A large fruit hawthorn heteropolysaccharide, a preparation method and application thereof

Abstract

This invention relates to the pharmaceutical field, specifically to a large-fruited hawthorn heteropolysaccharide, its preparation method, and its applications. The large-fruited hawthorn heteropolysaccharide comprises galacturonic acid, glucuronic acid, mannose, xylose, arabinose, glucose, galactose, fucose, and rhamnose; the weight-average molecular weight of the large-fruited hawthorn heteropolysaccharide is 81.6 kDa–157.3 kDa. The product of this invention possesses a unique structure characterized by a complex monosaccharide composition, low molecular weight, suitable uronic acid content, and low esterification degree. Compared to CP (concentrated glycosides) with a simple structure and high esterification, it exhibits superior antioxidant and bile acid binding capabilities. Compared to hawthorn polysaccharides with low uronic acid content and high molecular weight, its enzyme inhibitory activity is significantly enhanced. The product of this invention shows broad prospects in the development of drugs for regulating blood glucose / lipids, antioxidation, and prevention of metabolic syndrome.

Description

Technical Field

[0001] This invention relates to the field of pharmaceuticals, specifically to a heteropolysaccharide from hawthorn fruit, its preparation method, and its application. Background Technology

[0002] Large-fruited hawthorn [Malus doumeri (Bois) Chev], also known as Guangshanzha, Wuchanzi (Guangxi Miao language), Maquedun, Mojitun (Guangxi Zhuang language), and Saduo, is a plant belonging to the genus Malus Mill in the family Rosaceae. In Guangxi, China, it is commonly called "large fruit hawthorn" and is included in the "Guangxi Traditional Chinese Medicine Standards" (1990 edition). It is one of the 40 authentic medicinal materials produced in Guangxi, possessing both medicinal and edible characteristics. Large-fruited hawthorn is mainly distributed in southern China. Its taste is slightly sour and astringent, and it is usually consumed after processing in the form of candied fruit, fruit wine, and fruit vinegar. Modern pharmacological studies have shown that large-fruited hawthorn has effects such as promoting sleep, regulating immunity, antioxidation, antitumor, and antibacterial properties. These effects are attributed to its rich bioactive components, including phenols, flavonoids, polysaccharides, and alkaloids. Among them, polysaccharide compounds stand out due to their structural complexity and multifunctional properties, gradually becoming a key focus of research. Studies have shown that plant polysaccharides possess a variety of beneficial functional activities for human health, such as immunomodulation, anti-inflammation, and regulation of glucose and lipid metabolism, demonstrating great potential in the prevention of chronic metabolic diseases.

[0003] However, existing commercial pectin polysaccharides (such as those from apples) and commercial hawthorn polysaccharides have the following limitations:

[0004] (1) Commercial pectin polysaccharides are mainly composed of galacturonic acid, with a simple structure, limited types of monosaccharides, and a wide molecular weight distribution (usually greater than 200 kDa), resulting in weak functional activities (such as antioxidant and bile acid binding capacity).

[0005] (2) Commercial hawthorn polysaccharides are usually extracted from common hawthorn. Their monosaccharide composition is mainly glucose and arabinose, with low uronic acid content (usually less than 30%) and high molecular weight (often exceeding 200 kDa). This makes them insufficient in inhibiting α-amylase and α-glucosidase, and difficult to effectively regulate blood sugar and blood lipids.

[0006] In addition, some commercial polysaccharides have a high degree of esterification (usually greater than 50%) and a low proportion of free carboxyl groups, which limits their charge density and biological activity.

[0007] Therefore, developing a novel heteropolysaccharide with a unique monosaccharide composition, a specific molecular weight range, and low esterification to address the insufficient functional activity of polysaccharides in existing technologies has become an urgent need in this field. This invention extracts and isolates a novel heteropolysaccharide MDPS from hawthorn, which contains various monosaccharides (such as galacturonic acid, glucuronic acid, mannose, etc.), with a weight-average molecular weight of 81.6-157.3 kDa. It also exhibits moderate uronic acid content and low esterification, demonstrating significantly superior effects compared to commercial products in terms of antioxidant activity, bile acid binding, and enzyme inhibition, and possesses broad market prospects. Summary of the Invention

[0008] To address the aforementioned technical problems, this invention provides a large-fruited hawthorn heteropolysaccharide, its preparation method, and its application.

[0009] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0010] A method for preparing hawthorn heteropolysaccharide includes the following steps: crushing hawthorn fruit and mixing it with citric acid solution, heating and extracting, combining the extracts, filtering, concentrating the filtrate, adding ethanol for precipitation, collecting the precipitate, removing the protein in the precipitate with Sevag reagent; dialyzing and freeze-drying the solution after removing the protein to obtain the hawthorn heteropolysaccharide.

[0011] Further, the large-fruited hawthorn fruit was pulverized and mixed with a citric acid solution with a mass-volume concentration of 3%–5%. The mixture was then treated in a water bath at 80–90°C for 1–3 hours. This process was repeated twice. The extracts were combined, filtered, and the filtrate was concentrated to half its original volume at 50°C to obtain a concentrated solution. An equal volume of 95% ethanol was added to the concentrated solution, and the mixture was allowed to stand at 3–5°C for 12–48 hours to precipitate the polysaccharides. The precipitate was collected, and the proteins in the precipitate were removed using Sevag reagent. The solution after protein removal was dialyzed and freeze-dried to obtain the large-fruited hawthorn heteropolysaccharide.

[0012] Furthermore, the hawthorn heteropolysaccharide comprises galacturonic acid, glucuronic acid, mannose, xylose, arabinose, glucose, galactose, fucose, and rhamnose; the weight-average molecular weight of the hawthorn heteropolysaccharide is 81.6 kDa to 157.3 kDa.

[0013] Further, the molar ratio of galacturonic acid, glucuronic acid, mannose, xylose, arabinose, glucose, galactose, fucose and rhamnose is (21.3-25.9):(21.2-25.4):(12.8-15.6):(8.7-10.8):(8.6-11.2):(8.2-10.1):(7.4-9.3):(1.3-1.6):(1.1-1.4).

[0014] Furthermore, the hawthorn heteropolysaccharide contains four structural domains: homogalacturonic acid polysaccharide, rhamnogalacturonic acid polysaccharide, glucuronic acid xylan, and glucomannan; the total sugar content of the hawthorn heteropolysaccharide is ≥89.26%, the uronic acid content is 42.61-48.92%, and the degree of esterification is 22.31-29.64%.

[0015] Furthermore, the hawthorn heteropolysaccharide contains the following structural domains:

[0016] a) Homogalacturonic acid polysaccharide domain, whose main chain is composed of →4)-α-D-GalAp-(1→ repeating units, in which the carboxyl groups of some galacturonic acid residues are methylated, and the molar percentage of free carboxyl groups is higher than that of methylated carboxyl groups.

[0017] b) Rhamnogalacturonic acid polysaccharide-I domain, whose main chain is composed of →4)-α-D-GalAp-(1→2)-α-L-Rhap-(1→ repeating units, wherein the rhamnosine residues of the main chain are attached to side chains, the side chains are mainly composed of arabinofuranose, and the molar ratio of total sugar in the side chains to rhamnosine residues in the main chain (Ara+Gal) / Rha is 11.63 to 23.16;

[0018] c) A glucuronic acid arabinoxylan domain, the main chain of which is composed of β-1,4 linked xylose residues, and the main chain is connected to side chains containing arabinose and glucuronic acid.

[0019] d) Glucomannan domain, whose backbone is composed of glucose residues and mannose residues linked by β-glycosidic bonds.

[0020] This invention provides the application of the above-mentioned hawthorn heteropolysaccharide in the preparation of antioxidant agents and / or bile acid binders.

[0021] This invention provides the application of the above-mentioned hawthorn heteropolysaccharide in the preparation of α-amylase and / or α-glucosidase inhibitors.

