Bamboo cane polysaccharide compound as well as preparation method and application thereof

Abstract

The invention relates to a bamboo cane polysaccharide compound as well as a preparation method and application thereof. The method comprises the following steps: performing ultrafiltration, dialysis or alcohol precipitation separation on Songxi hundred-year sugarcane brown sugar or bamboo sugarcane stem juice obtained by perennial root growth in 2-291 years to obtain a bamboo sugarcane polysaccharide compound in a specific molecular weight range. The bamboo cane polysaccharide compound has treatment effects of reducing fasting blood-glucose, improving glucose tolerance, improving glucose metabolism disorder and improving renal function impairment in a db / db diabetic mouse model, and has diabetes prevention effects of controlling mouse weight and weight increase amplitude, improving glucose metabolism disorder and promoting energy metabolism in a high fat diet induced obesity C57 mouse. Therefore, the invention is beneficial to design and research and development of health-care foods and medicines for preventing and / or treating diabetes mellitus based on the bamboo cane polysaccharide compound.

Description

[0001] This application claims the priority benefit of the Chinese invention patent with application number 2023109990097 filed with the State Intellectual Property Office of China on August 9, 2023, and invention name “Songxi bamboo cane polysaccharide compounds and preparation methods and uses thereof”, the entire contents of which are incorporated herein by reference. Technical Field

[0002] The present invention relates to the technical field of preparation and application of plant-derived polysaccharide compounds, and more specifically to a bamboo cane polysaccharide compound separated and purified from bamboo cane brown sugar or bamboo cane stem juice, a preparation method thereof, and use thereof in preparing a medicine for preventing and / or treating diabetes or a health food for assisting in lowering blood sugar. Background Art

[0003] Diabetes is a chronic metabolic disease characterized by hyperglycemia, and its types are mainly divided into type I diabetes (insulin-dependent diabetes), type II diabetes (non-insulin-dependent diabetes) and gestational diabetes. Among them, type II diabetes patients account for about 90% of the total number of diabetic patients, and most of them are over 35 years old. Most diabetic patients often have varying degrees of acute complications or chronic complications due to persistent hyperglycemia and long-term metabolic disorders. Acute complications include ketoacidosis, lactic acidosis, and non-ketotic hyperosmolar syndrome, which can be life-threatening; while chronic complications include diabetic nephropathy, diabetic eye disease, diabetic foot, cardiovascular and cerebrovascular damage, and nervous system damage and dysfunction and failure, which can seriously endanger physical health. At present, the main treatment for patients with type I diabetes and type II diabetes in the middle and late stages is frequent subcutaneous injection of insulin. This treatment method not only brings pain and inconvenience to patients, but also easily induces problems such as hypoglycemia and obesity. Therefore, the development of oral insulin or oral hypoglycemic drugs is currently a popular strategy for treating diabetes.

[0004] Bamboo sugarcane (Saccharum sinense Roxb.) is a perennial plant of the genus Saccharum in the Poaceae family. Bamboo sugarcane is an ancient local original species in China, mainly distributed in Zhejiang, Jiangxi, Hunan, Fujian, Guangdong, Guangxi, Sichuan and Yunnan, China. Fujian Songxi bamboo sugarcane retains the oldest original species in China. It is the only variety in the world with underground runners and bamboo whip-like roots. It has strong perennial roots and stress resistance, and is suitable for long-term perennial root cultivation. The "Centennial Sugarcane" (bamboo sugarcane brand) in Songxi, Fujian has retained perennial roots for nearly 300 years and still has considerable yield. In November 2021, the bamboo sugarcane cultivation system of Songxi, Fujian was included in the sixth batch of China's important agricultural cultural heritage.

[0005] Fujian Songxi "Centennial Sugarcane" brown sugar is made using traditional ancient methods. Brown sugar has the effects of expelling cold and warming the stomach, promoting blood circulation and removing blood stasis, strengthening the spleen and warming the stomach, and nourishing qi and blood. As early as June 27, 1963, Fujian Daily reported that Songxi "Centennial Sugarcane" brown sugar was honored as the "monthly sugar" for pregnant women. This shows that bamboo cane brown sugar contains biologically active ingredients that can improve the body immunity of pregnant women.

[0006] There is no research on the hypoglycemic effect of polysaccharides in bamboo cane. Summary of the invention

[0007] The invention performs ultrafiltration, dialysis or alcohol precipitation separation on Songxi "Centennial Sugarcane" brown sugar or bamboo sugarcane stalk juice obtained from 2 to 291 years of perennial root growth to obtain bamboo sugarcane polysaccharide compounds in a specific molecular weight range, thereby eliminating the interference of small molecules such as sucrose during application.

[0008] The invention obtains bamboo cane polysaccharide compounds with a molecular weight greater than 1 kDa for the first time through ultrafiltration, dialysis or alcohol precipitation methods, and gel permeation chromatography (GPC) analysis shows that the polysaccharides have a molecular weight range of 1-400 kDa and 400-5000 kDa.

[0009] The present invention is the first to study the therapeutic effects of bamboo cane polysaccharide compounds with molecular weights of 1-400 kDa and 400-5000 kDa in reducing fasting blood sugar, improving glucose tolerance, improving glucose metabolism disorders, and improving renal function damage in db / db diabetic mouse models, and the diabetes prevention effects of controlling mouse body weight and body weight gain, improving glucose metabolism disorders, and promoting energy metabolism in high-fat diet-induced obese C57 mice. Therefore, the present invention is helpful for designing and developing health foods and drugs for preventing and / or treating diabetes based on bamboo cane polysaccharide compounds.

[0010] The present invention discloses a new hypoglycemic mechanism of the bamboo cane polysaccharide compounds of the present invention. The bamboo cane polysaccharide compounds can upregulate the sugar metabolism pathways related to glycolysis and gluconeogenesis, promote the secretion of glucagon-like peptide-1 (GLP-1) by intestinal cells, and the target of the hypoglycemic effect is not only in the intestine, but distributed in the body.

[0011] One of the purposes of the present invention is to provide a bamboo cane polysaccharide compound.

[0012] The second object of the present invention is to provide a method for preparing the bamboo cane polysaccharide compound.

[0013] The third object of the present invention is to provide a use of the bamboo cane polysaccharide compound in preparing a drug for preventing and / or treating diabetes or a health food for assisting in lowering blood sugar.

[0014] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are particularly adopted:

[0015] On one hand, the present invention provides a bamboo cane polysaccharide compound, which is obtained by separating bamboo cane juice to remove small molecular compounds with a molecular weight of less than 1 kDa. The molecular weight range of the bamboo cane polysaccharide compound is 1-400 kDa and 400-5000 kDa. Preferably, the molecular weight range of the bamboo cane polysaccharide compound is 1-200 kDa and 500-4000 kDa after gel permeation chromatography (GPC) analysis, more preferably 1.5-100 kDa and 500-3000 kDa, more preferably 1.5-70 kDa and 670-3000 kDa, more preferably 1.5-20 kDa and 670-3000 kDa.

[0016] In some embodiments, polysaccharides in the molecular weight range of 1.5-20 kDa account for 40%-90%, polysaccharides in the molecular weight range of 10-400 kDa account for 10%-30%, and polysaccharides in the molecular weight range of 400-5000 kDa account for 0%-30%.

