Lucid ginseng polysaccharide substance, preparation method and application thereof, and medicine for preventing and treating diabetes or nephropathy

Abstract

The invention provides a radix scrophulariae polysaccharide substance, a preparation method and application thereof and a medicine for preventing and treating diabetes or nephropathy, and belongs to the technical field of medicinal chemistry. The Lindera arboricola polysaccharide substance provided by the invention comprises five Lindera arboricola homogeneous polysaccharides and five peracetylated Lindera arboricola homogeneous polysaccharides, and the Lindera arboricola polysaccharide substance has a relatively good prevention and treatment effect on diabetes and nephropathy, is good in safety, can ensure the safety of long-term medication of patients with diabetes and diabetic nephropathy, and can be used for preventing and treating diabetes and nephropathy. Good development and application prospects are realized.

Description

Technical Field

[0001] The invention relates to the technical field of medicinal chemistry, and in particular to Radix Glehniae polysaccharides, a preparation method and application thereof, and drugs for preventing and treating diabetes or nephropathy. Background Art

[0002] Diabetes is a common metabolic disease worldwide, characterized by persistent hyperglycemia, which can lead to serious microvascular complications such as diabetic nephropathy (DN). In the early stages of diabetes, mesangial cells (MCs) undergo abnormal proliferation and dysfunction in a hyperglycemic environment, which can cause extensive changes in kidney structure and may eventually lead to proteinuria and renal failure, which can then evolve into DN.

[0003] Oxidative stress plays a vital role in the course of DN. Oxidative stress refers to the excessive production of oxidants such as reactive oxygen species (ROS) in the body, which causes an imbalance in the body's redox state. ROS will produce a series of oxidation products, including malondialdehyde (MDA), etc. Excessive levels of ROS will cause the loss of many functions in the cells, leading to the occurrence of the disease. In order to remove excessive ROS, the body will produce antioxidant enzymes such as superoxide dismutase (SOD) to antagonize ROS and restore the balance of the body's redox system. Therefore, the body's oxidation level can be indirectly reflected by detecting the content of SOD, MDA and ROS.

[0004] At present, the prevention and treatment methods and drug development for DN have not reached the ideal level. The existing treatment methods have limited effects and are usually accompanied by obvious side effects. It is of great significance to discover highly effective and safe drugs from traditional Chinese medicine and natural products. Summary of the invention

[0005] The purpose of the present invention is to provide Radix Glehniae polysaccharides and their preparation methods and applications, as well as drugs for preventing and treating diabetes or nephropathy. The Radix Glehniae polysaccharides provided by the present invention have good preventive and therapeutic effects on diabetes and nephropathy and are safe.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides a Radix Glehniae polysaccharide substance, including Radix Glehniae homogeneous polysaccharide or fully acetylated Radix Glehniae homogeneous polysaccharide, wherein the Radix Glehniae homogeneous polysaccharide has a structure shown in Formula 1, Formula 2, Formula 3, Formula 4 or Formula 5, and the fully acetylated Radix Glehniae homogeneous polysaccharide has a structure shown in Formula C1, Formula C2, Formula C3, Formula C4 or Formula C5:

[0008]

[0009]

[0010]

[0011] The present invention provides a method for preparing the polysaccharide substance of Radix Glehniae described in the above technical solution, comprising the following steps:

[0012] Using low-grade alcohol aqueous solution to extract Radix Glehniae to obtain alcohol-extracted medicinal residues;

[0013] Extracting the alcohol-extracted medicinal residue with water, and concentrating the obtained water extract to obtain a water-extracted concentrated solution;

[0014] Deproteinizing the water-extracted concentrated solution, and concentrating the obtained deproteinized liquid to obtain a deproteinized concentrated solution;

[0015] Using water as an eluent, subjecting the deproteinized concentrated solution to macroporous adsorption resin column chromatography separation, and concentrating the obtained eluate to obtain a column chromatography separation concentrated solution;

[0016] Mixing the lower alcohol with the column chromatography separation concentrate, subjecting the obtained first mixed solution to a first alcohol precipitation to obtain a first supernatant; the volume fraction of the lower alcohol in the first mixed solution is 20 to 35%;

[0017] The first supernatant is concentrated and then mixed with a lower alcohol, and the obtained second mixed solution is subjected to a second alcohol precipitation to obtain a second supernatant; the volume fraction of the lower alcohol in the second mixed solution is 55-60%;

[0018] The second supernatant is concentrated and then mixed with a lower alcohol, and the obtained third mixed solution is subjected to a third alcohol precipitation to obtain a water-soluble polysaccharide component of Radix Glehniae; the volume fraction of the lower alcohol in the third mixed solution is 90-95%;

[0019] The water-soluble polysaccharide component of Radix Glehniae, an acetylation agent and an organic solvent are mixed, and an acetylation reaction is performed in the presence of a catalyst to obtain a fully acetylated water-soluble polysaccharide component of Radix Glehniae;

[0020] Using a petroleum ether-ethyl acetate mixed solvent as an eluent, the fully acetylated Radix Glehniae water-soluble polysaccharide component is subjected to silica gel column chromatography to obtain a first fraction, a second fraction, a third fraction and a fourth fraction in sequence, wherein the first fraction contains a fully acetylated Radix Glehniae uniform polysaccharide with a structure shown in formula C1, the second fraction contains a fully acetylated Radix Glehniae uniform polysaccharide with a structure shown in formula C2, the third fraction contains a fully acetylated Radix Glehniae uniform polysaccharide with a structure shown in formula C3, and the fourth fraction contains a fully acetylated Radix Glehniae uniform polysaccharide with a structure shown in formula C4 and a fully acetylated Radix Glehniae uniform polysaccharide with a structure shown in formula C5;

[0021] The fourth fraction is subjected to ODS reverse phase column chromatography separation using a methanol-water mixed solvent as an eluent to obtain a fifth fraction and a sixth fraction in sequence, wherein the fifth fraction contains a fully acetylated radix spatholobiformis homogeneous polysaccharide having a structure represented by formula C4, and the sixth fraction contains a fully acetylated radix spatholobiformis homogeneous polysaccharide having a structure represented by formula C5;

[0022] Using a chloroform-methanol mixed solvent as an eluent, the first fraction, the second fraction, the third fraction, the fifth fraction and the sixth fraction are subjected to gel chromatography column chromatography purification to obtain fully acetylated radix radix polysaccharides with a structure represented by formula C1, fully acetylated radix radix polysaccharides with a structure represented by formula C2, fully acetylated radix radix polysaccharides with a structure represented by formula C3, fully acetylated radix radix polysaccharides with a structure represented by formula C4 and fully acetylated radix radix polysaccharides with a structure represented by formula C5, respectively;

[0023] The fully acetylated ginseng uniform polysaccharide with a structure shown in formula C1, the fully acetylated ginseng uniform polysaccharide with a structure shown in formula C2, the fully acetylated ginseng uniform polysaccharide with a structure shown in formula C3, the fully acetylated ginseng uniform polysaccharide with a structure shown in formula C4 and the fully acetylated ginseng uniform polysaccharide with a structure shown in formula C5 are mixed with organic solvents respectively, and hydrolyzed in the presence of an alkaline reagent to obtain the ginseng uniform polysaccharide with a structure shown in formula 1, the ginseng uniform polysaccharide with a structure shown in formula 2, the ginseng uniform polysaccharide with a structure shown in formula 3, the ginseng uniform polysaccharide with a structure shown in formula 4 and the ginseng uniform polysaccharide with a structure shown in formula 5 respectively.

[0024] Preferably, the acetylation agent includes one or more of acetic anhydride, glacial acetic acid, acetyl chloride, acetyl bromide and ethyl acetate; the catalyst includes one or more of pyridine, p-dimethylaminopyridine, 4-pyrrolidinylpyridine, triethylamine, sodium acetate and trifluoromethylsulfonate; the acetylation reaction temperature is 50-70°C and the reaction time is 12-36h.

[0025] Preferably, the volume ratio of petroleum ether to ethyl acetate in the petroleum ether-ethyl acetate mixed solvent is 1:0.1-1.

[0026] Preferably, the volume ratio of methanol to water in the methanol-water mixed solvent is 1:1-2.

[0027] Preferably, the volume ratio of chloroform to methanol in the chloroform-methanol mixed solvent is 1:0.1-1.

[0028] Preferably, the alkaline reagent includes one or more of potassium carbonate, sodium carbonate, sodium bicarbonate, sodium hydroxide and potassium hydroxide, and the temperature of the hydrolysis reaction is 20-25° C. and the time is 6-18 hours.

[0029] The present invention provides the use of the Radix Glehniae polysaccharides described in the above technical solution in the preparation of medicines or functional foods for preventing and treating diabetes or kidney disease.

[0030] Preferably, the diabetes is type II diabetes; the kidney disease is diabetic nephropathy, acute renal failure, chronic renal failure, nephrotic syndrome or glomerulonephritis, and the pathogenesis of the diabetic nephropathy is oxidative stress.

[0031] The present invention provides a medicine for preventing and treating diabetes or nephropathy, comprising an active ingredient and a pharmaceutically acceptable excipient, wherein the active ingredient is the Radix Glehniae polysaccharide substance described in the above technical solution.

[0032] Beneficial effects: The Radix Glehniae polysaccharides provided by the present invention include five Radix Glehniae homogeneous polysaccharides and five fully acetylated Radix Glehniae homogeneous polysaccharides, and the Radix Glehniae polysaccharides have good preventive and therapeutic effects on diabetes and nephropathy, and have good safety. Specifically, in the embodiments of the present invention, activity screening was performed on rat glomerular mesangial cells induced by high sugar. The results showed that the Radix Glehniae homogeneous polysaccharides and fully acetylated Radix Glehniae homogeneous polysaccharides provided by the present invention both had obvious protective effects on rat glomerular mesangial cells induced by high sugar, and could increase the SOD content in rat glomerular mesangial cells, and reduce the MDA and ROS content, and the effect was significant, indicating that the Radix Glehniae homogeneous polysaccharides can alleviate the intracellular oxidative stress level, thereby preventing and treating diabetes and nephropathy, and can be used to prepare drugs or functional foods for preventing and treating diabetes and nephropathy; and toxicity tests have confirmed that the Radix Glehniae polysaccharides provided by the present invention have good safety, can ensure the safety of long-term medication for patients with diabetes and diabetic nephropathy, and have good development and application prospects.

