Gelidium polysaccharide XEP-70, preparation method therefor, and use thereof

Abstract

The present application relates to the technical field of natural polymers and biological pharmacy, and specifically relates to a Gelidium polysaccharide XEP-70, a preparation method therefor, and a use thereof. The Gelidium polysaccharide XEP-70 provided by the present application comprises the following sugar residues and glycosidic bond types: →4)-α-D-Glcp-(1→, →4,6)-α-D-Glcp-(1→, →4)-α-D-GlcpA-(1→, →2)α-D-Manp(1→, →2,6)-α-D-Manp-(1→, →6)-β-D-Manp-(1→, →6)-α-D-Galp-(1→, →2,6)-β-D-Galf-(1→, and β-D-Xylp-(1→. The Gelidium polysaccharide XEP-70 has a weight-average molecular weight of 430.98 kDa. The Gelidium polysaccharide XEP-70 enhances cellular antioxidant capacity by activating the Nrf2-Keap1-ARE signaling pathway, thereby effectively protecting LX-2 cells from hydrogen peroxide-induced oxidative damage.

Description

A kind of stone flower polysaccharide XEP-70 and its preparation method and application

[0001] This application claims priority to the Chinese patent application filed with the Patent Office of China on March 1, 2024, with application number CN202410238888.6 and invention name “A kind of stone flower polysaccharide XEP-70 and its preparation method and application”, the entire contents of which are incorporated by reference into this application. Technical Field

[0002] The present application belongs to the field of natural polymers and biopharmaceutical technology, and specifically relates to a stone flower polysaccharide XEP-70 and its preparation method and application. Background Art

[0003] In biological systems, elevated levels of reactive oxygen species (ROS) and decreased antioxidant mechanisms lead to cellular oxidative stress. Normally, ROS are generated during mitochondrial oxygen metabolism and participate in cellular signaling. However, excessive ROS production and impaired antioxidant capacity can induce structural damage to cells and destroy tissues. This is thought to be associated with the development of many chronic diseases, such as inflammation, diabetes, cancer, and aging.

[0004] Hydrogen peroxide is one of the main causes of cellular oxidative stress and is commonly used to assess antioxidant capacity and the ability of cells to clear ROS. Studies have shown (DK Ingawale, SK Mandlik, SR Naik, Models of hepatotoxicity and the underlying cellular, biochemical and immunological mechanism(s): a critical discussion, Environ Toxicol Pharmacol 37(1)(2014)118-33; M. Parola, G. Robino, Oxidative stress-related molecules and liver fibrosis, J Hepatol 35(2)(2001)297-306.) that liver cell damage is closely related to increased ROS, and oxidative stress is considered to be one of the pathological mechanisms leading to the occurrence and development of various liver diseases. Therefore, antioxidants are considered to be a good strategy for treating liver diseases.

[0005] Although synthetic antioxidants can prevent free radical chain reactions, they almost inevitably have side effects and may cause liver damage and cancer. Therefore, it is crucial to develop natural antioxidants that can protect the body from free radical damage and delay the progression of liver disease.

[0006] Summary of the Invention

[0007] The purpose of this application is to provide a stone flower polysaccharide XEP-70 and its preparation method and application. Stone flower polysaccharide XEP-70 is a natural polymer that can enhance the antioxidant capacity of cells and effectively protect against oxidative damage, especially effectively protecting liver cells from hydrogen peroxide-induced oxidative damage.

[0008] In order to achieve the above-mentioned object, the present application provides a stone flower polysaccharide XEP-70, wherein the sugar residues and glycosidic bond types of the stone flower polysaccharide XEP-70 include: →4)-α-D-Glcp-(1→, →4,6)-α-D-Glcp-(1→, →4)-α-D-GlcpA-(1→, →2)α-D-Manp(1→, →2,6)-α-D-Manp-(1→, →6)-β-D-Manp-(1→, →6)-α-D-Galp-(1→, →2,6)-β-D-Galf-(1→, β-D-Xylp-(1→;

[0009] The weight average molecular weight of the Shihua polysaccharide XEP-70 is 430.98 kDa.

