A green plum polysaccharide component, a preparation method and application thereof

Abstract

This invention belongs to the field of medicinal chemistry, specifically relating to a polysaccharide component from green plums, its preparation method, and its application. The invention first involves ethanol extraction of green plums (a dual-purpose medicinal and edible variety), separating the ethanol extract and the ethanol-extracted residue. The residue is then subjected to water extraction to remove protein, yielding a water-soluble crude extract. The ethanol extract and the water-soluble crude extract are then separated by macroporous adsorption resin column chromatography using pure water as the eluent, yielding a pure water eluent. The pure water eluent of the water-soluble crude extract is further fractionated and precipitated with alcohol to obtain different water-soluble polysaccharide alcohol-precipitated fractions. The ethanol-extracted polysaccharide component is obtained from the pure water eluent of the ethanol extract. The fractionated and ethanol-extracted polysaccharide components from green plums prepared using the method of this invention can be used to prepare drugs for the prevention and treatment of diabetes and kidney disease, exhibiting high safety and promising development and application prospects.

Description

Technical Field

[0001] This invention belongs to the field of medicinal chemistry technology, specifically relating to a plum polysaccharide component, its preparation method, and its application. Background Technology

[0002] Diabetes mellitus is a syndrome caused by insufficient insulin secretion or utilization, leading to disorders in the metabolism of carbohydrates, fats, and proteins. Diabetic nephropathy (DN) is a common complication of diabetes and a leading cause of death in end-stage renal disease patients. The early and significant pathological features of DN are glomerular hypertrophy due to mesangial proliferation and increased glomerular filtration rate due to impaired mesangial cell (MC) contractile function. MCs are among the most functionally active intrinsic cells in the glomerulus and are also the main target cells for various pathogenic factors. The pathogenesis of DN is complex, with oxidative stress and glucose metabolism disorders being the main mechanisms. Oxidative stress refers to the excessive production of oxidants such as reactive oxygen species (ROS) in the body, causing an imbalance in the redox state. ROS produces a series of oxidation products, including malondialdehyde (MDA). Excessive ROS levels can cause the loss of many cellular functions, leading to the disease. To eliminate excess reactive oxygen species (ROS), the body produces antioxidant enzymes and non-enzymatic agents, such as superoxide dismutase (SOD), catalase (from micrococcus lysodeiktic, CAT), and glutathione (GSH), to antagonize ROS and restore the balance of the body's redox system. Therefore, detecting the levels of SOD, MDA, and ROS can indirectly reflect the body's oxidation level.

[0003] Current clinical treatment of diabetic nephropathy (DN) primarily relies on combination therapy. However, these drugs only slow the progression of DN, failing to cure it, and often cause serious adverse reactions. Therefore, discovering highly effective and safe drugs from traditional Chinese medicine and natural products is of great significance. The important role of plant-derived active polysaccharides in the prevention and treatment of diabetes and its complications has become a hot research topic. As a product with both medicinal and edible properties, plum has significant medicinal value. Obtaining polysaccharides from plum that have good preventive and therapeutic effects on diabetes and kidney disease would contribute to the development of adjuvant therapeutic drugs. Summary of the Invention

[0004] In view of this, the purpose of this invention is to provide a plum polysaccharide component, its preparation method, and its application. The plum polysaccharide component prepared by this invention has good preventive and therapeutic effects on diabetes and kidney disease, and is highly safe.

[0005] This invention provides a method for preparing a polysaccharide component from green plums, wherein the polysaccharide component comprises a water-soluble polysaccharide fractionated by alcohol precipitation and / or an alcohol-extracted polysaccharide component, and the method for preparing the alcohol-extracted polysaccharide component includes the following steps: The green plum was extracted with a low-grade alcohol-water solution to obtain alcohol-extracted residue and alcohol extract. The alcohol extract was concentrated and then subjected to deproteinization treatment to obtain a deproteinized alcohol-soluble component. The deproteinized alcohol-soluble component was separated by a macroporous adsorption resin column, eluted with water, and the resulting water eluent was dried to obtain the alcohol-extracted polysaccharide component. The water-soluble polysaccharide fractionated alcohol precipitation component includes one or more of a first water-soluble polysaccharide alcohol precipitation component, a second water-soluble polysaccharide alcohol precipitation component, and a third water-soluble polysaccharide alcohol precipitation component, and the preparation method includes the following steps: The alcohol-extracted residue was subjected to water extraction to obtain an aqueous extract; The aqueous extract was concentrated and then subjected to deproteinization treatment to obtain a deproteinized water-soluble component. The deproteinized water-soluble component was separated by a macroporous adsorption resin column and eluted with water. The resulting water eluent was concentrated and mixed with a lower alcohol for a first alcohol precipitation. A first supernatant and a first precipitate were separated. The first precipitate was dried to obtain the first water-soluble polysaccharide alcohol-precipitated component. The volume fraction of the lower alcohol in the system during the first alcohol precipitation was 20-35%. The first supernatant was concentrated and then mixed with a lower alcohol for a second alcohol precipitation. The second supernatant and the second precipitate were separated. The second precipitate was dried to obtain the second water-soluble polysaccharide alcohol-precipitated component. The volume fraction of the lower alcohol in the system during the second alcohol precipitation was 55-65%. The second supernatant was concentrated and mixed with a lower alcohol for a third alcohol precipitation to obtain a third precipitate. The third precipitate was dried to obtain a third water-soluble polysaccharide alcohol precipitate component. The volume fraction of the lower alcohol in the system during the third alcohol precipitation was 75-85%.

[0006] Preferably, the volume fraction of the lower alcohol in the lower alcohol aqueous solution is 40-95%; the lower alcohol aqueous solution is an ethanol aqueous solution.