[0033] The present invention provides a method for preparing the polysaccharide substance of Radix Glehniae. Firstly, Radix Glehniae, a variety of Radix Glehniae for both medicinal and edible use, is subjected to alcohol extraction. The obtained alcohol-extracted medicinal residue is subjected to water extraction and protein removal treatment. The water-soluble crude extract (i.e., deproteinized concentrated solution) is subjected to macroporous adsorption resin column chromatography separation using water as an eluent to remove pigments and other low-polarity small molecular components. The obtained pure water elution component (i.e., column chromatography separation concentrated solution) is subjected to specific alcohol precipitation to obtain the Radix Glehniae water-soluble polysaccharide component. Then, the Radix Glehniae water-soluble polysaccharide component is fully acetylated to obtain The fully acetylated Radix Glehniae water-soluble polysaccharide component is then subjected to silica gel column chromatography, ODS reverse phase column chromatography, and gel chromatography column chromatography to obtain five fully acetylated Radix Glehniae uniform polysaccharides (respectively abbreviated as LLTP-C1, LLTP-C2, LLTP-C3, LLTP-C4, and LLTP-C5), and then deacetylated by hydrolysis reaction to obtain five Radix Glehniae uniform polysaccharides (respectively abbreviated as LLTP-1, LLTP-2, LLTP-3, LLTP-4, and LLTP-5). The method provided by the present invention is simple to operate and easy to achieve large-scale production. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 This is the infrared spectrum of fully acetylated Radix Glehniae homogeneous polysaccharide;

[0035] Figure 2 This is the infrared spectrum of the uniform polysaccharide of Radix Ginseng;

[0036] Figure 3 This is the HPLC gel phase chromatogram of the uniform polysaccharide of Radix Glehniae;

[0037] Figure 4 This is a graph showing the effects of different time periods and different concentrations of high-glucose culture medium on the proliferation of rat glomerular mesangial cells in Test Example 2;

[0038] Figure 5 This is a graph showing the effects of five fully acetylated Radix Glehniae homogeneous polysaccharides in Test Example 2 on the cell viability of normal rat glomerular mesangial cells;

[0039] Figure 6 This is a graph showing the inhibitory effects of the five fully acetylated Radix Glehniae homogeneous polysaccharides in Test Example 2 on the proliferation of rat glomerular mesangial cells induced by high glucose;

[0040] Figure 7 This is a graph showing the effects of five fully acetylated Radix Glehniae homogeneous polysaccharides in Test Example 2 on the SOD content in rat glomerular mesangial cells induced by high glucose;

[0041] Figure 8 This is a graph showing the effects of five fully acetylated Radix Glehniae homogeneous polysaccharides in Test Example 2 on the MDA content in rat glomerular mesangial cells induced by high glucose;

[0042] Fig. 9 This is a graph showing the effects of five fully acetylated Radix Glehniae homogeneous polysaccharides in Test Example 2 on the ROS content in rat glomerular mesangial cells induced by high glucose;

[0043] Fig.10 This is a graph showing the effects of five Radix Glehniae homogeneous polysaccharides on the cell viability of normal rat glomerular mesangial cells in Test Example 3;

[0044] Fig.11 This is a graph showing the inhibitory effect of the homogeneous polysaccharides of five Radix Glehniae on the proliferation of rat glomerular mesangial cells induced by high glucose in Test Example 3;

[0045] Fig.12 This is a graph showing the effects of five Radix Glehniae homogeneous polysaccharides on the SOD content in rat glomerular mesangial cells induced by high glucose in Test Example 3;

[0046] Fig.13 This is a graph showing the effects of five types of Radix Glehniae homogeneous polysaccharides on the MDA content in rat glomerular mesangial cells induced by high glucose in Test Example 3;

[0047] Fig.14 This is a graph showing the effects of the five types of Radix Glehniae homogeneous polysaccharides in Test Example 3 on the ROS content in rat glomerular mesangial cells induced by high glucose. DETAILED DESCRIPTION

[0048] The present invention provides a Radix Glehniae polysaccharide substance, which comprises five Radix Glehniae uniform polysaccharides and five fully acetylated Radix Glehniae uniform polysaccharides, wherein the Radix Glehniae uniform polysaccharides have a structure shown in Formula 1, Formula 2, Formula 3, Formula 4 or Formula 5, and the fully acetylated Radix Glehniae uniform polysaccharides have a structure shown in Formula C1, Formula C2, Formula C3, Formula C4 or Formula C5:

[0049]

[0050]

[0051]

[0052] In the present invention, the weight average molecular weight (M) of the Radix Glehniae homogeneous polysaccharide having the structure shown in Formula 1, Formula 2, Formula 3, Formula 4 or Formula 5 is w ) is in the range of 3000 to 9000, and the number average molecular weight (M n ) is in the range of 2000 to 6000, and the polydispersity index (M w / M n ) is 1.346-1.820; the specific indicators of the homogeneous polysaccharides from various ginsengs are shown in Table 1 in Example 1 and will not be repeated here.

[0053] In the present invention, in the Radix Glehniae homogeneous polysaccharide having the structure shown in Formula 1, Formula 2, Formula 3, Formula 4 or Formula 5, the monosaccharides are preferably linked by α-1→2, α-1→4, or β-1→4 bonds.

[0054] In the present invention, the linking method of the Radix Glehniae homogeneous polysaccharide having the structure shown in Formula 1 is as follows:

[0055] [→1)-β-D-Fruf-(2→1)-α-D-Glup-(2→]n=22.

[0056] In the present invention, the linking method of the Radix Glehniae homogeneous polysaccharide having the structure shown in Formula 2 is as follows:

[0057] [→1)-β-D-Fruf-(2→1)-α-D-Glup-(4→1)-β-D-Galp-(4→]n=14.

[0058] In the present invention, the linking method of the Radix Glehniae homogeneous polysaccharide having the structure shown in Formula 3 is as follows:

[0059] [→1)-β-D-Fruf-(2→1)-α-D-Glup-(4→1)-β-D-Galp-(4→1)-β-D-Galp-(4→]n=11.

[0060] In the present invention, the linking method of the Radix Glehniae homogeneous polysaccharide having the structure shown in Formula 4 is as follows:

[0061] [→1)-β-D-Fruf-(2→1)-α-D-Glup-(4→1)-β-D-Galp-(4→1)-β-D-Galp-(4→1)-β-D-Galp-(4→]n=5.

[0062] In the present invention, the linking method of the Radix Glehniae homogeneous polysaccharide having the structure shown in Formula 5 is as follows:

[0063] [→1)-β-D-Fruf-(2→1)-α-D-Glup-(4→1)-β-D-Galp-(4→1)-β-D-Galp-(4→1)-β-D-Galp-(4→1)-β-D-

[0064] Galp-(4→]n=3.

[0065] In the present invention, in the linkage mode of the above-mentioned homogeneous polysaccharides from various ginseng regions, n represents the number of corresponding repeating units.

[0066] The present invention provides a method for preparing the polysaccharide substance of Radix Glehniae described in the above technical solution, comprising the following steps:

[0067] Using low-grade alcohol aqueous solution to extract Radix Glehniae to obtain alcohol-extracted medicinal residues;

[0068] Extracting the alcohol-extracted medicinal residue with water, and concentrating the obtained water extract to obtain a water-extracted concentrated solution;

[0069] Deproteinizing the water-extracted concentrated solution, and concentrating the obtained deproteinized liquid to obtain a deproteinized concentrated solution;

[0070] Using water as an eluent, subjecting the deproteinized concentrated solution to macroporous adsorption resin column chromatography separation, and concentrating the obtained eluate to obtain a column chromatography separation concentrated solution;

[0071] Mixing the lower alcohol with the column chromatography separation concentrate, subjecting the obtained first mixed solution to a first alcohol precipitation to obtain a first supernatant; the volume fraction of the lower alcohol in the first mixed solution is 20 to 35%;

[0072] The first supernatant is concentrated and then mixed with a lower alcohol, and the obtained second mixed solution is subjected to a second alcohol precipitation to obtain a second supernatant; the volume fraction of the lower alcohol in the second mixed solution is 55-60%;

[0073] The second supernatant is concentrated and then mixed with a lower alcohol, and the obtained third mixed solution is subjected to a third alcohol precipitation to obtain a water-soluble polysaccharide component of Radix Glehniae; the volume fraction of the lower alcohol in the third mixed solution is 90-95%;

[0074] The water-soluble polysaccharide component of Radix Glehniae, an acetylation agent and an organic solvent are mixed, and an acetylation reaction is performed in the presence of a catalyst to obtain a fully acetylated water-soluble polysaccharide component of Radix Glehniae;

[0075] Using a petroleum ether-ethyl acetate mixed solvent as an eluent, the fully acetylated Radix Glehniae water-soluble polysaccharide component is subjected to silica gel column chromatography to obtain a first fraction, a second fraction, a third fraction and a fourth fraction in sequence, wherein the first fraction contains a fully acetylated Radix Glehniae uniform polysaccharide with a structure shown in formula C1, the second fraction contains a fully acetylated Radix Glehniae uniform polysaccharide with a structure shown in formula C2, the third fraction contains a fully acetylated Radix Glehniae uniform polysaccharide with a structure shown in formula C3, and the fourth fraction contains a fully acetylated Radix Glehniae uniform polysaccharide with a structure shown in formula C4 and a fully acetylated Radix Glehniae uniform polysaccharide with a structure shown in formula C5;

[0076] The fourth fraction is subjected to ODS reverse phase column chromatography separation using a methanol-water mixed solvent as an eluent to obtain a fifth fraction and a sixth fraction in sequence, wherein the fifth fraction contains a fully acetylated radix spatholobiformis homogeneous polysaccharide having a structure represented by formula C4, and the sixth fraction contains a fully acetylated radix spatholobiformis homogeneous polysaccharide having a structure represented by formula C5;

[0077] Using a chloroform-methanol mixed solvent as an eluent, the first fraction, the second fraction, the third fraction, the fifth fraction and the sixth fraction are subjected to gel chromatography column chromatography purification to obtain fully acetylated radix radix polysaccharides with a structure represented by formula C1, fully acetylated radix radix polysaccharides with a structure represented by formula C2, fully acetylated radix radix polysaccharides with a structure represented by formula C3, fully acetylated radix radix polysaccharides with a structure represented by formula C4 and fully acetylated radix radix polysaccharides with a structure represented by formula C5, respectively;

[0078] The fully acetylated ginseng uniform polysaccharide with a structure shown in formula C1, the fully acetylated ginseng uniform polysaccharide with a structure shown in formula C2, the fully acetylated ginseng uniform polysaccharide with a structure shown in formula C3, the fully acetylated ginseng uniform polysaccharide with a structure shown in formula C4 and the fully acetylated ginseng uniform polysaccharide with a structure shown in formula C5 are mixed with organic solvents respectively, and hydrolyzed in the presence of an alkaline reagent to obtain the ginseng uniform polysaccharide with a structure shown in formula 1, the ginseng uniform polysaccharide with a structure shown in formula 2, the ginseng uniform polysaccharide with a structure shown in formula 3, the ginseng uniform polysaccharide with a structure shown in formula 4 and the ginseng uniform polysaccharide with a structure shown in formula 5 respectively.