[0010] Preferably, the total sugar content of the stone polysaccharide XEP-70 is 99.05 wt.%, and the uronic acid content is 13.09 wt.%.

[0011] Preferably, the monosaccharide of the Shihua polysaccharide XEP-70 consists of mannose, galacturonic acid, glucose, galactose and xylose.

[0012] Preferably, the molar ratio of mannose, galacturonic acid, glucose, galactose and xylose is 39.28:16.09:17.56:23.20:3.87.

[0013] The present application also provides a method for preparing the agarwood polysaccharide XEP-70 described in the above technical solution, comprising the following steps:

[0014] After hot extraction of the stone flower with water, the solid-liquid separation is carried out, and the obtained aqueous solution is concentrated to obtain a crude stone flower polysaccharide solution;

[0015] subjecting the crude polysaccharide solution of the stone flower to alcohol precipitation, collecting the precipitate and freeze-drying it to obtain crude polysaccharide of the stone flower;

[0016] The crude polysaccharide of the stone flower is separated and purified using a DEAE anion exchange column; the separation and purification comprises: redissolving the crude polysaccharide of the stone flower in water to prepare an aqueous solution, loading the DEAE anion exchange column, eluting with water and 0.5 mol / L NaCl salt solution in sequence, collecting the eluted fractions of the 0.5 mol / L NaCl salt solution, dialyzing, and freeze-drying to obtain the stone flower polysaccharide XEP-1;

[0017] The agarwood polysaccharide XEP-1 was redissolved in water, and anhydrous ethanol was added to make the final concentration of ethanol 70% v / v. After standing at 0-4° C. for 8-48 hours, the precipitate was collected by centrifugation and dried to obtain the agarwood polysaccharide XEP-70.

[0018] Preferably, the number of times of adding water for hot extraction is 3 times, and the mass volume ratio of the stone flower to water is 1 kg:10 L each time the water is added for hot extraction;

[0019] The temperature of the water-adding heat extraction is 90-100°C;

[0020] The concentration temperature is 95°C;

[0021] The final concentration of ethanol in the alcohol precipitation is 80% v / v;

[0022] The dialysis includes distilled water dialysis and running water dialysis; the molecular weight cut-off of the distilled water dialysis is 3500Da, and the dialysis time is 24h; the molecular weight cut-off of the running water dialysis is 1000-10000Da, and the dialysis time is 24h;

[0023] The freeze-drying conditions are: pressure of 10 to 30 MPa and temperature of -60 to -80°C.

[0024] The present application also provides the use of the stone flower polysaccharide XEP-70 described in the above technical solution in the preparation of antioxidant products.

[0025] Preferably, the antioxidant product comprises a drug for preventing and / or treating oxidative damage.

[0026] Preferably, the effective concentration of XEP-70 in the drug is 25 to 100 μg / mL.

[0027] Preferably, the oxidative damage comprises H2O2-induced oxidative damage. Beneficial effects:

[0028] The present application obtains a polysaccharide XEP-70 from stone flower, and finds that the sugar residues and glycosidic bond types of the polysaccharide include: →4)-α-D-Glcp-(1→, →4,6)-α-D-Glcp-(1→, →4)-α-D-GlcpA-(1→, →2)α-D-Manp(1→, →2,6)-α-D-Manp-(1→, →6)-β-D-Manp-(1→, →6)-α-D-Galp-(1→, →2,6)-β-D-Galf-(1→, β-D-Xylp-(1→, with a weight average molecular weight of 430.98 kDa. The polysaccharide XEP-70 can enhance cell Antioxidant capacity, effectively protecting against oxidative damage, especially effectively protecting liver cells from hydrogen peroxide-induced oxidative damage. This application also used the hydrogen peroxide-induced LiemingXu-2 cell (LX-2) oxidative damage model to evaluate the potential protective effect of XEP-70 against oxidative damage to liver cells. The results showed that the stone flower polysaccharide XEP-70 can enhance the cellular antioxidant capacity by activating the Nrf2-Keap1-ARE signaling pathway, thereby effectively protecting LX-2 cells from hydrogen peroxide-induced oxidative damage. Compared with other stone flower polysaccharides, XEP-70 showed the best antioxidant activity in free radical scavenging experiments and is a potential natural antioxidant. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments.