[0007] Preferably, the water extraction is a hot water extraction, the temperature of the hot water extraction is 90~95℃, and the number of hot water extractions is 2~4 times.

[0008] Preferably, the deproteinization treatment after concentration of the alcohol extract and the deproteinization treatment after concentration of the water extract are both carried out using the Sevag method. The Sevag reagent used in the Sevag method includes chloroform and n-butanol, and the volume ratio of chloroform to n-butanol is (2~5):1.

[0009] Preferably, the macroporous adsorption resin column used for separating the deproteinized alcohol-soluble component and the deproteinized water-soluble component is an AB-8 macroporous adsorption resin column.

[0010] Preferably, the lower alcohol used in the first, second, and third alcohol precipitations is ethanol.

[0011] Preferably, the volume fraction of the lower alcohol in the first alcohol precipitation system is 25-30%, the volume fraction of the lower alcohol in the second alcohol precipitation system is 60%, and the volume fraction of the lower alcohol in the third alcohol precipitation system is 80%.

[0012] The present invention provides a plum polysaccharide component obtained by the preparation method described above, wherein the plum polysaccharide component includes a water-soluble polysaccharide fractionated by alcohol precipitation and / or an alcohol-soluble polysaccharide component.

[0013] This invention provides the application of the polysaccharide component of the medicinal and edible plum described above in the preparation of drugs for the prevention and treatment of diabetes or kidney disease.

[0014] Preferably, the diabetes is type II diabetes; the nephropathy includes at least one of diabetic nephropathy, acute renal failure, chronic renal failure, nephrotic syndrome, and glomerulonephritis; the mechanism of the diabetic nephropathy is oxidative stress.

[0015] Compared with the prior art, the present invention has the following beneficial effects: This invention provides a method for preparing plum polysaccharide components, wherein the plum polysaccharide components include water-soluble polysaccharide fractions with graded alcohol precipitation and / or alcohol-soluble polysaccharide components. The plum polysaccharide components (water-soluble polysaccharide fractions with graded alcohol precipitation and alcohol-soluble polysaccharide components) prepared using the method of this invention have good effects on the prevention or treatment of diabetes and kidney disease, and are highly safe.

[0016] In this embodiment of the invention, rat glomerular mesangial cells were screened for activity using high glucose-induced glomerular mesangial cells. The results showed that the 80% alcohol-precipitated fraction and alcohol-extracted polysaccharide fraction of the water-soluble extract of the medicinal and edible plum prepared in this invention both had significant protective effects on high glucose-induced rat glomerular mesangial cells, increasing the intracellular SOD content and decreasing the MDA and ROS content. This indicates that the alcohol-precipitated fraction and alcohol-extracted polysaccharide fraction of the water-soluble extract of the medicinal and edible plum can alleviate intracellular oxidative stress levels, thereby preventing and treating diabetes and nephropathy. They can be used to prepare drugs or health products for the prevention and treatment of diabetes and nephropathy. Furthermore, toxicity tests confirmed that the alcohol-precipitated fraction and alcohol-extracted polysaccharide fraction of the water-soluble extract of the plum have high safety, ensuring the safety of long-term use by patients with diabetes and nephropathy, and have good development and application prospects. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 The graph shows the proliferation of rat glomerular mesangial cells under different time and concentrations of high glucose culture medium as described in Example 1. Figure 2 The figure shows the effect of different ethanol-precipitated and ethanol-extracted components of plum polysaccharides on the cell viability of normal rat glomerular mesangial cells in Example 1. Figure 3 The diagram shows the inhibitory effect of different ethanol-precipitated and ethanol-extracted components of plum polysaccharides on the proliferation of high-glucose-induced rat glomerular mesangial cells in Example 1. Figures 4-6 The figure shows the effect of the 80% polysaccharide precipitate and alcohol extract of green plum from Example 1 on the levels of SOD, MDA, and ROS in high-glucose-induced rat glomerular mesangial cells. Figure 4 For SOD, Figure 5 For MDA, Figure 6 For ROS. Detailed Implementation

[0019] This invention provides a method for preparing a polysaccharide component from green plums. The polysaccharide component from green plums includes a water-soluble polysaccharide fractionated by alcohol precipitation and / or an alcohol-soluble polysaccharide component. The method for preparing the water-soluble polysaccharide fractionated by alcohol precipitation includes the following steps: The green plum was extracted with a low-grade alcohol-water solution to obtain alcohol-extracted residue and alcohol extract. The alcohol-extracted residue was subjected to water extraction to obtain an aqueous extract; The aqueous extract was concentrated and then subjected to deproteinization treatment to obtain a deproteinized water-soluble component. The deproteinized water-soluble component was separated by a macroporous adsorption resin column and eluted with water. The resulting water eluent was concentrated and mixed with a lower alcohol for a first alcohol precipitation. A first supernatant and a first precipitate were separated. The first precipitate was dried to obtain the first water-soluble polysaccharide alcohol-precipitated component. The volume fraction of the lower alcohol in the system during the first alcohol precipitation was 20-35%. The first supernatant was concentrated and then mixed with a lower alcohol for a second alcohol precipitation. The second supernatant and the second precipitate were separated. The second precipitate was dried to obtain the second water-soluble polysaccharide alcohol-precipitated component. The volume fraction of the lower alcohol in the system during the second alcohol precipitation was 55-65%. The second supernatant was concentrated and then mixed with a lower alcohol for a third alcohol precipitation. The third precipitate was obtained by separation and drying to obtain the third water-soluble polysaccharide alcohol precipitate component. The volume fraction of the lower alcohol in the system during the third alcohol precipitation was 75-85%. The preparation method of the alcohol-soluble polysaccharide component includes the following steps: The alcohol extract was concentrated and then subjected to deproteinization treatment to obtain a deproteinized alcohol-soluble component. The deproteinized alcohol-soluble component was separated by passing it through a macroporous adsorption resin column, eluted with water, and the resulting water eluent was dried to obtain the alcohol-soluble polysaccharide component.