[0017] In some embodiments, the bamboo cane juice is derived from an aqueous solution of bamboo cane brown sugar or bamboo cane stem juice.

[0018] Preferably, the origin of the bamboo sugarcane is not limited, and is preferably from Guangdong, Guangxi, Yunnan, Sichuan, Songxi County, Fujian, etc., preferably Songxi County, Fujian.

[0019] The separation process is not particularly limited and can be any means known in the art that can separate polysaccharides of a certain molecular weight to obtain desired polysaccharides of a certain molecular weight range. In some embodiments, the separation process includes one or more of ultrafiltration, dialysis and alcohol precipitation.

[0020] The bamboo cane polysaccharide compound of the present invention is a water-soluble polysaccharide compound obtained by removing small molecules such as sucrose below 1 kDa from Songxi "century-old sugarcane" brown sugar aqueous solution or bamboo cane stem juice through ultrafiltration, dialysis or alcohol precipitation (molecular weight range 1-400 kDa and 400-5000 kDa, preferably, the bamboo cane polysaccharide compound is analyzed by gel permeation chromatography (GPC) to have a molecular weight range of 1-200 kDa and 500-4000 kDa, more preferably a molecular weight range of 1.5-100 kDa and 500-3000 kDa, more preferably a molecular weight range of 1.5-70 kDa and 670-3000 kDa, more preferably a molecular weight range of 1.5-20 kDa and 670-3000 kDa).

[0021] Another aspect of the present invention provides a method for preparing the bamboo cane polysaccharide compound, the method comprising the following steps:

[0022] The bamboo cane polysaccharide compound is obtained by separating and treating the aqueous solution of bamboo cane brown sugar or the bamboo cane stem juice to remove small molecular compounds with a molecular weight below 1 kDa, and then drying.

[0023] The separation process is not particularly limited, and can be any means known in the art that can separate according to molecular weight to obtain a desired molecular weight polysaccharide of a certain range. In some embodiments, the separation process includes one or more of ultrafiltration, dialysis and alcohol precipitation, such as ultrafiltration, or dialysis, or dialysis and further alcohol precipitation after dialysis.

[0024] In some embodiments, the aqueous solution of bamboo cane brown sugar or bamboo cane stalk juice further comprises the step of centrifuging or filtering the aqueous solution of bamboo cane brown sugar or bamboo cane stalk juice to remove insoluble matter before separation.

[0025] In some embodiments, the concentration of the aqueous solution of brown sugar is 100-200 mg / mL.

[0026] In some embodiments, the drying is freeze-drying, and concentration may be performed before drying.

[0027] Figure 1 The process flow chart of preparing the bamboo cane polysaccharide compound of the present invention according to one embodiment of the present invention is shown. However, the present invention is not limited thereto.

[0028] In one embodiment, the method for preparing the bamboo cane polysaccharide compound comprises:

[0029] 1) dissolving bamboo cane brown sugar (e.g., Songxi "Centennial Sugarcane") with ultrapure water to obtain an aqueous solution of bamboo cane brown sugar with a concentration of 100-200 mg / mL;

[0030] 2) The aqueous solution of bamboo cane brown sugar is centrifuged or filtered to remove insoluble matter to obtain a clear solution;

[0031] 3) treating the clear liquid obtained in step 2) with an ultrafiltration membrane with a molecular weight cutoff of 1 kDa to obtain a retentate;

[0032] 4) Concentrating and freeze-drying the retentate obtained in step 3) to obtain bamboo cane polysaccharide compounds. According to the test, the molecular weight of the bamboo cane polysaccharide compounds ranges from 1 to 400 kDa and 400 to 5000 kDa.

[0033] Preferably, during the dissolution in step 1), magnetic stirring or 60° C. water can be used to promote the dissolution;

[0034] Preferably, in step 2), a ceramic membrane is used for filtration treatment;

[0035] Preferably, the ultrafiltration membrane in step 3) is a rolled ultrafiltration membrane, and the pressure of the ultrafiltration membrane treatment is 1-2 kg / cm 3 ;

[0036] Preferably, the bamboo cane polysaccharide compound in step 4) is analyzed by gel permeation chromatography (GPC) to have a molecular weight range of 1-200 kDa and 500-4000 kDa, more preferably a molecular weight range of 1.5-100 kDa and 500-3000 kDa, more preferably a molecular weight range of 1.5-70 kDa and 670-3000 kDa, and more preferably a molecular weight range of 1.5-20 kDa and 670-3000 kDa.

[0037] After ultrafiltration membrane treatment, GPC testing can be performed to determine the effect of removing small molecule impurities.

[0038] In another embodiment, the preparation method of the bamboo cane polysaccharide compound comprises:

[0039] a) dissolving bamboo cane brown sugar (e.g., Songxi "Centennial Sugarcane") with ultrapure water to form an aqueous solution of bamboo cane brown sugar with a concentration of 100-200 mg / mL;

[0040] b) the aqueous solution of bamboo cane brown sugar is centrifuged or filtered to remove insoluble matter to obtain a clear solution;

[0041] c) dialyzing the clear solution obtained in step b) through a dialysis bag with a molecular weight cutoff of 1 kDa to obtain a retentate;

[0042] d) freeze-drying the retentate obtained in step c) to obtain bamboo cane polysaccharide compounds.

[0043] According to the test, the molecular weight of the bamboo cane polysaccharide compound is in the range of 1-400 kDa, for example.

[0044] Preferably, in step c), the dialyzation is performed in flowing water for 2-5 days.

[0045] Preferably, the retentate obtained in step c) is further subjected to graded alcohol precipitation and freeze-dried to obtain bamboo cane polysaccharide compounds.

[0046] Preferably, the final ethanol concentrations of the graded alcohol precipitation are 40%, 60% and 80% respectively.

[0047] It is divided into three components by graded alcohol precipitation method: a) 40% alcohol precipitated polysaccharide (the molecular weight range is, for example, 1-400 kDa), b) 60% alcohol precipitated polysaccharide (the molecular weight range is, for example, 1-400 kDa), c) 80% alcohol precipitated polysaccharide (the molecular weight range is, for example, 1-20 kDa). However, the present invention is not limited thereto.

[0048] In another aspect, the present invention provides the use of the bamboo cane polysaccharide compound in preparing a medicine for preventing and / or treating diabetes or a health food for assisting in lowering blood sugar.

[0049] The bamboo cane polysaccharide compounds exert a hypoglycemic effect by upregulating sugar metabolism pathways related to glycolysis and gluconeogenesis and insulin secretion signal pathways (such as glucagon-like peptide-1).

[0050] Drugs for preventing and / or treating diabetes can be prepared into various pharmaceutical preparations, and health foods that assist in lowering blood sugar can be made into various industrial foods and health products, which can include various oral forms, including but not limited to pills, beverages, etc.