[0079] The polysaccharide components isolated from the medicinal and edible plants have clear safety and almost no toxic side effects; some medicinal and edible plant polysaccharides have unique biological activities. Lycopus lucidus Turcz. is a perennial herbaceous plant of the genus Lycopus in the family Labiatae. As a medicinal and edible plant in western Yunnan, it has the effects of invigorating qi, strengthening the spleen and tonifying the kidney. The present invention extracts and separates from the medicinal and edible variety Lycopus lucidus Turcz. to prepare the uniform polysaccharide and fully acetylated uniform polysaccharide, which have significant therapeutic effects on diabetic nephropathy (DN), especially DN caused by oxidative stress, and has the characteristics of being non-toxic to rat glomerular mesangial cells, indicating that the uniform polysaccharide and fully acetylated uniform polysaccharide can be used to prevent and treat DN caused by oxidative stress, and have good development and application prospects. The preparation method of the uniform polysaccharide and fully acetylated uniform polysaccharide of Lycopus lucidus Turcz. according to the present invention is described in detail below.

[0080] The present invention uses a low-grade alcohol aqueous solution to extract the Radix Glehniae to obtain an alcohol-extracted medicinal residue. The present invention preferably extracts the underground part (i.e., rhizome) of the Radix Glehniae with alcohol, and the rhizome of the Radix Glehniae is preferably washed and dried in sequence with the alcohol in advance; the present invention has no special limitation on the washing; the drying preferably includes natural air-drying or drying, and the drying temperature is preferably 40 to 50°C. The present invention preferably cuts the dried Radix Glehniae rhizome into small pieces of 2 cm in length for alcohol extraction.

[0081] The present invention preferably mixes ground ginseng with lower alcohol for alcohol extraction. In the present invention, the lower alcohol in the lower alcohol aqueous solution is preferably ethanol; the volume fraction of the lower alcohol aqueous solution is preferably 60-95%, more preferably 80-95%, further preferably 90-95%, and further preferably 93-95%; in the embodiment of the present invention, edible alcohol (ethanol volume fraction is 95%) is specifically used to extract the ground ginseng. The alcohol extraction of the present invention is preferably cold soaking extraction, specifically the alcohol extraction is carried out under room temperature; the number of alcohol extractions is preferably 2-4 times, and more preferably 3 times. The material-liquid ratio of each alcohol extraction of the present invention is independently preferably 2kg: 3-6L, and more preferably 2kg: 4-4.5L, and the material-liquid ratio is specifically the ratio of the amount of the material to be extracted to the lower alcohol aqueous solution; the time of each alcohol extraction is independently preferably 24-36h, more preferably 24-30h, and further preferably 24-26h. The present invention specifically filters out the drug residue after each alcohol extraction for the next alcohol extraction, and the drug residue filtered out after the last alcohol extraction is used as the alcohol extraction drug residue for subsequent water extraction. The present invention preferably uses cold leaching extraction to perform alcohol extraction, which can extract and remove small molecular components with low polarity, and is conducive to the preparation of subsequent water-soluble polysaccharide components.

[0082] After obtaining the alcohol-extracted medicinal residue, the present invention performs water extraction on the alcohol-extracted medicinal residue to obtain a water extract. In the present invention, the water extraction is preferably reflux extraction or cooking extraction, and the temperature of the reflux extraction is preferably 90-95°C; the number of water extractions is preferably 2-4 times, and more preferably 3 times. The material-liquid mass ratio of each water extraction of the present invention is independently preferably 1:2-5, and more preferably 1:3-3.5, and the material-liquid mass ratio specifically refers to the mass ratio of the material to be water-extracted and the water used for water extraction; the time of each water extraction is independently preferably 2-3h, and more preferably 2.5h. The present invention specifically filters out the medicinal residue after each water extraction for the next water extraction, and combines the solutions obtained after filtering out the medicinal residue at the end of each water extraction as a water extract. The present invention preferably uses reflux extraction or cooking extraction for water extraction, which is conducive to completely extracting the water-soluble components in the alcohol-extracted medicinal residue.

[0083] After obtaining the water extract, the present invention concentrates the water extract (referred to as the first concentration) to obtain a water extract concentrate. In the present invention, the first concentration is preferably reduced pressure concentration, the temperature of the first concentration is preferably 60-70° C., more preferably 60-65° C.; the first concentration is preferably to concentrate the water extract to 10-20% of the original volume (i.e., the volume of the water extract concentrate is 10-20% of the volume of the water extract, preferably 15%).

[0084] After obtaining the water extract concentrate, the present invention performs a deproteinization treatment on the water extract concentrate to obtain a deproteinized liquid. In the present invention, the deproteinization method is preferably the Sevag method, the Sevag reagent used for the deproteinization is preferably a chloroform-n-butanol mixture, and the number of deproteinization treatments is preferably 2 to 4 times, and more preferably 3 times. In the present invention, the volume ratio of chloroform and n-butanol in the Sevag reagent used for each deproteinization treatment is independently preferably 2 to 5:1, and more preferably 3.5 to 4:1; the volume ratio of the Sevag reagent used for each deproteinization treatment to the material to be deproteinized is independently preferably 1 to 2:1, and more preferably 1 to 1.5:1.

[0085] After obtaining the deproteinized liquid, the present invention concentrates the deproteinized liquid (referred to as the second concentration) to obtain a deproteinized concentrated liquid. In the present invention, the second concentration is preferably reduced pressure concentration, the temperature of the second concentration is preferably 60 to 70° C., more preferably 60 to 65° C.; the second concentration is preferably to concentrate the deproteinized liquid until there is no n-butanol.

[0086] After obtaining the deproteinized concentrated solution, the present invention uses water as an eluent to perform macroporous adsorption resin column chromatography on the deproteinized concentrated solution, and collects the eluate. In the present invention, the macroporous adsorption resin used for the macroporous adsorption resin column chromatography separation preferably includes AB-8 macroporous adsorption resin or 101 macroporous adsorption resin, and more preferably AB-8 macroporous adsorption resin. When performing macroporous adsorption resin column chromatography separation, the present invention preferably adopts a wet loading method, specifically directly loading the deproteinized concentrated solution. The present invention uses water as an eluent to perform macroporous adsorption resin column chromatography on the deproteinized concentrated solution, which is convenient for separating the polysaccharide components in the ginseng.

[0087] After obtaining the eluate, the present invention concentrates the eluate (referred to as the third concentration) to obtain a column chromatography separation concentrate. The present invention has no particular limitation on the method of the third concentration, as long as a large amount of water in the eluate can be removed; the third concentration is preferably to concentrate the eluate to 5-10% of the original volume (i.e., the volume of the column chromatography separation concentrate is 5-10% of the volume of the eluate).

[0088] After obtaining the column chromatography separation concentrate, the present invention mixes a lower alcohol (referred to as the first lower alcohol) with the column chromatography separation concentrate, and performs the first alcohol precipitation on the obtained first mixed solution to obtain a first supernatant; the volume fraction of the lower alcohol in the first mixed solution is 20-35%. In the present invention, the first lower alcohol preferably includes methanol and / or ethanol, more preferably ethanol; in the embodiment of the present invention, edible alcohol is specifically used. In the present invention, the first lower alcohol is preferably used in the form of a first lower alcohol aqueous solution. The present invention does not specifically limit the concentration of the first lower alcohol aqueous solution, and the volume fraction requirement of the first lower alcohol during the first alcohol precipitation can be met. In the present invention, the number of the first alcohol precipitation is preferably 2 to 4 times, more preferably 3 times; the volume ratio of the first lower alcohol aqueous solution used in each first alcohol precipitation to the material to be precipitated is preferably 1.5 to 3:1, more preferably 1.5 to 2:1. In the present invention, the conditions for each first alcohol precipitation independently include: the volume fraction of the first lower alcohol in the first mixed solution is preferably 25-30%; the temperature is preferably 4-8°C, more preferably 4-6°C. In the embodiment of the present invention, the first alcohol precipitation is specifically carried out in a refrigerator; the time is preferably 12-24h, more preferably 12-18h. In the embodiment of the present invention, the lower alcohol in the first mixed solution is ethanol, and the volume fraction of ethanol in the first mixed solution is 30%.

[0089] After obtaining the first supernatant, the present invention concentrates the first supernatant (recorded as the fourth concentration) and mixes it with a lower alcohol, and performs a second alcohol precipitation on the obtained second mixed solution to obtain a second supernatant; the volume fraction of the lower alcohol in the second mixed solution is 55-60%. In the present invention, the fourth concentration is preferably reduced pressure concentration, and the temperature of the fourth concentration is preferably 60-70°C, more preferably 65°C; the fourth concentration is specifically to remove most of the water in the first supernatant. In the present invention, the second lower alcohol preferably includes methanol and / or ethanol, more preferably ethanol; in the embodiment of the present invention, edible alcohol is specifically used. In the present invention, the second lower alcohol is preferably used in the form of a second lower alcohol aqueous solution. The present invention does not specifically limit the concentration of the second lower alcohol aqueous solution, and the volume fraction requirement of the second lower alcohol during the second alcohol precipitation can be met. In the present invention, the number of the second alcohol precipitation is preferably 2-4 times, more preferably 3 times; the volume ratio of the second lower alcohol aqueous solution used in each second alcohol precipitation to the material to be precipitated is preferably 1.5-3:1, more preferably 1.5-2:1. In the present invention, the conditions for each second alcohol precipitation independently include: the volume fraction of the second lower alcohol in the second mixed solution is preferably 58-60%; the temperature is preferably 4-8°C, more preferably 4-6°C. In the embodiment of the present invention, the second alcohol precipitation is specifically carried out in a refrigerator; the time is preferably 12-24h, more preferably 12-18h. In the embodiment of the present invention, the lower alcohol in the second mixed solution is ethanol, and the volume fraction of ethanol in the second mixed solution is 60%.

