A novel polysaccharide extracted from dried plum, its preparation method and uses

Abstract

This invention provides a novel polysaccharide extracted from dried plum (Prunus mume), its preparation method, and its uses. The monosaccharide molar fraction in the Prunus mume polysaccharide is as follows: 0.5%-1.0 mol% fucose, 1.0%-2.0 mol% rhamnose, 35%-37 mol% arabinose, 15%-17 mol% galactose, 9.0%-11 mol% glucose, 2.0%-3.0 mol% xylose, 6%-8 mol% mannose, 23%-25 mol% galacturonic acid, and 1.0%-2.0 mol% glucuronic acid. The Prunus mume polysaccharide isolated and purified by this invention exhibits excellent antioxidant activity. Furthermore, based on the complex acidic heteropolysaccharide structure of the Prunus mume polysaccharide, especially its high proportion of uronic acid and branched structure, it possesses potential for various biological activities and has extremely high industrial utilization value.

Description

Technical Field

[0001] This invention relates to the field of polysaccharide preparation technology, and in particular to a novel polysaccharide extracted from dried plum, its preparation method, and its uses. Background Technology

[0002] Ume (dried plum) is the nearly mature fruit of the plum tree (Prunus mume), a plant belonging to the genus Prunus in the Rosaceae family. It is produced through low-temperature drying or smoking and is a traditional Chinese medicinal material that is both food and medicine. In traditional Chinese medicine theory, ume is neutral in nature, sour and astringent in taste, and enters the liver, spleen, lung, and large intestine meridians. It has traditional effects such as astringing the lungs and relieving cough, astringing the intestines and stopping diarrhea, promoting body fluid production and quenching thirst, and expelling roundworms and relieving pain. In clinical practice, it is often used to treat chronic cough due to lung deficiency, chronic diarrhea and dysentery, thirst due to deficiency heat, and vomiting due to roundworm infestation.

[0003] Modern pharmacological studies have shown that the active components of dried plum (Prunus mume) are complex and diverse, mainly including organic acids (such as citric acid and malic acid), flavonoids, phenylpropanoids, terpenes, and polysaccharides. Among them, organic acids are considered to be important material basis for the thirst-quenching, antibacterial, and anti-inflammatory effects of dried plum, while flavonoids have antioxidant activity. However, systematic research on its polysaccharide components is relatively limited and insufficient. Existing literature reports mostly on crude extracts or preliminarily purified fractions of dried plum polysaccharides, without refining and purifying them. Furthermore, there is a lack of analysis of their fine chemical structures (such as monosaccharide composition, glycosidic bond linkage, and spatial conformation), and systematic characterization of their physicochemical properties such as molecular weight distribution, spatial morphology, and crystallization characteristics is also inadequate. This lack of clear structure and ambiguous mechanism of action severely restricts the in-depth development and application of dried plum polysaccharides as functional factors or drug lead compounds.

[0004] Inflammation and oxidative stress are important pathophysiological processes in the body, interconnected and mutually causal, and are key factors leading to and exacerbating many chronic diseases. Excessive or uncontrolled inflammatory responses and oxidative damage are closely related to the development and progression of metabolic diseases (such as atherosclerosis and diabetes), neurodegenerative diseases (such as Alzheimer's disease), autoimmune diseases, tumors, and fibrotic lesions in multiple organs. Currently used anti-inflammatory drugs (such as nonsteroidal anti-inflammatory drugs and glucocorticoids) and synthetic antioxidants have significant side effects and poor long-term safety. Therefore, finding and developing novel natural products with high efficiency, low toxicity, and antioxidant and anti-inflammatory activities from natural resources has become an important direction for current drug development and functional food development. Summary of the Invention

[0005] In view of the shortcomings of the prior art described above, the purpose of this invention is to provide a novel polysaccharide extracted from dried plum, its preparation method and uses, to solve the problem that the prior art cannot effectively refine and purify dried plum polysaccharides, which limits the in-depth development and application of dried plum polysaccharides.

[0006] To achieve the above and other related objectives, the present invention provides a novel polysaccharide extracted from dried plum, its preparation method, and its uses.

[0007] The first aspect of this invention provides a plum polysaccharide, wherein the monosaccharide molar fraction is: 0.5%-1.0 mol% fucose, 1.0%-2.0 mol% rhamnose, 35%-37 mol% arabinose, 15%-17 mol% galactose, 9.0%-11 mol% glucose, 2.0%-3.0 mol% xylose, 6%-8 mol% mannose, 23%-25 mol% galacturonic acid, and 1.0%-2.0 mol% glucuronic acid.

[0008] Preferably, the monosaccharide molar fraction in the plum polysaccharide is: 0.7%-0.8 mol% fucose, 1.4%-1.5 mol% rhamnose, 36%-36.5 mol% arabinose, 16%-16.5 mol% galactose, 9.5%-10 mol% glucose, 2.5%-3.0 mol% xylose, 6.5%-7.0 mol% mannose, 24.5%-25 mol% galacturonic acid, and 1.0%-1.5 mol% glucuronic acid.

[0009] More preferably, the monosaccharide molar fraction in the plum polysaccharide is: 0.76 mol% fucose, 1.42 mol% rhamnose, 36.39 mol% arabinose, 16.1 mol% galactose, 9.8 mol% glucose, 2.81 mol% xylose, 6.95 mol% mannose, 24.66 mol% galacturonic acid, and 1.1 mol% glucuronic acid.

[0010] Preferably, the weight-average molecular weight of the plum polysaccharide is 20-30 kDa; for example, it can be 20 kDa, 22 kDa, 24 kDa, 26 kDa, 28 kDa, or 30 kDa.

[0011] More preferably, the weight-average molecular weight of the plum polysaccharide is 20-25 kDa.

[0012] Preferably, the polydispersity index of the plum polysaccharide is <1.25.

[0013] More preferably, the polydispersity index of the plum polysaccharide is 1-1.2.