[0022] This invention provides the application of the above-mentioned hawthorn heteropolysaccharide in the preparation of pharmaceuticals for regulating blood sugar and / or blood lipids.

[0023] This invention also provides the application of the above-mentioned hawthorn heteropolysaccharide in the preparation of medicines for the prevention or adjunctive treatment of metabolic syndrome-related diseases; the metabolic syndrome-related diseases include any one or more of diabetes, hyperlipidemia, and obesity.

[0024] In summary, due to the adoption of the above technical solution, the beneficial effects of the present invention are:

[0025] The MDPS of this invention, through its unique structural design, exhibits a complex monosaccharide composition, low molecular weight, suitable uronic acid content, and low esterification degree, among other comprehensive characteristics. This structural advantage enables it to successfully overcome the limitations of commercial polysaccharides: on the one hand, compared to commercial polysaccharides (CP) with simple structures and high esterification degrees, the complex structure and low esterification degree of MDPS endow it with superior antioxidant activity and bile acid binding capacity; on the other hand, compared to commercial hawthorn polysaccharides (CHPS) with low uronic acid content and high molecular weight, the moderate uronic acid content and low molecular weight of MDPS synergistically enhance its enzyme inhibitory activity. Therefore, MDPS shows broader application prospects in the preparation of antioxidants, bile acid binders, blood glucose / lipid regulators, and drugs for the prevention of metabolic syndrome-related diseases such as diabetes and hyperlipidemia. Attached Figure Description

[0026] Figure 1 The following are characterization diagrams of the basic physicochemical properties of MDPS in Example 1, including: (A) gel permeation chromatogram of MDPS and dextran standard curve used to calculate relative molecular weight; (B) UV-Vis absorption spectrum of MDPS (200-700 nm); (C) Fourier transform infrared spectrum of MDPS (500-4000 cm⁻¹). -1 (D) Variation of the maximum absorption wavelength (λmax) of the MDPS-Congo red complex in NaOH solutions of different concentrations;

[0027] Figure 2 This is a schematic diagram of the structures of HG(A), RG-I(B), GAX(C) and GM(D) in Example 1;

[0028] Figure 3 The image shows the NMR spectrum of MDPS in Example 1, where (A) 1 HNMR; (B) 13 CNMR;

[0029] Figure 4 The images shown are SEM images of MDPS in Example 1, where (AB) are scanning electron microscope images of MDPS at different magnifications; (C) are two-dimensional morphological images of MDPS obtained by atomic force microscopy; and (D) are three-dimensional morphological images of MDPS obtained by atomic force microscopy.

[0030] Figure 5 The thermal property analysis diagram of MDPS in Example 1;

[0031] Figure 6The image shows the in vitro antioxidant activity results of MDPS in Example 1, where (A) the scavenging rate of DPPH free radicals by MDPS, commercial hawthorn polysaccharide, commercial pectin polysaccharide, and ascorbic acid (positive control); and (B) the scavenging rate of ABTS by MDPS, commercial hawthorn polysaccharide, commercial pectin polysaccharide, and ascorbic acid (positive control). + Free radical scavenging rate;

[0032] Figure 7 The figure shows the in vitro lipid-lowering and blood glucose-lowering potential of MDPS in Example 1. (A) Binding ability of MDPS, commercial hawthorn polysaccharide, commercial pectin polysaccharide and cholestyramine (positive control) to glycocholic acid; (B) Binding ability of MDPS, commercial hawthorn polysaccharide, commercial pectin polysaccharide and cholestyramine (positive control) to taurocholic acid; (C) Inhibition rate of MDPS, commercial hawthorn polysaccharide, commercial pectin polysaccharide and acarbose (positive control) to α-amylase; (D) Inhibition rate of MDPS, commercial hawthorn polysaccharide, commercial pectin polysaccharide and acarbose (positive control) to α-glucosidase. Detailed Implementation

[0033] Example 1

[0034] 1. Introduction

[0035] This study extracted and isolated a novel heteropolysaccharide, named MDPS, from *Malus doumeri* (Bois) Chev., and systematically characterized its natural structure using gel permeation chromatography, liquid chromatography, nuclear magnetic resonance spectroscopy, spectroscopic analysis, and atomic force microscopy. Based on this, MDPS was compared with commercial pectin polysaccharides and commercial hawthorn polysaccharides in multiple dimensions, focusing on evaluating their antioxidant capacity, bile acid binding capacity, and hypoglycemic potential based on α-amylase / α-glucosidase inhibition kinetics, thereby elucidating the relationship between MDPS structure and biological function. This research is expected to provide a scientific basis for the high-value utilization of *Malus doumeri* resources, promote its application in drug development, and provide a new case study for structure-activity relationship research of plant polysaccharides.

[0036] 2. Materials and Methods

[0037] 2.1. Materials

[0038] Large-fruited hawthorn (Malus doumeri (bois) Chev. fruit (M. doumeri) was sourced from Jingxi City, Baise City, Guangxi Zhuang Autonomous Region (23°34'N, 106°28'E, altitude 798.8 m), and supplied by a food company in Jingxi. Soluble starch, total sugar content assay kits, and total bile acid (TBA) content assay kits were purchased from Beijing Solarbio Technology Co., Ltd. (Beijing, China). The Bradford method protein quantification kit was purchased from Wuhan Servicebio Biotechnology Co., Ltd. Monosaccharide standards, ascorbic acid (VC), sodium taurocholate, sodium glycylcholate, 1-phenyl-3-methyl-5-pyrazolone (PMP), cholestyramine, p-nitrophenyl-α-D-glucopyranoside (PNPG), 2,2-diaza-3-ethylbenzothiazole-6-sulfonic acid (ABTS), 3,5-dinitrosalicylic acid (DNS), acarbose, α-glucosidase, and α-amylase (derived from porcine pancreas) were purchased from Shanghai Yuanye Biotechnology Co., Ltd. (Shanghai, China). Citric acid was purchased from Shanghai Maclean Biochemical Co., Ltd. (Shanghai, China). Commercial pectin polysaccharide (CP, derived from apples) and commercial hawthorn polysaccharide (CHPS) were purchased from Hefei BoMei Biotechnology Co., Ltd. and Shaanxi Tianen Biotechnology Co., Ltd., respectively. CHPS was purified before use. Other chemicals were of analytical grade.

[0039] 2.2. Preparation of MDPS

[0040] The dried hawthorn fruit was crushed and mixed with 4% (w / v) citric acid solution. The mixture was extracted in a water bath at 85°C for 2 hours, and the extraction was repeated twice. The extracts were combined and filtered under reduced pressure using a Buchner funnel to remove the suspended solids. The filtrate was concentrated to half its original volume using a rotary evaporator at 50°C. An equal volume of 95% ethanol was added to the concentrate and the mixture was allowed to stand at 4°C for 24 hours to precipitate the polysaccharides. The mixture was centrifuged at 6000 rpm for 20 minutes and the supernatant was discarded. The collected precipitate was then treated with Sevag reagent to remove the protein. Finally, the solution was dialyzed (molecular weight cutoff 8000-14000 Da) and freeze-dried to obtain MDPS. The yield was calculated according to formula [1]:

[0041] [1]

[0042] 2.3. Determination of Chemical Properties

[0043] The protein content of MDPS was determined using the Bradford method with a protein quantification kit and bovine serum albumin as the standard. Total sugar content was determined using a corresponding kit with anhydrous glucose as the standard. Uronic acid content was determined using the carbazole-sulfuric acid method with galacturonic acid as the standard. The absorbance of GalA standard solution and MDPS solution at 525 nm was measured, and the uronic acid content was calculated using a standard curve.