[0090] After obtaining the second supernatant, the present invention concentrates the second supernatant (recorded as the fifth concentration) and mixes it with a lower alcohol, and performs a third alcohol precipitation on the obtained third mixed solution to obtain a water-soluble polysaccharide component of Radix Glehniae; the volume fraction of the lower alcohol in the third mixed solution is 90-95%. In the present invention, the fifth concentration is preferably a reduced pressure concentration, and the temperature of the fifth concentration is preferably 60-70°C, more preferably 65°C; the fifth concentration is specifically to remove most of the water in the second supernatant. In the present invention, the third lower alcohol preferably includes methanol and / or ethanol, more preferably ethanol; in an embodiment of the present invention, edible alcohol is specifically used. In the present invention, the number of the third alcohol precipitation is preferably 2-4 times, more preferably 3 times; the volume ratio of the third lower alcohol used in each third alcohol precipitation to the material to be precipitated is preferably 1.5-3:1, more preferably 1.5-2:1. In the present invention, the conditions of each third alcohol precipitation independently include: the volume fraction of the third lower alcohol in the third mixed solution is preferably 90-95%, more preferably 90-92%; the temperature is preferably 4-8°C, more preferably 4-6°C. In the embodiment of the present invention, the third alcohol precipitation is specifically carried out in a refrigerator; the time is preferably 12-24h, more preferably 12-18h. In the embodiment of the present invention, the lower alcohol in the third mixed solution is ethanol, and the volume fraction of ethanol in the third mixed solution is 90%. The present invention preferably washes and dries the alcohol precipitate obtained after the third alcohol precipitation in sequence to obtain the water-soluble polysaccharide component of Radix Glehniae; the reagent used for the washing is preferably an ethanol aqueous solution with a volume fraction of 95%, more preferably edible alcohol, and the number of washings is preferably 2-3 times; the drying is preferably freeze-drying or spray-drying, more preferably freeze-drying.

[0091] After obtaining the water-soluble polysaccharide component of Radix Glehniae, the present invention mixes the water-soluble polysaccharide component of Radix Glehniae, an acetylating agent and an organic solvent, and performs an acetylation reaction in the presence of a catalyst to obtain a fully acetylated water-soluble polysaccharide component of Radix Glehniae. In the present invention, the acetylating agent preferably includes one or more of acetic anhydride, glacial acetic acid, acetyl chloride, acetyl bromide and ethyl acetate, more preferably acetic anhydride; the catalyst preferably includes one or more of pyridine, p-dimethylaminopyridine, 4-pyrrolidinylpyridine, triethylamine, sodium acetate and trifluoromethylsulfonate, more preferably pyridine; the organic solvent preferably includes dimethyl sulfoxide (DMSO) or pyridine, more preferably DMSO. When pyridine is used as the organic solvent, it can also act as a catalyst, and no additional catalysts need to be added. In the present invention, taking the acetylation agent as acetic anhydride and the catalyst as pyridine as an example, the amount ratio of the ginseng water-soluble polysaccharide component, the acetylation agent and the catalyst is preferably 3.5g:80-100mL:5-15mL, more preferably 3.5g:100mL:10mL; the present invention does not specifically limit the amount of the organic solvent, as long as it can ensure the smooth progress of the acetylation reaction. In the present invention, the temperature of the acetylation reaction is preferably 50-70°C, more preferably 55-60°C; the time is preferably 12-36h, more preferably 20-24h; the acetylation reaction is preferably carried out under stirring. After the acetylation reaction, the present invention preferably adds water to the obtained product system to quench the excess acetylation agent, spin-dry the obtained feed liquid and then add water to dissolve it, extract the obtained solution with ethyl acetate, and spin-dry the obtained ethyl acetate phase to obtain the fully acetylated ginseng water-soluble polysaccharide component.

[0092] After obtaining the fully acetylated Radix Glehniae water-soluble polysaccharide component, the present invention uses a petroleum ether-ethyl acetate mixed solvent as an eluent to separate the fully acetylated Radix Glehniae water-soluble polysaccharide component by silica gel column chromatography to obtain a first fraction, a second fraction, a third fraction, and a fourth fraction in sequence, wherein the first fraction contains a fully acetylated Radix Glehniae uniform polysaccharide having a structure shown in formula C1, the second fraction contains a fully acetylated Radix Glehniae uniform polysaccharide having a structure shown in formula C2, the third fraction contains a fully acetylated Radix Glehniae uniform polysaccharide having a structure shown in formula C3, and the fourth fraction contains a fully acetylated Radix Glehniae uniform polysaccharide having a structure shown in formula C4 and a fully acetylated Radix Glehniae uniform polysaccharide having a structure shown in formula C5. In the present invention, the volume ratio of petroleum ether to ethyl acetate in the petroleum ether-ethyl acetate mixed solvent is preferably 1:0.1-1, and more preferably 1:0.8-1.

[0093] After obtaining the fourth fraction, the present invention uses a methanol-water mixed solvent as an eluent to separate the fourth fraction by ODS reverse phase column chromatography to obtain a fifth fraction and a sixth fraction in sequence, wherein the fifth fraction contains a fully acetylated radix spatholobiformis polysaccharide having a structure shown in formula C4, and the sixth fraction contains a fully acetylated radix spatholobiformis polysaccharide having a structure shown in formula C5. In the present invention, the volume ratio of methanol to water in the methanol-water mixed solvent is preferably 1:1 to 2, and more preferably 1:1 to 1.2.

[0094] After obtaining the first fraction, the second fraction, the third fraction, the fifth fraction and the sixth fraction, the present invention uses a chloroform-methanol mixed solvent as an eluent, and purifies the first fraction, the second fraction, the third fraction, the fifth fraction and the sixth fraction by gel chromatography column chromatography, respectively, to obtain fully acetylated radix radix polysaccharides with a structure shown in formula C1, fully acetylated radix radix polysaccharides with a structure shown in formula C2, fully acetylated radix radix polysaccharides with a structure shown in formula C3, fully acetylated radix radix polysaccharides with a structure shown in formula C4 and fully acetylated radix radix polysaccharides with a structure shown in formula C5. In the present invention, the volume ratio of chloroform to methanol in the chloroform-methanol mixed solvent is preferably 1:0.1-1, and more preferably 1:0.8-1.

[0095] After obtaining fully acetylated ginseng uniform polysaccharide with a structure shown in formula C1, fully acetylated ginseng uniform polysaccharide with a structure shown in formula C2, fully acetylated ginseng uniform polysaccharide with a structure shown in formula C3, fully acetylated ginseng uniform polysaccharide with a structure shown in formula C4 and fully acetylated ginseng uniform polysaccharide with a structure shown in formula C5, the present invention mixes the fully acetylated ginseng uniform polysaccharide with a structure shown in formula C1, fully acetylated ginseng uniform polysaccharide with a structure shown in formula C2, fully acetylated ginseng uniform polysaccharide with a structure shown in formula C3, fully acetylated ginseng uniform polysaccharide with a structure shown in formula C4 and fully acetylated ginseng uniform polysaccharide with a structure shown in formula C5 with an organic solvent respectively, and performs a hydrolysis reaction in the presence of an alkaline reagent to obtain fully acetylated ginseng uniform polysaccharide with a structure shown in formula 1, fully acetylated ginseng uniform polysaccharide with a structure shown in formula 2, fully acetylated ginseng uniform polysaccharide with a structure shown in formula 3, fully acetylated ginseng uniform polysaccharide with a structure shown in formula 4 and fully acetylated ginseng uniform polysaccharide with a structure shown in formula 5 respectively. In the present invention, the alkaline reagent preferably includes one or more of potassium carbonate, sodium carbonate, sodium bicarbonate, sodium hydroxide and potassium hydroxide, more preferably potassium carbonate; the organic solvent preferably includes methanol or ethanol, more preferably methanol. In the present invention, the molar ratio of each fully acetylated Radix Glehniae homogeneous polysaccharide to the alkaline reagent is preferably 1:3-5, more preferably 1:3-3.5; the present invention has no special limitation on the amount of the organic solvent, as long as it can ensure the smooth progress of the hydrolysis reaction. In the present invention, the temperature of the hydrolysis reaction is preferably 20-25°C, more preferably room temperature; the time is preferably 6-18h, more preferably 10-12h. After the hydrolysis reaction, the present invention preferably centrifuges the obtained feed liquid, discards the supernatant, washes the precipitate with methanol and then dissolves it in water, and then performs dialysis and drying in sequence to obtain each fully acetylated Radix Glehniae homogeneous polysaccharide. In the present invention, the dialysis bag used for dialysis is preferably a cellulose dialysis bag, and the molecular weight cutoff of the dialysis bag is preferably 500-1000D; the dialysis fluid used for dialysis is preferably water, more preferably distilled water; the dialysis time is preferably 12 hours, and the dialysis fluid is replaced every 2 hours. In the present invention, the drying is preferably spray drying or freeze drying, more preferably freeze drying.

[0096] The present invention provides the use of the Radix Glehniae polysaccharides described in the above technical solution in the preparation of a medicine or functional food for preventing and treating diabetes or nephropathy. In the present invention, the diabetes is preferably type II diabetes; the nephropathy is preferably diabetic nephropathy, acute renal failure, chronic renal failure, nephrotic syndrome or glomerulonephritis, and the pathogenesis of the diabetic nephropathy is preferably oxidative stress.

[0097] In the present invention, the drug preferably includes an active ingredient and a pharmaceutically acceptable excipient. In the present invention, the active ingredient in the drug is the Radix Glehniae polysaccharide substance described in the above technical solution, and more preferably is one or more of the Radix Glehniae homogeneous polysaccharide with a structure shown in Formula 1, the Radix Glehniae homogeneous polysaccharide with a structure shown in Formula 2, the Radix Glehniae homogeneous polysaccharide with a structure shown in Formula 3, the Radix Glehniae homogeneous polysaccharide with a structure shown in Formula 4, the Radix Glehniae homogeneous polysaccharide with a structure shown in Formula 5, the fully acetylated Radix Glehniae homogeneous polysaccharide with a structure shown in Formula C1, the fully acetylated Radix Glehniae homogeneous polysaccharide with a structure shown in Formula C2, the fully acetylated Radix Glehniae homogeneous polysaccharide with a structure shown in Formula C3, the fully acetylated Radix Glehniae homogeneous polysaccharide with a structure shown in Formula C4, and the fully acetylated Radix Glehniae homogeneous polysaccharide with a structure shown in Formula C5. In the present invention, the pharmaceutically acceptable excipients preferably include pharmaceutically acceptable carriers and / or excipients; the content of the active ingredient in the drug is preferably 0.1-99.9wt%, more preferably 1-90wt%, and further preferably 10-80wt%; the present invention has no special restrictions on the pharmaceutically acceptable carrier, and a pharmaceutically acceptable carrier well known to those skilled in the art can be used; the present invention has no special restrictions on the pharmaceutically acceptable excipient, and a pharmaceutically acceptable excipient well known to those skilled in the art can be used, which may specifically include mannitol, magnesium stearate, starch or cyclodextrin. In the present invention, the dosage form of the drug preferably includes an injection or an oral preparation. In the present invention, when the drug is used to prevent and treat diabetes, the effective dose of the drug is preferably 50-200mg, and the method of taking the drug is preferably oral; when the drug is used to prevent and treat kidney disease, the effective dose of the drug is preferably 100mg, and the method of taking the drug is preferably oral.