[0030] Figure 1 shows the results of uniformity and molecular weight determination of different stone flower polysaccharides;

[0031] Figure 2 shows the Fourier transform infrared spectroscopy analysis results of different stone flower polysaccharides;

[0032] Figure 3 shows the results of scanning electron microscopy (SEM) analysis of XEP-70. The magnification of Figure A is 400 times, and the magnification of Figure B is 4000 times.

[0033] Figure 4 shows the results of atomic force microscopy analysis of XEP-70; C is a two-dimensional image; D is a three-dimensional image;

[0034] Figure 5 shows the results of nuclear magnetic resonance analysis of stone flower polysaccharide XEP-70 ( 1 HNMR);

[0035] Figure 6 shows the results of nuclear magnetic resonance analysis of stone flower polysaccharide XEP-70 ( 13 CNMR);

[0036] Figure 7 shows the results of the free radical scavenging ability test of different stone flower polysaccharides; all data are expressed as mean ± SD (n = 4), A to D are DPPH free radical, ABTS free radical, O 2- Free radicals and OH - Free radical; E is reducing power;

[0037] Figure 8 shows the effects of different concentrations of XEP-70 on LX-2 cell viability; all data are expressed as mean ± SD (n = 4), compared with H2O2, *p < 0.05, **p < 0.01;

[0038] Figure 9 shows the effects of different concentrations of H2O2 on LX-2 cell viability; all data are expressed as mean ± SD (n = 4), compared with H2O2, *p < 0.05, **p < 0.01, ***p < 0.001;

[0039] Figure 10 shows the antioxidant activity test results (cell viability) of different concentrations of XEP-70 against H2O2 oxidative damage model; all data are expressed as mean ± SD (n = 4), compared with H2O2, *p < 0.05, **p < 0.01, ***p < 0.001;

[0040] Figure 11 shows the results of oxidative stress levels and antioxidant enzyme activity tests in the H2O2 oxidative damage model induced by different concentrations of XEP-70. A to G represent MDA content, LDH activity, SOD activity, T-AOC activity, GSH activity, SOD activity, and CAT activity, respectively. All data are expressed as mean ± SD (n = 4). Compared with H2O2, *p < 0.05, **p < 0.01, ***p < 0.001.

[0041] Figure 12 shows the results of Western blotting;

[0042] Figure 13 shows the effect of XEP-70 on the expression of key proteins in the Nrf2-Keap1-ARE signaling pathway in the H2O2 oxidative damage model; compared with H2O2, *p<0.05, **p<0.01, ***p<0.001. DETAILED DESCRIPTION

[0043] The present application provides a stone flower polysaccharide XEP-70, wherein the sugar residues and glycosidic bond types of the stone flower polysaccharide XEP-70 include: →4)-α-D-Glcp-(1→, →4,6)-α-D-Glcp-(1→, →4)-α-D-GlcpA-(1→, →2)α-D-Manp(1→, →2,6)-α-D-Manp-(1→, →6)-β-D-Manp-(1→, →6)-α-D-Galp-(1→, →2,6)-β-D-Galf-(1→, β-D-Xylp-(1→;

[0044] The weight average molecular weight of the Shihua polysaccharide XEP-70 is 430.98 kDa.

[0045] In the present application, the total sugar content of the stone flower polysaccharide XEP-70 is preferably 99.05wt.%, and the uronic acid content is preferably 13.09wt.%; the monosaccharides of the stone flower polysaccharide XEP-70 are preferably composed of mannose, galacturonic acid, glucose, galactose and xylose; the molar ratio of mannose, galacturonic acid, glucose, galactose and xylose is preferably 39.28:16.09:17.56:23.20:3.87.