[0044] 2.4. Molecular weight determination

[0045] The molecular weight of MDPS was determined by gel permeation chromatography (GPC). The GPC system consisted of a Waters 515 HPLC pump, two Agilent mixed columns, and a Waters 2414 RI detector. The mobile phase was 0.1 mol / L NaNO3 and 550 ppm NaN3, and the flow rate was 1 mL / min. Calibration curves were plotted using a series of dextran standards.

[0046] 2.5. Determination of Monosaccharide Composition

[0047] The monosaccharide composition of MDPS was determined by high-performance liquid chromatography (HPLC). MDPS were hydrolyzed with 4M trifluoroacetic acid at 90°C for 12 h, and then dried under N2. The dried hydrolysis product was redissolved in water, and the extract (methanol:acetonitrile:water = 2:2:1, v / v / v) was added and the mixture was shaken to mix. After sonication and centrifugation in an ice-water bath, the supernatant was dried under N2, redissolved in acetonitrile and water (1:1, v / v), and the supernatant was collected by centrifugation again. Detection was performed using an UPLC BEH Amide column and an electrospray ionization source. The chromatographic conditions were: injection volume 5 μL, column temperature 55 °C, mobile phase A was an aqueous solution containing 25 mM CH3COONH4 and 25 mM NH4OH, and mobile phase B was 100% acetonitrile.

[0048] 2.6. Ultraviolet-Visible Spectroscopy Analysis

[0049] The UV-Vis spectrum of MDPS solution (0.5 mg / mL) was scanned in the wavelength range of 190–700 nm using a UV-Vis spectrophotometer.

[0050] 2.7. Congo Red Experiment

[0051] Congo red reagent can form a complex with polysaccharides having a triple helix structure, causing a red shift in their maximum absorption wavelength, which can be used to determine the polysaccharide backbone. MDPS solution, Congo red solution, and sodium hydroxide solution were mixed in a 2:1:1 (v / v / v) ratio to achieve final concentrations of 1 mg / mL for MDPS and 40 μg / mL for Congo red, and final concentrations of 0, 0.1, 0.2, 0.3, 0.4, and 0.5 mol / L for NaOH. After standing for 10 min, the maximum absorption wavelength in the 400-700 nm range was measured using a microplate reader.

[0052] 2.8. Fourier Transform Infrared Spectroscopy and Determination of Esterification Degree

[0053] MDPS and dry potassium bromide were ground in an agate mortar at a ratio of 1:100 and then pressed into tablets. The FT-IR spectra of the MDPS samples were recorded using a Fourier transform infrared spectroscopy (FT-IR) instrument with a resolution of 4 cm⁻¹. -1 The wavelength range is 400-4000 cm. -1 The degree of esterification is calculated according to formula [2] (Qin et al., 2019):

[0054] [2]

[0055] Where A 1740 and A 1635 1740 cm respectively -1 and 1635 cm -1 Peak area at that location.

[0056] 2.9. Nuclear Magnetic Resonance Spectroscopy Analysis

[0057] One-dimensional NMR spectra were obtained using a Bruker AVANCE NEO 600M nuclear magnetic resonance spectrometer equipped with a QXI1H / 31P / 13C / 15N 5mm quad-resonance inverse detection probe. The polysaccharide was fully dissolved in deuterium oxide and then loaded into an NMR tube for measurement. Chemical shifts are expressed as δ (ppm), and data were analyzed using MestReNova software.

[0058] 2.10. Microscopic Analysis

[0059] The surface morphology of MDPS was analyzed using a Prisma E scanning electron microscope. Lyophilized polysaccharides were dispersed on a conductive adhesive and gold-plated under vacuum for 60 seconds. Morphological features were observed at magnifications of 500× and 1000×. Diluted MDPS solutions were dropped onto mica sheets, and the ultrastructure of the samples was analyzed using atomic force microscopy.

[0060] 2.11. Thermal Analysis

[0061] The thermal behavior of MDPS was analyzed using a thermogravimetric analyzer. 10 mg of sample was placed in an alumina crucible and heated from 40 °C to 650 °C at a rate of 10 °C / min under a nitrogen atmosphere.

[0062] 2.12. Antioxidant activity

[0063] 2.12.1. DPPH free radical scavenging experiment

[0064] Different concentrations of MDPS solutions (0.0125 to 0.20 mg / mL) were mixed with 0.2 mM DPPH ethanol solution at a volume ratio of 3:1 and mixed by pipetting. After being placed in the dark for 30 minutes, the absorbance was measured at 517 nm. Different concentrations of ascorbic acid were used as positive controls. Ascorbic acid, CHPS, and CP were all treated in the same way as MDPS. The scavenging activity of polysaccharides and ascorbic acid against DPPH free radicals was calculated according to formula [3].

[0065] 2.12.2. ABTS + Free radical scavenging experiment

[0066] Equal volumes of 7 mM ABTS + ABTS was prepared by mixing the solution with 2.45 mM K2S2O8 solution and incubating overnight in the dark. + Stock solution. The next day, dilute ABTS with PBS buffer. + The mother liquor was prepared so that its absorbance at 734 nm was 0.70 ± 0.02, thus obtaining ABTS. + Working solutions. Prepare MDPS solutions of different concentrations (0.25-2.75 mg / mL) using PBS buffer. Take 180 μL of ABTS. + The working solution was prepared by adding 20 μL of MDPS solution, allowing it to stand in the dark for 6 min, and then measuring the absorbance at 734 nm. Different concentrations of ascorbic acid were used as positive controls. Ascorbic acid, CHPS, and CP were all treated in the same way as MDPS. The effect of polysaccharides or ascorbic acid on ABTS was calculated according to formula [3]. + Free radical scavenging activity.

[0067] [3]

[0068] Where A1 contains the sample and DPPH / ABTS + The absorbance of the working solution mixture; A2 is the absorbance of the mixture containing the sample and PBS buffer instead of DPPH / ABTS. + The absorbance of the working solution; A3 contains only DPPH / ABTS + The absorbance of the working solution; A4 is the absorbance containing only PBS buffer.

[0069] 2.13. Bile acid conjugation experiment

[0070] The bile acid binding capacity of MDPS was determined using sodium taurocholate and sodium glycocholate. MDPS, CHPS and CP solutions of different concentrations were prepared, and an equal volume of 0.25 M HCl was added to adjust the pH to 2.0 ± 0.2. The solutions were incubated for 60 minutes at 37°C in a constant temperature shaker (100 rpm) to simulate gastric digestion. Preheated sodium taurocholate / sodium glycocholate working solution (1 mM) was added to the gastric digested samples to adjust the pH to 6.8 ± 0.2. After simulated intestinal digestion under the same conditions, the samples were centrifuged at 10000 rpm for 15 minutes and the supernatant was collected. Cholestyramine of different concentrations was used as a positive control and treated in the same way as MDPS. The concentration of bile acids in the supernatant was measured using a bile acid content detection kit, the content was calculated according to the standard curve, and the binding rate of each bile acid was calculated according to formula [4]:

[0071] [4]

[0072] 2.14. Determination of blood glucose lowering potential

[0073] 2.14.1. α-Amylase Inhibitory Effect

[0074] MDPS solutions with concentrations of 12-20 mg / mL and α-amylase solutions with concentrations of 0.4 U / mL were prepared using PBS buffer. 100 μL of MDPS solution was mixed with an equal volume of α-amylase solution and incubated at 37 °C for 10 min. Then, 200 μL of 1% (w / v) soluble starch solution was added. At this point, the enzyme activity in the reaction system was 20 U / g substrate (starch). After further incubation at 37 °C for 10 min, 200 μL of 1% (w / v) DNS was added, and the reaction was immediately terminated by heating in a water bath for 5 min. The mixture was diluted with 1 mL of PBS buffer and the absorbance was measured at 540 nm. Acarbose at different concentrations was used as a positive control. Acarbose, CHPS, and CP were all treated in the same way as MDPS. The inhibition rate of polysaccharide or acarbose against α-amylase was calculated according to formula [5].