[0020] Unless otherwise specified, all materials and equipment used in this invention are commercially available products in the field.

[0021] Polysaccharide components isolated from plants that are both medicinal and edible have a proven safety profile with virtually no toxic side effects; some polysaccharides from these plants possess unique biological activities. Green plum (Prunus mume) is a plant belonging to the Rosaceae family. Prunus mume The mature fruit of *Prunus serrulata* (also known as plum), has the effects of invigorating qi, strengthening the spleen, and tonifying the kidneys. This invention extracts and separates water-soluble polysaccharide fractions and alcohol-extracted polysaccharide fractions from the medicinal and edible variety *Prunus serrulata*. These fractions show significant therapeutic effects on diabetic nephropathy (DN) caused by oxidative stress, and are non-toxic to rat glomerular mesangial cells. This indicates that the water-soluble polysaccharide fractions and alcohol-extracted polysaccharide fractions from *Prunus serrulata* can be used to prevent and treat DN caused by oxidative stress, and have good development and application prospects.

[0022] This invention uses a low-grade alcohol-water solution to extract green plums, obtaining alcohol-extracted residue and alcohol extract.

[0023] In this invention, the green plums are preferably washed before use; the invention preferably uses mature green plums, i.e., the pulp with the pit. The invention also preferably involves breaking open the washed green plums for alcohol extraction.

[0024] In this invention, the volume fraction of the lower alcohol in the lower alcohol aqueous solution is preferably 40-95%; the lower alcohol aqueous solution is preferably an ethanol aqueous solution.

[0025] In this invention, the alcohol extraction is preferably a cold maceration extraction, specifically involving mixing the green plums with a low-grade alcohol aqueous solution and soaking them at room temperature (25°C). The number of alcohol extractions is preferably 2-4 times, specifically 3 times; the volume fraction of the ethanol aqueous solution used in each extraction is preferably 80-95%, specifically 95%; the ratio of ethanol aqueous solution to green plums used in each extraction is preferably (3-6) L:2 kg, specifically 3.5 L:2 kg or 2 L:1 kg; the extraction time for each extraction is preferably 24-36 h, specifically 24 h. Specifically, after each extraction, the residue is filtered out before the next extraction. The residue filtered out after the final extraction is used as the alcohol extraction residue. The solutions obtained after filtering out the residue from each extraction are combined to form the alcohol extract. This invention uses a cold maceration extraction method for alcohol extraction, which can extract small molecule components with low polarity.

[0026] The present invention involves water extraction of the alcohol-extracted residue to obtain an aqueous extract.

[0027] In this invention, distilled water is preferably used for water extraction; the water extraction is preferably hot water extraction, and the number of hot water extractions is preferably 2 to 4 times, specifically 3 times; the method of each hot water extraction is independently preferred to be reflux extraction or boiling extraction, and the temperature of the reflux extraction is preferably 90 to 95°C; the mass of water used in each hot water extraction is preferably 2 to 5 times the mass of the material to be extracted, specifically 2.5 times or 3 times; the time of each hot water extraction is independently preferred to be 2 to 3 hours, specifically 2.5 hours. Specifically, after each hot water extraction, the residue is filtered out before the next hot water extraction, and the solutions obtained after filtering out the residue from each hot water extraction are combined as the water extract. This invention preferably uses reflux extraction or boiling extraction for hot water extraction, which can extract the water-soluble components in the alcohol-extracted residue as completely as possible.

[0028] The present invention concentrates the water extract and then performs deproteinization treatment to obtain a deproteinized water-soluble component.

[0029] In this invention, the concentration of the water extract is preferably vacuum concentration, and the vacuum concentration temperature is preferably 60~70℃, specifically 65℃; the concentration is preferably to concentrate the water extract to 10~20% of its original volume (i.e., the volume of the resulting concentrated water extract is 10~20% of the volume of the water extract).

[0030] In this invention, the deproteinization treatment preferably employs the Sevag method, and the number of deproteinization treatments is preferably 2 to 4 times, specifically 3 times; the Sevag reagent used in the Sevag method preferably includes chloroform and n-butanol, and the volume ratio of chloroform to n-butanol in each deproteinization treatment is preferably (2 to 5):1, specifically 4:1; the volume ratio of the Sevag reagent to the material to be deproteinized in each deproteinization treatment is preferably (1 to 2):1, specifically 1:1.

[0031] In this invention, the deproteinization process preferably further includes: reconcentrating the obtained deproteinized solution. The reconcentration is preferably vacuum concentration, and the vacuum concentration temperature is preferably 60-70°C, more preferably 60-65°C; the reconcentration is preferably to concentrate the deproteinized solution until no n-butanol is present.

[0032] In this invention, the deproteinized water-soluble component is separated by a macroporous adsorption resin column, eluted with water, and the resulting eluent is concentrated and mixed with a lower alcohol for a first alcohol precipitation. A first supernatant and a first precipitate are obtained, and the first precipitate is dried to obtain a first water-soluble polysaccharide alcohol-precipitated component. The volume fraction of the lower alcohol in the system during the first alcohol precipitation is 20-35%. The first supernatant was concentrated and then mixed with a lower alcohol for a second alcohol precipitation. The second supernatant and the second precipitate were separated. The second precipitate was dried to obtain the second water-soluble polysaccharide alcohol-precipitated component. The volume fraction of the lower alcohol in the system during the second alcohol precipitation was 55-65%. The second supernatant was concentrated and mixed with a lower alcohol for a third alcohol precipitation to obtain a third precipitate. The third precipitate was dried to obtain a third water-soluble polysaccharide alcohol precipitate component. The volume fraction of the lower alcohol in the system during the third alcohol precipitation was 75-85%.