[0046] The present application also preferably provides a method for preparing the agarwood polysaccharide XEP-70 described in the above technical solution, comprising the following steps:

[0047] After hot extraction of the stone flower with water, the solid-liquid separation is carried out, and the obtained aqueous solution is concentrated to obtain a crude stone flower polysaccharide solution;

[0048] subjecting the crude polysaccharide solution of the stone flower to alcohol precipitation, collecting the precipitate and freeze-drying it to obtain crude polysaccharide of the stone flower;

[0049] The crude polysaccharide of the stone flower is separated and purified using a DEAE anion exchange column; the separation and purification comprises: redissolving the crude polysaccharide of the stone flower in water to prepare an aqueous solution, loading the DEAE anion exchange column, eluting with water and 0.5 mol / L NaCl salt solution in sequence, collecting the eluted fractions of the 0.5 mol / L NaCl salt solution, dialyzing, and freeze-drying to obtain the stone flower polysaccharide XEP-1;

[0050] The agarwood polysaccharide XEP-1 was redissolved in water, and anhydrous ethanol was added to make the final concentration of ethanol 70% v / v. After standing at 0-4° C. for 8-48 hours, the precipitate was collected by centrifugation and dried to obtain the agarwood polysaccharide XEP-70.

[0051] In the present application, a mixture of lithospermum and water is mixed, and solid-liquid separation is performed after heat extraction. The resulting aqueous solution is concentrated to obtain a lithospermum crude polysaccharide solution. In the present application, the number of heat extractions is preferably 3 times, and the mass volume ratio of lithospermum to water is preferably 1kg:10L during each heat extraction. The temperature of the heat extraction in the present application is preferably 90-100°C, more preferably 95°C; the temperature of the concentration is preferably 95°C; and the multiple of the concentration is preferably 9 times. The method of solid-liquid separation in the present application is preferably filtration.

[0052] After obtaining the crude polysaccharide solution of stone flower, the present application performs alcohol precipitation on the crude polysaccharide solution of stone flower, collects the precipitate and then freeze-dries it to obtain crude polysaccharide of stone flower. In the present application, the final concentration of ethanol in the alcohol precipitation is preferably 80% v / v. The method of collecting the precipitate described in the present application is preferably centrifugation; the speed of the centrifugation is preferably 4500 rpm, and the time is preferably 15 minutes. The freeze-drying pressure described in the present application is preferably 10 to 30 MPa, more preferably 15 to 25 MPa, and more preferably 20 MPa; the freeze-drying temperature is preferably -60 to -80°C, and more preferably -70°C.

[0053] After obtaining the crude polysaccharide of stone flower, the present application uses a DEAE anion exchange column to separate and purify the crude polysaccharide of stone flower, redissolves the crude polysaccharide of stone flower in water to prepare an aqueous solution, loads the DEAE anion exchange column, and elutes with water and 0.5 mol / L NaCl salt solution in sequence, and collects the eluted components of the 0.5 mol / L NaCl salt solution. In the present application, the DEAE anion exchange column preferably includes a DEAE cellulose column. The present application preferably mixes the crude polysaccharide of stone flower and water in a mass volume ratio of 4 mg:1 mL to prepare the aqueous solution. During elution in the present application, the volume ratio of the aqueous solution, elution water and 0.5 mol / L NaCl salt solution for elution is preferably 1:40:40. The water in the present application is preferably distilled water. The present application uses a 0.5 mol / L NaCl salt solution for elution, which can remove highly polar impurities while obtaining acidic polysaccharides.