[0075] 2.14.2. α-Glucosidase Inhibitory Effect

[0076] According to the preliminary experiment, the test concentrations of MDPS were 0.0156, 0.0625, 0.25, 1 and 4 mg / mL. α-glucosidase was dissolved in PBS buffer to prepare an enzyme solution with a concentration of 0.5 U / mL. 100 μL of MDPS solution was mixed with an equal volume of α-glucosidase solution and incubated at 37 °C for 10 min. Then, 100 μL of 5 mM PNPG solution was added. At this time, the enzyme activity in the reaction system was 332 U / g substrate. After further incubation at 37 °C for 20 min, 250 μL of 0.2 M sodium carbonate solution was added to terminate the reaction, and the absorbance was measured at 405 nm. Acarbose at different concentrations was used as a positive control. Acarbose, CHPS and CP were treated in the same way as MDPS. The inhibition rate of polysaccharide or acarbose on α-glucosidase was calculated according to formula [5].

[0077] [5]

[0078] Where A1 is the absorbance of a mixture containing the sample, α-amylase / α-glucosidase, and soluble starch / PNPG; A2 is the absorbance of a mixture containing the sample and soluble starch / PNPG; A3 is the absorbance of a mixture containing α-amylase / α-glucosidase and soluble starch / PNPG; and A4 is the absorbance of a solution containing only soluble starch / PNPG.

[0079] 2.15. Statistical Analysis

[0080] All experiments were repeated three times using freshly prepared samples, and data are presented as mean ± standard deviation. Statistical analysis was performed using GraphPad Prism 8.3.0, and data plots were generated using Origin 2018.

[0081] 3. Results and Discussion

[0082] 3.1. Chemical Composition of MDPS

[0083] 3.1.1. Yield and Chemical Properties

[0084] Table 1. Yield and chemical properties of M. doumeri heteropolysaccharides

[0085]

[0086] The extraction rate, total sugar, protein, uronic acid content, and degree of esterification of MDPS are summarized in Table 1. The yield of MDPS obtained from dried M. doumeri powder was 6.38% ± 0.19%, with total sugar and protein contents of 90.32% ± 2.01% and 0.43% ± 0.02%, respectively. The uronic acid content and degree of esterification of MDPS were 44.05% ± 3.33% and 25.65% ± 2.91%, respectively, indicating that a large number of carboxyl groups were in a free state and would ionize to release H+. + This demonstrates that MDPS is an acidic polysaccharide. The high proportion of free carboxyl groups indicates a large amount of negative charge in the MDPS aqueous solution, and the high charge density suggests strong hydrophilicity and metal chelating ability. Furthermore, negatively charged polysaccharides readily interact with receptors on the surface of immune cells, possessing potential immunomodulatory capabilities (Z. Cui et al., 2025).

[0087] 3.1.2. Molecular weight

[0088] Table 2. Molecular weight of M. doumeri heteropolysaccharides

[0089]

[0090] Note: Mw and Mn are the weight-average molecular weight and number-average molecular weight, respectively. Mw / Mn is the molecular weight distribution.

[0091] The weight-average molecular weight, number-average molecular weight, and Z-average molecular weight of MDPS were determined by GPC, such as... Figure 1 As shown in A and Table 2, the chromatographic peaks present a main single peak that is basically symmetrical, with a small leading shoulder at the low molecular weight edge (starting region). The Mw / Mn ratio (i.e., polydispersity index) of MDPS is 1.447, indicating that its molecular weight distribution is relatively narrow and the main polymer chain length is relatively uniform. However, the presence of the shoulder in the low molecular weight region suggests that the sample still contains a small amount of components with a molecular weight lower than the main peak. Overall, MDPS exhibits apparent homogeneity on the molecular weight scale, but its intrinsic components are heterogeneous. In addition, the linear regression equation established based on the standards ( The molecular weight of MDPS was further confirmed by an R² value of 0.9949. The results showed that the Mw, Mn, and Mz of MDPS were 110.114, 76.124, and 199.396 kDa, respectively. Compared with polysaccharides from other plant sources (112-384 kDa), the molecular weight of MDPS is relatively low. This may be because the low pH during preparation favors the protonation and dissolution of the polysaccharide matrix, leading to a decrease in its molar mass.

[0092] 3.1.3. Monosaccharide Composition

[0093] Table 3 Monosaccharide composition of M. doumeri heteropolysaccharides

[0094]

[0095] As shown in Table 3, MDPS is mainly composed of galacturonic acid and glucuronic acid, with molar percentages of 23.46% ± 0.16 and 23.19% ± 0.34, respectively. These two uronic acids are key components of acidic polysaccharides. The high content of uronic acids (totaling 46.65%) gives the polysaccharide a negative charge, thus MDPS is an acidic polysaccharide. The relatively high content of GalA suggests the possible presence of homogalacturonic acid polysaccharide chains (…). Figure 2 A). In addition, the total neutral sugar content is 52.36%, including mannose, glucose, galactose, arabinose, and xylose. Trace components include 1.26% rhamnose and 1.29% fucose. Galactose and arabinose may be related to the side chains of the rhamnogalacturonic acid polysaccharide-I domain, while rhamnose is often a characteristic component of the RG-I domain backbone. Figure 2 B). Their presence suggests the possible presence of RG-I domains. Glucose has diverse origins, possibly from cellulose or starch dextran, or as part of the heteropolysaccharide backbone or side chain. Fucose is often used as a modifying sugar at the end of the side chain of complex polysaccharides. The results indicate that MDPS is a highly complex acidic heteropolysaccharide composed of multiple monosaccharides, with no single monosaccharide being absolutely dominant. MDPS may contain both HG and RG-I domains. The relative contents of HG and RG-I domains in MDPS were estimated from the monosaccharide composition using the following formula:

[0096] HG (%) = GalA – Rha

[0097] RG-I (%) = 2 × Rha + Ara + Gal

[0098] The results showed that MDPS consisted of 22.20% HG and 20.62% RG-I. The remaining portion likely consisted of glucuronide arabinoxylan and glucomannan. Since xylose and mannose are core markers of GAX and GM, respectively, they belong to the GAX main chain and the GM main chain (…). Figure 2 (CD). Furthermore, to estimate the extent of extension of the neutral side chain linked to RG-I, the ratio of its side-chain sugar to the main chain repeating unit was calculated, i.e., (Gal+Ara) / Rha. The MDPS value was 14.37, indicating a high degree of branching.

[0099] 3.2. Structural characteristics of MDPS

[0100] 3.2.1. Ultraviolet-Visible Spectroscopy and Fourier Transform Infrared Spectroscopy Analysis