[0051] Beneficial effects:

[0052] The bamboo cane polysaccharide compound of the present invention is obtained by graded separation and purification from Songxi "Centennial Sugarcane" brown sugar aqueous solution or bamboo cane stalk juice using a simple and effective polysaccharide extraction process and purification method. The experimental results of db / db diabetic mice show that the bamboo cane polysaccharide compounds prepared by different perennial root years and different processes of the present invention have the ability to improve the glucose tolerance of diabetic mice, can upregulate the sugar metabolism pathway related to glycolysis and gluconeogenesis and the insulin secretion signal pathway; long-term administration can effectively improve the sugar metabolism disorder of db / db mice, alleviate the urine protein excretion of db / db mice, have a certain recovery effect on kidney damage, and can improve the abnormal blood lipid metabolism of db / db mice. The experimental results of obese C57 mice induced by high-fat diet show that the bamboo cane polysaccharide compound of the present invention can effectively control the weight and weight gain of mice without affecting appetite; can improve the sugar metabolism disorder of mice, and promote the energy metabolism of mice. Therefore, the bamboo cane polysaccharide compound of the present invention has the potential to prevent and / or treat diabetes, and is expected to be developed into a health product or medicine for diabetes.

[0053] The present invention has been described in detail above, but the above embodiments are only illustrative in nature and are not intended to limit the present invention. In addition, this article is not limited by any theory described in the above prior art or invention content or the following examples. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 It is a process flow chart for preparing bamboo cane polysaccharide compounds.

[0055] Figure 2 These are the GPC spectra of bamboo cane brown sugar raw materials and bamboo cane polysaccharide compounds prepared therefrom. A is the GPC spectrum of bamboo cane brown sugar; B is the GPC spectrum of bamboo cane polysaccharide compounds prepared by ultrafiltration; C is the GPC spectrum of bamboo cane polysaccharide compounds prepared by dialysis; and D is the GPC spectrum of bamboo cane polysaccharide compounds prepared by alcohol precipitation.

[0056] Figure 3 The results of the oral glucose tolerance test after the bamboo cane polysaccharide compound prepared by the ultrafiltration method of the present invention was administered to db / db mice. A is the effect of the bamboo cane ultrafiltration polysaccharide compound on the fasting blood glucose of mice; B is the effect of the bamboo cane ultrafiltration polysaccharide compound on the blood glucose change curve within 2 hours of oral glucose administration of mice; C is the effect of the bamboo cane ultrafiltration polysaccharide compound on the glucose tolerance of mice. (*P<0.05, compared with the model group)

[0057] Figure 4 The results of the oral glucose tolerance test after the bamboo cane polysaccharide compound prepared by the dialysis method of the present invention was administered to db / db mice. A is the effect of the bamboo cane dialysis polysaccharide compound on the fasting blood glucose of mice; B is the effect of the bamboo cane dialysis polysaccharide compound on the blood glucose change curve within 2 hours of oral glucose administration of mice; C is the effect of the bamboo cane dialysis polysaccharide compound on the glucose tolerance of mice. (*P<0.05, **P<0.01, compared with the model group)

[0058] Figure 5 The results of the oral glucose tolerance test after the bamboo cane polysaccharide compound prepared by the alcohol precipitation method of the present invention was administered to db / db mice. A is the effect of the bamboo cane alcohol precipitation polysaccharide compound on the fasting blood glucose of mice; B is the effect of the bamboo cane alcohol precipitation polysaccharide compound on the blood glucose change curve within 2 hours of oral glucose administration of mice; C is the effect of the bamboo cane alcohol precipitation polysaccharide compound on the glucose tolerance of mice. (*P<0.05, **P<0.01, ***P<0.001, compared with the model group)

[0059] Figure 6 The results of the glucose tolerance test after intraperitoneal injection of the bamboo cane polysaccharide compound prepared by the dialysis method of the present invention to db / db mice. A is the effect of the bamboo cane dialysis polysaccharide compound on the fasting blood glucose of mice; B is the effect of the bamboo cane dialysis polysaccharide compound on the blood glucose change curve within 2 hours after intraperitoneal injection of glucose in mice; C is the effect of the bamboo cane dialysis polysaccharide compound on the glucose tolerance of mice. (*P<0.05, **P<0.01, compared with the model group)

[0060] Figure 7 The effect of long-term administration of the bamboo cane polysaccharide compound prepared by the dialysis method of the present invention on the glucose metabolism of db / db mice. A is the random blood sugar level test after long-term administration; B is the effect of long-term administration of bamboo cane dialysis polysaccharide compound on the fasting blood sugar of mice; C is the effect of long-term administration of bamboo cane dialysis polysaccharide compound on the blood sugar change curve within 2 hours of oral glucose administration of mice; D is the effect of long-term administration of bamboo cane dialysis polysaccharide compound on glucose tolerance of mice. (*P<0.05, **P<0.01, compared with the model group)

[0061] Figure 8This is the effect of long-term administration of the bamboo cane polysaccharide compound prepared by the dialysis method of the present invention on the recovery of renal function damage in db / db mice. (*P<0.05, **P<0.01, compared with the model group)

[0062] Fig. 9 This is the effect of long-term administration of the bamboo cane polysaccharide compound prepared by dialysis method on blood lipid metabolism of db / db mice. (*P<0.05, compared with the model group)

[0063] Fig.10 The effect of long-term administration of the bamboo cane polysaccharide compound prepared by ultrafiltration method of the present invention on the body weight, body weight growth rate and food intake of DIO mice. (*P<0.05, **P<0.01, ***P<0.001, compared with the model group)

[0064] Fig.11 This is the blood sugar response of DIO mice after first administration of bamboo cane ultrafiltered polysaccharide compounds. (*P<0.05, **P<0.01, compared with the model group)

[0065] Fig.12 The effects of long-term administration of bamboo cane polysaccharide compounds prepared by the ultrafiltration method of the present invention on glucose metabolism in DIO mice. A is the random blood sugar level test after long-term administration of bamboo cane ultrafiltered polysaccharide compounds; B is the effect of long-term administration of bamboo cane ultrafiltered polysaccharide compounds on fasting blood sugar in mice; C is the effect of long-term administration of bamboo cane ultrafiltered polysaccharide compounds on the blood sugar change curve within 2 hours of oral glucose administration in mice; D is the effect of long-term administration of bamboo cane ultrafiltered polysaccharide compounds on glucose tolerance in mice; E is the effect of long-term administration of bamboo cane ultrafiltered polysaccharide compounds on the blood sugar change curve within 2 hours of intraperitoneal injection of insulin in mice; F is the effect of long-term administration of bamboo cane ultrafiltered polysaccharide compounds on insulin tolerance in mice; G is the effect of long-term administration of bamboo cane ultrafiltered polysaccharide compounds on the blood sugar change curve within 2 hours of intraperitoneal injection of sodium pyruvate in mice; H is the effect of long-term administration of bamboo cane ultrafiltered polysaccharide compounds on pyruvate tolerance in mice. (*P<0.05, **P<0.01, ***P<0.001, compared with the model group)

[0066] Fig.13 This is the effect of long-term administration of the bamboo cane polysaccharide compound prepared by ultrafiltration method in the present invention on the energy metabolism of DIO mice. (**P<0.01, compared with the model group)

[0067] Fig.14 The invention relates to the influence of the bamboo cane polysaccharide compounds prepared by the dialysis method on the intracellular sugar metabolism pathway.