[0014] Most preferably, the polydispersity index of the plum polysaccharide is 1.1-1.2.

[0015] Preferably, the glycosidic bond linkages in the plum polysaccharide include: 15%-18 mol% t-Araf, 3.0%-5.0 mol% 3-Araf, 2.0%-4.0 mol% t-Galp-UA, 2.0%-4.0 mol% t-Galp, 6.0%-8.0 mol% 5-Araf, 2.0%-3.0 mol% 4-Manp, 25%-30 mol% 4-Galp-UA, 20%-23 mol% 4-Galp, 3.0%-5.0 mol% 4-Glcp, 1.0%-3.0 mol% 2,5-Araf, 4.0%-6.0 mol% 2,3,5-Araf, and 2.0%-4.0 mol% 2,3,4-Arap.

[0016] More preferably, the glycosidic bond linkages in the plum polysaccharide include: 16%-17 mol% t-Araf, 3.5%-4.0 mol% 3-Araf, 3.0%-3.5 mol% t-Galp-UA, 3.0%-3.5 mol% t-Galp, 7.0%-7.5 mol% 5-Araf, 2.5%-3.0 mol% 4-Manp, 27%-28 mol% 4-Galp-UA, 20.5%-21.5 mol% 4-Galp, 4.5%-5.0 mol% 4-Glcp, 1.5%-2.0 mol% 2,5-Araf, 5.0%-5.5 mol% 2,3,5-Araf, and 3.0%-3.5 mol% 2,3,4-Arap.

[0017] More preferably, the glycosidic bond linkages in the plum polysaccharide include: 16.85 mol% t-Araf, 3.77 mol% 3-Araf, 3.12 mol% t-Galp-UA, 3.14 mol% t-Galp, 7.33 mol% 5-Araf, 2.61 mol% 4-Manp, 27.4 mol% 4-Galp-UA, 21 mol% 4-Galp, 4.59 mol% 4-Glcp, 1.93 mol% 2,5-Araf, 5.02 mol% 2,3,5-Araf, and 3.24 mol% 2,3,4-Arap.

[0018] The above t-Araf represents a terminal arabinofuranose residue, 3-Araf represents a linkage position at the 3rd carbon atom of the arabinofuranose, t-Galp-UA represents a terminal galacturonic acid residue, t-Galp represents a terminal galactopyranose residue, 5-Araf represents a linkage position at the 5th carbon atom of the arabinofuranose, 4-Manp represents a linkage position at the 4th carbon atom of the mannopyranose, and 4-Galp-UA represents a linkage position at the galacturonic acid pyranose. At the 4th carbon atom of the sugar, 4-Galp represents the linkage position at the 4th carbon atom of galactopyranose, 4-Glcp represents the linkage position at the 4th carbon atom of glucopyranose, 2,5-Araf represents the linkage position at the 2nd and 5th carbon atom of arabinopyranose, 2,3,5-Araf represents the linkage position at the 2nd, 3rd, and 5th carbon atom of arabinopyranose, and 2,3,4-Arap represents the linkage position at the 2nd, 3rd, and 4th carbon atom of arabinopyranose.

[0019] Preferably, the plum polysaccharide comprises the following structural fragments:

[0020] .

[0021] The above structural fragment indicates that the plum polysaccharide includes a main chain and two branches. On the main chain, the C4 of mannopyranose is connected to the C1 of the preceding sugar; the C1 (α-configuration) of mannopyranose is connected to the C4 of D-glucopyranose; the C1 (β-configuration) of D-glucopyranose is connected to the C4 of D-galacturonic acid pyranose; the C1 (α-configuration) of D-galacturonic acid pyranose is connected to the C4 of D-galacturonic acid pyranose; the C1 (α-configuration) of D-galacturonic acid pyranose is connected to the C4 of D-galacturonic acid pyranose; the C1 (α-configuration) of D-galacturonic acid pyranose is connected to the C4 of D-galacturonic acid pyranose; and the C1 (α-configuration) of D-galacturonic acid pyranose is connected to the C3 of α-L-arabinofuranose. The α-L-arabinofuranose in the main chain is connected via 2... The 5th position is connected to two branches; on the first branch, the C1 (α configuration) of L-arabinofuranose is connected to the C3 of L-arabinofuranose, the C1 (α configuration) of L-arabinofuranose is connected to the C5 of L-arabinofuranose, and the C1 (α configuration) of L-arabinofuranose is connected to the C5 of α-L-arabinofuranose in the main chain; on the second branch, the C1 (α configuration) of L-arabinofuranose is connected to the C4 of D-galactopyranose, the C1 (β configuration) of D-galacturonic acid pyranose is connected to the C4 of D-galacturonic acid pyranose, and the C1 (β configuration) of D-galacturonic acid pyranose is connected to the C2 of α-L-arabinofuranose in the main chain.

[0022] The second aspect of the present invention provides a method for preparing the above-mentioned plum polysaccharide, the method comprising: treating plum fruit with alcohol to defatt the precipitate, extracting the precipitate with water to obtain an extract, and precipitating the extract with alcohol to obtain crude plum polysaccharide; subjecting the crude plum polysaccharide to protein removal, defatting, and decolorization sequentially to obtain purified plum polysaccharide; subjecting the purified plum polysaccharide to ion exchange column chromatography to obtain first purified plum polysaccharide; and subjecting the first purified plum polysaccharide to gel filtration column chromatography to obtain finally purified plum polysaccharide.

[0023] Preferably, the alcohol treatment is performed by immersion in ethanol.

[0024] More preferably, the alcohol treatment is performed using anhydrous ethanol.

[0025] Preferably, the solid-liquid ratio of plum fruit to alcohol during the alcohol treatment is 50-150 g / L; for example, it can be 50 g / L, 80 g / L, 100 g / L, 120 g / L, or 150 g / L.

[0026] More preferably, the solid-liquid ratio of plum fruit to alcohol during the alcohol treatment is 80-120 g / L.