[0101] UV-Vis spectroscopy analysis indicates that ( Figure 1 B) MDPS exhibits a weak absorption peak at 280 nm, indicating the presence of a very small amount of protein, consistent with the protein content analysis results. The FT-IR spectrum of MDPS is as follows: Figure 1 As shown in C. Overall, MDPS exhibits a typical carbohydrate absorption band, ranging from 500 to 4000 cm⁻¹. -1 3419 cm -1 The broad absorption band in the vicinity is attributed to the stretching vibration of -OH groups in the molecular structure, indicating the presence of hydrogen bonds between or within molecules. Polysaccharide molecules contain a large number of hydroxyl groups (-OH), especially on the sugar ring and in structural water. Hydroxyl groups on the molecular chain readily form intermolecular hydrogen bonds (e.g., between sugar chains) or intramolecular hydrogen bonds (e.g., within the sugar ring). The broader the peak, the stronger the hydrogen bond network. 2930 cm⁻¹ -1 The absorption peak at 1082 cm⁻¹ is attributed to the CH stretching vibrations, including CH, CH₂, and CH₃. Typically, this peak occurs at 1082 cm⁻¹. -1 Sharp and strong absorption peaks at this point typically point to the pyranose backbone, such as in HG. However, in MDPS, the absorption peak shifts to a lower wavenumber (1080 cm⁻¹). -1 This indicates an increase in furanose content and a change in glycosidic bond angles, with the main chain twisting due to side chain connections. 929 cm -1 The absorption peaks at 929 and 1080 cm⁻¹ are attributed to the ring stretching vibration of α-Araf, indicating the presence of arabinofuranose side chains, characteristic of RG-I and GAX side chains. Further calculations yielded peaks at 929 and 1080 cm⁻¹. -1 The peak area ratio was 1.07, indicating a relatively abundant side-chain structure in MDPS, consistent with the results of monosaccharide analysis. (851 cm⁻¹) -1 and 763 cm -1 The absorption peaks belong to the characteristic peaks of the C1-H deformation vibration of the α-glycosidic bond and the breathing vibration and out-of-plane bending of the α-pyranose ring, respectively, confirming the presence of the HG backbone (→4)-α-GalA-(1→). These results further confirm that MDPS is a mixture of polysaccharide molecules with different chemical structures, consistent with the results of monosaccharide composition. At 1635 cm⁻¹ -1 The nearby absorption peak is due to the stretching vibration of the carbonyl group (C=O) of the free carboxyl group (-COOH) superimposed with the HOH bending vibration of bound water. 1744 cm⁻¹ -1The absorption peak at 1635 cm⁻¹ confirms that some carboxyl groups are methylated (-COOCH₃). -1 Peak strength greater than 1744 cm -1 The high peak intensity indicates a low degree of esterification and a high proportion of free carboxyl groups. (1414 cm⁻¹) -1 The weak peak at that point is mainly due to the in-plane bending vibration of the OH group of the free carboxyl group (-COOH), which also confirms this inference.

[0102] 3.2.2. Congo Red Analysis

[0103] Congo red can form a complex with polysaccharides possessing a triple helix structure, causing a red shift in the maximum absorption wavelength of this complex in alkaline solutions. Therefore, it can be used to verify the presence of a triple helix structure in polysaccharides. Figure 1 As shown in Figure D, compared with the control group, the MDPS-Congo red complex was observed to have... A significant red shift was observed in sodium hydroxide solutions of varying concentrations, indicating the presence of a triple helix structure in MDPS. The complex... It depends on the integrity of the Congo red conjugated system. However, with the increase of NaOH concentration, the alkaline environment destroys the conjugated structure of Congo red, breaking down the bond between MDPS and Congo red, leading to... Blue shift.

[0104] 3.2.3. Nuclear Magnetic Resonance Analysis

[0105] Figure 3 The NMR spectrum of MDPS is shown. (The polysaccharide is...) 1 The 1H NMR spectrum is mainly concentrated in the region of δ 3.0-5.6 ppm, which is the core region of the sugar ring hydrogen ( Figure 3A). Signals at δ 5.25, 5.03, 5.00, and 4.93 are attributed to anomeric protons. A typical and strong signal appears at δ 5.25, with a single peak indicating an α-configuration, pointing to the H-1 of the GalA residue in the HG or RG-I domain. Within the dense signal clusters from 3.51 to 4.71 ppm, some signals with slightly higher chemical shifts may originate from anomeric protons in specific environments within RG-I (Deng et al., 2025; H. Wang et al., 2023). Similar to previous work, characteristic signals associated with the methoxy group in the HG domain appear around 3.7 ppm, while the signal at δ 1.91 is attributed to the C5 methyl proton of Rha, suggesting the presence of the RG-I domain. The signal concentrated in the 4.56–4.60 ppm range indicates the possible presence of β-glycosidic anomeric protons, possibly belonging to β-Xyl, β-Man, and β-Glc, thus supporting the presence of GAX and GM. The signal in the 3.51–3.98 ppm range belongs to the region of non-anomeric cyclic protons and oxygen-linked methylene / methoxymethylene protons; this region exhibits complex and highly overlapping signals. MDPS 13 CNMR spectroscopy ( Figure 3 B) Contains 99.62 ppm of anomeric carbon signals, 61.00 and 60.31 ppm of primary alcohol carbon signals, and C-2, C-3, C-4, and C-5 carbon signals appearing in the 67.67–76.49 ppm region. Signals from methyl esters linked to α-D-GalAp units can be attributed to the vicinity of δ 52.8 ppm.

[0106] 3.2.4. Morphology

[0107] The surface morphology and microstructure of freeze-dried MDPS were observed using SEM. Figure 4 AB analysis revealed that MDPS exhibited irregular flake-like structures with a randomly distributed, interwoven, and stacked network, resulting in a relatively loose structure, similar in morphology to Lactarius hatsudake Tanaka polysaccharide. The MDPS surface was relatively smooth, becoming more pronounced with increasing magnification. No obvious impurities were observed, but pores of varying sizes were present. The presence of these pores can be attributed to both internal and external factors. Internally, the high carboxyl group content in MDPS induced a relatively strong negative charge and interfered with interchain bonding, leading to a porous surface. Externally, the freezing process before freeze-drying likely caused ice crystal growth, which punctured the flake-like polysaccharide, leaving uneven pores after sublimation. Notably, the flake structure of MDPS was relatively intact, possibly related to its relatively small molecular weight.

[0108] Figure 4 CD shows the AFM image of MDPS. MDPS exhibits a granular, aggregated morphology, likely due to multi-chain aggregated nanoclusters ranging in size from tens to hundreds of nanometers, a typical AFM characteristic of heteropolysaccharide mixtures. Because heteropolysaccharides consist of multiple linear chains, they readily form aggregated particles through hydrogen bonds and hydrophobic interactions. The roughness of MDPS was evaluated using average roughness and root-mean-square roughness, with Ra and Rq values ​​of 20.17 nm and 25.91 nm, respectively. The results indicate that the surface of MDPS is uneven, with some relatively prominent peaks or relatively concave valleys. Furthermore, the height distribution on its surface may not be perfectly symmetrical; peaks may be sharper than valleys, or vice versa. This may be because MDPS has a high degree of branching, making it difficult to form a tightly stacked, flat surface during deposition or drying, resulting in a relatively loose structure.

[0109] 3.2.5. Thermal Performance Analysis

[0110] The thermal stability of MDPS was studied using thermogravimetric-differential thermogravimetric analysis. Figure 5 The mass curves show that MDPS can be divided into three stages within the temperature range of 40-650 °C. The first stage corresponds to the evaporation of water molecules, ranging from 40 °C to 180 °C. MDPS contains a large number of hydrophilic groups that can absorb water, so its mass decreases with water evaporation, with an initial weight loss of approximately 8.14%. In the second stage, from 180 °C to 350 °C, the mass of MDPS decreases significantly, corresponding to the depolymerization of MDPS to produce small volatile molecules. In this process, polysaccharide molecules undergo depolymerization and decomposition, first forming oligosaccharides and monosaccharides, and then further decomposing to produce water vapor and carbon dioxide, which escape, with a weight loss of approximately 41.83%. The third stage is the carbonization stage, mainly producing gaseous compounds and solid carbon, occurring between 350 °C and 650 °C, with a weight loss of approximately 30.89%. Throughout the entire process, the total weight loss is approximately 80% (H. Wang et al., 2023). The derivative thermogravimetric curve represents the first derivative of weight loss with time; the larger the y-value, the higher the degradation rate. The DTG curve shows that the maximum decomposition rate is 0.625 mg / min at 255.5 ℃.

[0111] 3.2.6. Structural Domain Feature Analysis

[0112] Based on NMR and monosaccharide composition data, MDPS was confirmed to contain four main structural domains, which together constitute its unique heteropolysaccharide backbone. Figure 2As shown, each structural domain has specific repeating units and sidechain features:

[0113] a) Homogalacturonic acid polysaccharide (HG) domain: The main chain consists of repeating units of →4)-α-D-GalAp-(1→, where the carboxyl groups of some galacturonic acid residues are methylated, but the molar percentage of free carboxyl groups is higher than that of methylated carboxyl groups (degree of esterification 25.65% ± 2.91%). This feature endows MDPS with high charge density, enhancing its hydrophilicity and metal ion chelating ability.