[0054] After obtaining the elution fraction of 0.5 mol / L NaCl salt solution, the present application dialyzes the elution fraction of the 0.5 mol / L NaCl salt solution, and obtains stone flower polysaccharide XEP-1 after freeze-drying. In the present application, the dialysis preferably includes distilled water dialysis and running water dialysis. The present application preferably performs distilled water dialysis and running water dialysis on the NaCl salt solution component in sequence; the molecular weight cutoff of the distilled water dialysis is preferably 3500 Da; the time of the distilled water dialysis is preferably 24 hours, and it is more preferred that fresh distilled water is replaced every 3 hours of dialysis; the molecular weight cutoff of the running water dialysis is preferably 1000-10000 Da, more preferably 2000-8000 Da, more preferably 3000-6000 Da, and most preferably 3500 Da; the time of the running water dialysis is preferably 24 hours. The method of running water dialysis in the present application is preferably to use tap water with a certain flow rate for dialysis; the flow rate of the tap water is preferably 300 mL / min. The freeze-drying pressure of the present application is preferably 10 to 30 MPa, more preferably 15 to 25 MPa, and more preferably 20 MPa; the freeze-drying temperature is preferably -60 to -80°C, and more preferably -70°C.

[0055] In the preparation of XEP-70, the polysaccharide from the calcareous flower is extracted with hot water and precipitated with alcohol, yielding a crude polysaccharide yield of 8.62%. The crude polysaccharide is further purified via a DEAE anion exchange column to a 0.5 mol / L NaCl fraction, yielding 2.48% XEP-1. XEP-1 is then purified by ethanol precipitation, yielding 0.83% XEP-70. After obtaining XEP-70, the present applicant studied the structural characteristics of XEP-70 by nuclear magnetic resonance, scanning electron microscopy and atomic force microscopy, and found that its sugar residues and glycosidic bond types include: →4)-α-D-Glcp-(1→, →4,6)-α-D-Glcp-(1→, →4)-α-D-GlcpA-(1→, →2)α-D-Manp(1→, →2,6)-α-D-Manp-(1→, →6)-β-D-Manp-(1→, →6)-α-D-Galp-(1→, →2,6)-β-D-Galf-(1→, β-D- Xylp-(1→, with a weight-average molecular weight of 430.98 kDa. The present application also used the hydrogen peroxide-induced LiemingXu-2 cell (LX-2) oxidative damage model to evaluate its potential protective effect against oxidative damage to hepatocytes. The results showed that the obtained stone flower polysaccharide XEP-70 can enhance the cellular antioxidant capacity by activating the Nrf2-Keap1-ARE signaling pathway, thereby effectively protecting LX-2 cells from hydrogen peroxide-induced oxidative damage. Compared with other stone flower polysaccharides, XEP-70 showed the best antioxidant activity in free radical scavenging experiments and is a potential natural antioxidant.

[0056] In view of the effects of the stone flower polysaccharide XEP-70 provided in this application, the use of the stone flower polysaccharide XEP-70 in the preparation of antioxidant products also falls within the scope of protection of this application. In this application, the antioxidant product preferably includes a drug for preventing and / or treating oxidative damage; the oxidative damage preferably includes hepatocyte oxidative damage; the oxidative damage preferably includes H2O2-induced oxidative damage; the effective concentration of stone flower polysaccharide XEP-70 in the drug described in this application is preferably 25 to 100 μg / mL, and more preferably 50 μg / mL. This application constructs a hydrogen peroxide-induced LiemingXu-2 cell (LX-2) oxidative damage model to evaluate the potential protective effect of stone flower polysaccharide XEP-70 on hepatocyte oxidative damage. The results show that stone flower polysaccharide XEP-70 can enhance the cellular antioxidant capacity by activating the Nrf2-Keap1-ARE signaling pathway, thereby effectively protecting LX-2 cells from hydrogen peroxide-induced oxidative damage.

[0057] In order to further illustrate the present application, the following detailed description of a stone polysaccharide XEP-70 provided in the present application and its preparation method and application is provided in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present application.

