11±0.79## Pioglitazone hydrochloride group 5.5 7.31±0.86## Radix ophiopogonis oligosaccharide low-dose group 112 8.32±1.65# Radix ophiopogonis oligosaccharide medium-dose group 225 7.03±0.99## Radix ophiopogonis oligosaccharide high-dose group 450 6.32±0.61##△

[0082] Note: Compared with the normal control group: **P < 0.01; Compared with the model group: #P < 0.05; ##P < 0.01; Compared with the metformin hydrochloride group: △P < 0.05; △△P < 0.01

[0083] 3.4 Effects of Ophiopogon japonicus oligosaccharides on the expression of InsR, IRS-1 / IRS-2 in pancreatic islet cells, liver, skeletal muscle and fat in spontaneously diabetic rats. The results are shown in Tables 10-13.

[0084] Table 10 Effects of Ophiopogon japonicus oligosaccharides on the expression of InsR and IRS-1 / IRS-2 in pancreatic islet cells of type II diabetic rats

[0085]

[0086] Note: Compared with the normal control group: **P<0.01; Compared with the model group: #P<0.05; ##P<0.01; Compared with the metformin hydrochloride group: △P<0.05; △△P<0.01; Compared with the pioglitazone hydrochloride group: ※P<0.05, ※※P<0.01.

[0087] Table 10 shows that InsR positive expression in islet cells is brown and diffusely distributed throughout the islets. Compared with the normal control group, InsR positive expression was significantly reduced in the model group. Compared with the model group, InsR positive expression was not increased in the pioglitazone hydrochloride group and the metformin hydrochloride group. The low, medium, and high dose groups of Ophiopogon japonicus oligosaccharide showed significantly stronger InsR positive expression in islet cells than the model group, exhibiting a dose-dependent relationship. Moreover, the InsR positive expression in islet cells in the medium and high dose groups was significantly higher than that in the metformin hydrochloride and pioglitazone hydrochloride groups (P < 0.05), indicating that Ophiopogon japonicus oligosaccharide has a promoting effect on InsR expression in islet cells of diabetic rats, and is superior to that of metformin hydrochloride and pioglitazone hydrochloride.

[0088] IRS-1 positive expression in pancreatic islet cells is brown and distributed in the cytoplasm surrounding the islets. Compared with the normal control group, IRS-1 positive expression was significantly reduced in the model group. Compared with the model group, IRS-1 positive expression was not increased in the pioglitazone hydrochloride group and the metformin hydrochloride group. The low, medium, and high dose groups of Ophiopogon japonicus oligosaccharide showed significantly stronger IRS-1 positive expression in pancreatic islet cells than the model group, exhibiting a dose-dependent relationship, indicating that Ophiopogon japonicus oligosaccharide promotes IRS-1 expression in pancreatic islet cells of diabetic rats.

[0089] Tables 11, 12, and 13 show that compared with the normal control group, the expression levels of InsR and IRS-1 / IRS-2 proteins in the liver, skeletal muscle, and adipose tissue of the model group were significantly increased, indicating that the model exhibited significant peripheral insulin resistance and was successful. After administration, compared with the model group, the expression of InsR and IRS-1 / IRS-2 proteins in the liver, skeletal muscle, and adipose tissue of the low, medium, and high dose groups of Ophiopogon japonicus oligosaccharide was significantly increased in a dose-dependent manner, suggesting that Ophiopogon japonicus oligosaccharide has a significant inhibitory effect on peripheral insulin resistance. The upregulation of InsR and IRS-1 / IRS-2 protein expression in the liver, skeletal muscle, and adipose tissue by the medium and high dose groups of Ophiopogon japonicus oligosaccharide was significantly stronger than that by pioglitazone hydrochloride and metformin hydrochloride (P < 0.05 or 0.01).

[0090] Table 11 Effects of Ophiopogon japonicus oligosaccharides on the expression of InsR and IRS-1 / IRS-2 proteins in the liver of type II diabetic rats

[0091]

[0092] Note: Compared with the normal control group: **P<0.01; Compared with the model group: #P<0.05; ##P<0.01; Compared with the metformin hydrochloride group: △P<0.05; △△P<0.01; Compared with the pioglitazone hydrochloride group: ※P<0.05, ※※P<0.01.

[0093] Table 12 Effects of Ophiopogon japonicus oligosaccharides on the expression of InsR and IRS-1 / IRS-2 proteins in skeletal muscle of type II diabetic rats

[0094]

[0095] Note: Compared with the normal control group: **P<0.01; Compared with the model group: #P<0.05; ##P<0.01; Compared with the metformin hydrochloride group: △P<0.05; △△P<0.01; Compared with the pioglitazone hydrochloride group: ※P<0.05, ※※P<0.01.

[0096] Table 13 Effects of Ophiopogon japonicus oligosaccharides on the expression of adipose-derived InsR and IRS-1 / IRS-2 proteins in type II diabetic rats

[0097]

[0098] Note: Compared with the normal control group: **P<0.01; Compared with the model group: #P<0.05; ##P<0.01; Compared with the metformin hydrochloride group: △P<0.05; △△P<0.01; Compared with the pioglitazone hydrochloride group: ※P<0.05, ※※P<0.01.