[0114] b) Rhamnogalacturonic acid polysaccharide-I (RG-I) domain: The main chain consists of repeating units of →4)-α-D-GalAp-(1→2)-α-L-Rhap-(1→), with side chains mainly composed of arabinofuranose attached to the rhamnose residues. The molar ratio of total sugar in the side chains to rhamnose residues in the main chain (Ara+Gal) / Rha is 14.37, indicating a high degree of branching, which may promote its interaction with bile acids or enzymes.

[0115] c) Glucuronoarabinoxylan (GAX) domain: The main chain consists of xylose residues linked by β-1,4 linkages, and the side chains contain arabinose and glucuronic acid. This domain increases the complexity of the polysaccharide and may affect enzyme activity through competitive binding.

[0116] d) Glucomannan (GM) domain: The main chain is composed of glucose residues and mannose residues linked by β-glycosidic bonds, contributing the neutral sugar portion of MDPS (mannose content 14.02 ± 0.22) and possibly enhancing its structural rigidity.

[0117] The synergistic effect of these four domains explains the multifunctional activities of MDPS, such as antioxidant, bile acid binding, and enzyme inhibition. For example, the high proportion of free carboxyl groups in HG and RG-I is directly related to their antioxidant capacity, while the β-glycosidic bond structure of GAX and GM may facilitate binding to the active sites of α-amylase and α-glucosidase.

[0118] In summary, the structural features of MDPS were fully revealed through multi-dimensional characterization. Its unique domain composition, low molecular weight, low degree of esterification, and porous morphology jointly support its excellent biological activity.

[0119] 3.3. Functional Characteristics of MDPS

[0120] 3.3.1. Antioxidant activity

[0121] Plant polysaccharides are effective biological antioxidants. Among them, acidic polysaccharides (especially those rich in uronic acids) have attracted much attention due to their association with various important biological activities, such as antioxidant activity. Therefore, the antioxidant activity of three polysaccharides was evaluated using in vitro antioxidant methods.

[0122] DPPH radicals are stable free radicals and have been widely used to evaluate the scavenging effects of compounds on free radicals. For example... Figure 6 As shown in Figure A, the DPPH scavenging activity of ascorbic acid and MDPS exhibits a concentration-dependent trend. Among the three polysaccharides, MDPS shows relatively strong DPPH scavenging activity, achieving a scavenging rate of 38.9% at a concentration of 0.1 mg / mL. When the concentration of MDPS reaches 0.2 mg / mL, its scavenging rate is close to 80%, while the DPPH free radical scavenging activity of CHPS and CP is not significant at the same concentration. The DPPH scavenging activity of MDPS is closely related to its structural characteristics. Studies have shown that the amount of negative charge carried by polysaccharides is one of the key factors affecting their antioxidant capacity. The higher the negative charge density, the more conducive it is to stabilizing free radical intermediates, thereby effectively preventing the initiation and propagation of oxidation chain reactions. In addition, MDPS is rich in uronic acid, and the carboxyl groups on the uronic acid residues can not only directly scavenge free radicals, but also inhibit metal ion-catalyzed Fenton or Fenton-like reactions by chelating metal ions, thereby blocking the oxidation chain reaction.

[0123] ABTS of MDPS, CHPS and CP were measured. + Free radical scavenging activity was compared with that of ascorbic acid. Figure 6 B). All three polysaccharides have the ability to scavenge ABTS. + The scavenging capacity of free radicals is dose-dependent. The scavenging rate of MDPS steadily increased with increasing concentration, from 0.25 mg / mL to 2.75 mg / mL, showing a significant increase in activity. In contrast, the scavenging rates of CHPS and CP increased slowly with increasing concentration, but their overall values ​​remained lower than those of MDPS, indicating that CHPS has a lower ABTS (absence of free radicals). + The free radical scavenging activity was weaker than that of MDPS, but similar to that of DPPH. At a concentration of 2.75 mg / mL, the scavenging abilities of MDPS, CHPS, and CP reached their maximum values ​​of 94.24%, 42.44%, and 14.68%, respectively.

[0124] 3.3.2. Bile acid binding capacity

[0125] Bile acids, deoxycholic acid, glycocholic acid, and taurocholic acid are the main bile acids synthesized in the human body. Excessive reabsorption of bile acids in the intestines promotes the liver's synthesis of new bile acids from cholesterol, leading to high blood cholesterol levels. Dietary fiber has been widely reported to have the ability to bind bile acids, a property believed to be related to its lipid-lowering effects. The bile acid binding capacity of MDPS, CHPS, and CP was evaluated, with cholestyramine as a positive control. The results are as follows: Figure 7 As shown in Figures AB, the results indicate that all three polysaccharides possess some binding capacity for glycocholic acid and taurocholic acid, but both are lower than that of cholestyramine, as the latter is a widely used bile acid chelating agent. The binding capacity of all three polysaccharides increases with increasing concentration. At the same concentration, the bile acid binding capacity of the polysaccharides is ranked in descending order as MDPS > CHPS > CP. MDPS showed a binding capacity of 35.51% for glycocholic acid and 53.67% for taurocholic acid at 20 mg / mL. Notably, in the concentration range of 12–20 mg / mL, the binding capacity of MDPS for taurocholic acid is close to that of cholestyramine. In contrast, other polysaccharides typically require concentrations of 50 mg / mL or higher to achieve equivalent taurocholic acid binding capacity (W. Xu et al., 2020). This study suggests that polysaccharides affect bile acids in two ways. On the one hand, polysaccharides can directly bind to bile acids, preventing their reabsorption. On the other hand, most polysaccharides can alter the gut microbiota, thereby affecting the synthesis, conversion, and reabsorption of bile acids. The results indicate that MDPS can effectively bind to bile acids and reduce their reabsorption, showing promise for lowering cholesterol levels and potentially improving health.

[0126] 3.3.3. Inhibitory activities of α-amylase and α-glucosidase

[0127] Plant polysaccharides can mitigate postprandial blood glucose spikes by regulating the activity of specific enzymes or delaying intestinal glucose absorption. For example, α-amylase inhibitors can prevent a sharp rise in blood glucose by slowing the rate of starch digestion; inhibition of α-glucosidase can reduce glucose release, thereby preventing blood glucose spikes. The inhibition rates of three polysaccharides and acarbose on α-amylase are shown in the figure. Figure 7As shown in C. Acarbose is a known potent inhibitor, so the inhibitory effects of MDPS, CHPS, and CP were lower than that of the positive control. The inhibitory effect of MDPS on α-amylase increased slowly with increasing concentration. At a polysaccharide concentration of 20 mg / mL, the inhibition rate was close to 40%, indicating that MDPS has certain α-amylase inhibitory activity. In contrast, the inhibitory effects of CHPS and CP were weaker. Therefore, MDPS is the polysaccharide with relatively prominent α-amylase inhibitory activity among the three. All three polysaccharides have certain inhibitory abilities against α-glucosidase, but the inhibition rates of different polysaccharides against α-glucosidase vary significantly ( Figure 7 (D) When the polysaccharide concentration was 4 mg / mL, MDPS showed the best inhibitory effect on α-glucosidase, with an inhibition rate of 84.04%. CHPS and CP followed, with inhibition rates of 52.04% and 22.56%, respectively. In summary, MDPS is the most effective polysaccharide among the three in inhibiting α-glucosidase activity. Studies have shown that polysaccharides can inhibit α-amylase and α-glucosidase activity through various mechanisms, such as competitive binding to the enzyme's active site and altering the enzyme's conformation to weaken its catalytic ability. Their inhibitory effect is regulated by the polysaccharide structure; low molecular weight polysaccharides are more likely to bind to the enzyme's active site.