[0068] Fig.15 This is the effect of the bamboo cane polysaccharide compound prepared by the dialysis method of the present invention on the secretion of GLP-1 in mouse intestinal organoids. (***P<0.001, compared with the model group) DETAILED DESCRIPTION

[0069] The present invention is further described below in conjunction with examples. It should be noted that the following examples are provided for illustrative purposes only and do not constitute a limitation on the scope of protection claimed for the present invention.

[0070] Unless otherwise specified, the raw materials, reagents, methods, etc. used in the examples are conventional raw materials, reagents, methods in the art.

[0071] Example

[0072] The reagents and equipment used include:

[0073] Experimental materials: Bamboo sugarcane brown sugar from different perennial root growth years (2 years, 10 years, 30 years, 291 years) all came from Wantong Centennial Sugarcane and Vegetable Professional Cooperative in Songxi County.

[0074] Experimental reagents: Sodium nitrate, sodium chloride, anhydrous ethanol, etc. are all domestic reagents of analytical grade.

[0075] Experimental equipment: Classic Di ultrapure water machine (ELGA); multifunctional spiral membrane pilot test equipment; multifunctional spiral membrane pilot equipment; Spectra Max M4 multifunctional plate reader (Molecular Devices); Waters high performance liquid phase system (Waters); SECURA225D-1CN electronic analytical balance (Sartorius); GL-21M high-speed centrifuge (Xiangyi Company); high-speed refrigerated centrifuge MultifugeX3R (Thermo Fisher Scientific); rotary evaporator N-1100 (Shanghai Ailang Instrument Co., Ltd.); vacuum refrigerated centrifugal concentrator (Labconco, USA); low-temperature freeze dryer FD-5 (Beijing Boyikang Experimental Instrument Co., Ltd.).

[0076] Example 1: Preparation of bamboo cane polysaccharide compounds by ultrafiltration

[0077] 1) Dissolution: Weigh 100g of brown sugar from bamboo cane, add ultrapure water to 1L, and stir with magnetic force for about 30 minutes.

[0078] 2) Centrifugation: Remove insoluble matter by centrifugation and take the supernatant. The centrifugation conditions are 9000rpm, 20℃, 10min. The supernatant is subjected to GPC analysis to compare the effect of removing small molecule impurities ( Figure 2 (A).

[0079] 3) Filtration: First, filter the centrifugal supernatant obtained in step 2) through a 0.22 μm filter membrane; then filter the filtrate through a 1 kDa cutoff spiral membrane filter with a pressure controlled at 1 kg / cm 3After replacing about 5L of liquid for ultrapurification, the retentate and filtrate were collected, and the two components were tested by GPC to determine the effect of removing small molecule impurities ( Figure 2 (B).

[0080] 4) Concentrate 1 L of the retentate obtained in step 3) to 50 mL at 55° C., freeze-dry, and obtain bamboo cane polysaccharide compounds, and calculate the yield; concentrate 5 L of the filtrate to 120 mL at 55° C., freeze-dry, and obtain a small molecule component with sucrose as the main component.

[0081] Example 2: Pilot-scale preparation of bamboo cane polysaccharide compounds by ultrafiltration

[0082] 1) Dissolution: weigh 10-20 kg of brown sugar from bamboo cane, add ultrapure water to 100-200 kg.

[0083] 2) Filtration: 120 mesh sieve filtration, the solution was filtered through a ceramic membrane (200 nm pore size).

[0084] 3) Spiral membrane filtration: The filtrate from step 2) was treated with a 1 kDa spiral membrane and eluted with 4-5 times the volume of ultrapure water (about 1 t), and the pressure was controlled at 2 kg / cm 3 Within 1000 ℃, collect the filtrate and retentate, and perform GPC test on the two components to determine the effect of removing small molecule impurities ( Figure 2 (B).

[0085] 4) Concentrating 250 kg of the retentate obtained in step 3) to 2.5 L, freeze-drying, obtaining bamboo cane polysaccharide compounds, and calculating the yield; concentrating 1 t of the filtrate to 100 kg, spray-drying, obtaining a small molecule component with sucrose as the main component.

[0086] Example 3: Preparation of bamboo cane polysaccharide compounds by dialysis

[0087] Weigh 100g of bamboo cane brown sugar and dissolve it in ultrapure water to a concentration of 100-200mg / mL. Use a dialysis bag with a molecular weight cutoff of 1kDa to dialyze in flowing water for 2-5 days, and use the phenol-sulfuric acid method to detect the dialysis effect. The purpose of dialysis is mainly to remove small molecular oligosaccharides with a molecular weight of less than 1kDa and other substances that affect the activity. After the dialysis is completed, the retentate sample is freeze-dried and its yield is calculated. GPC detection is used to determine the effect of removing small molecule impurities ( Figure 2 Middle C).

[0088] Example 4: Preparation of bamboo cane polysaccharide compounds by alcohol precipitation

[0089] The retentate obtained by dialysis in Example 3 was added with anhydrous ethanol for graded alcohol precipitation, with the final ethanol concentrations being 40%, 60% and 80% respectively. Each alcohol precipitation component was centrifuged at 4700rpm for 20min. Each precipitated component was first dissolved in water, then centrifuged to remove insoluble matter, and then freeze-dried. GPC analysis was performed to determine the effect of removing small molecule impurities ( Figure 2 (middle D).

[0090] Example 5: Study on the therapeutic effect of bamboo cane polysaccharide compounds on type II diabetes model db / db mice

[0091] The following animal experiments were carried out using the bamboo cane polysaccharide compounds of Example 1, Example 2, Example 3 and Example 4.

[0092] 1. Experiment and Methods

[0093] 1.1 Effect of single administration of bamboo cane polysaccharides on glucose tolerance in db / db mice

[0094] 1.1.1 Animal grouping

[0095] SPF male 6-week-old db / db mice were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd. (Batch No.: SCXK(Su)2018-0008). After one week of adaptive feeding, random blood glucose was measured, and the model animals were randomly divided into a model control group and an experimental group after the unsuccessful modeling animals were eliminated for the in vivo hypoglycemic activity investigation of bamboo cane polysaccharide compounds obtained from different perennial root growth years and different preparation methods.

[0096] 1.1.2 Oral glucose tolerance test after single administration of bamboo cane polysaccharide compounds

[0097] The mice in the experimental group were gavaged with ultrafiltered bamboo cane polysaccharide, dialyzed bamboo cane polysaccharide, and alcohol-precipitated bamboo cane polysaccharide (all prepared with sterile water as solvent), and the mice in the control group were gavaged with an equal volume of blank solvent, followed by an oral glucose tolerance test (OGTT): fasting for 6 hours, and fasting blood glucose was measured; each group of mice was gavaged with 0.8 g / kg glucose (dosing volume was 5 mL / kg), and 0, 15, 30, 60, 90 and 120 min were set as glucose loading time points, and the blood glucose concentration in the blood samples was measured by blood glucose test strips, the blood glucose data were statistically analyzed, the glucose tolerance curve was drawn, and the area under the curve (Area under the curve, AUC) of the blood glucose curve of each group was calculated.