[0033] In this invention, the macroporous adsorption resin column is preferably an AB-8 macroporous adsorption resin column. The separation using the macroporous adsorption resin column preferably employs a wet loading method, specifically, directly loading the deproteinized water-soluble components after significant water removal. This invention utilizes an isogradient water elution method to facilitate the separation of polysaccharide components from the medicinal and edible variety, plum.

[0034] The present invention does not have special requirements for the concentration of the water washing solution, as long as it can remove a large amount of water from the water washing solution (flux PMP). Specifically, the fluid PMP can be concentrated to 5-10% of its original volume (that is, the fluid PMP is concentrated to obtain a fluid PMP concentrate, the volume of which is 5-10% of the volume of the fluid PMP).

[0035] In this invention, the lower alcohol is preferably methanol or ethanol; the lower alcohol is preferably used in the form of an aqueous solution, specifically industrial alcohol. The volume fraction of the lower alcohol in the system during the first alcohol precipitation is preferably 25% or 30% (the resulting precipitated alcohol fraction is denoted as PMP-30%).

[0036] In this invention, the first alcohol precipitation is preferably performed 2 to 4 times, specifically 3 times; the volume ratio of the alcohol precipitation reagent to the material to be precipitated during each first alcohol precipitation is preferably (1.5 to 3):1, more preferably (1.5 to 2):1; the temperature of each alcohol precipitation is preferably 4 to 8°C, specifically 4°C; in this embodiment of the invention, the alcohol precipitation is specifically carried out in a refrigerator; the time for each alcohol precipitation is preferably 12 to 24 hours, more preferably 12 to 18 hours (standing overnight).

[0037] In this invention, the method for drying the first precipitate is preferably freeze drying.

[0038] In this invention, the volume fraction of the lower alcohol in the system during the second alcohol precipitation is preferably 60% (the resulting alcohol precipitation component is denoted as PMP-60%).

[0039] In this invention, the volume fraction of the lower alcohol in the system during the third alcohol precipitation is preferably 80% (the resulting alcohol precipitation component is denoted as PMP-80%).

[0040] In this invention, the conditions for concentrating the first and second supernatants are preferably the same as those for concentrating the water wash solution, and will not be repeated here. Except for the volume fraction of lower alcohols in the system, the conditions for the second and third alcohol precipitation are preferably the same as those for the first alcohol precipitation, and will not be repeated here.

[0041] In this invention, the preparation method of the alcohol-extracted polysaccharide component includes the following steps: The alcohol extract was concentrated and then subjected to deproteinization treatment to obtain a deproteinized alcohol-soluble component. The deproteinized alcohol-soluble component was separated by a macroporous adsorption resin column, eluted with water, and the resulting water eluent was dried to obtain the alcohol-extracted polysaccharide component (denoted as PMP-A).

[0042] In this invention, the alcohol extract is the alcohol extract obtained by extracting green plums using a low-grade alcohol-water solution. The deproteinization process preferably further includes: re-concentrating the obtained deproteinized solution. The re-concentration is preferably vacuum concentration, and the vacuum concentration temperature is preferably 60-70°C, more preferably 60-65°C; the re-concentration is preferably to concentrate the deproteinized solution until no n-butanol is present.

[0043] In this invention, the method of deproteinization is preferably the same as that used in the preparation of water-soluble polysaccharide fractionated alcohol precipitation components, and will not be described in detail here.

[0044] In this invention, the method of separation by macroporous adsorption resin column is preferably the same as that used in the preparation of water-soluble polysaccharide fractionated alcohol precipitation components, and will not be described in detail here.

[0045] In this invention, the drying process is preferably freeze-drying.

[0046] The present invention provides a plum polysaccharide component obtained by the preparation method described above, wherein the plum polysaccharide component includes a water-soluble polysaccharide fractionated by alcohol precipitation and / or an alcohol-soluble polysaccharide component.

[0047] This invention provides the application of the plum polysaccharide component described in the above technical solution in the preparation of drugs for the prevention and treatment of diabetes or kidney disease.

[0048] In this invention, the diabetes is preferably type II diabetes; the kidney disease preferably includes one or more of diabetic nephropathy, acute renal failure, chronic renal failure, nephrotic syndrome, and glomerulonephritis; the mechanism for preventing and treating diabetes or kidney disease is related to alleviating oxidative stress.

[0049] In this invention, the drug preferably comprises an active ingredient and pharmaceutically acceptable excipients. The active ingredient in the drug is the plum polysaccharide component, and the pharmaceutically acceptable excipients include pharmaceutically acceptable carriers and / or excipients. The content of the active ingredient in the drug is preferably 0.1~99.9 wt%, more preferably 1~90 wt%. This invention does not have special requirements for the pharmaceutically acceptable carrier; any pharmaceutically acceptable carrier well-known to those skilled in the art can be used. This invention also does not have special requirements for the pharmaceutically acceptable excipients; any pharmaceutically acceptable excipient well-known to those skilled in the art can be used, specifically including mannitol, magnesium stearate, starch, or cyclodextrin.

[0050] In this invention, the dosage form of the drug preferably includes an injection or an oral preparation. When the drug is used to prevent and treat diabetes, the effective dose of the drug is preferably 0.5~1 mg / kg, and the method of administration is preferably oral; when the drug is used to prevent and treat diabetic nephropathy, the effective dose of the drug is preferably 1 mg / kg, and the method of administration is preferably oral.