[0098] The present invention has no special limitation on the application of the Radix Glehniae polysaccharides in the preparation of functional foods for preventing and treating diabetes or kidney disease, and the method well known to those skilled in the art can be used.

[0099] The present invention provides a drug for preventing and treating diabetes or kidney disease, comprising an active ingredient and a pharmaceutically acceptable excipient, wherein the active ingredient is the Radix Glehniae polysaccharide substance described in the above technical solution. The type and content of the active ingredient, the type of pharmaceutically acceptable excipient, the dosage form, the effective dose and the administration method of the drug of the present invention are preferably consistent with the above technical solution, and will not be repeated here.

[0100] The technical solutions in the present invention will be described clearly and completely below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0101] The petroleum ether, ethyl acetate and methanol used in the following experiments of the present invention are all industrial-grade reagents and are used after re-distillation; the separation materials used include: dextran gel Sephadex LH-20 (Amersham Biosciences, Sweden), reverse phase material ODS and silica gel (300-400 mesh); the ginseng used was collected from Songgui Town, Heqing County, Dali Bai Autonomous Prefecture, Yunnan Province in November 2022.

[0102] Preparation Example 1

[0103] The steps of preparing 90% alcohol precipitation component of water-soluble polysaccharide of Radix Glehniae using Radix Glehniae as raw material are as follows:

[0104] The rhizomes of Radix Glehniae were washed and dried naturally. 1.7 kg of the dried rhizomes of Radix Glehniae were weighed, cut into 2 cm long segments, added with 3.5 L of edible alcohol (ethanol volume fraction was 95%), and extracted at room temperature (25°C) for 24 h. After each extraction, the residue was filtered out and the next extraction was repeated. After three extractions, the residue was filtered out. 5 L of distilled water was added to the residue, and water extraction was performed for 3 h under boiling (95°C). After each extraction, the residue was filtered out and the next extraction was repeated. After three extractions, the water extracts were combined to obtain the total water extract. The extract is concentrated under reduced pressure at 65° C. to obtain a water extract concentrate (the volume of the water concentrate is 15% of the total volume of the water extract); the water extract concentrate is subjected to a deproteinization treatment for 3 times by the Sevag method, wherein the Sevag reagent used in each deproteinization treatment is a mixture of chloroform and n-butanol in a volume ratio of 4:1, and the volume ratio of the Sevag reagent to the material to be deproteinized is 1:1 in each deproteinization treatment, and then the obtained deproteinization solution is concentrated under reduced pressure at 65° C. until there is no n-butanol, to obtain a deproteinization concentrate;

[0105] The protein-removed concentrated solution is subjected to macroporous adsorption resin column chromatography separation, specifically, wet loading is adopted, the macroporous adsorption resin used is AB-8 macroporous resin, pure water is used as eluent, the eluate is collected and recorded as fraction LLP, and then the fraction LLP is concentrated under reduced pressure at 65° C. to obtain a pure water elution portion (the volume of the pure water elution portion is 5% of the volume of the fraction LLP);

[0106] Adding an edible alcohol aqueous solution to the pure water elution portion, placing the obtained mixed material in a refrigerator, and performing a first alcohol precipitation under static conditions, collecting the supernatant after each first alcohol precipitation to perform the next first alcohol precipitation, performing a total of 3 first alcohol precipitations, combining all precipitates to record the first precipitation, and recording the supernatant obtained from the last first alcohol precipitation as the first supernatant; wherein, the conditions for each first alcohol precipitation are: the volume ratio of the edible alcohol aqueous solution to the material to be precipitated is 1.5:1, the volume fraction of ethanol in the mixed material obtained after the edible alcohol aqueous solution and the material to be precipitated is 30%, the precipitation temperature is 4°C, and the precipitation time is 12h;

[0107] The first supernatant was concentrated under reduced pressure at 65°C to remove most of the water, and then an edible alcohol aqueous solution was added, and the obtained mixed material was placed in a refrigerator, and a second alcohol precipitation was performed under static conditions. After each second alcohol precipitation, the supernatant was collected for the next second alcohol precipitation, and the second alcohol precipitation was performed 3 times in total. All precipitates were combined and recorded as the second precipitation, and the supernatant obtained from the last second alcohol precipitation was recorded as the second supernatant; wherein, the conditions for each second alcohol precipitation were: the volume ratio of the edible alcohol aqueous solution to the material to be precipitated was 1.5:1, the volume fraction of ethanol in the mixed material obtained after the edible alcohol aqueous solution and the material to be precipitated was 60%, the precipitation temperature was 4°C, and the precipitation time was 12h;

[0108] The second supernatant is concentrated under reduced pressure at 65°C to remove most of the water, and then edible alcohol is added, and the resulting mixed material is placed in a refrigerator and subjected to a third alcohol precipitation under static conditions. After each third alcohol precipitation, the supernatant is collected for the next third alcohol precipitation, and a total of three third alcohol precipitations are performed, and all precipitates are combined and recorded as the third precipitation; wherein, the conditions for each third alcohol precipitation are as follows: the volume ratio of edible alcohol to the material to be precipitated is 1.5:1, the volume fraction of ethanol in the mixed material obtained after mixing the edible alcohol and the material to be precipitated is 90%, the precipitation temperature is 4°C, and the precipitation time is 12h; the third precipitate is washed three times with edible alcohol, and then freeze-dried to obtain a 90% alcohol precipitation component of the water-soluble polysaccharide of Radix Glehniae (referred to as LLP-90%).

[0109] Example 1

[0110] Weigh 3.5 g of 90% alcohol precipitation component of Radix Glehniae water-soluble polysaccharide (LLP-90%) in a round-bottom flask, add dimethyl sulfoxide (DMSO) until the solid is completely dissolved, then add 100 mL of acetic anhydride, and add 10 mL of pyridine as a catalyst, and stir the reaction at 60 ° C for 24 hours; after the reaction is completed, add distilled water to quench the excess acetic anhydride, spin dry the obtained feed liquid and add water to dissolve it, place the obtained solution in a 500 mL separatory funnel, add ethyl acetate to extract 3 times, pour out the upper ethyl acetate phase, spin dry to obtain the fully acetylated Radix Glehniae water-soluble polysaccharide 90% alcohol precipitation component (referred to as LLP-C90%);

[0111] The LLP-C90% is subjected to silica gel column chromatography separation, wherein the eluent used in the silica gel column chromatography separation is a petroleum ether-ethyl acetate mixed solvent (the volume ratio of petroleum ether to ethyl acetate is 1:1), and a first fraction, a second fraction, a third fraction and a fourth fraction are obtained in sequence, wherein the first fraction contains the first fully acetylated Radix Glehniae uniform polysaccharide (recorded as LLTP-C1), the second fraction contains the second fully acetylated Radix Glehniae uniform polysaccharide (recorded as LLTP-C2), the third fraction contains the third fully acetylated Radix Glehniae uniform polysaccharide (recorded as LLTP-C3), and the fourth fraction contains the fourth fully acetylated Radix Glehniae uniform polysaccharide (recorded as LLTP-C4) and the fifth fully acetylated Radix Glehniae uniform polysaccharide (recorded as LLTP-C5);

[0112] The fourth fraction is subjected to ODS reverse phase column chromatography separation, wherein the eluent used in the ODS reverse phase column chromatography separation is a methanol-water mixed solvent (the volume ratio of methanol to water is 1:1), to obtain a fifth fraction and a sixth fraction in sequence, wherein the fifth fraction contains LLTP-C4, and the sixth fraction contains LLTP-C5;

[0113] The first fraction, the second fraction, the third fraction, the fifth fraction and the sixth fraction are subjected to gel chromatography column chromatography purification to remove pigments and other unnecessary impurities, wherein the eluent used in the gel chromatography column chromatography purification is a chloroform-methanol mixed solvent (the volume ratio of chloroform to methanol is 1:1), and then freeze-dried to obtain LLTP-C1, LLTP-C2, LLTP-C3, LLTP-C4 and LLTP-C5 respectively;

[0114] The LLTP-C1, LLTP-C2, LLTP-C3, LLTP-C4 and LLTP-C5 are placed in round-bottom flasks respectively, methanol is added until completely dissolved, and then K2CO3 is added (the molar ratio of the K2CO3 to the corresponding fully acetylated Radix Glehniae uniform polysaccharide is 3:1), and the reaction is stirred for 12 hours at room temperature; after the reaction is completed, the obtained liquid is centrifuged, the supernatant is discarded, the precipitate is washed with methanol 3 times, and then water is added to dissolve, and dialyzed for 12 hours using a cellulose dialysis bag (molecular weight cutoff of 500-1000D), the dialysis fluid used for the dialysis is distilled water, and the dialysis fluid is replaced every 2 hours; after the dialysis is completed, freeze-drying is performed to obtain the first Radix Glehniae uniform polysaccharide (recorded as LLTP-1), the second Radix Glehniae uniform polysaccharide (recorded as LLTP-2), the third Radix Glehniae uniform polysaccharide (recorded as LLTP-3), the fourth Radix Glehniae uniform polysaccharide (recorded as LLTP-4) and the fifth Radix Glehniae uniform polysaccharide (recorded as LLTP-5), respectively.

[0115] Figure 1 This is the infrared spectrum of fully acetylated Radix Glehniae homogeneous polysaccharide. Figure 2This is the infrared spectrum of the uniform polysaccharide of Radix Glehniae. The results show that there are characteristic absorption peaks of sugar compounds, among which: Figure 2 Medium 3400cm -1 The broad and blunt absorption peak near is the -OH stretching vibration absorption peak on the sugar ring, and Figure 1 The absorption peak here disappears obviously, indicating that the acetylation is complete. In addition, in the characteristic region, 2900cm -1 The absorption peak near is the CH stretching vibration in the sugar structure; 1600cm -1 The nearby absorption peak is generated by C=O stretching vibration; 1300cm -1 The absorption peak near is caused by CH deformation vibration. -1 Within the fingerprint area below 1000cm -1 The three absorption peaks near 850-930 cm are the characteristic absorption peaks of pyranose COC, indicating that it contains pyranose; -1 The absorption peak at 830 cm indicates that it contains β-glycosidic bonds; -1 The absorption peak near 600cm indicates that it contains α-glycosidic bond; -1 The absorption peak at indicated that it contained furanose.

[0116] Test Example 1

[0117] The uniform polysaccharides LLTP-1, LLTP-2, LLTP-3, LLTP-4 and LLTP-5 of Radix Glehniae were respectively subjected to HPLC gel chromatography detection, wherein the detection conditions included: using a gel exclusion chromatography column Ohpak SB-804 (300×7.5mm) and a differential refractometer; the column temperature was 45°C, the injection volume was 100μL, the mobile phase was an aqueous solution containing NaN3 and NaNO3 (the concentration of NaN3 in the mobile phase was 0.02wt%, and the concentration of NaNO3 was 0.1wt%), the flow rate was 0.6mL / min, and the isocratic elution was 75min.