[0099] Example 5: General Pharmacological Study of Ophiopogon japonicus Oligosaccharides

[0100] A single oral administration of 320, 960, and 2880 mg / kg of Ophiopogon japonicus oligosaccharide to Kunming mice had no significant effect on spontaneous activity or the subthreshold hypnotic effect of sodium pentobarbital. A single oral administration of 66, 200, and 600 mg / kg to anesthetized Beagle dogs had no significant effect on their respiratory or cardiovascular systems.

[0101] Example 6: Toxicological Study of Ophiopogon japonicus Oligosaccharides

[0102] Ophiopogon japonicus oligosaccharide was administered orally to Kunming mice at a maximum concentration of 320 mg / mL and a maximum volume of 40 mL / kg. The mice were 12.8 g / kg (376 times the clinically intended dose) and observed for 14 days. During this period, no mice died, their mental and behavioral condition was good, their skin and fur were clean, their urination and defecation were normal, their weight gain was normal, and their food intake was not abnormal. No organ abnormalities were found during gross anatomical examination after the observation period. A single oral administration of 8.8 g / kg (258 times the intended human clinical dose) to Beagle dogs showed no significant toxicity. SD rats were administered 2.25, 6.75, and 13.5 g / kg of Ophiopogon japonicus oligosaccharide orally for three consecutive months; the animals were in good general condition, and no toxic reactions related to the test product were observed in hematological, blood biochemical, and histopathological examinations. Beagle dogs were administered Ophiopogon japonicus oligosaccharide (660, 1980, and 5940 mg / kg, representing 10, 30, and 90 times the effective canine dose, and 20, 60, and 120 times the intended human clinical dose, respectively) orally for nine months; the animals were in good general condition, and no toxic reactions related to the test product were observed in hematological, blood biochemical, and histopathological examinations.

[0103] Example 7: Preparation of Ophiopogon japonicus Oligosaccharide Tablets

[0104] Formula: 20kg of Ophiopogon japonicus oligosaccharides, 2.4kg of microcrystalline cellulose, and 0.1kg of low-substituted hydroxypropyl cellulose.

[0105] Preparation method: Mix the above materials together evenly; granulate, dry and sizing with 50% ethanol as binder; add 0.5% magnesium stearate by weight of granules and mix evenly; compress into tablets at a dosage of 450mg / tablet and coat to obtain the final product.

[0106] Example 8: Preparation of Ophiopogon japonicus Oligosaccharide Chewable Tablets

[0107] Formula: 35kg of Ophiopogon japonicus oligosaccharides, 12.5kg of microcrystalline cellulose, 12.5kg of mannitol, 0.6kg of sweet orange powder flavoring, 0.6kg of magnesium stearate, and 10.5kg of 95% ethanol.

[0108] Preparation method: pass the oligosaccharide of Ophiopogon japonicus, microcrystalline cellulose and mannitol through an 80-mesh sieve, mix well, add 10.5 kg of 95% ethanol, make soft material, granulate through a 20-mesh sieve, dry at 50℃, granulate, add sweet orange powder flavoring and magnesium stearate, mix well, compress into tablets (0.9 g / tablet), coat with film, and package to obtain the product.

[0109] Example 9: Ophiopogon japonicus oligosaccharide effervescent granules

[0110] Acid-containing granules: 17.5 kg of Ophiopogon japonicus oligosaccharide, 6.25 kg of dextrin, 6.25 kg of mannitol, 13.185 kg of lactose, 15.75 kg of citric acid, 0.1575 kg of sucralose, and 3 kg of 95% ethanol.

[0111] Alkaline granules: 17.5 kg of Ophiopogon japonicus oligosaccharide, 6.25 kg of dextrin, 6.25 kg of mannitol, 15.75 kg of sodium bicarbonate, 0.1575 kg of sucralose, and 7 kg of 95% ethanol.

[0112] Preparation: Ophiopogon japonicus oligosaccharide, dextrin, and mannitol are passed through an 80-mesh sieve, mixed well, and then lactose, citric acid, and sucralose are added. The mixture is then softened with 95% ethanol, granulated through a 20-mesh sieve, dried at 50℃, and granulated to obtain acidic granules. Ophiopogon japonicus oligosaccharide, dextrin, and mannitol are passed through an 80-mesh sieve, mixed well, and then sodium bicarbonate and sucralose are added. The mixture is then softened with 95% ethanol, granulated through a 20-mesh sieve, dried at 50℃, and granulated to obtain alkali-containing granules. The acidic granules, alkali-containing granules, and 0.315 kg of sweet orange powder flavoring are mixed well and packaged (3.5 g / bag) to obtain the final product.

Claims

1. A method for preparing Ophiopogon japonicus oligosaccharides, characterized in that, Includes the following steps: Take an appropriate amount of Ophiopogon japonicus, extract it twice with water at a material-to-liquid ratio of 1:6-10, and at an extraction temperature of 90°C. At -100℃, for 1-2 hours each time, concentrate the filtrate to obtain an extract; add 40% ethanol for alcohol precipitation, concentrate the filtrate to obtain an extract; add 80% ethanol for alcohol precipitation, filter, wash the precipitate with 95% ethanol, and dry to obtain the final product.

2. The preparation method according to claim 1, characterized in that, The material-to-liquid ratio is 1:10, the extraction temperature is 100℃, and the extraction time is 2 hours each time.

3. The preparation method according to claim 1, characterized in that, In the step of concentrating the filtrate to prepare the extract, the relative density of the extract is greater than or equal to 1.

25.

4. The preparation method according to claim 1 or 2, characterized in that, The drying method for the step of drying the precipitate is hot air drying at 55°C.

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