[0051] In Example 1 of this invention, the medicinal and edible variety of plum is first subjected to alcohol extraction to obtain an alcohol extract and alcohol-extracted residue. The alcohol-extracted residue is then subjected to water extraction and protein removal treatment, and placed in a macroporous adsorption resin column for elution with pure water to remove pigments and other low-polarity small molecule components, yielding a pure water eluted fraction, which is the crude polysaccharide of the water-soluble extract of the medicinal and edible plum. Then, the crude polysaccharide of the water-soluble extract of the medicinal and edible plum is further subjected to fractional alcohol precipitation to obtain a supernatant and alcohol precipitate. This further separates the active substances in the crude polysaccharide of the water-soluble extract of the medicinal and edible plum, yielding a first water-soluble polysaccharide alcohol-precipitated fraction (PMP-30%), a second water-soluble polysaccharide alcohol-precipitated fraction (PMP-60%), and a third water-soluble polysaccharide alcohol-precipitated fraction (PMP-80%).

[0052] In Example 2 of this invention, the medicinal and edible variety of plum is first subjected to alcohol extraction to obtain an alcohol extract. After protein removal treatment, the alcohol extract is placed in a macroporous adsorption resin column and eluted with pure water to remove pigments and other low-polarity small molecule components, resulting in a pure water eluted component, which is the alcohol-extracted polysaccharide of the medicinal and edible plum variety.

[0053] To further illustrate the present invention, the following detailed description of the plum polysaccharide components, their preparation methods, and applications provided by the present invention, in conjunction with the accompanying drawings and embodiments, is provided but should not be construed as limiting the scope of protection of the present invention.

[0054] In the embodiments of the present invention, the chloroform, n-butanol, and ethanol used are all industrial-grade reagents and are used after redistillation.

[0055] Example 1 The steps for preparing water-soluble polysaccharide fractions (water-soluble polysaccharide fractions) from green plums are as follows: Weigh 2 kg of ripe plums, break them open, and soak them in 3.5 L of 95% ethanol solution for 24 h. Repeat this process three times, and filter out the residue. Add 5 L of distilled water to the residue and heat at 95 °C for 3 h. Repeat this process three times to obtain an aqueous extract. Concentrate the aqueous extract under reduced pressure at 65 °C to obtain a concentrated aqueous extract. Remove protein from the concentrated aqueous extract using the Sevag method (chloroform to n-butanol volume ratio = 4:1), with the Sevag reagent volume ratio to the concentrated aqueous extract being 1:1. Concentrate the resulting aqueous solution under reduced pressure at 65 °C three times to obtain the supernatant.

[0056] After concentration of the supernatant under reduced pressure at 65 °C, it was separated by AB-8 macroporous resin column chromatography. The sample was loaded wet and eluted with pure water at a gradient to obtain the PMP fraction. The PMP fraction was then concentrated under reduced pressure at 65 °C to obtain the pure water elution fraction. 30% (v / v) ethanol was added to the pure water elution fraction for alcohol precipitation, and the mixture was allowed to stand overnight at 4 °C to obtain supernatant A and the first precipitate. Supernatant A was concentrated under reduced pressure at 65 °C and then further precipitated with 60% (v / v) ethanol, and allowed to stand overnight at 4 °C to obtain supernatant B and the second precipitate. Supernatant B was further concentrated under reduced pressure at 65 °C and then precipitated with 80% (v / v) ethanol to obtain the supernatant and the third precipitate. The three precipitates were washed three times with 95% ethanol solution and then freeze-dried to obtain water-soluble polysaccharide fractions of the medicinal and edible plum, which were designated as the first water-soluble polysaccharide fraction (PMP-30%), the second water-soluble polysaccharide fraction (PMP-60%), and the third water-soluble polysaccharide fraction (PMP-80%).

[0057] Example 2 The steps for preparing plum alcohol-soluble polysaccharide (a component of alcohol-soluble polysaccharide) from green plums are as follows: Weigh 2 kg of ripe plums, break them open, and soak them in 4 L of 95% ethanol solution for 24 h. Repeat this process three times, and filter out the residue. Concentrate the ethanol extract under reduced pressure at 65 °C to obtain a concentrated ethanol extract. Remove protein from the concentrated ethanol extract using the Sevag method (chloroform to n-butanol volume ratio = 4:1), with the Sevag reagent volume ratio to the concentrated ethanol extract being 1:1. Concentrate the resulting ethanol solution under reduced pressure at 65 °C, repeating this process three times to obtain the supernatant.

[0058] The supernatant was concentrated under reduced pressure at 65 °C, then separated by AB-8 macroporous resin column chromatography. The sample was loaded wet and eluted with pure water at a gradient to obtain a fraction. This fraction was concentrated under reduced pressure at 65 °C to obtain the pure water eluted fraction. The pure water eluted fraction was concentrated under reduced pressure at 65 °C and then freeze-dried to obtain the polysaccharide fraction (PMP-A) from the medicinal and edible variety, plum.

[0059] Test Example 1 Effects of water-soluble polysaccharides from the medicinal and edible variety *Prunus mume* fractionated by alcohol precipitation and alcohol-extracted polysaccharides on high-glucose-induced proliferation of rat glomerular mesangial cells 1. Establishing a high-glucose model experiment: Experimental Groups: Low-glucose control group (Control): Glu content was 5.5 mmol / L; High glucose control group (1~5): Glu content was 20, 25, 30, 35, and 40 mmol / L.

[0060] Cell culture time: 24 h, 36 h, 48 h, 60 h.