[0118] Figure 3 The HPLC gel phase chromatogram of the uniform polysaccharides of Radix Glehniae (LLTP-1, LLTP-2, LLTP-3, LLTP-4, LLTP-5), in which the small absorption peak near 37min is the solvent peak of the mobile phase. Table 1 shows the molecular weight data of the uniform polysaccharides of Radix Glehniae LLTP-1, LLTP-2, LLTP-3, LLTP-4, LLTP-5. The results show that the relative molecular weight range of the uniform polysaccharides of Radix Glehniae LLTP-1, LLTP-2, LLTP-3, LLTP-4, LLTP-5 is 3000 to 9000.

[0119] Table 1 Molecular weight of uniform polysaccharide from Radix Glehniae

[0120]

[0121]

[0122] Using one-dimensional NMR spectra ( 1 H-NMR, 13 C-NMR) and two-dimensional nuclear magnetic resonance spectra (HSQC, HMBC, TOCSY) were used to assign chemical shifts to the sugar residues of the uniform polysaccharide of Radix Glehniae. The HMBC two-dimensional nuclear magnetic resonance spectrum was then used to determine the connection sequence between the sugar residues of the uniform polysaccharide of Radix Glehniae. The chemical structure of the uniform polysaccharide of Radix Glehniae was inferred. The chemical shifts of the sugar residues in the uniform polysaccharides LLTP-1, LLTP-2, LLTP-3, LLTP-4 and LLTP-5 were 1 H-NMR (800 MHZ, D2O) and 13 C-NMR (200 MHZ, D2O) data are shown in Tables 2 to 6. As shown in Tables 2 to 6, the uniform polysaccharides LLTP-1, LLTP-2, LLTP-3, LLTP-4 and LLTP-5 of Radix Glehniae have the structures shown in Formula I, Formula II, Formula III, Formula IV and Formula V, respectively.

[0123] Table 2 Chemical shifts of sugar residues in LLTP-1

[0124]

[0125] Table 3 Chemical shifts of sugar residues in LLTP-2

[0126]

[0127] Table 4 Chemical shifts of sugar residues in LLTP-3

[0128]

[0129] Table 5 Chemical shifts of sugar residues in LLTP-4

[0130]

[0131]

[0132] Table 6 Chemical shifts of sugar residues in LLTP-5

[0133]

[0134] Test Example 2

[0135] This test case studies the effect of fully acetylated Radix Glehniae homogeneous polysaccharide on high glucose-induced proliferation of rat glomerular mesangial cells, as follows:

[0136] 1. Establish high sugar model experiment:

[0137] Experimental groups:

[0138] Low-sugar control group (Control): Glu content was 5.5mmol / L.

[0139] High-glucose control group (1-5): Glu content was 20mmol / L, 25mmol / L, 30mmol / L, 35mmol / L, and 40mmol / L.

[0140] Cell culture time: 24h, 36h, 48h, 60h.

[0141] Experimental content:

[0142] CCK-8 method was used to detect cell viability. According to the above experimental groups, 96-well plates were divided, and each group was repeated for 5 wells. The cell concentration in each well was 5×10 4 100 μL of cell culture medium with a concentration of 100 μL cells / mL was added for 24 h, and the old culture medium was discarded. The corresponding new culture medium was added according to the experimental group. The culture plate was taken out after 24 h, 36 h, 48 h, and 60 h of culture, and 10 μL CCK-8 reagent was added to each well. The plate was placed in a CO2 incubator for 1 h. The wavelength of the microplate reader was set to 450 nm, and the absorbance value (OD) was measured. The proliferation of rat glomerular mesangial cells in culture medium with different glucose contents at different time points was judged according to cell viability. Cell viability was calculated according to the following formula:

[0143] Cell viability = [OD(drug added)-OD(blank)] / [OD(Control)-OD(blank)]

[0144] Figure 4 The figure is a graph showing the proliferation of rat glomerular mesangial cells at different times and different concentrations of high-glucose culture medium in test example 2 (data representation is mean ± SEM, n = 3, *p < 0.05, **p < 0.01, ***p < 0.001 vs. Control). The results show that within 24 to 60 hours, the culture medium with high glucose content has a certain promoting effect on the proliferation of cells, making the cells proliferate. Among them, under the condition of 25mmol / L glucose content, HBZY-1 cells are cultured for 36h, and the cell growth promoting effect is the strongest. Therefore, the optimal glucose concentration of the culture medium required for high-sugar induced HBZY-1 cell growth is 25mmol / L, and the optimal culture time is 36h.

[0145] 2. Experiment on the effect of fully acetylated Radix Glehniae homogeneous polysaccharide on normal rat glomerular mesangial cells:

[0146] Experimental groups:

[0147] Blank group: no cells were implanted, and the medium contained low-glucose complete medium (5.5mmol / LGlu).

[0148] Control group: cells were implanted and the cell culture medium was low-glucose complete medium (5.5mmol / LGlu).

[0149] Drug group: cells were implanted, and 5 kinds of fully acetylated Radix Glehniae polysaccharides were first dissolved in DMSO and then dissolved in low-glucose complete culture medium to make their concentrations 0.05 mg / mL, 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, and 0.4 mg / mL.

[0150] Experimental content:

[0151] CCK-8 method was used to detect cell viability. According to the above experimental groups, 96-well plates were divided, and each group was repeated for 5 wells. The cell concentration in each well was 5×10 4 100 μL of cell culture medium with a concentration of 100 μL cells / mL was added for 24 h, and the old culture medium was discarded. The corresponding new culture medium was added according to the experimental grouping. After 48 h of culture, the culture plate was taken out and 10 μL CCK-8 reagent was added to each well. The plate was placed in a CO2 incubator for 1 h. The wavelength of the microplate reader was set to 450 nm, and the absorbance value (OD) was measured. The toxicity of different concentrations of fully acetylated Radix Glehniae homogeneous polysaccharide to normal HBZY-1 cells was determined based on cell viability. Cell viability was calculated according to the following formula:

[0152] Cell viability = [OD(drug added)-OD(blank)] / [OD(Control)-OD(blank)]

[0153] Figure 5 This is a graph showing the effects of the five fully acetylated Radix Glehniae uniform polysaccharides in Test Example 2 on the cell viability of normal rat glomerular mesangial cells (data expressed as mean±SEM, n=3, *p<0.05, **p<0.01vs.Control). The results showed that the five fully acetylated Radix Glehniae uniform polysaccharides had no inhibitory effect on normal HBZY-1 cells within the set concentration range, indicating that the set concentration range can be used for the next activity test.

[0154] 3. Experiment on the effect of fully acetylated Radix Glehniae homogeneous polysaccharide on the proliferation of rat glomerular mesangial cells induced by high glucose:

[0155] Experimental groups:

[0156] Blank group: no cells were implanted, and the medium contained low-glucose complete medium (5.5mmol / LGlu).

[0157] Low-glucose control group (NG): cells were implanted and the cell culture medium was low-glucose complete medium (5.5mmol / LGlu).

[0158] High glucose control group (HG): cells were implanted and the cell culture medium was high glucose complete medium (25mmol / LGlu).

[0159] Drug group: cells were implanted, and 5 kinds of fully acetylated Radix Glehniae polysaccharides were first dissolved in DMSO and then dissolved in low-glucose complete culture medium to make their concentrations 0.05 mg / mL, 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, and 0.4 mg / mL.

[0160] Experimental content:

[0161] CCK-8 method was used to detect cell viability. According to the above experimental groups, 96-well plates were divided, and each group was repeated for 5 wells. The cell concentration in each well was 5×10 4 100 μL of cell culture medium with a concentration of 100 μL cells / mL was added for 24 h, and the old culture medium was discarded. The corresponding new culture medium was added according to the experimental grouping. After 48 h of culture, the culture plate was taken out and 10 μL CCK-8 reagent was added to each well. The plate was placed in a CO2 incubator for 1 h. The wavelength of the microplate reader was set to 450 nm, and the absorbance value (OD) was measured. According to the cell viability, it was determined whether different concentrations of fully acetylated Radix Glehnialiensis homogeneous polysaccharide had an inhibitory effect on the proliferation of HBZY-1 cells induced by high sugar. The cell viability was calculated according to the following formula:

[0162] Cell viability = [OD(drug added) - OD(blank)] / [OD(HG) - OD(blank)]

[0163] Figure 6 The figure shows the inhibitory effect of the five fully acetylated Radix Glehniae homogeneous polysaccharides in Test Example 2 on the proliferation of rat glomerular mesangial cells induced by high glucose (data are expressed as mean±SEM, n=3, *p<0.05, **p<0.01 vs. HG., ## p<0.01vs.NG.). The results showed that the five fully acetylated Radix Glehniae polysaccharides had significant differences in the inhibitory effects on the proliferation of HBZY-1 cells induced by high glucose. In the concentration range of 0.25-4 mg / mL, LLTP-C1, LLTP-C2, LLTP-C3, LLTP-C4 and LLTP-C5 all had significant inhibitory effects on the proliferation of HBZY-1 cells induced by high glucose in a concentration-dependent manner, and LLTP-C1 and LLTP-C5 had the strongest inhibitory effects.

[0164] 4. Experiment on the effect of fully acetylated Radix Glehniae homogeneous polysaccharide on the content of SOD, MDA and ROS in rat glomerular mesangial cells induced by high glucose:

[0165] Experimental groups:

[0166] Low-glucose control group (NG): cells were implanted and the cell culture medium was low-glucose complete medium (5.5mmol / LGlu).

[0167] High glucose control group (HG): cells were implanted and the cell culture medium was high glucose complete medium (25mmol / LGlu).

[0168] Positive control group (Tempol): cells were seeded and Tempol was dissolved in high-glucose complete medium (25 mmol / L Glu) to a concentration of 100 μmol / L.

[0169] Drug group: cells were implanted and five fully acetylated Radix Glehniae polysaccharides were dissolved in high-glucose medium at concentrations of 0.5 times their IC 50 or maximum inhibitory concentration, 0.75 times IC 50 or maximum inhibitory concentration, IC 50 or maximum inhibitory concentration, 1.5 times IC 50 or maximum inhibitory concentration, 2 times IC 50 Or maximum inhibitory concentration (mg / mL).