[0027] Preferably, the solid-liquid ratio of the precipitate to water during water extraction is 20-80 g / L; for example, it can be 20 g / L, 30 g / L, 40 g / L, 50 g / L, 60 g / L, 70 g / L, or 80 g / L.

[0028] More preferably, the solid-liquid ratio of the precipitate to water during water extraction is 40-60 g / L.

[0029] Preferably, the water extraction temperature is 50-80℃; for example, it can be 50℃, 60℃, 70℃, or 80℃.

[0030] Preferably, the water extraction time is 2-5 hours; for example, it can be 2 hours, 3 hours, 4 hours, or 5 hours.

[0031] Preferably, the preparation method further includes combining the extracts after water extraction and concentrating the extracts.

[0032] Preferably, the concentration of ethanol in the extract and the total ethanol solution during the alcohol precipitation is 70%-90%; for example, it can be 70%, 80%, or 90%.

[0033] Preferably, the temperature during alcohol precipitation is 1-10℃; for example, it can be 1℃, 2℃, 4℃, 6℃, 8℃, or 10℃.

[0034] More preferably, the temperature during alcohol precipitation is 2-6°C.

[0035] In this invention, the crude polysaccharide of dried plum is first dissolved in water, and then subsequent processes such as protein removal, defatting, and decolorization are carried out.

[0036] Preferably, the protein removal process includes adding protease to the crude polysaccharide of dried plum for enzymatic hydrolysis, and then adding Sevag reagent to remove protein residue.

[0037] More preferably, the protein removal process involves dissolving crude polysaccharide from dried plums in water and then adding protease for enzymatic hydrolysis.

[0038] More preferably, the mass-to-volume ratio of the crude polysaccharide of dried plum to water is 10-20 g / L; for example, it can be 10 g / L, 11 g / L, 12 g / L, 13 g / L, 14 g / L, 15 g / L, 16 g / L, 17 g / L, 18 g / L, 19 g / L, or 20 g / L.

[0039] More preferably, the protease includes any one or both of papain and complex protease.

[0040] More preferably, the ratio of the protease to the crude polysaccharide of dried plum is 1:50-80.

[0041] More preferably, the Sevag test solution is a solution of chloroform and n-butanol in a 4:1 ratio.

[0042] The Sevag solution needs to be added to the reaction in multiple cycles until no protein precipitation occurs.

[0043] Preferably, the defatting is performed by adding petroleum ether to the polysaccharide solution after protein removal for extraction and defatting.

[0044] More preferably, during the defatting process, the amount of petroleum ether added is 10-20 mL, based on 1 g of crude plum polysaccharide.

[0045] Preferably, the decolorization is performed by adding macroporous resin to the defatted polysaccharide solution.

[0046] More preferably, the macroporous resin is AB-8 type macroporous resin.

[0047] More preferably, in the decolorization process, based on 1g of crude polysaccharide from dried plum, the amount of macroporous resin added is 20-50mL.

[0048] More preferably, the preparation method further includes dialysis of the filtrate after decolorization, and freeze-drying the liquid in the dialysis bag after dialysis.

[0049] Preferably, the ion exchange column is an anion exchange column.

[0050] More preferably, the functional group of the ion exchange column is DEAE-diethylaminoethyl.

[0051] More preferably, the matrix of the ion exchange column is agarose.

[0052] Preferably, the eluent for the ion exchange column chromatography comprises water and a 0.1-0.5M aqueous solution of NaCl.

[0053] More preferably, the eluent for the ion exchange column chromatography includes water, 0.1 M NaCl aqueous solution, 0.2 M NaCl aqueous solution, and 0.3 M NaCl aqueous solution.

[0054] Preferably, the elution flow rate of the ion exchange column chromatography is 1-6 mL / min; for example, it can be 1 mL / min, 2 mL / min, 3 mL / min, 4 mL / min, 5 mL / min, or 6 mL / min.

[0055] More preferably, the elution flow rate of the ion exchange column chromatography is 3-5 mL / min.

[0056] More preferably, the preparation method further includes dialysis of the eluent after ion exchange column chromatography, and freeze-drying the liquid in the dialysis bag after dialysis to obtain the first purified plum polysaccharide.

[0057] Preferably, the gel filter column is a dextran-crosslinked acrylamide composite gel column.

[0058] Preferably, the eluent for the gel filtration column chromatography is water.

[0059] Preferably, the elution flow rate of the gel filtration column chromatography is 0.5-5 mL / min; for example, it can be 0.5 mL / min, 1 mL / min, 2 mL / min, 3 mL / min, 4 mL / min, or 5 mL / min.

[0060] A third aspect of the present invention provides the use of the above-mentioned plum polysaccharide in the preparation of products for antioxidant, immunomodulatory and / or anti-inflammatory purposes.

[0061] A fourth aspect of the present invention provides an antioxidant, immunomodulatory, and / or anti-inflammatory product, wherein the active ingredient of the antioxidant product includes the aforementioned plum polysaccharide.

[0062] As described above, the novel polysaccharide extracted from dried plum, its preparation method, and its uses, according to the present invention, have the following beneficial effects:

[0063] (1) This invention uses water extraction and alcohol precipitation, protein removal, defatting and decolorization and other impurity removal processes, and multi-step chromatography purification processes such as ion exchange column chromatography and gel filtration column chromatography to obtain a novel polysaccharide of plum with narrow molecular weight distribution and uniform structure. Its physicochemical properties and structure were characterized, laying the foundation for subsequent activity research and quality standard establishment.

[0064] (2) The preparation method of this invention has clear steps and well-defined parameters, which can achieve the stable preparation of the novel polysaccharide of dried plum.