[0098] The dosing schedule is as follows:

[0099] (1) Model control group: Each mouse was gavaged with an equal amount of sterile water, with a dosing volume of 10 mL / kg;

[0100] (2) Low-dose group of 2-year-old bamboo cane ultrafiltered polysaccharide: Each mouse was intragastrically administered with 25 mg / kg of 2-year-old bamboo cane ultrafiltered polysaccharide (E2JL-25 mg / kg), with a dosing volume of 10 mL / kg;

[0101] (3) Medium-dose group of 2-year-old bamboo cane ultrafiltered polysaccharide: Each mouse was intragastrically administered with 50 mg / kg of 2-year-old bamboo cane ultrafiltered polysaccharide (E2JL-50 mg / kg), with a dosing volume of 10 mL / kg;

[0102] (4) High-dose group of 2-year-old bamboo cane ultrafiltered polysaccharide: Each mouse was intragastrically administered with 100 mg / kg of 2-year-old bamboo cane ultrafiltered polysaccharide (E2JL-100 mg / kg), with a dosing volume of 10 mL / kg;

[0103] (5) Low-dose group of 30-year-old bamboo cane ultrafiltered polysaccharide: Each mouse was intragastrically administered with 25 mg / kg of 30-year-old bamboo cane ultrafiltered polysaccharide (E30JL-25 mg / kg), with a dosing volume of 10 mL / kg;

[0104] (6) 30-year-old bamboo cane ultrafiltered polysaccharide medium-dose group: Each mouse was intragastrically administered with 50 mg / kg of 30-year-old bamboo cane ultrafiltered polysaccharide (E30JL-50 mg / kg), with a dosing volume of 10 mL / kg;

[0105] (7) High-dose group of 30-year-old bamboo cane ultrafiltered polysaccharide: Each mouse was intragastrically administered with 100 mg / kg of 30-year-old bamboo cane ultrafiltered polysaccharide (E30JL-100 mg / kg), with a dosing volume of 10 mL / kg;

[0106] (8) 10-year-old bamboo cane dialysis polysaccharide group: Each mouse was intragastrically administered with 100 mg / kg of 10-year-old bamboo cane dialysis polysaccharide (E10-100 mg / kg), with a dosing volume of 10 mL / kg;

[0107] (9) 30-year-old bamboo cane dialyzed polysaccharide group: Each mouse was intragastrically administered with 100 mg / kg of 30-year-old bamboo cane dialyzed polysaccharide (E30-100 mg / kg), with a dosing volume of 10 mL / kg;

[0108] (10) 291-year-old bamboo cane dialyzed polysaccharide group: Each mouse was intragastrically administered with 100 mg / kg of 291-year-old bamboo cane dialyzed polysaccharide (E291-100 mg / kg), with a dosing volume of 10 mL / kg;

[0109] (11) Bamboo cane 40% alcohol precipitation polysaccharide group (10-year-old bamboo cane root dialysis polysaccharide further alcohol precipitation): each mouse was gavaged with 100 mg / kg bamboo cane 40% alcohol precipitation polysaccharide (E40% alcohol precipitation-100 mg / kg), and the administration volume was 10 mL / kg;

[0110] (12) Bamboo cane 60% alcohol-precipitated polysaccharide group (40% alcohol-precipitated polysaccharide was further precipitated with alcohol): Each mouse was intragastrically administered with 100

[0111] mg / kg bamboo cane 60% alcohol precipitation polysaccharide (E60% alcohol precipitation-100mg / kg), the administration volume is 10

[0112] mL / kg;

[0113] (13) Bamboo cane 80% alcohol-precipitated polysaccharide group (60% alcohol-precipitated polysaccharide was further precipitated with alcohol): Each mouse was gavaged with 100

[0114] mg / kg bamboo cane 80% alcohol precipitation polysaccharide (E80% alcohol precipitation-100mg / kg), the administration volume is 10

[0115] mL / kg.

[0116] 1.1.3 Glucose tolerance test after single intraperitoneal administration of bamboo cane polysaccharide compounds

[0117] The mice in the experimental group were gavaged with bamboo sugarcane dialyzed polysaccharide compounds (all prepared with sterile water as solvent), and the mice in the control group were gavaged with the same volume of blank solvent, followed by intraperitoneal glucose tolerance test (IPGTT): fasting for 6 hours, and fasting blood glucose was measured; each group of mice was intraperitoneally injected with 0.8g / kg glucose (dosing volume was 5mL / kg), and 0, 15, 30, 60, 90 and 120min were set as glucose loading time points, and the blood glucose concentration in the blood samples was measured by blood glucose test strips, the blood glucose data were statistically analyzed, the glucose tolerance curve was drawn, and the AUC of the blood glucose curve of each group was calculated.

[0118] The dosing schedule is as follows:

[0119] (1) Model control group: Each mouse was gavaged with an equal amount of sterile water, with a dosing volume of 10 mL / kg;

[0120] (2) 10-year-old bamboo cane dialysis polysaccharide group: Each mouse was intragastrically administered with 100 mg / kg of 10-year-old bamboo cane dialysis polysaccharide (E10-100 mg / kg), with a dosing volume of 10 mL / kg;

[0121] (3) 30-year-old bamboo cane dialyzed polysaccharide group: Each mouse was intragastrically administered with 100 mg / kg of 30-year-old bamboo cane dialyzed polysaccharide (E30-100 mg / kg), with a dosing volume of 10 mL / kg;

[0122] (4) 291-year-old bamboo cane dialyzed polysaccharide group: Each mouse was intragastrically administered with 100 mg / kg of 291-year-old bamboo cane dialyzed polysaccharide (E291-100 mg / kg), with a dosing volume of 10 mL / kg.

[0123] 1.2 Long-term efficacy evaluation of bamboo cane polysaccharide compounds

[0124] 1.2.1 Animal grouping and drug administration

[0125] SPF male 5-6 week old db / db mice were purchased from Jiangsu Huachuang Xinnuo Pharmaceutical Technology Co., Ltd. (Batch No.: SCXK(Su)2020-0009). After 10 weeks of feeding, random blood glucose was measured, and the model animals were randomly divided into a model control group, a 10-year-old bamboo cane dialysis polysaccharide group (E10), and a 291-year-old bamboo cane dialysis polysaccharide group (E291) after the animals failed to establish the model. The mice were given intragastric administration every day for four weeks (the polysaccharides were prepared with sterile water as the solvent), and the model group was given an equal volume of blank solvent to evaluate the in vivo hypoglycemic efficacy of 10-year-old and 291-year-old bamboo cane dialysis polysaccharides.

[0126] The dosing schedule is as follows:

[0127] (1) Model control group: Each mouse was gavaged with an equal amount of sterile water, with a volume of 10 mL / kg, once a day;

[0128] (2) 10-year-old bamboo cane dialysis polysaccharide group: Each mouse was intragastrically administered with 100 mg / kg of 10-year-old bamboo cane dialysis polysaccharide (E10), with a dosing volume of 10 mL / kg, once a day;

[0129] (3) 291-year-old bamboo cane dialyzed polysaccharide group: Each mouse was intragastrically administered with 100 mg / kg of 291-year-old bamboo cane dialyzed polysaccharide (E291), with a dosing volume of 10 mL / kg, once a day.