[0058] Example 1

[0059] Preparation of Geum japonicum polysaccharide

[0060] 1. Materials and Reagents

[0061] Lithops were collected from the Laji Mountain habitat in Qinghai Province, China; DEAE cellulose was from Yuanye Biotechnology Co., Ltd. (Shanghai, China); BCA and T-AOC were from Bio-Thera (Shanghai) Biotechnology Co., Ltd.; CAT, MDA, ROS, GSH, LDH, and SOD were purchased from Nanjing Jiancheng Bioengineering Co., Ltd. (Nanjing, China); LX-2 cells (CL-0560) and DMEM (PM1500210) were provided by Procell (Wuhan, China); FBS (10099-141C) was provided by Gibco (New York, USA).

[0062] All antibodies used in the experiments were from Abcam (Cambridge, UK), except for goat anti-rabbit antibody (HRP) (AS1107) which was purchased from Aspen (Wuhan, China);

[0063] All other chemical reagents were of analytical grade;

[0064] 2. Extraction and purification of stone flower polysaccharide

[0065] (1) Extract the agave pepo three times with boiling water at 90-100°C, with a solid-liquid ratio of 1 kg:10 L each time, and then concentrate at 95°C to a volume of about 1 liter to obtain a agave pepo crude polysaccharide solution. The agave pepo crude polysaccharide solution was mixed with ethanol to a final ethanol concentration of 80% v / v, allowed to stand at 4°C for 24 hours, centrifuged at 4500 rpm for 15 minutes, and freeze-dried (cold trap temperature -70°C, vacuum degree 10 Pa) for 24 hours to obtain agave pepo crude polysaccharide (XEP) with a yield of 8.62%;

[0066] (2) 200 mg of the XEP obtained in step (1) was dissolved in 5 mL of distilled water and then loaded onto a DEAE-cellulose column (20 mm × 20 cm) at a flow rate of 1.8 mL / min. The column was eluted with 200 mL of distilled water and 200 mL of 0.5 mol / L NaCl salt solution, respectively. 7.4 mL of the eluate was collected in each test tube and the sugar content was determined by the phenol-sulfuric acid method. After collection, the main fraction was dialyzed against distilled water for 24 h (molecular weight cut-off 3500 Da, static dialysis, with new distilled water replaced every 3 h), then dialyzed against flowing water (molecular weight cut-off 3500 Da, tap water was filled into the container, a dialysis bag was placed, and tap water was added to the container at a low flow rate until excess tap water overflowed) for 24 h, and then freeze-dried (cold trap temperature -70°C, vacuum degree 10 Pa) for 24 h. The polysaccharide eluted with 0.5 mol / L NaCl salt solution was collected and named XEP-1 with a yield of 2.48%;

[0067] (3) The XEP-1 obtained in step (2) was dissolved in deionized water to a concentration of 10 mg / mL, and then anhydrous ethanol was slowly added to the XEP-1 solution to a final ethanol concentration of 50% (v / v); the solution was stored at 4°C for 48 hours, and then centrifuged at 12,000 rpm for 15 minutes. The precipitate was collected and recorded as agar-agar polysaccharide XEP-50, with a yield of 0.57%;

[0068] The XEP-1 obtained in step (2) was dissolved in deionized water to a concentration of 10 mg / mL, and then anhydrous ethanol was slowly added to the XEP-1 solution to a final ethanol concentration of 70% (v / v); the solution was stored at 4°C for 48 hours, and then centrifuged at 12,000 rpm for 15 minutes to collect the precipitate, which was designated as agar-agar polysaccharide XEP-70, with a yield of 0.83%;

[0069] The XEP-1 obtained in step (2) was dissolved in deionized water to a concentration of 10 mg / mL, and then anhydrous ethanol was slowly added to the XEP-1 solution to a final ethanol concentration of 90% (v / v). The solution was stored at 4°C for 48 hours and then centrifuged at 12,000 rpm for 15 minutes. The precipitate was collected and recorded as agar-agar polysaccharide XEP-90, with a yield of 0.37%.