[0065] (3) The polysaccharide obtained by the preparation method of the present invention is a natural product with better biocompatibility and safety than chemical drugs. Studies have shown that it has excellent antioxidant activity. In addition, based on the complex acidic heteropolysaccharide structure of polysaccharide, especially the high proportion of uronic acid and branched structure, it has potential biological activities, such as potential application value in immune regulation, anti-tumor, and anti-inflammatory. Attached Figure Description

[0066] Figure 1 The image shown is the elution profile of the DEAE Seplife FF anion exchange column used for the purification of ume polysaccharides in this invention.

[0067] Figure 2 The image shown is the elution diagram of Sephacryl S-400 HR gel column for the purification of ume polysaccharide in this invention.

[0068] Figure 3 The image shown is a scanning electron microscope image of the plum polysaccharide in this invention.

[0069] Figure 4 The image shown is an XRD diffraction image of the plum polysaccharide in this invention.

[0070] Figure 5 The image shown is the UV-Vis spectrum of the plum polysaccharide in this invention.

[0071] Figure 6 The image shown is an infrared spectrum of the plum polysaccharide from this invention.

[0072] Figure 7 The image shown is a chromatographic result of the detection of polysaccharides from dried plums in this invention.

[0073] Figure 8 The image shown is a GC-MS detection result of the polysaccharide in dried plum in this invention.

[0074] Figure 9 The image shows the nuclear magnetic resonance (NMR) of the polysaccharide ume in this invention. 1 H-NMR spectrum.

[0075] Figure 10The image shows the nuclear magnetic resonance (NMR) of the polysaccharide ume in this invention. 13 C-NMR spectrum.

[0076] Figure 11 The image shown is the COSY NMR spectrum of the plum polysaccharide in this invention.

[0077] Figure 12 The image shown is the NOESY NMR spectrum of the polysaccharide from the plum in this invention.

[0078] Figure 13 The image shown is the HSQC nuclear magnetic resonance spectrum of the plum polysaccharide in this invention.

[0079] Figure 14 The image shown is the nuclear magnetic resonance (HMBC) spectrum of the plum polysaccharide in this invention.

[0080] Figure 15 The image shown is the DEPT135 nuclear magnetic resonance spectrum of the plum polysaccharide in this invention.

[0081] Figure 16 The image shown is the TOCSY NMR spectrum of the polysaccharide from the plum in this invention.

[0082] Figure 17 The results shown are from the DPPH free radical scavenging experiment of the polysaccharide in plum in this invention. Detailed Implementation

[0083] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0084] It should be noted that the process equipment or apparatus not specifically mentioned in the following embodiments are all conventional equipment or apparatus in the art.

[0085] Furthermore, it should be understood that the existence of other method steps before or after the combined steps, or the insertion of other method steps between these explicitly mentioned steps, does not preclude the presence of other method steps before or after the combined steps, unless otherwise stated. It should also be understood that the combined connection relationship between one or more devices / apparatus mentioned in this invention does not preclude the existence of other devices / apparatus before or after the combined devices / apparatus, or the insertion of other devices / apparatus between these explicitly mentioned devices / apparatus. Moreover, unless otherwise stated, the numbering of each method step is merely a convenient tool for identifying each method step, and not for limiting the order of the method steps or defining the scope of the invention. Changes or adjustments to their relative relationships, without substantially altering the technical content, should also be considered within the scope of the invention.

[0086] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention; in the specification and claims of the present invention, unless otherwise expressly stated in the text, the singular forms "a", "an" and "this" include the plural forms.

[0087] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the present invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, apparatus, and materials used in the embodiments, based on the knowledge of the prior art possessed by one of ordinary skill in the art and the description of this invention, any prior art methods, apparatus, and materials similar to or equivalent to those described, apparatus, and materials in the embodiments of this invention may be used to implement the present invention.

[0088] This invention provides a novel plum polysaccharide comprising various monosaccharides including fucose, rhamnose, arabinose, galactose, glucose, xylose, mannose, galacturonic acid, and glucuronic acid. In particular, it contains a high proportion of uronic acids and a branched structure, classifying it as an acidic heteropolysaccharide. Based on its high proportion of uronic acids (especially galacturonic acid), it exhibits a high negative charge density and strong binding ability to cations / receptors, thus possessing immunomodulatory, anti-inflammatory, and antitumor activities. Furthermore, highly branched polysaccharides typically have more exposed active hydroxyl groups and reducing ends, and the carboxyl groups in the uronic acids can chelate pro-oxidative metal ions, thereby granting it antioxidant activity. Therefore, this plum polysaccharide has broad application prospects. In addition, this invention also provides a method for preparing the plum polysaccharide, specifically including the following steps:

[0089] S1. Take dried plum fruit, add anhydrous ethanol at a mass-to-volume ratio of 90-110 g / L, stir at 20-30℃ for 1-3 h to defatting, and collect the precipitate by centrifugation.

[0090] S2. Add water to the precipitate at a mass-to-volume ratio of 40-60 g / L, stir at 55-65℃ for 2-5 hours, and collect the supernatant by centrifugation.

[0091] S3. After concentrating the supernatant, add anhydrous ethanol to obtain the total solution. Ensure that the concentration of anhydrous ethanol in the total solution is 75%-85%. Let it stand at 2-6℃ to precipitate, and freeze-dry the precipitate to obtain crude polysaccharide from dried plum.

[0092] S4. Add water to the crude polysaccharide of dried plum at a mass-volume ratio of 12-17 g / L. Add protease at a mass ratio of protease: crude polysaccharide of dried plum = 1:55-65. Enzymatically hydrolyze at 35-40℃ for 10-15 h to remove protein. Then, repeatedly treat with chloroform: n-butanol 4:1 to remove protein residue and retain the aqueous phase.

[0093] S5. Add petroleum ether to the aqueous phase for extraction and defatting, retain the aqueous phase, and add 10-20 mL of petroleum ether based on 1 g of crude polysaccharide of dried plum.

[0094] S6. Add AB-8 type macroporous resin to the defatted aqueous phase for decolorization. Based on 1g of crude polysaccharide of dried plum, the amount of macroporous resin added is 30-40mL. Filter to remove the resin and obtain the filtrate.