[0130] 1.2.2 Random blood glucose test in mice

[0131] Blood was collected from the tail vein and the blood glucose concentration in the blood sample was measured using a blood glucose test strip.

[0132] 1.2.3 Oral glucose tolerance test

[0133] Same as 1.1.2 in Example 5.

[0134] 1.2.4 Sample collection and processing

[0135] The urine of mice was collected for 24 hours, centrifuged at 4°C, 2500 rpm, for 10 minutes, and the supernatant was taken for urine biochemical detection. After the experiment reached the end point, the mice were fasted for 6 hours, and the mice were weighed and blood was collected according to the fasting order.

[0136] 1.2.5 Detection of blood lipid biochemical indicators

[0137] The triglyceride (TG) level in serum samples was measured using a kit (Nanjing Jiancheng). The operating steps were as follows: 2 μL of serum and corresponding standard were added to the ELISA plate, distilled water was used as a blank control, and then 200 μL of working solution was added, mixed, incubated at 37°C for 10 min, and the absorbance of each well was read at a wavelength of 510 nm using an ELISA reader.

[0138] 1.2.6 Serum creatinine test

[0139] A kit (Nanjing Jiancheng) was used to measure the creatinine (CRE) level in serum samples. The operating steps were as follows: 6 μL of serum and corresponding standard were added to the ELISA plate, distilled water was used as a blank control, 180 μL of working solution 1 was added, mixed, incubated at 37°C for 5 min, and the absorbance value A1 of each well was measured at a wavelength of 546 nm using an ELISA reader; then 60 μL of working solution 2 was added, mixed, incubated at 37°C for 5 min, and the absorbance value A2 of each well was measured at a wavelength of 546 nm using an ELISA reader.

[0140] 1.3 Data Analysis

[0141] The statistical graphs in this study were generated using GraphPad Prism software, and the quantitative data were expressed as Mean ± SEM. The two-tailed T test (Student's ttest) was used to compare the significant differences among the groups, and p < 0.05 was considered to be statistically significant.

[0142] 2. Experimental results

[0143] 2.1 Single administration of bamboo cane polysaccharides can effectively improve oral glucose tolerance in db / db mice

[0144] The results of OGTT after a single administration of bamboo cane dialyzed polysaccharide compounds showed that the bamboo cane ultrafiltered polysaccharide E2JL at a dose of 100 mg / kg could significantly reduce the fasting blood glucose of mice, and at a dose of 50 mg / kg could significantly improve the glucose tolerance of mice; the bamboo cane ultrafiltered polysaccharide E30JL at a dose of 25 mg / kg and 100 mg / kg could significantly reduce the fasting blood glucose of mice ( Figure 3 ). Bamboo cane dialysis polysaccharide E10 at a dose of 100 mg / kg can significantly reduce fasting blood glucose and improve glucose tolerance in mice; bamboo cane dialysis polysaccharide E30 at a dose of 100 mg / kg can significantly reduce fasting blood glucose in mice; bamboo cane dialysis polysaccharide E291 at a dose of 100 mg / kg can significantly reduce fasting blood glucose and improve glucose tolerance in mice ( Figure 4). 40% alcohol-precipitated polysaccharide from bamboo cane can significantly reduce fasting blood sugar and improve glucose tolerance in mice at a dose of 100 mg / kg; 60% alcohol-precipitated polysaccharide from bamboo cane can significantly improve glucose tolerance in mice at a dose of 100 mg / kg; 80% alcohol-precipitated polysaccharide from bamboo cane can significantly improve glucose tolerance in mice at a dose of 100 mg / kg ( Figure 5 ).

[0145] The above results indicate that bamboo cane polysaccharide compounds prepared from different perennial root years and different processes have a good ability to improve glucose tolerance in diabetic mice after a single administration.

[0146] 2.2 Single administration of bamboo cane polysaccharides can effectively improve intraperitoneal glucose tolerance in db / db mice

[0147] The results of IPGTT after a single administration of bamboo cane polysaccharides showed that compared with OGTT, bamboo cane dialysis polysaccharide E10 at a dose of 100 mg / kg could still significantly improve the glucose tolerance of mice; bamboo cane dialysis polysaccharide E30 at a dose of 100 mg / kg could still significantly improve the glucose tolerance of mice; bamboo cane dialysis polysaccharide E291 at a dose of 100 mg / kg could still significantly improve the glucose tolerance of mice ( Figure 6 ).

[0148] The above results indicate that the targets of bamboo cane polysaccharide compounds for hypoglycemic effect are distributed in the body.

[0149] 2.3 Long-term administration of bamboo shoot dialysis polysaccharides can effectively improve glucose metabolism disorders in db / db mice

[0150] Random blood glucose results showed that after one week of administration, bamboo cane dialysis polysaccharides E10 and E291 could significantly reduce the random blood glucose levels of mice. OGTT results showed that bamboo cane dialysis polysaccharides E10 and E291 could significantly reduce the fasting blood glucose of mice and significantly improve glucose tolerance ( Figure 7 ).

[0151] The above results indicate that long-term administration of bamboo shoot dialysis polysaccharide compounds can effectively improve the glucose metabolism disorder in db / db mice.

[0152] 2.4 Long-term administration of bamboo cane dialysis polysaccharides can effectively improve diabetic complications in db / db mice 2.4.1 Long-term administration of bamboo cane dialysis polysaccharides can effectively improve kidney damage in db / db mice

[0153] The results of urine biochemical tests showed that compared with the model group, bamboo cane dialysis polysaccharides E10 and E291 could significantly reduce the 24h urine protein excretion level of mice; bamboo cane dialysis polysaccharides E291 could significantly reduce the ratio of microalbumin to creatinine in mice; bamboo cane dialysis polysaccharides E10 and E291 could significantly reduce the CRE level of mice ( Figure 8 ).

[0154] The above experimental results suggest that bamboo cane dialyzed polysaccharide compounds can alleviate the urinary protein excretion of db / db mice and have a certain recovery effect on kidney damage.

[0155] 2.4.2 Long-term administration of bamboo cane dialysis polysaccharides can effectively improve dyslipidemia in db / db mice

[0156] The results of blood lipid index detection showed that bamboo sugarcane dialysis polysaccharides E10 and E291 could significantly reduce the serum TG level of mice ( Fig. 9 ).

[0157] The above experimental results suggest that bamboo cane dialyzed polysaccharide compounds can improve the abnormal lipid metabolism of db / db mice.