[0070] Test Example 1

[0071] Structural characteristics of agarwood polysaccharide

[0072] The following tests were performed using the agarwood polysaccharide XEP-50, agarwood polysaccharide XEP-70 and agarwood polysaccharide XEP-90 obtained in Example 1 as test samples:

[0073] 1. Homogeneity and Molecular Weight Determination

[0074] Molecular weight was measured using SEC-MALLS-RI: Shodex OH-pak SB-805, 804, and 803 columns (8 mm × 300 mm inner diameter; Showa Denko K.K., Tokyo, Japan) were connected in series. The sample (1 mg) was dissolved in 1 mL of 0.1 mol / L NaNO3 aqueous solution and then passed through the membrane (0.45 μm). The column temperature was 45°C, the injection volume was 100 μL, the mobile phase consisted of 0.02% NaN3 and 0.1 mol / L NaNO3, and the flow rate was 0.4 mL / min. Data were processed using ASTRA 6.1 (Wyatt Technology, USA). The results showed that XEP-50 had a single elution peak with a molar mass of 322.25 kDa; XEP-70 had two elution peaks with a molar mass of 430.98 kDa; and XEP-90 had two elution peaks with a molar mass of 76.36 kDa (Figure 1).

[0075] 2. Chemical composition and monosaccharide composition analysis

[0076] 2.1 The total content of polysaccharides was calculated using the phenol-sulfuric acid method with six monosaccharides as reference substances and the correction coefficient of the monosaccharides (M. Dubois, K. Gilles, J. K. Hamilton, P. A. Rebers, F. Smith, A colorimetric method for the determination of sugars, Nature 168(4265)(1951)167; F. Yue, J. Zhang, J. Xu, T. Niu, X. Lu, M. Liu, Effects of monosaccharide composition on quantitative analysis of total sugar content by phenol-sulfuric acid method, Front Nutr 9(2022)963318.). The results are shown in Table 1.

[0077] 2.2 Using galacturonic acid as a reference, the m-hydroxybiphenyl method was used to quantify the content of uronic acid (N. Blumenkrantz, G. Asboe-Hansen, New method for quantitative determination of uronic acids, Anal Biochem 54(2)(1973)484-9.). The results are shown in Table 1.

[0078] 2.3 Using bovine serum albumin as a reference, the content of uronic acid was determined by the Coomassie brilliant blue method (MM Bradford, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Analytical Biochemistry 72(1)(1976)248-254.). The results are shown in Table 1.

[0079] 2.4 The sample to be tested (3 mg) was dissolved in 1 mL of 2 M trifluoroacetic acid (TFA) and sealed in an ampoule at 120°C for two hours. The remaining TFA was then removed with anhydrous ethanol, and the solution was evaporated to dryness below 70°C. The polysaccharide hydrolysate was derivatized with 0.5 mL of 0.3 M NaOH and 0.5 mL of 0.5 M PMP at 70°C for 30 minutes. The mixture was neutralized with 0.5 mL of 0.3 M HCl and extracted three times with chloroform. The derivatization was performed using an Ultimate 3000 ultra-high performance liquid chromatography system using a Kinetex C18 column (1004.6 mmi.d.) with a guard column. Ultraviolet absorbance at a wavelength of 245 nm was used to record the process, and the monosaccharide composition was determined based on the peak elution time and peak area. The results are shown in Table 1.

[0080] Table 1 Chemical composition and monosaccharide composition of stone flower polysaccharide Note: ND means not detected, and the percentage of monosaccharide is molar percentage.

[0081] As shown in Table 1, the total sugar contents of XEP-50, XEP-70, and XEP-90 were 89.00%, 99.05%, and 82.42%, respectively. The uronic acid contents of XEP-50, XEP-70, and XEP-90 were 1.13%, 13.09%, and 7.22%, respectively. The protein content of XEP-50 was 0.91%, while no protein was detected in XEP-70 and XEP-90; XEP-50 mainly contained mannose (55.00%), galacturonic acid (2.56%), glucose (26.48%), galactose (13.96%) and xylose (2.00%); XEP-70 mainly contained mannose (39.28%), galacturonic acid (16.09%), glucose (17.56%), galactose (23.20%) and xylose (3.87%); XEP-90 mainly contained mannose (32.63%), galacturonic acid (8.64%), glucose (15.65%), galactose (34.67%), xylose (1.17%) and arabinose (7.24%).