[0170] Experimental content:

[0171] Determination of SOD and MDA content:

[0172] Divide the 12-well plate into groups according to the above experiments, repeat 3 wells in each group, and add cells at a concentration of 2×10 5 2mL of cell culture medium with a concentration of 10 cells / mL was added for 24 hours. The old culture medium was discarded and the corresponding new culture medium was added according to the experimental grouping. After culturing for another 36 hours, the cells were collected into a 1.5mL centrifuge tube and the cells were shattered using a 200W ultrasonic instrument. The cells were ultrasonicated for 3 seconds each time, and then ultrasonicated for 3 seconds after 10 seconds. This was repeated 30 times. After ultrasonication, the cells were placed in a high-speed centrifuge and centrifuged for 10 minutes at a speed of 8000r / min and a temperature of 4°C. The supernatant was taken and placed on ice for testing.

[0173] A protein standard curve was drawn using the BCA kit. The sample protein was added with reagents according to the kit. After incubation at 37°C for 30 min, the absorbance value was measured at 560 nm to obtain the sample protein concentration.

[0174] According to the instructions of the SOD kit, reagents were added in sequence and reacted at 37°C for 30 minutes. The absorbance (A) of each group was measured at 560 nm. ΔA assay = A assay - A control, ΔA blank = A1 blank - A2 blank, and the inhibition percentage was calculated according to the following formula:

[0175] Inhibition percentage = (ΔA blank - ΔA measurement) ÷ ΔA blank × 100%

[0176] SOD activity was calculated according to the following formula:

[0177] SOD activity (U / mgprot) = [inhibition percentage ÷ (1-inhibition percentage) × V 反总 ]÷V 样 ×Cpr×F 稀释倍数

[0178] According to the MDA kit, the corresponding reagents were added in sequence, the water bath was set to 100°C, and the mixture was taken out after 60 minutes, cooled in ice water, and centrifuged at 1000g and room temperature for 10 minutes. The 96-well plate was divided into experimental groups, with 3 plate wells repeated in each group, and 200 μL of supernatant was added to each well. The wavelengths of the microplate reader were set to 532 nm and 600 nm, respectively, and the absorbance values ​​(A) of each group were measured, and ΔA532 = A532 assay - A532 blank, ΔA600 = A600 assay - A600 blank were calculated, and ΔA was calculated according to the following formula:

[0179] ΔA=ΔA532-ΔA600

[0180] The MDA content was calculated according to the following formula:

[0181] MDA content (nmol / mgprot) = 53.763 × ΔA ÷ Cpr × F 稀释倍数

[0182] ROS content determination:

[0183] Divide the 12-well plate into groups according to the above experiments, repeat 3 wells in each group, and add cells at a concentration of 2×10 5 Cells / mL of cell culture medium were added in 2mL, cultured for 24h, the old culture medium was discarded, and the corresponding new culture medium was added according to the experimental groups. After culturing for another 36h, the cells were collected into a 1.5mL centrifuge tube, PBS was added and blown evenly, 10μL of cell suspension was aspirated into a cell counting plate, covered with a coverslip, and the counting principle of "counting the top but not the bottom, and counting the left but not the right" was followed to calculate the number of cells according to the following formula:

[0184] Cell number (cells / mL) = average number of cells in 4 16-square grids × 10 4 × Dilution factor

[0185] Load the probe according to the ROS kit instructions. Add the probe-loaded cell suspension to a 96-well plate, with 3 replicates per group and 200 μL per well. Measure the fluorescence value at an excitation wavelength of 488 nm and an emission wavelength of 525 nm. Calculate the relative ROS value according to the following formula:

[0186] Relative amount of ROS = fluorescence value / cell number (Cells / mL)

[0187] Figure 7 This is a graph showing the effects of the five fully acetylated Radix Glehniae homogeneous polysaccharides in Test Example 2 on the SOD content in rat glomerular mesangial cells induced by high glucose. Figure 8 This is a graph showing the effects of the five fully acetylated Radix Glehniae homogeneous polysaccharides in Test Example 2 on the MDA content in rat glomerular mesangial cells induced by high glucose. Fig. 9 This is a graph showing the effects of five fully acetylated Radix Glehniae homogeneous polysaccharides in Test Example 2 on the ROS content in rat glomerular mesangial cells induced by high glucose. Figure 7 to Figure 9 Data are presented as mean ± SEM, n = 3, *p < 0.05, **p < 0.01 vs. HG., ## p<0.01vs.NG. The results showed that the five fully acetylated Radix Glehniae polysaccharides increased the SOD content in HBZY-1 induced by high sugar, and reduced the MDA and ROS content. This indicates that the fully acetylated Radix Glehniae polysaccharides provided by the present invention can alleviate the intracellular oxidative stress level, thereby preventing and treating diabetes and nephropathy, and can be used to prepare drugs or functional foods for preventing and treating diabetes and nephropathy.

[0188] Test Example 3

[0189] This test case studies the effect of Radix Glehniae Polysaccharide on the proliferation of rat glomerular mesangial cells induced by high glucose, as follows:

[0190] 1. Experiment on the effect of Radix Ginseng homogeneous polysaccharide on normal rat glomerular mesangial cells:

[0191] Experimental groups:

[0192] Blank group: no cells were implanted, and the medium contained low-glucose complete medium (5.5mmol / LGlu).

[0193] Control group: cells were implanted and the cell culture medium was low-glucose complete medium (5.5mmol / LGlu).

[0194] Drug group: cells were implanted, and 5 kinds of Radix Glehniae polysaccharides were dissolved in low-sugar complete culture medium to a concentration of 0.01 mg / mL, 0.0625 mg / mL, 0.125 mg / mL, 0.25 mg / mL, and 0.25 mg / mL.

[0195] Experimental content:

[0196] CCK-8 method was used to detect cell viability. According to the above experimental groups, 96-well plates were divided, and each group was repeated for 5 wells. The cell concentration in each well was 5×104 cells / mL of cell culture medium 100μL, culture for 24h, discard the old culture medium, add the corresponding new culture medium according to the experimental group, take out the culture plate after 48h of culture, add 10μL CCK-8 reagent to each well, put it in a CO2 incubator for 1h, set the wavelength of the microplate reader to 450nm, and measure the absorbance value (OD). According to the cell viability, it is judged whether different concentrations of ginseng homogeneous polysaccharide are toxic to normal HBZY-1 cells. Calculate the cell viability according to the following formula:

[0197] Cell viability = [OD(drug added)-OD(blank)] / [OD(Control)-OD(blank)]

[0198] Fig.10 This is a graph showing the effects of the five types of Radix Glehniae homogeneous polysaccharides in Test Example 3 on the cell viability of normal rat glomerular mesangial cells (data expressed as mean±SEM, n=3, *p<0.05, **p<0.01vs.Control.). The results showed that the five types of Radix Glehniae homogeneous polysaccharides had no inhibitory effect on normal HBZY-1 cells within the set concentration range, indicating that the set concentration range can be used for the next activity test.

[0199] 2. Experiment on the effect of Radix Glehniae homogeneous polysaccharide on the proliferation of rat glomerular mesangial cells induced by high glucose:

[0200] Experimental groups:

[0201] Blank group: no cells were implanted, and the medium contained low-glucose complete medium (5.5mmol / LGlu).

[0202] Low-glucose control group (NG): cells were implanted and the cell culture medium was low-glucose complete medium (5.5mmol / LGlu).

[0203] High glucose control group (HG): cells were implanted and the cell culture medium was high glucose complete medium (25mmol / LGlu).

[0204] Drug group: cells were implanted, and 5 types of Radix Glehniae polysaccharides were dissolved in high-glucose culture medium to concentrations of 0.01 mg / mL, 0.0625 mg / mL, 0.125 mg / mL, 0.25 mg / mL, and 0.25 mg / mL.

[0205] Experimental content:

[0206] CCK-8 method was used to detect cell viability. According to the above experimental groups, 96-well plates were divided, and each group was repeated for 5 wells. The cell concentration in each well was 5×10 4100 μL of cell culture medium with a concentration of 100 μL cells / mL was added for 24 h, and the old culture medium was discarded. The corresponding new culture medium was added according to the experimental grouping. After 48 h of culture, the culture plate was taken out and 10 μL CCK-8 reagent was added to each well. The plate was placed in a CO2 incubator for 1 h. The wavelength of the microplate reader was set to 450 nm, and the absorbance value (OD) was measured. According to the cell viability, it was determined whether different concentrations of ginseng homogeneous polysaccharide had an inhibitory effect on the proliferation of HBZY-1 cells induced by high sugar. The cell viability was calculated according to the following formula:

[0207] Cell viability = [OD(drug added) - OD(blank)] / [OD(HG) - OD(blank)]

[0208] Fig.11 The figure shows the inhibitory effect of the homogeneous polysaccharides of five Radix Glehniae on the proliferation of rat glomerular mesangial cells induced by high glucose in Test Example 3 (data are expressed as mean±SEM, n=3, *p<0.05, **p<0.01 vs. HG., ## p<0.01vs.NG.), the results showed that the five polysaccharides had a certain inhibitory effect on the proliferation of HBZY-1 cells induced by high glucose. Among them, LLTP-1 and LLTP-3 had a significant inhibitory effect on the proliferation of HBZY-1 cells induced by high glucose in the concentration range of 0.25-4 mg / mL, which was concentration-dependent, and LLTP-3 had the strongest inhibitory effect.

[0209] 3. Experiment on the effect of Radix Glehniae homogeneous polysaccharide on the content of SOD, MDA and ROS in rat glomerular mesangial cells induced by high glucose:

[0210] Experimental groups:

[0211] Low-glucose control group (NG): cells were implanted and the cell culture medium was low-glucose complete medium (5.5mmol / LGlu).

[0212] High glucose control group (HG): cells were implanted and the cell culture medium was high glucose complete medium (25mmol / LGlu).

[0213] Positive control group (Tempol): cells were seeded and Tempol was dissolved in high-glucose complete medium (25 mmol / L Glu) to a concentration of 100 μmol / L.

[0214] Drug group: cells were implanted and the five ginseng polysaccharides were dissolved in high-glucose medium at concentrations of 0.5 times their IC 50 or maximum inhibitory concentration, 0.75 times IC 50 or maximum inhibitory concentration, IC 50 or maximum inhibitory concentration, 1.5 times IC50 or maximum inhibitory concentration, 2 times IC 50 Or maximum inhibitory concentration (mg / mL).

[0215] Experimental content:

[0216] Determination of SOD and MDA content:

[0217] Divide the 12-well plate into groups according to the above experiments, repeat 3 wells in each group, and add cells at a concentration of 2×10 5 2mL of cell culture medium with a concentration of 10 cells / mL was added for 24 hours. The old culture medium was discarded and the corresponding new culture medium was added according to the experimental grouping. After culturing for another 36 hours, the cells were collected into a 1.5mL centrifuge tube and the cells were shattered using a 200W ultrasonic instrument. The cells were ultrasonicated for 3 seconds each time, and then ultrasonicated for 3 seconds after 10 seconds. This was repeated 30 times. After ultrasonication, the cells were placed in a high-speed centrifuge and centrifuged for 10 minutes at a speed of 8000r / min and a temperature of 4°C. The supernatant was taken and placed on ice for testing.