[0128] 4. Conclusion

[0129] This study obtained the acidic heteropolysaccharide MDPS from the fruit of *Metasequoia glyptostroboides* via water extraction and alcohol precipitation, and systematically characterized its structural features and bioactivity. The results showed that the weight-average molecular weight of MDPS was 110.114 kDa, slightly lower than that of polysaccharides from other plant sources. MDPS was composed of galacturonic acid, glucuronic acid, mannose, xylose, arabinose, glucose, galactose, fucose, and rhamnose in a molar ratio of 23.46: 23.19: 14.02: 9.66: 9.67: 9.03: 8.43: 1.29: 1.26, with a uronic acid content of approximately 45%, confirming it as an acidic heteropolysaccharide. MDPS also contained four structural domains: homogalacturonic acid polysaccharide chains, rhamnogalacturonic acid polysaccharide-I, glucuronic acid arabinoxylan, and glucomannan. SEM results showed that MDPS were irregularly shaped flakes with pores on the surface but a relatively intact overall structure, which may be related to its small molecular weight. Its average roughness and root mean square roughness were 20.17 nm and 25.91 nm, respectively, which may be related to its high degree of branching.

[0130] To further investigate the bioactivity of MDPS, commercial hawthorn polysaccharides and commercial pectin polysaccharides were introduced for comparison. In vitro antioxidant assays showed that MDPS exhibited stronger antioxidant activity than commercial hawthorn polysaccharides and commercial pectin polysaccharides, achieving a DPPH scavenging rate of 77.06% at a concentration of 0.2 mg / mL and a ABTS scavenging rate of 2.75 mg / mL. + The free radical scavenging rate reached 94.24%. Regarding bile acid binding, MDPS showed superior binding effects to both glycocholic acid and taurocholic acid compared to commercial hawthorn polysaccharides and pectin polysaccharides, with the strongest binding capacity for taurocholic acid, achieving a binding rate of 53.67% at a concentration of 20 mg / mL, approaching that of the positive control cholestyramine. Furthermore, MDPS inhibited the activity of α-amylase and α-glucosidase, an effect possibly related to its specific structural domain competitively binding to the active sites of these enzymes or altering enzyme conformation. In summary, MDPS exhibits diverse functional properties, demonstrating good potential in antioxidation, blood glucose regulation (inhibiting postprandial blood glucose spikes), and blood lipid regulation (promoting bile acid excretion).

[0131] Example 2

[0132] Preparation of MDPS

[0133] Dried hawthorn berries were pulverized and mixed with a 3% (w / v) citric acid solution. The mixture was extracted in an 80°C water bath for 3 hours, repeated twice. The extracts were combined and filtered under reduced pressure using a Buchner funnel to remove suspended solids. The filtrate was concentrated to half its original volume using a rotary evaporator at 50°C. An equal volume of 95% ethanol was added to the concentrate, and the mixture was allowed to stand at 3°C ​​for 48 hours to precipitate the polysaccharides. The mixture was centrifuged at 6000 rpm for 20 minutes, and the supernatant was discarded. Subsequently, the collected precipitate was treated with Sevag's reagent to remove proteins. Finally, the solution was dialyzed (molecular weight cutoff 8000-14000 Da) and freeze-dried to obtain MDPS.

[0134] Example 3

[0135] Preparation of MDPS

[0136] Dried hawthorn berries were pulverized and mixed with a 5% (w / v) citric acid solution. The mixture was extracted in a 90°C water bath for 1 hour, repeated twice. The extracts were combined and filtered under reduced pressure using a Buchner funnel to remove suspended solids. The filtrate was concentrated to half its original volume using a rotary evaporator at 50°C. An equal volume of 95% ethanol was added to the concentrate, and the mixture was allowed to stand at 5°C for 12 hours to precipitate the polysaccharides. The mixture was centrifuged at 6000 rpm for 20 minutes, and the supernatant was discarded. Subsequently, the collected precipitate was treated with Sevag's reagent to remove proteins. Finally, the solution was dialyzed (molecular weight cutoff 8000-14000 Da) and freeze-dried to obtain MDPS.

[0137] Comparative Example 1:

[0138] The difference from Example 1 is that Comparative Example 1 uses pure water extraction. The specific steps are as follows: large-fruited hawthorn fruit is crushed and mixed with pure water (without citric acid) at the same solid-liquid ratio, and extracted in a water bath at 85°C for 2 hours, repeated twice. Subsequent steps (filtration, concentration, precipitation with an equal volume of 95% ethanol at 4°C, protein removal, dialysis, and freeze-drying) are completely consistent with Example 1 of the present invention.

[0139] Comparative Example 2

[0140] The difference from Example 1 is that Comparative Example 2 uses low-concentration citric acid extraction. The specific steps are as follows: large-fruited hawthorn fruit is crushed and mixed with 1% (w / v) citric acid solution. The extraction and all subsequent steps are completely consistent with Example 1 of the present invention.

[0141] Comparative Example 3

[0142] The difference from Example 1 is that Comparative Example 3 uses low-concentration ethanol precipitation. The specific steps are as follows: the extraction and concentration steps are completely consistent with those of Example 1 of the present invention, but in the precipitation step, an equal volume of 50% ethanol (instead of 95%) is added to the concentrate, and it is also left to stand at 4°C for 24 hours. The subsequent steps are completely consistent with those of Example 1 of the present invention.

[0143] Comparative experiments: Structural features and functional characteristics of Examples 1-3, Comparative Examples 1-3, and commercial products

[0144] To demonstrate the technical effectiveness of this invention, the MDPS prepared in Examples 1-3, the polysaccharides prepared in Comparative Examples 1-3, and commercial pectin polysaccharides (CP) and commercial hawthorn polysaccharides (CHPS) were compared and analyzed. The results are shown in Tables 4 and 5.

[0145] Table 4: Comparison of structural characteristics of Examples 1-3, Comparative Examples 1-3, and commercial pectin polysaccharides and commercial hawthorn polysaccharides

[0146]

[0147] Table 5: Comparison of functional properties of Examples 1-3, Comparative Examples 1-3, and commercial pectin polysaccharides and commercial hawthorn polysaccharides

[0148]

[0149] Analysis and Summary:

[0150] 1. The complexity of monosaccharide composition and structure

[0151] The MDPS (Examples 1-3) prepared under specific extraction conditions (3%-5% citric acid) of this invention have a complex and balanced composition of nine monosaccharides, rich in galacturonic acid and glucuronic acid (total uronic acid content 42.61%–48.92%). In contrast, the uronic acid content of the products from Comparative Example 1 (pure water extraction) and Comparative Example 2 (1% citric acid) was significantly reduced (28.37% and 36.82%, respectively). Commercial products (CP and CHPS) suffer from either a lack of monosaccharide variety or low uronic acid content. This demonstrates that the extraction conditions of this invention are key to obtaining a complex and balanced heteropolysaccharide structure.

[0152] 2. Molecular weight and distribution

[0153] The weight-average molecular weight of the MDPS in this invention ranges from 81.6 to 157.3 kDa, exhibiting a narrow distribution. In contrast, the molecular weights of Comparative Examples 1 and 2 increased to 235.8 kDa and 185.4 kDa, respectively, closer to the high molecular weight range of commercial polysaccharides (CP 285.7 kDa, CHPS 350.2 kDa). Comparative Example 3 (50% ethanol precipitation) contained numerous impurities, making accurate determination of uniform molecular weight impossible; however, its functional activity decreased significantly, suggesting that low precipitation efficiency may lead to component impurities or altered degree of polymerization. The data indicate that the synergistic effect of 3%-5% citric acid extraction and 95% ethanol precipitation used in this invention is essential for achieving a lower and more uniform molecular weight, which is crucial for bioactivity.