[0061] Experiment content: Cell viability was assessed using the CCK-8 assay. Cells were divided into 96-well plates according to the experimental groups described above, with 5 replicates per group. Cell concentration was 5 x 10⁶ cells per well. 4 100 μL of cell culture medium per 100 cells / mL was added and cultured for 24 h. The old culture medium was discarded, and fresh culture medium was added according to the experimental groups. After 24 h, 36 h, 48 h, and 60 h of culture, the culture plates were removed, and 10 μL of CCK-8 reagent was added to each well. The plates were then incubated in a CO2 incubator for 1 h. The absorbance (OD) was measured using a microplate reader set to 450 nm. Cell viability was used to determine the proliferation of rat glomerular mesangial cells at different time points and in cultures with different glucose concentrations. Cell viability was calculated using the following formula: Cell viability = [OD(drug-doped) - OD(blank)] / [OD(control) - OD(blank)].

[0062] Figure 1 Figure 1 shows the proliferation of rat glomerular mesangial cells in high-glucose culture media at different times and concentrations; Data represent mean ± SEM, n = 3. p <0.05, p <0.01, p <0.001 vs Control. The results showed that, within 24–60 h, high glucose concentrations in the culture medium promoted cell proliferation, resulting in cells in a proliferative state. The strongest cell growth promotion effect was observed when HBZY-1 cells were cultured for 36 h at a glucose concentration of 25 mmol / L. Therefore, the optimal glucose concentration for high glucose-induced HBZY-1 cell growth is 25 mmol / L, and the optimal culture time is 36 h.

[0063] 2. Effects of polysaccharide fractionated alcohol precipitation on normal rat glomerular mesangial cells: Experimental Groups: Control group: No cells were seeded, and the medium contained low glucose (5.5 mmol / L Glu). Control group: Cells were seeded and cultured in a low-glucose complete medium (5.5 mmol / L Glu). Drug group: Cells were seeded and the four plum polysaccharide components were dissolved in low-sugar complete culture medium to a concentration of 0.125 mg / mL, 0.25 mg / mL, 0.5 mg / mL, 1 mg / mL, and 2 mg / mL.

[0064] Experiment content: Cell viability was assessed using the CCK-8 assay. Cells were divided into 96-well plates according to the experimental groups described above, with 5 replicates per group. Cell concentration was 5 x 10⁶ cells per well. 4 100 μL of cell culture medium (cells / mL) was added and cultured for 24 h. The old culture medium was discarded, and fresh culture medium was added according to the experimental groups. After 48 h of culture, the culture plate was removed, and 10 μL of CCK-8 reagent was added to each well. The plate was then incubated in a CO2 incubator for 1 h. The absorbance (OD) was measured using a microplate reader set to 450 nm. Cell viability was used to determine whether different alcohol-precipitated components and different concentrations of plum polysaccharide were toxic to normal HBZY-1 cells. Cell viability was calculated using the following formula: Cell viability = [OD(drug-doped) - OD(blank)] / [OD(control) - OD(blank)].

[0065] Figure 2 Figure showing the effects of different ethanol-precipitated and ethanol-extracted components of plum polysaccharides on the cell viability of normal rat glomerular mesangial cells; Data represent mean ± SEM, n = 3. p <0.05, p <0.01 vs Control. The results showed that the polysaccharide fraction extracted from green plum and the fractionated alcohol-precipitated water-soluble polysaccharide fraction from green plum had no inhibitory effect on normal HBZY-1 cells within the set concentration range (Cell Viability>IC50), indicating that the set concentration range can be used for the next activity test.

[0066] 3. Effects of polysaccharide fractions and alcohol-extracted fractions on high-glucose-induced proliferation of rat glomerular mesangial cells: Experimental Groups: Control group: No cells were seeded, and the medium contained low glucose (5.5 mmol / L Glu). Low glucose control group (NG): Cells were seeded and cultured in low glucose complete medium (5.5 mmol / L Glu). High glucose control group (HG): Cells were seeded and cultured in high glucose complete medium (25 mmol / L Glu). Drug group: Cells were seeded and the four polysaccharide precipitates of plum were dissolved in high-glucose culture medium to a concentration of 0.125, 0.25 mg / mL, 0.5 mg / mL, 1 mg / mL and 2 mg / mL.

[0067] Experiment content: Cell viability was assessed using the CCK-8 assay. Cells were divided into 96-well plates according to the experimental groups described above, with 5 replicates per group. Cell concentration was 5 x 10⁶ cells per well. 4 100 μL of cell culture medium per cell / mL was added and cultured for 24 h. The old culture medium was discarded, and fresh culture medium was added according to the experimental groups. After 48 h of culture, the culture plate was removed, and 10 μL of CCK-8 reagent was added to each well. The plate was then incubated in a CO2 incubator for 1 h. The absorbance (OD) was measured using a microplate reader set to 450 nm. Cell viability was used to determine whether different alcohol-precipitated components and different concentrations of plum polysaccharide inhibited the proliferation of high-glucose-induced HBZY-1 cells. Cell viability was calculated using the following formula: Cell viability = [OD(drug-doped) - OD(blank)] / [OD(HG) - OD(blank)].