[0095] S7. The filtrate is placed in a dialysis bag with a molecular weight of 2000-5000 Da and dialyzed with running water for 30-50 hours. The liquid in the dialysis bag is concentrated and freeze-dried to obtain the purified plum polysaccharide.

[0096] S8. Add water to the purified plum polysaccharide at a solid-liquid ratio of 20-30 g / L, centrifuge and collect the supernatant.

[0097] S9. Load the supernatant into a DEAE anion exchange column and perform gradient elution with water, 0.1 M NaCl, 0.2 M NaCl and 0.3 M NaCl solutions in sequence. Collect the main elution peak of 0.2 M NaCl and concentrate to obtain the concentrate.

[0098] S10. The concentrated solution is placed in a dialysis bag with a molecular weight of 2000-5000 Da and dialyzed with running water for 30-50 hours. The solution in the dialysis bag is concentrated and freeze-dried to obtain the first purified plum polysaccharide.

[0099] S11. Add water to the first purified plum polysaccharide at a solid-liquid ratio of 0.02-0.08 g / L, centrifuge and collect the supernatant.

[0100] S12. Load the supernatant onto a dextran-crosslinked acrylamide composite gel column, elute with water, collect the main peak, concentrate to obtain a concentrate, and freeze-dry to obtain purified plum polysaccharide.

[0101] Example 1

[0102] This embodiment 1 provides a method for extracting and purifying polysaccharides from dried plums, specifically including the following steps:

[0103] S1. Extraction of crude polysaccharides from dried plums

[0104] Weigh 500g of dried plum fruit and pulverize it through a 60-mesh sieve. Add 5L of anhydrous ethanol, stir at room temperature for 2 hours to defatted the fruit, centrifuge and discard the supernatant, evaporate the solvent from the precipitate. Add 10L of pure water to the precipitate, extract by stirring in a 60℃ water bath for 4 hours, centrifuge and collect the supernatant. Repeat the extraction once with the residue. Combine the two extracts and concentrate them to approximately 1L by rotary evaporation at 50℃. Slowly add 4L of anhydrous ethanol (final concentration 80%) while stirring, and let stand at 4℃ for 12 hours. Centrifuge at 8000g for 15 minutes to collect the precipitate, freeze-dry the precipitate to obtain approximately 40.6g of crude plum polysaccharide extract.

[0105] S2, Purification of crude polysaccharide extract from dried plum

[0106] Take 30g of the crude polysaccharide extract obtained in step S1 above and dissolve it in 2L of pure water. Add 0.5g each of papain and complex protease (purchased from Sigma-Aldrich, catalog number P5147) and enzymatically hydrolyze at 37℃ for 12h to remove protein. Repeatedly treat with the Sevag method (chloroform: n-butanol = 4:1) to remove protein residue. Add 500mL of petroleum ether to the aqueous phase for extraction and defatting. Then add 1L of macroporous resin AB-8 and stir slowly at room temperature for 12h to adsorb pigments. Filter to remove resin, and put the filtrate into a dialysis bag with a molecular weight cutoff of 3000 Da and dialyze against running water for 48 hours. Concentrate the dialysate and freeze-dry to obtain approximately 7.1g of purified crude polysaccharide from dried plum.

[0107] S3, Ion exchange column chromatography purification

[0108] Take 5g of the purified crude polysaccharide obtained in step S2 above, dissolve it in 200mL of pure water, centrifuge at 10000g for 10 minutes, and collect the supernatant. Load the sample onto a DEAE Seplife FF anion exchange column (26 mm × 400 mm) pre-equilibrated with pure water. Perform a stepwise gradient elution with 400 mL each of pure water, 0.1 M NaCl, 0.2 M NaCl, and 0.3 M NaCl solutions at a flow rate of 4 mL / min, collecting one tube every 15 mL. The polysaccharide content of each tube is determined using the sulfuric acid-phenol method: take 1.0 mL of eluent in a test tube, add 1.0 mL of 5% phenol solution, and vortex to mix. Quickly add 5.0 mL of concentrated sulfuric acid and immediately vortex to mix. Let stand at room temperature for 10 minutes, then place in a 30℃ water bath for 20 minutes. After cooling to room temperature, measure the absorbance at a wavelength of 490 nm and plot the elution curve. The elution curve is shown in the figure. Figure 1 As shown. The components corresponding to the main peak of the acidic polysaccharide with the highest absorbance of the main active fraction of plum polysaccharide were collected by elution with 0.2 M NaCl, combined and concentrated, dialyzed with a 3000 Da dialysis bag to remove salt, and lyophilized to obtain approximately 1.368 g of ion-exchange purified polysaccharide.

[0109] S4. Gel filtration column chromatography purification

[0110] Take 1 g of the ion-exchange purified polysaccharide obtained in step S4 above, dissolve it in 20 mL of pure water, centrifuge at 10000 g for 10 minutes, and collect the supernatant. Load the sample into a Sephacryl S-400 HR gel column (26 mm × 1000 mm), and perform isocratic elution with 1000 mL of pure water as the mobile phase at a flow rate of 1.0 mL / min, collecting one tube every 10 mL. Detect the elution using the sulfuric acid-phenol method described above and plot the elution curve as shown in the figure. Figure 2 As shown. The components corresponding to the main peak (symmetric single peak) were collected, combined, concentrated, and freeze-dried to obtain approximately 200 mg of the final purified plum polysaccharide.

[0111] Test section

[0112] Physicochemical characterization of ume polysaccharides:

[0113] (1) The morphology of the polysaccharide from dried plum was observed using a scanning electron microscope.

[0114] Specifically: The polysaccharide sample was passed through a 100-mesh sieve, a small amount was placed on conductive carbon tape, sputter-coated with gold, and then scanned and photographed using an electron microscope. The morphology image at a magnification of 5K is shown below. Figure 3 As shown in the figure, the polysaccharide sample is in the form of flakes with a rough surface and a uniform morphology.