[0158] Example 6: Study on the metabolic improvement effect of bamboo cane polysaccharides on diet-induced obesity (DIO) C57 mice

[0159] 1. Experiment and Methods

[0160] 1.1 Animal grouping and drug administration

[0161] SPF male 8-10 week old C57BL / 6J mice were purchased from Jiangsu Huachuang Xinnuo Pharmaceutical Technology Co., Ltd. (Batch No.: SCXK (Su) 2020-0009). After one week of adaptive feeding, the mice were weighed and randomly divided into blank control group, model control group, 2-year-old bamboo cane ultrafiltered polysaccharide low-dose administration group (E2JL-L), 2-year-old bamboo cane ultrafiltered polysaccharide high-dose administration group (E2JL-H), 30-year-old bamboo cane ultrafiltered polysaccharide low-dose administration group (E30JL-L) and 30-year-old bamboo cane ultrafiltered polysaccharide high-dose administration group (E30JL-H). The blank control group was given a low-fat diet LFD (10% Kcal, purchased from Research diet, D12450J), and the model control group and the treatment group were given a high-fat diet HFD (60% Kcal, purchased from Research diet, D12492). The drug was administered continuously for 8 weeks (all polysaccharides were prepared with sterile water as the solvent) for the purpose of prevention to investigate whether the two bamboo cane ultrafiltered polysaccharides, E2JL and E30JL, could improve a series of obesity-related metabolic disorder phenotypes induced by a high-fat diet.

[0162] The dosing schedule is as follows:

[0163] (1) Blank control group: Each mouse was gavaged with an equal amount of sterile water, with a dosing volume of 10 mL / kg, once a day;

[0164] (2) Model control group: Each mouse was gavaged with an equal amount of sterile water, with a volume of 10 mL / kg, once a day;

[0165] (3) Low-dose 2-year-old bamboo cane ultrafiltered polysaccharide group: Each mouse was intragastrically administered with 100 mg / kg of 2-year-old bamboo cane ultrafiltered polysaccharide (E2JL-L), with a dosing volume of 10 mL / kg, once a day;

[0166] (4) 2-year-old bamboo cane ultrafiltered polysaccharide high-dose administration group: each mouse was gavaged with 500 mg / kg of 2-year-old bamboo cane ultrafiltered polysaccharide (E2JL-H), the administration volume was 10 mL / kg, and the administration was once a day; (5) 30-year-old bamboo cane ultrafiltered polysaccharide low-dose administration group: each mouse was gavaged with 100 mg / kg of 30-year-old bamboo cane ultrafiltered polysaccharide (E30JL-L), the administration volume was 10 mL / kg, and the administration was once a day;

[0167] (6) High-dose 30-year-old bamboo cane ultrafiltered polysaccharide group: Each mouse was intragastrically administered with 500 mg / kg of 30-year-old bamboo cane ultrafiltered polysaccharide (E30JL-H), with a dosing volume of 10 mL / kg, once a day.

[0168] 1.2 Monitoring of mouse weight and food intake

[0169] During the administration period, the body weight of mice in each group was weighed and recorded at a fixed time every week. The weight of mouse feed was measured at the same time point for 4 consecutive days every week to calculate the daily food intake of each mouse.

[0170] 1.3 Sugar metabolism capacity test

[0171] (1) Blood glucose response after the first dose

[0172] Blood was collected from the tail vein 2h and 24h after the first administration, and the blood glucose concentration in the blood samples was measured using blood glucose test strips.

[0173] (2) Random blood glucose test

[0174] Same as 1.2.2 in Example 5.

[0175] (3) Oral glucose tolerance test

[0176] Same as 1.1.2 in Example 5.

[0177] (4) Insulin sensitivity test

[0178] Intraperitoneal insulin tolerance test (IPITT) was performed on mice. The test was performed after fasting for 4 hours, and 0.75 IU / kg insulin (dosage volume was 5 mL / kg) was given by intraperitoneal injection. 0, 15, 30, 60, 90 and 120 min were set as insulin loading time points. The blood glucose concentration in the blood sample was measured by blood glucose test strips, the blood glucose data was statistically analyzed, the blood glucose curve was drawn, and the AUC of each group of blood glucose curves was calculated.

[0179] (5) Pyruvate tolerance test

[0180] The mice were subjected to an intraperitoneal pyruvate tolerance test (IPPTT). The test was conducted after an overnight fast of 16 hours, and 2 g / kg sodium pyruvate (dosing volume was 5 mL / kg) was intraperitoneally injected. 0, 15, 30, 60, 90 and 120 min were set as pyruvate loading time points. The blood glucose concentration in the blood sample was measured by blood glucose test strips, the blood glucose data was statistically analyzed, the blood glucose curve was drawn, and the AUC of the blood glucose curve of each group was calculated.

[0181] 1.4 Energy metabolism detection

[0182] The rectal temperature of the mice was measured 4-5 cm away from the anal sphincter using a small animal temperature maintenance instrument.

[0183] 1.5 Data Analysis

[0184] Same as 1.3 in Example 5.

[0185] 2. Experimental results

[0186] 2.1 Bamboo cane ultrafiltration polysaccharides can effectively control the body weight of DIO mice

[0187] During the 8-week continuous administration, the body weight and weight gain of the mice in the bamboo cane ultrafiltered polysaccharide group were effectively controlled, and the appetite was not affected ( Fig.10 ).

[0188] 2.2 Bamboo cane ultrafiltration polysaccharide compounds can effectively improve glucose metabolism disorders in DIO mice

[0189] The blood glucose levels of mice in each group were measured 2 hours after the first administration. The results showed that a single administration of bamboo cane ultrafiltered polysaccharide had the effect of lowering blood glucose. The blood glucose level test results 24 hours after the first administration showed that the blood glucose levels of mice in the administration group were still significantly reduced, indicating that the blood glucose-lowering effect of bamboo cane ultrafiltered polysaccharide is sustained ( Fig.11 ).

[0190] Random blood glucose results showed that the bamboo cane ultrafiltered polysaccharide E2JL could significantly reduce the random blood glucose level of mice at a dose of 500mg / kg; the bamboo cane ultrafiltered polysaccharide E30JL could significantly reduce the random blood glucose level of mice at a dose of 100mg / kg and 500mg / kg. The fasting blood glucose results showed that E2JL could significantly reduce the fasting blood glucose of mice at a dose of 100mg / kg and 500mg / kg; E30JL could significantly reduce the fasting blood glucose of mice at a dose of 100mg / kg and 500mg / kg. The OGTT results showed that E2JL could significantly reduce the fasting blood glucose and improve glucose tolerance of mice at a dose of 100mg / kg and 500mg / kg; E30JL could significantly reduce the fasting blood glucose and improve glucose tolerance of mice at a dose of 100mg / kg and 500mg / kg. IPITT results showed that E2JL at a dose of 100mg / kg and 500mg / kg could significantly improve insulin resistance and insulin sensitivity in mice; E30JL at a dose of 100mg / kg and 500mg / kg could significantly improve insulin resistance and insulin sensitivity in mice. IPPTT results showed that E2JL at a dose of 100mg / kg and 500mg / kg could significantly reduce the gluconeogenesis capacity of mice and improve the abnormal gluconeogenesis in mice; E30JL at a dose of 100mg / kg and 500mg / kg could significantly reduce the gluconeogenesis capacity of mice and improve the abnormal gluconeogenesis in mice ( Fig.12 ).

[0191] The above experimental results show that bamboo cane ultrafiltered polysaccharide compounds can effectively improve the glucose metabolism disorder of DIO mice.

[0192] 2.3 Ultrafiltered polysaccharides from bamboo cane can promote energy metabolism in DIO mice

[0193] The results of rectal temperature test showed that E2JL at a dose of 100 mg / kg could significantly increase the core rectal temperature of mice, indicating that bamboo cane ultrafiltered polysaccharides can promote the energy metabolism of DIO mice ( Fig.13 ).