[0068] Figure 3 Figure showing the inhibitory effects of different alcohol-precipitated and alcohol-extracted components of plum polysaccharides on high glucose-induced proliferation of rat glomerular mesangial cells; Data represent mean ± SEM, n = 3. p <0.05, p <0.01 vs HG. ## p <0.01 vs NG. The results showed that the four plum polysaccharide components inhibited the proliferation of high-glucose-induced HBZY-1 cells. Among them, PMP-A, PMP-30%, PMP-60%, and PMP-80% significantly inhibited the proliferation of high-glucose-induced HBZY-1 cells in a concentration range of 0.125–2 mg / mL, showing a concentration-dependent effect, with PMP-80% exhibiting the strongest inhibitory effect. All four polysaccharide components were non-toxic and did not reach the IC50 threshold. 50 While the values ​​were not statistically significant, the 80% and PMP-A components showed lower toxicity to normal HBZY-1 cells and better inhibition of abnormal proliferation due to high glucose levels compared to the 60% and 30% components. Therefore, these two components were chosen for subsequent experiments.

[0069] 4. Effects of 80% alcohol-precipitated polysaccharide fraction and alcohol-extracted polysaccharide fraction PMP-A on SOD, MDA, and ROS levels in high glucose-induced rat glomerular mesangial cells: Experimental Groups: Low glucose control group (NG): Cells were seeded and cultured in low glucose complete medium (5.5 mmol / L Glu). High glucose control group (HG): Cells were seeded and cultured in high glucose complete medium (25 mmol / L Glu). Positive control group (nitrogen oxide free radical piperidine alcohol, Tempol): cells were seeded and Tempol was dissolved in high glucose complete medium (25 mmol / L Glu) to a concentration of 100 μmol / L; Drug group: Cells were seeded and two plum polysaccharide components were dissolved in high-glucose culture medium to make their concentrations 0.125, 0.25, 0.5, 0.75 and 1 times their IC50 values.

[0070] Experiment content: SOD and MDA content determination Divide the cells into 12-well plates according to the experimental groups described above, with 3 replicates per group. Add cells to each well at a concentration of 1.5 × 10⁻⁶ cells. 5 2 mL of cell culture medium was added to each cell group (cells / mL), and the cells were cultured for 24 h. The old culture medium was discarded, and fresh culture medium was added according to the experimental groups. After culturing for another 36 h, the cells were collected into 1.5 mL centrifuge tubes. The cells were then sonicated for 3 seconds each time, followed by a 10-second interval and another 3-second sonication, and this process was repeated 30 times. After sonication, the cells were placed in a high-speed centrifuge at 8000 rpm and 4 °C for 10 min. The supernatant was collected and placed on ice for analysis.

[0071] Following the SOD kit instructions, reagents were added sequentially, and the reaction was carried out at 37 °C for 30 min. The absorbance (A) of each group was measured at 560 nm, and the results were calculated. A measurement = A measurement - A control A blank = A1 blank - A2 blank. The inhibition percentage is calculated using the following formula: Inhibition percentage = ( A blank - (A measurement) ÷ A blank × 100%; SOD activity is calculated using the following formula: SOD activity (U / mg prot) = [Inhibition percentage ÷ (1 - Inhibition percentage) × V] 反总]÷1mL÷(N×Vsample÷Vtotalsample)×F=10×inhibitionpercentage÷(1-inhibitionpercentage)×F÷N; In the formula, N is the number of cells and F is the dilution factor.

[0072] Add the appropriate reagents sequentially according to the MDA kit. Set the water bath temperature to 100 °C and incubate for 60 min. After incubation, remove the plate and cool it in ice water. Centrifuge at 10000 g at room temperature for 10 min. Divide the 96-well plates according to the experimental groups, with 3 replicates per group. Add 200 μL of supernatant to each well. Set the microplate reader to wavelengths of 532 nm and 600 nm, and measure the absorbance (A) for each group. Calculate the absorbance values. A532 = A532 determination - A532 blank. A600 = A600 measurement - A600 blank, and calculated according to the following formula. A: A = A532- A600; Calculate the MDA content using the following formula: MDA content (nmol / mg prot) = 53.763 × A÷N 细胞数量 ×F 稀释倍数 .

[0073] ROS content determination Divide the cells into 12-well plates according to the experimental groups described above, with 3 replicates per group. Add cells to each well at a concentration of 1.5 x 10⁻⁶ cells. 5 Incubate 2 mL of cell culture medium (cells / mL) for 24 h, discard the old medium, add fresh medium according to the experimental groups, and incubate for another 36 h. Collect cells into 1.5 mL centrifuge tubes, add PBS, and disperse thoroughly. Load probes according to the ROS kit instructions. Add the probe-loaded cell suspension to a confocal microscopy glass dish, allow it to adhere completely, and observe directly using a laser confocal microscope. Perform three replicates per group, and obtain three fields of view for each sample.

[0074] ROS values ​​were observed directly using a laser confocal microscope, and the average fluorescence intensity was measured using ImageJ plotting software.

[0075] Figures 4-6 The graph shows the effects of 80% polysaccharide precipitate and ethanol extract from green plum on the levels of SOD, MDA, and ROS in high-glucose-induced rat glomerular mesangial cells. (Data represent mean ± SEM, n = 3) p <0.05, p <0.01 vs HG. ## p <0.01 vs NG. The results showed that both plum polysaccharide components increased the SOD content and decreased the MDA and ROS content in HBZY-1 cells induced by high sugar. This indicates that the 80% alcohol-precipitated and alcohol-extracted polysaccharide components of the water-soluble polysaccharide from the medicinal and edible plum variety can alleviate intracellular oxidative stress levels, thereby preventing and treating diabetes and kidney disease, and can be used to prepare drugs or health products for the prevention and treatment of diabetes and kidney disease.