[0115] (2) X-ray diffraction was used to observe the crystal form of plum polysaccharide.

[0116] Specifically: The purified polysaccharide sample is dried, pulverized, and passed through a 100-mesh sieve. 20 mg of the sample is weighed onto the stage, pressed firmly, spread evenly, and then analyzed using the instrument. For example... Figure 4 As shown, the XDR spectrum of this plum polysaccharide exhibits typical amorphous diffuse peaks typical of polysaccharides.

[0117] (3) The ultraviolet-visible spectrum of ume polysaccharide was tested.

[0118] The polysaccharides in dried plums were scanned using a UV-Vis spectrophotometer. Specifically, a small amount of polysaccharide sample was weighed and dissolved in pure water to prepare a 5 mg / mL polysaccharide solution, which was then tested using a UV-Vis spectrophotometer. Figure 5 As shown, the UV-Vis spectrum of ume polysaccharide shows no absorption in the 260-280 nm range, indicating that it has good purity and effectively removes common impurities such as nucleic acids, proteins, and polyphenols.

[0119] (4) The infrared spectrum of plum polysaccharide was tested.

[0120] The polysaccharides in dried plums were tested using Fourier transform infrared spectroscopy. Specifically, a small amount of polysaccharide sample was weighed, mixed with 200 mg of potassium bromide, pressed into 1 mm thick sheets, and then analyzed using the instrument. Figure 6 As shown, the infrared spectrum reveals a distinct polysaccharide characteristic peak at 3334.75 cm⁻¹. -1 This is the absorption peak of the stretching vibration of OH, a characteristic peak of carbohydrates. It is located at 2925.7 cm⁻¹. -1 The absorption peak at 1012.98 cm⁻¹ is attributed to the CH stretching vibration. -1 There is an absorption peak at this point, which is attributed to the stretching vibration of CO.

[0121] (5) The weight-average molecular weight and polydispersity index of plum polysaccharide were tested.

[0122] Its weight-average molecular weight (Mw) was determined to be 23.594 kDa and its polydispersity index (Mw / Mn) was 1.16 by gel permeation chromatography (SEC-MALLS-RI).

[0123] Structural analysis of ume polysaccharides:

[0124] (1) The composition and percentage content of monosaccharides were determined using a Thermo ICS 5000+ ion chromatography system.

[0125] Monosaccharide components were analyzed and detected using an electrochemical detector on a Thermo ICS 5000+ ion chromatography system (ICS 5000+, Thermo Fisher Scientific, USA). A clean chromatographic vial was used. 3 mg of polysaccharide sample was weighed and added to 1 mL of 2M TFA acid solution. The vial was heated at 121°C for 2 hours. Nitrogen gas was purged, and the sample was dried. The vial was then washed with 99.99% methanol and dried again. This methanol washing process was repeated 2-3 times. The sample was dissolved in sterile water and transferred to a chromatographic vial for analysis.

[0126] An anion exchange column (Dionex™ CarboPac™ PA20 (150*3.0mm, 10μm) liquid chromatography column) was used, with an injection volume of 5μl. Mobile phase A (H2O), mobile phase B (0.1M NaOH), mobile phase C (0.1 M NaOH, 0.2M NaAc), flow rate 0.5 ml / min; column temperature 30℃; elution gradient: 0 min A phase / B phase / C phase (95:5:0, V / V / V), 26 min A phase / B phase / C phase (85:5:10, V / V / V), 42 min A phase / B phase / C phase (85:5:10, V / V / V), 42.1 min A phase / B phase / C phase (60:0:40, V / V / V), 52 min A phase / B phase / C phase (60:40:0, V / V / V), 52.1 min A phase / B phase / C phase (95:5:0, V / V / V), 60 min A phase / B phase / C phase (95:5:0, V / V / V).

[0127] Chromatogram as shown Figure 7 The total content of each monosaccharide component shown is 822.9776. By comparing the peak times of each monosaccharide with the standard peak times, the chromatographic peaks were assigned. Based on the proportion of the chromatographic peak area of ​​each monosaccharide to the total chromatographic peak area, the percentage of each monosaccharide in the plum polysaccharide was calculated as shown in Table 1.

[0128] Table 1. Mass percentage of each monosaccharide in plum polysaccharide

[0129]

[0130] Note: The Chinese names corresponding to the English abbreviations of monosaccharides in Table 1 are: Fuc-fucose, Ara-arabinose, Rha-rhamnose, Gal-galactose, Glc-glucose, Xyl-xylose, Man-mannose, Gal-UA-galacturonic acid, and Glc-UA-glucuronic acid.

[0131] (2) Methylation analysis was performed using GC-MS.

[0132] S1, Sample Pretreatment

[0133] (1) Weigh a small amount of sample (about 5 mg), add 1 ml of pure water to dissolve it, add 1 ml of 100 mg / ml 1-cyclohexyl-2-morpholinoethyl carbodiimide methyl p-toluenesulfonate, and react for 2 h.

[0134] (2) Add 1 ml of 2 M imidazole, divide the sample into two equal parts, add 1 ml of 30 mg / ml NaBH4 and 1 ml of 30 mg / ml NaBD4 respectively, and react for 3 h.

[0135] (3) Add 100 μl of glacial acetic acid to terminate the reaction. After dialyzing for 48 h, freeze-dry the sample for further processing.

[0136] (4) Add 500 μl of DMSO to the sample to dissolve it.

[0137] (5) Add 1 mg NaOH and incubate for 30 min.

[0138] (6) Add 50 μl of iodomethane solution and react for 1 h.

[0139] (7) Add 1 ml of water and 2 ml of dichloromethane, vortex to mix, centrifuge, and discard the aqueous phase. Repeat the water washing 3 times.

[0140] (8) Take out the lower layer of dichloromethane phase and dry it with nitrogen.

[0141] (9) Add 100 μl of 2M TFA and react at 121℃ for 90 min.