[0194] Example 7: Effects of bamboo cane polysaccharides on intracellular transcriptome

[0195] The experiment was carried out using the bamboo cane polysaccharide compound of Example 3.

[0196] Human malignant melanoma A375 cells in the exponential growth phase were plated into 12-well plates. After the cells adhered to the wall, different concentrations of bamboo cane polysaccharide compounds were added. After culturing for 24 hours, the cells were collected by trypsin digestion, TRIzol reagent was added to lyse the cells, and the samples were quickly frozen in liquid nitrogen. The samples were sent to RNAseq technical service company for sample RNAseq sequencing analysis.

[0197] RNAseq data analysis: Download the human genome reference index file and human reference genome annotation information file from Hisat2 and UCSC official websites respectively; use Hisat2, Samtools, Feature Counts and other software to perform sequence alignment, format conversion, index construction and generate expression matrix files; use R software for sugar metabolism pathway enrichment analysis.

[0198] The results showed that bamboo cane polysaccharides can upregulate the glucose metabolism pathways related to glycolysis and gluconeogenesis as well as the insulin secretion signaling pathway ( Fig.14 ).

[0199] Example 8: Effects of bamboo cane polysaccharides on the secretion of glucagon-like peptide-1 (GLP-1) in mouse intestinal organoids

[0200] The experiment was carried out using the bamboo cane polysaccharide compound of Example 3.

[0201] 1. Experiment and Methods

[0202] Intestinal organoids from 6-8 week old C57 mice were isolated and a blank control group, a low-dose (1000 μg / mL) 10-year-old bamboo cane dialysis polysaccharide administration group, and a high-dose (5000 μg / mL) 10-year-old bamboo cane dialysis polysaccharide administration group were set up. After 2 days of culture, the drug was administered for 2 consecutive days. On the 5th day, after starvation with DMEM sugar-free medium for 30 minutes, 1% sitagliptin (DPP4 inhibitor) was added and administered to conduct a GLP-1 secretion experiment. After 1 hour, the samples were collected, centrifuged at 4°C, 3000 rpm, and the supernatant was taken. The mouse GLP-1 kit (Shanghai Tongwei) was used to detect the level of GLP-1 secreted by the organoids under different treatments, and the BCA protein quantification / concentration assay kit (Meilun Bio) was used to determine the protein concentration for calibration.

[0203] 2. Experimental results

[0204] The experimental results showed that 10-year-old bamboo root dialysis polysaccharide at a dose of 5000 μg / mL could significantly promote the level of GLP-1 secretion in organoids ( Fig.15 ).

[0205] The above results suggest that bamboo shoot dialyzate polysaccharide compounds may exert hypoglycemic effects through GLP-1.

[0206] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit them. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that the technical solutions described in the above embodiments may be modified, or some or all of the technical features thereof may be replaced by equivalents, without departing from the spirit and essence of the claims of the present invention; and these modifications or replacements are still within the scope defined by the claims of the present invention.

Claims

1. A bamboo cane polysaccharide compound obtained by separating bamboo cane juice and removing small molecular compounds with a molecular weight of less than 1 kDa, wherein the molecular weight of the bamboo cane polysaccharide compound ranges from 1 to 400 kDa and 400 to 5000 kDa. Preferably, the molecular weight of the bamboo cane polysaccharide compound is in the range of 1 to 200 kDa and 500 to 4000 kDa as analyzed by gel permeation chromatography (GPC), more preferably in the range of 1.5 to 100 kDa and 500 to 3000 kDa, more preferably in the range of 1.5 to 70 kDa and 670 to 3000 kDa, and even more preferably in the range of 1.5 to 20 kDa and 670 to 3000 kDa.

2. The bamboo cane polysaccharide compound according to claim 1, wherein The bamboo cane juice comes from an aqueous solution of bamboo cane brown sugar or bamboo cane stem juice. Preferably, the bamboo cane is produced in Guangdong, Guangxi, Yunnan, Sichuan, Songxi County, Fujian, etc., preferably Songxi County, Fujian.

3. The bamboo cane polysaccharide compound according to claim 1, wherein The separation treatment includes one or more treatment methods of ultrafiltration, dialysis and alcohol precipitation.

4. A method for preparing the bamboo cane polysaccharide compound according to any one of claims 1 to 3, the method comprising the following steps: The bamboo cane polysaccharide compound is obtained by separating and treating the aqueous solution of bamboo cane brown sugar or the bamboo cane stem juice to remove small molecular compounds with a molecular weight below 1 kDa, and then drying.

5. The preparation method according to claim 4, wherein: The method further comprises the step of centrifuging or filtering the aqueous solution of bamboo cane brown sugar or the bamboo cane stalk juice to remove insoluble matter before the separation treatment. Preferably, the drying is freeze-drying, and concentration is performed before drying.

6. The preparation method according to claim 4, wherein The preparation method of the bamboo cane polysaccharide compound comprises: 1) dissolving brown sugar from bamboo cane with ultrapure water to obtain an aqueous solution of brown sugar from bamboo cane with a concentration of 100-200 mg / mL; 2) The aqueous solution of bamboo cane brown sugar is centrifuged or filtered to remove insoluble matter to obtain a clear solution; 3) treating the clear liquid obtained in step 2) with an ultrafiltration membrane with a molecular weight cutoff of 1 kDa to obtain a retentate; 4) concentrating and freeze-drying the retentate obtained in step 3) to obtain bamboo cane polysaccharide compounds; Preferably, the ultrafiltration membrane in step 3) is a rolled ultrafiltration membrane, and the pressure of the ultrafiltration membrane treatment is 1-2 kg / cm 3 .

7. The preparation method according to claim 4, wherein The preparation method of the bamboo cane polysaccharide compound comprises: a) dissolving brown sugar from bamboo cane with ultrapure water to form an aqueous solution of brown sugar from bamboo cane with a concentration of 100-200 mg / mL; b) the aqueous solution of bamboo cane brown sugar is centrifuged or filtered to remove insoluble matter to obtain a clear solution; c) dialyzing the clear solution obtained in step b) through a dialysis bag with a molecular weight cutoff of 1 kDa to obtain a retentate; d) freeze-drying the retentate obtained in step c) to obtain bamboo cane polysaccharide compounds; Preferably, in step c), the dialyzation is performed in flowing water for 2-5 days.

8. The preparation method according to claim 7, wherein: Step c) the retentate obtained is further subjected to graded alcohol precipitation and freeze-dried to obtain bamboo cane polysaccharide compounds; Preferably, the final ethanol concentrations of the graded alcohol precipitation are 40%, 60% and 80% respectively.

9. Use of the bamboo cane polysaccharide compound according to any one of claims 1 to 3 in the preparation of a drug for preventing and / or treating diabetes or a health food for assisting in lowering blood sugar.

10. The use according to claim 9, wherein The bamboo cane polysaccharide compounds exert blood sugar lowering effects by upregulating sugar metabolism pathways related to glycolysis and gluconeogenesis and insulin secretion signal pathways.

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