[0154] 3. Glucuronic acid content and degree of esterification

[0155] The MDPS of this invention achieves an ideal combination of high uronic acid content (>42%) and low esterification degree (<30%), ensuring high charge density. The esterification degree of Comparative Example 1 (pure water extraction) is as high as 58.42%, and that of Comparative Example 2 (low acid extraction) is 38.76%, both significantly higher than that of this invention. This indicates that insufficient or absent citric acid concentration leads to increased esterification degree and reduced free carboxyl groups in the extracted polysaccharides. While commercial CP has high uronic acid content, its esterification degree is excessively high (72.48%), while CHPS has both indicators being low. The process of this invention precisely controls the acidity and charge state of the polysaccharides.

[0156] 4. Structural Domain and Branching Degree

[0157] Structural analysis revealed that the MDPS of this invention possesses multi-domain characteristics, including HG, RG-I, GAX, and GM, with the RG-I domain exhibiting high branching ((Ara+Gal) / Rha = 11.63–23.16). This highlights the advantages of the method of this invention in obtaining polysaccharides with specific higher-order structural and functional conformations.

[0158] 5. Antioxidant activity

[0159] Regarding antioxidant capacity, the present invention exhibits a MDPS (DPPH) scavenging rate of 74.83%–78.51%, and an ABTS scavenging rate of 100%. + The scavenging rate (91.47%–95.76%) was significantly superior to all comparative examples and commercial products. Comparative Examples 1 and 2 showed moderate activity, but were still far below that of this invention, while the activity of Comparative Example 3 decreased dramatically to a level close to that of commercial products. This directly demonstrates that deviations from the extraction and precipitation process parameters (such as acidity and alcohol concentration) of this invention will lead to a significant deterioration in antioxidant activity.

[0160] 6. Bile acid conjugation

[0161] The MDPS of this invention exhibits superior binding capacity for both bile acids. Most importantly, its binding rate for taurocholic acid (50.96%–55.43%) is close to that of the positive control drug cholestyramine at 20 mg / mL. The binding capacity of Comparative Examples 1, 2, and 3 showed varying degrees of decrease, with Comparative Example 1 (pure water extraction) showing the most significant decrease. This strongly demonstrates that the acidic extraction process of this invention is indispensable for activating and exposing the bile acid binding sites of the polysaccharide.

[0162] 7. Inhibitory activity of glucose-lowering enzymes

[0163] The MDPS of this invention exhibits superior inhibitory activity against α-amylase and α-glucosidase, especially against α-glucosidase, with an inhibition rate (80.25%–86.09%) significantly higher than that of commercial products and all comparative examples. The inhibitory activities of Comparative Examples 1, 2, and 3 show a regular decrease and are all lower than those of the embodiments of this invention. This clearly demonstrates that the specific molecular weight, charge characteristics, and structural complexity of the polysaccharide obtained by this invention are the structural basis for its potent enzyme inhibitory activity, and the acquisition of this structure is strictly dependent on the preparation process of this invention.

[0164] In summary, polysaccharides prepared under unoptimized conditions such as pure water, low-concentration acid, or low-concentration ethanol exhibit significant defects in structural integrity, molecular weight, charge properties, and final functional activity. However, by employing the core technology of this invention—"3%-5% citric acid extraction combined with 95% ethanol precipitation"—a novel large-fruited hawthorn heteropolysaccharide (MDPS) with a complex monosaccharide composition, moderately low molecular weight, high free carboxyl content, and synergistic multi-domain structure was successfully prepared. This unique structure enables it to exhibit comprehensive and significantly superior bioactivity compared to comparative studies and existing commercial products in areas such as antioxidant activity, bile acid binding, and inhibition of key glucose-metabolizing enzymes, thus demonstrating clear and promising application prospects in drug development.

[0165] The above description is a detailed description of the preferred embodiments of the present invention. However, the embodiments are not intended to limit the scope of the patent application of the present invention. All equivalent changes or modifications made under the technical spirit of the present invention should fall within the patent scope covered by the present invention.

Claims

1. A method for preparing heteropolysaccharides from large-fruited hawthorn, characterized in that, Includes the following steps: The large-fruited hawthorn fruit was crushed and mixed with a citric acid solution with a mass-volume concentration of 3%–5%. The mixture was then treated in a water bath at 80–90°C for 1–3 hours. This process was repeated twice. The extracts were combined, filtered, and the filtrate was concentrated to half its original volume at 50°C to obtain a concentrated solution. An equal volume of 95% ethanol was added to the concentrated solution, and the mixture was allowed to stand at 3–5°C for 12–48 hours to precipitate the polysaccharides. The precipitate was collected, and the proteins in the precipitate were removed using Sevag reagent. The solution after protein removal was dialyzed and freeze-dried to obtain the large-fruited hawthorn heteropolysaccharide.

2. The hawthorn heteropolysaccharide obtained by the preparation method according to claim 1, characterized in that, The large-fruited hawthorn heteropolysaccharide contains galacturonic acid, glucuronic acid, mannose, xylose, arabinose, glucose, galactose, fucose, and rhamnose; the weight-average molecular weight of the large-fruited hawthorn heteropolysaccharide is 81.6 kDa to 157.3 kDa.

3. The hawthorn heteropolysaccharide according to claim 2, characterized in that, The molar ratio of galacturonic acid, glucuronic acid, mannose, xylose, arabinose, glucose, galactose, fucose and rhamnose is (21.3–25.9):(21.2–25.4):(12.8–15.6):(8.7–10.8):(8.6–11.2):(8.2–10.1):(7.4–9.3):(1.3–1.6):(1.1–1.4).

4. The hawthorn heteropolysaccharide according to claim 2, characterized in that, The large-fruited hawthorn heteropolysaccharide contains four structural domains: homogalacturonic acid polysaccharide, rhamnogalacturonic acid polysaccharide-I, glucuronic acid arabinoxylan, and glucomannan; the total sugar content of the large-fruited hawthorn heteropolysaccharide is ≥89.26%, the uronic acid content is 42.61-48.92%, and the degree of esterification is 22.31-29.64%.

5. The hawthorn heteropolysaccharide according to claim 4, characterized in that, The large-fruited hawthorn heteropolysaccharide contains the following structural domains: a) Homogalacturonic acid polysaccharide domain, whose main chain is composed of →4)-α-D-GalAp-(1→ repeating units, in which the carboxyl groups of some galacturonic acid residues are methylated, and the molar percentage of free carboxyl groups is higher than that of methylated carboxyl groups. b) Rhamnogalacturonic acid polysaccharide-I domain, whose main chain is composed of →4)-α-D-GalAp-(1→2)-α-L-Rhap-(1→ repeating units, wherein the rhamnosine residues of the main chain are attached to side chains, the side chains are mainly composed of arabinofuranose, and the molar ratio of total sugar in the side chains to rhamnosine residues in the main chain (Ara+Gal) / Rha is 11.63 to 23.16; c) A glucuronic acid arabinoxylan domain, the main chain of which is composed of β-1,4 linked xylose residues, and the main chain is connected to side chains containing arabinose and glucuronic acid. d) Glucomannan domain, whose backbone is composed of glucose residues and mannose residues linked by β-glycosidic bonds.

6. The use of hawthorn heteropolysaccharide according to any one of claims 2-5 in the preparation of antioxidant agents and / or bile acid binders.

7. The use of hawthorn heteropolysaccharide according to any one of claims 2-5 in the preparation of α-amylase and / or α-glucosidase inhibitors.

8. The use of hawthorn heteropolysaccharide according to any one of claims 2-5 in the preparation of pharmaceuticals for regulating blood sugar and / or blood lipids.

9. The use of hawthorn heteropolysaccharide according to any one of claims 2-5 in the preparation of a medicine for the prevention or adjunctive treatment of metabolic syndrome-related diseases; wherein the metabolic syndrome-related diseases include any one or more of diabetes mellitus, hyperlipidemia, and obesity.

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