[0142] (10) Evaporate at 30℃.

[0143] (11) Add 50 μl of 2 M ammonia water and 50 μl of 1 M NaBD4, mix well, and react at room temperature for 2.5 h.

[0144] (12) Add 20 μl of acetic acid to terminate the reaction, blow dry with nitrogen, wash twice with 250 μl of methanol, and blow dry with nitrogen.

[0145] (13) Add 250 μl of acetic anhydride, vortex to mix, and react at 100 °C for 2.5 h.

[0146] (14) Add 1 ml of water and let stand for 10 min.

[0147] (15) Add 500 μl of dichloromethane, vortex to mix, centrifuge, discard the aqueous phase, and wash with water 3 times.

[0148] (16) Take the lower layer of dichloromethane phase as the sample to be tested.

[0149] S2 and GC-MS detection were performed using an Agilent Technologies Inc. (CA, USA) 6890A-5977B GC-MS system with an autosampler model G4567A. Chromatographic conditions: The chromatographic system used was an Agilent 6890A gas chromatograph (Agilent Technologies, USA); column: BPX70 (30m × 0.25 mm × 0.25 µm, SGE, Australia). The injection volume was 1 μl, the split ratio was 10:1, and the carrier gas was high-purity helium. The column oven was initially set at 140℃ and held for 2.0 min, then programmed to increase to 230℃ at a rate of 3℃ / min and held for 3 min. Mass spectrometry conditions: The mass spectrometry system used was an Agilent 5977B quadrupole mass spectrometer (Agilent Technologies, USA), equipped with an electron impact ionization (EI) source and a MassHunter workstation. Electron impact ionization (EI) was used, and the analyte was detected in full scan mode. The ion source temperature was 200℃, the MS quadrupole temperature was 110℃, the ionization energy was 50 EV, the transfer line temperature was 210℃, and the mass scan range (m / z) was 50-350. The H-chromatogram and D-chromatogram obtained after detection and analysis are shown below. Figure 8 As shown in Table 2, the methylation analysis results of the ume polysaccharides were obtained, and the results of the relative molar ratio of methylated monosaccharides and the relative molar ratio of monosaccharide composition were shown in Table 3.

[0150] Table 2. Methylation analysis of ume polysaccharides

[0151]

[0152] Table 3 Comparison of methylation and monosaccharide results

[0153]

[0154] Note: This table lists methylated derivatives that can be clearly identified and have a molar percentage > 3%. Trace amounts of Fuc, Rha, Xyl, etc., detected in monosaccharide compositions are not included because their derivative signals do not reach the reliable integration threshold.

[0155] (3) Nuclear magnetic resonance analysis of ume polysaccharides

[0156] 50 mg of purified polysaccharide was fully dissolved in D2O to prepare a polysaccharide solution with a concentration greater than or equal to 40 mg / mL. The solution was transferred to an NMR tube, with an addition volume of 0.5 mL. The NMR tube was then placed in an NMR spectrometer to scan one-dimensional 1H spectrum, 13C spectrum, and two-dimensional COSY, NOESY, HSQC, HMBC, DEPT-135, and TOCSY spectra. The detection results are shown below. Figures 9-16 As shown, hydrogen signals are mainly concentrated in... 1 In the 3.1–5.3 ppm region of the HNMR spectrum, the carbon signal is mainly distributed in... 13 The range of 55–110 ppm in CNMR spectra. 13 In the CNMR spectrum, multiple signals were observed in the anodic carbon region (90–110 ppm), with signals of 107.48 ppm, 104.33 ppm, 101.61 ppm, and 98.92 ppm corresponding to the anodic carbons of different sugar residues, indicating the presence of multiple glycosidic bond configurations in the sample. HSQC spectral analysis clearly identified the direct correlation signals between anodic hydrogen / carbon pairs (e.g., H1 / C1 at 5.02 / 104.39 ppm and 4.91 / 97.47 ppm) and related peaks at other sites (e.g., H2 / C2 and H3 / C3), thus enabling the systematic assignment of signals at various positions on different sugar rings. The long-range correlation peaks observed in the HMBC spectrum between the 4.91 ppm (H1) and 78.86 ppm (C4) sugar residues support a (1→4) linkage. Furthermore, COSY and TOCSY spectra revealed the proton coupling network within the sugar residues, and the correlations between adjacent protons such as A1–A2 and C1–C2, further verifying the proton signal assignment of each sugar ring. The trans-glycan ring nuclear Overhouse effect signals (such as D1–E5 and H1–F4) appearing in the NOESY spectrum provided conformational and connection sequence evidence for spatially close proton pairs. Combining the above multidimensional NMR analysis results with methylation analysis data, the sugar residue types, connection modes, and side chain structural characteristics of this polysaccharide can be systematically inferred.

[0157] The sugar residues and chemical shifts were obtained after analyzing the NMR results, as shown in Table 4.

[0158] Table 4. NMR analysis results of Prunus mume polysaccharides

[0159]

[0160] The structural units of this plum polysaccharide were obtained by combining NMR analysis results with methylation analysis results, as shown below:

[0161]

[0162] Study on the in vitro antioxidant activity of ume polysaccharides

[0163] Scavenging experiment of 1,1-diphenyl-2-trinitrophenylhydrazine (DPPH) radicals: Different concentrations (0.1, 0.5, 1.0, 2.0, 5.0 mg / mL) of ume polysaccharide solutions were prepared and reacted with DPPH ethanol solution for 30 minutes in the dark. The absorbance was measured at 517 nm, and the scavenging rate was calculated according to the following formula:

[0164] Clearance rate (%) = [1 - (absorbance of experimental group - absorbance of blank group) / absorbance of control group] × 100%;

[0165] In the experimental group, DPPH ethanol solution was added to the polysaccharide solution;

[0166] The control group used anhydrous ethanol instead of polysaccharide solution and added DPPH solution (to determine the initial absorbance of DPPH).