[0218] A protein standard curve was drawn using the BCA kit. The sample protein was added with reagents according to the kit. After incubation at 37°C for 30 min, the absorbance value was measured at 560 nm to obtain the sample protein concentration.

[0219] According to the instructions of the SOD kit, reagents were added in sequence and reacted at 37°C for 30 minutes. The absorbance (A) of each group was measured at 560 nm. ΔA assay = A assay - A control, ΔA blank = A1 blank - A2 blank, and the inhibition percentage was calculated according to the following formula:

[0220] Inhibition percentage = (ΔA blank - ΔA measurement) ÷ ΔA blank × 100%

[0221] SOD activity was calculated according to the following formula:

[0222] SOD activity (U / mgprot) = [inhibition percentage ÷ (1-inhibition percentage) × V 反总 ]÷V 样 ×Cpr×F 稀释倍数

[0223] According to the MDA kit, the corresponding reagents were added in sequence, the water bath was set to 100°C, and the mixture was taken out after 60 minutes, cooled in ice water, and centrifuged at 1000g and room temperature for 10 minutes. The 96-well plate was divided into experimental groups, with 3 plate wells repeated in each group, and 200 μL of supernatant was added to each well. The wavelengths of the microplate reader were set to 532 nm and 600 nm, respectively, and the absorbance values ​​(A) of each group were measured, and ΔA532 = A532 assay - A532 blank, ΔA600 = A600 assay - A600 blank were calculated, and ΔA was calculated according to the following formula:

[0224] ΔA=ΔA532-ΔA600

[0225] The MDA content was calculated according to the following formula:

[0226] MDA content (nmol / mgprot) = 53.763 × ΔA ÷ Cpr × F 稀释倍数

[0227] ROS content determination:

[0228] Divide the 12-well plate into groups according to the above experiments, repeat 3 wells in each group, and add cells at a concentration of 2×10 5 Cells / mL of cell culture medium were added in 2mL, cultured for 24h, the old culture medium was discarded, and the corresponding new culture medium was added according to the experimental groups. After culturing for another 36h, the cells were collected into a 1.5mL centrifuge tube, PBS was added and blown evenly, 10μL of cell suspension was aspirated into a cell counting plate, covered with a coverslip, and the counting principle of "counting the top but not the bottom, and counting the left but not the right" was followed to calculate the number of cells according to the following formula:

[0229] Cell number (cells / mL) = average number of cells in 4 16-square grids × 10 4 × Dilution factor

[0230] Load the probe according to the ROS kit instructions. Add the probe-loaded cell suspension to a 96-well plate, with 3 replicates per group and 200 μL per well. Measure the fluorescence value at an excitation wavelength of 488 nm and an emission wavelength of 525 nm. Calculate the relative ROS value according to the following formula:

[0231] Relative amount of ROS = fluorescence value / cell number (Cells / mL)

[0232] Fig.12 This is a graph showing the effects of the five Radix Glehniae homogeneous polysaccharides in Test Example 3 on the SOD content in rat glomerular mesangial cells induced by high glucose. Fig.13 This is a graph showing the effects of five Radix Glehniae homogeneous polysaccharides on the MDA content in rat glomerular mesangial cells induced by high glucose in Test Example 3. Fig.14 This is a graph showing the effects of five Radix Glehniae homogenous polysaccharides on the ROS content in rat glomerular mesangial cells induced by high glucose in Test Example 3. Figure 12 to Figure 14 Data are presented as mean ± SEM, n = 3, *p < 0.05, **p < 0.01 vs. HG., ##p<0.01vs.NG. The results showed that the five kinds of Radix Glehniae polysaccharides increased the SOD content in HBZY-1 induced by high sugar, and reduced the MDA and ROS content. Among them, LLTP-1, LLTP-3 and LLTP-4 had a certain concentration dependence, and LLTP-3 had the most obvious effect. It shows that the Radix Glehniae polysaccharide provided by the present invention can alleviate the intracellular oxidative stress level, thereby preventing and treating diabetes and nephropathy, and can be used to prepare drugs or functional foods for preventing and treating diabetes and nephropathy.

[0233] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A Radix Glehniae polysaccharide substance, comprising Radix Glehniae homogeneous polysaccharide or fully acetylated Radix Glehniae homogeneous polysaccharide, wherein the Radix Glehniae homogeneous polysaccharide has a structure shown in Formula 1, Formula 2, Formula 3, Formula 4 or Formula 5, and the fully acetylated Radix Glehniae homogeneous polysaccharide has a structure shown in Formula C1, Formula C2, Formula C3, Formula C4 or Formula C5:

2. The method for preparing the polysaccharide substance of Radix Glehniae according to claim 1, comprising the following steps: Using low-grade alcohol aqueous solution to extract Radix Glehniae to obtain alcohol-extracted medicinal residues; Extracting the alcohol-extracted medicinal residue with water, and concentrating the obtained water extract to obtain a water-extracted concentrated solution; Deproteinizing the water-extracted concentrated solution, and concentrating the obtained deproteinized liquid to obtain a deproteinized concentrated solution; Using water as an eluent, subjecting the deproteinized concentrated solution to macroporous adsorption resin column chromatography separation, and concentrating the obtained eluate to obtain a column chromatography separation concentrated solution; Mixing the lower alcohol with the column chromatography separation concentrate, subjecting the obtained first mixed solution to a first alcohol precipitation to obtain a first supernatant; the volume fraction of the lower alcohol in the first mixed solution is 20 to 35%; The first supernatant is concentrated and then mixed with a lower alcohol, and the obtained second mixed solution is subjected to a second alcohol precipitation to obtain a second supernatant; the volume fraction of the lower alcohol in the second mixed solution is 55-60%; The second supernatant is concentrated and then mixed with a lower alcohol, and the obtained third mixed solution is subjected to a third alcohol precipitation to obtain a water-soluble polysaccharide component of Radix Glehniae; the volume fraction of the lower alcohol in the third mixed solution is 90-95%; The water-soluble polysaccharide component of Radix Glehniae, an acetylation agent and an organic solvent are mixed, and an acetylation reaction is performed in the presence of a catalyst to obtain a fully acetylated water-soluble polysaccharide component of Radix Glehniae; Using a petroleum ether-ethyl acetate mixed solvent as an eluent, the fully acetylated Radix Glehniae water-soluble polysaccharide component is subjected to silica gel column chromatography to obtain a first fraction, a second fraction, a third fraction and a fourth fraction in sequence, wherein the first fraction contains a fully acetylated Radix Glehniae uniform polysaccharide with a structure shown in formula C1, the second fraction contains a fully acetylated Radix Glehniae uniform polysaccharide with a structure shown in formula C2, the third fraction contains a fully acetylated Radix Glehniae uniform polysaccharide with a structure shown in formula C3, and the fourth fraction contains a fully acetylated Radix Glehniae uniform polysaccharide with a structure shown in formula C4 and a fully acetylated Radix Glehniae uniform polysaccharide with a structure shown in formula C5; The fourth fraction is subjected to ODS reverse phase column chromatography separation using a methanol-water mixed solvent as an eluent to obtain a fifth fraction and a sixth fraction in sequence, wherein the fifth fraction contains a fully acetylated radix spatholobiformis homogeneous polysaccharide having a structure represented by formula C4, and the sixth fraction contains a fully acetylated radix spatholobiformis homogeneous polysaccharide having a structure represented by formula C5; Using a chloroform-methanol mixed solvent as an eluent, the first fraction, the second fraction, the third fraction, the fifth fraction and the sixth fraction are subjected to gel chromatography column chromatography purification to obtain fully acetylated radix radix polysaccharides with a structure represented by formula C1, fully acetylated radix radix polysaccharides with a structure represented by formula C2, fully acetylated radix radix polysaccharides with a structure represented by formula C3, fully acetylated radix radix polysaccharides with a structure represented by formula C4 and fully acetylated radix radix polysaccharides with a structure represented by formula C5, respectively; The fully acetylated ginseng uniform polysaccharide with a structure shown in formula C1, the fully acetylated ginseng uniform polysaccharide with a structure shown in formula C2, the fully acetylated ginseng uniform polysaccharide with a structure shown in formula C3, the fully acetylated ginseng uniform polysaccharide with a structure shown in formula C4 and the fully acetylated ginseng uniform polysaccharide with a structure shown in formula C5 are mixed with organic solvents respectively, and hydrolyzed in the presence of an alkaline reagent to obtain the ginseng uniform polysaccharide with a structure shown in formula 1, the ginseng uniform polysaccharide with a structure shown in formula 2, the ginseng uniform polysaccharide with a structure shown in formula 3, the ginseng uniform polysaccharide with a structure shown in formula 4 and the ginseng uniform polysaccharide with a structure shown in formula 5 respectively.

3. The preparation method according to claim 2, characterized in that: The acetylation agent includes one or more of acetic anhydride, glacial acetic acid, acetyl chloride, acetyl bromide and ethyl acetate; the catalyst includes one or more of pyridine, p-dimethylaminopyridine, 4-pyrrolidinopyridine, triethylamine, sodium acetate and trifluoromethylsulfonate; the acetylation reaction temperature is 50-70°C and the reaction time is 12-36h.

4. The preparation method according to claim 2, characterized in that: The volume ratio of petroleum ether to ethyl acetate in the petroleum ether-ethyl acetate mixed solvent is 1:0.1-1.

5. The preparation method according to claim 2, characterized in that: The volume ratio of methanol to water in the methanol-water mixed solvent is 1:1-2.

6. The preparation method according to claim 2, characterized in that: The volume ratio of chloroform to methanol in the chloroform-methanol mixed solvent is 1:0.1-1.

7. The preparation method according to claim 2, characterized in that: The alkaline reagent includes one or more of potassium carbonate, sodium carbonate, sodium bicarbonate, sodium hydroxide and potassium hydroxide. The temperature of the hydrolysis reaction is 20-25° C. and the time is 6-18 hours.

8. Use of the Radix Glehniae polysaccharides according to claim 1 in the preparation of medicines or functional foods for preventing and treating diabetes or kidney disease.

9. The use according to claim 8, characterized in that: The diabetes is type II diabetes; the kidney disease is diabetic nephropathy, acute renal failure, chronic renal failure, nephrotic syndrome or glomerulonephritis, and the pathogenesis of the diabetic nephropathy is oxidative stress.

10. A medicine for preventing and treating diabetes or kidney disease, comprising an active ingredient and a pharmaceutically acceptable excipient, wherein the active ingredient is the Radix Glehniae polysaccharide substance according to claim 1.

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