[0076] This invention provides a method for preparing polysaccharide components from a medicinal and edible plum variety. First, the medicinal and edible plum variety is subjected to ethanol extraction to obtain an ethanol extract and ethanol extract residue. The ethanol extract residue is then extracted with water to obtain an aqueous extract. After removing proteins, both the aqueous and ethanol extracts are placed in a macroporous adsorption resin column and eluted with pure water to remove pigments and other low-polarity small molecule components, yielding two pure water eluted fractions. The ethanol extract eluted with pure water yields the medicinal and edible plum ethanol-extracted polysaccharide component (PMP-A). The pure water eluted fraction of the aqueous extract is further subjected to fractional ethanol precipitation to obtain three different ethanol-precipitated fractions of the medicinal and edible plum variety: a first ethanol-precipitated fraction of the medicinal and edible plum variety (PMP-30%), a second ethanol-precipitated fraction of the medicinal and edible plum variety (PMP-60%), and a third ethanol-precipitated fraction of the medicinal and edible plum variety (PMP-80%). In this embodiment of the invention, rat glomerular mesangial cells were screened for activity using high-glucose-induced glomerular mesangial cells. The results showed that the alcohol-precipitated components of the medicinal and edible plum polysaccharide provided by this invention had a significant protective effect on high-glucose-induced rat glomerular mesangial cells, increasing the SOD content and decreasing the MDA and ROS content in the model cells. This indicates that the alcohol-precipitated components of the medicinal and edible plum polysaccharide can alleviate intracellular oxidative stress levels, thereby preventing and treating diabetes and nephropathy. It can be used to prepare drugs or health products for the prevention and treatment of diabetes and nephropathy. Furthermore, toxicity tests confirmed that the plum polysaccharide has high safety, ensuring the safety of long-term use by patients with diabetes and nephropathy, and has good development and application prospects.

[0077] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, not all embodiments. People can obtain other embodiments based on the present invention without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for preparing a plum polysaccharide component, wherein the plum polysaccharide component comprises a water-soluble polysaccharide fractionated by alcohol precipitation and / or an alcohol-extracted polysaccharide component, characterized in that, The preparation method of the alcohol-extracted polysaccharide component includes the following steps: The green plum was extracted with a low-grade alcohol-water solution to obtain alcohol-extracted residue and alcohol extract. The alcohol extract was concentrated and then subjected to deproteinization treatment to obtain a deproteinized alcohol-soluble component. The deproteinized alcohol-soluble component was separated by a macroporous adsorption resin column, eluted with water, and the resulting water eluent was dried to obtain the alcohol-extracted polysaccharide component. The water-soluble polysaccharide fractionated alcohol precipitation component includes one or more of a first water-soluble polysaccharide alcohol precipitation component, a second water-soluble polysaccharide alcohol precipitation component, and a third water-soluble polysaccharide alcohol precipitation component, and the preparation method includes the following steps: The alcohol-extracted residue was subjected to water extraction to obtain an aqueous extract; The aqueous extract was concentrated and then subjected to deproteinization treatment to obtain a deproteinized water-soluble component. The deproteinized water-soluble component was separated by a macroporous adsorption resin column and eluted with water. The resulting water eluent was concentrated and mixed with a lower alcohol for a first alcohol precipitation. A first supernatant and a first precipitate were separated. The first precipitate was dried to obtain the first water-soluble polysaccharide alcohol-precipitated component. The volume fraction of the lower alcohol in the system during the first alcohol precipitation was 20-35%. The first supernatant was concentrated and then mixed with a lower alcohol for a second alcohol precipitation. The second supernatant and the second precipitate were separated. The second precipitate was dried to obtain the second water-soluble polysaccharide alcohol-precipitated component. The volume fraction of the lower alcohol in the system during the second alcohol precipitation was 55-65%. The second supernatant was concentrated and mixed with a lower alcohol for a third alcohol precipitation to obtain a third precipitate. The third precipitate was dried to obtain a third water-soluble polysaccharide alcohol precipitate component. The volume fraction of the lower alcohol in the system during the third alcohol precipitation was 75-85%.

2. The preparation method according to claim 1, characterized in that, The volume fraction of the lower alcohol in the aqueous solution is 40-95%; the aqueous solution of the lower alcohol is an aqueous solution of ethanol.

3. The preparation method according to claim 1, characterized in that, The water extraction is a hot water extraction, the temperature of which is 90~95℃, and the number of hot water extractions is 2~4 times.

4. The preparation method according to claim 1, characterized in that, The deproteinization treatment of the concentrated alcohol extract and the deproteinization treatment of the concentrated water extract both adopt the Sevag method. The Sevag reagent used in the Sevag method includes chloroform and n-butanol, and the volume ratio of chloroform to n-butanol is (2~5):

1.

5. The preparation method according to claim 1, characterized in that, The macroporous adsorption resin column used for separating the deproteinized alcohol-soluble component and the deproteinized water-soluble component is an AB-8 macroporous adsorption resin column.

6. The preparation method according to claim 1, characterized in that, The lower alcohols used in the first, second, and third alcohol precipitations are all ethanol.

7. The preparation method according to claim 1 or 6, characterized in that, The volume fraction of lower alcohols in the first alcohol precipitation system is 25-30%, the volume fraction of lower alcohols in the second alcohol precipitation system is 60%, and the volume fraction of lower alcohols in the third alcohol precipitation system is 80%.

8. The plum polysaccharide component obtained by the preparation method according to any one of claims 1 to 7, wherein the plum polysaccharide component comprises a water-soluble polysaccharide graded alcohol precipitation component and / or an alcohol-soluble polysaccharide component.

9. The use of the plum polysaccharide component according to claim 8 in the preparation of a drug for the prevention and treatment of diabetes or kidney disease.

10. The application according to claim 9, characterized in that, The diabetes mellitus is type II diabetes mellitus; the kidney disease includes one or more of the following: diabetic nephropathy, acute renal failure, chronic renal failure, nephrotic syndrome, and glomerulonephritis.

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