[0167] The blank group consisted of polysaccharide solution with added anhydrous ethanol (used to subtract color interference from the sample itself).

[0168] The results are as follows Figure 17 As shown, the free radical scavenging rate gradually increases with the increase of polysaccharide concentration. At a concentration of 5 mg / mL, its DPPH free radical scavenging rate reaches 75.3%, showing significant concentration-dependent antioxidant activity, indicating that the plum polysaccharide of the present invention has excellent antioxidant activity.

[0169] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any form or substance. It should be noted that those skilled in the art can make various improvements and additions without departing from the method of the present invention, and these improvements and additions should also be considered within the scope of protection of the present invention. Any modifications, alterations, and equivalent changes made by those skilled in the art based on the above-disclosed technical content without departing from the spirit and scope of the present invention are equivalent embodiments of the present invention. Furthermore, any modifications, alterations, and evolutions made to the above embodiments based on the essential technology of the present invention still fall within the scope of the technical solution of the present invention.

Claims

1. A plum polysaccharide, characterized in that, The monosaccharide molar fractions in the plum polysaccharide are: 0.5%-1.0 mol% fucose, 1.0%-2.0 mol% rhamnose, 35%-37 mol% arabinose, 15%-17 mol% galactose, 9.0%-11 mol% glucose, 2.0%-3.0 mol% xylose, 6%-8 mol% mannose, 23%-25 mol% galacturonic acid, and 1.0%-2.0 mol% glucuronic acid.

2. The plum polysaccharide according to claim 1, characterized in that, The weight-average molecular weight of the plum polysaccharide is 20-30 kDa; And / or, the polydispersity index of the plum polysaccharide is <1.

25.

3. The plum polysaccharide according to claim 1, characterized in that, The glycosidic linkages in the plum polysaccharide include: 15%-18 mol% t-Araf, 3.0%-5.0 mol% 3-Araf, 2.0%-4.0 mol% t-Galp-UA, 2.0%-4.0 mol% t-Galp, 6.0%-8.0 mol% 5-Araf, 2.0%-3.0 mol% 4-Manp, 25%-30 mol% 4-Galp-UA, 20%-23 mol% 4-Galp, 3.0%-5.0 mol% 4-Glcp, 1.0%-3.0 mol% 2,5-Araf, 4.0%-6.0 mol% 2,3,5-Araf, and 2.0%-4.0 mol% 2,3,4-Arap.

4. The plum polysaccharide according to claim 1, characterized in that, The plum polysaccharide includes the following structural segments: 。 5. A method for preparing the polysaccharide of dried plum as described in any one of claims 1 to 4, characterized in that, The preparation method includes: treating plum fruit with alcohol to defatt the fruit and obtaining a precipitate; extracting the precipitate with water to obtain an extract; precipitating the extract with alcohol to obtain crude plum polysaccharide; subjecting the crude plum polysaccharide to protein removal, defatting, and decolorization sequentially to obtain purified plum polysaccharide; subjecting the purified plum polysaccharide to ion exchange column chromatography to obtain first purified plum polysaccharide; and subjecting the first purified plum polysaccharide to gel filtration column chromatography to obtain finally purified plum polysaccharide.

6. The preparation method according to claim 5, characterized in that, The alcohol treatment is performed by immersion in ethanol. And / or, the solid-liquid ratio of plum fruit to alcohol during the alcohol treatment is 50-150 g / L; And / or, the solid-liquid ratio of the precipitate to water during water extraction is 20-80 g / L; And / or, the water extraction temperature is 50-80℃, and the water extraction time is 2-5 hours; And / or, the concentration of ethanol in the extract and the total ethanol solution during the alcohol precipitation is 70%-90%; And / or, the temperature during alcohol precipitation is 1-10°C.

7. The preparation method according to claim 5, characterized in that, The protein removal process involves adding protease to the crude polysaccharide of dried plum for enzymatic hydrolysis, and then adding Sevag solution to remove protein residue. And / or, the defatting is performed by adding petroleum ether to the polysaccharide solution after protein removal for extraction and defatting; And / or, the decolorization is performed by adding macroporous resin to the defatted polysaccharide solution for decolorization; And / or, the ion exchange column is an anion exchange column; And / or, the eluent for the ion exchange column chromatography comprises water and a 0.1-0.5M aqueous solution of NaCl; And / or, the elution flow rate of the ion exchange column chromatography is 1-6 mL / min; And / or, the gel filter column is a dextran-crosslinked acrylamide composite gel column; And / or, the eluent for the gel filtration column chromatography is water; And / or, the elution flow rate of the gel filtration column chromatography is 0.5-5 mL / min.

8. The preparation method according to claim 7, characterized in that, The protein removal process involves dissolving crude polysaccharide from dried plums in water, then adding protease for enzymatic hydrolysis, wherein the mass-to-volume ratio of crude polysaccharide from dried plums to water is 10-20 g / L. And / or, the protease includes any one or both of papain and complex protease; And / or, the ratio of the amount of protease to crude polysaccharide of dried plum is 1:50-80; And / or, during the defatting process, the amount of petroleum ether added is 10-20 mL based on 1 g of crude polysaccharide from dried plum; And / or, the macroporous resin is AB-8 type macroporous resin; And / or, based on 1g of crude polysaccharide from dried plum, the amount of macroporous resin added in the decolorization process is 20-50mL; And / or, the functional group of the ion exchange column is DEAE-diethylaminoethyl; And / or, the eluent for the ion exchange column chromatography includes water, 0.1 M NaCl aqueous solution, 0.2 M NaCl aqueous solution, or 0.3 M NaCl aqueous solution.

9. The use of the plum polysaccharide as described in any one of claims 1 to 4 in the preparation of products for antioxidant, immunomodulatory and / or anti-inflammatory purposes.

10. A product with antioxidant, immunomodulatory, and / or anti-inflammatory properties, characterized in that, The active ingredient of the antioxidant product includes the plum polysaccharide as described in any one of claims 1 to 4.

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