[0082] 3. Fourier Transform Infrared Spectroscopy

[0083] The Fourier transform infrared spectra of the samples were recorded using a Fourier transform infrared spectrophotometer (Nicolet-IS50, USA): the samples (2 mg) were ground with KBr powder and condensed into pellets, which were then recorded on the Fourier transform infrared spectrometer (resolution, 4 cm-1) in the frequency range of 4000-400 cm-1 (mid-infrared region). The results showed that the Fourier transform infrared spectra of the XEP-50, XEP-70 and XEP-90 fractions were in the range of 4000-400 cm-1. -1The polysaccharide absorption peak is shown in the range of about 3428.23cm -1 (XEP-50), 3417.36cm -1 (XEP-70) and 3422.12cm -1 The characteristic strong absorption band at (XEP-90) represents the stretching vibration of the OH bond. The stretching vibration of the CH bond in the sugar ring is assigned to approximately 2924.01 cm -1 (XEP-50), 2927.40cm -1 (XEP-70) and 2927.90cm -1 (XEP-90) peak. 1643.71cm -1 (XEP-50), 1651.46cm -1 (XEP-70) and 1651.11cm -1 The band at (XEP-90) is due to the C=O stretching vibration of uronic acid. 1438.46 cm -1 (XEP-50),1438.09cm -1 (XEP-70) and 1411.75cm -1 The signal of (XEP-90) is considered to be the in-plane bending vibration of the CH bond. 1048.41 cm -1 (XEP-50), 1067.18cm -1 (XEP-70) and 1067.38cm -1 The band at (XEP-90) is the stretching vibration of the pyranose ring. 868.29cm -1 (XEP-50), 876.01cm -1 (XEP-70) and 869.74cm -1 The band at (XEP-90) indicates the presence of β-glycosidic bonds in these fractions. -1 (XEP-50), 876.01cm -1 (XEP-70) and 869.74cm -1 The absorption peaks at (XEP-90) are characteristic of mannose. It is worth mentioning that the absorption peaks at about 810 and 870 cm -1 The peaks at 37 and 39 appear simultaneously, representing the typical β-dominant configuration peak, which is composed of glucose and mannose in the form of pyranose (Figure 2).

[0084] 4. Scanning Electron Microscope (SEM) Analysis

[0085] XEP-70 was placed on a conductive adhesive and sprayed with gold. Scanning electron microscopy images of XEP-70 were recorded on a ZEISS Gemini SEM 300 (Zeiss, Germany). Images were observed at 400x and 4000x magnifications under a high vacuum environment at 3.0 kV. The results show that at 400x magnification, XEP-70 appears to be a stable irregular network structure. At 4000x magnification, the compact structure is composed of numerous lamellar structures and ribbon-like fibers (Figure 3).

[0086] 5. Atomic Force Microscopy (AFM) Analysis

[0087] XEP-70 was dissolved in ultrapure water to a final concentration of 5 g / mL. Then, 5 μL of the polysaccharide solution was placed on the surface of a pristinely cleaved mica sheet and allowed to dry at room temperature. The morphology of XEP-70 was examined using an atomic force microscope (DimensionIcon, Bruker, Germany) with a scan size of 600 nm and a scan frequency of 1.0 Hz. The AFM images of XEP-70 revealed numerous chain-like structures. Morphological analysis revealed that XEP-70 molecules exist as flexible chains with an average thickness of 0.863 nm, which is intermediate between single polysaccharide chains (approximately 0.1–1.0 nm) (Figure 4).

[0088] 6. Nuclear Magnetic Resonance Analysis

[0089] (1) XEP-70 (20 mg) was added to 0.5 mL of D2O (99.8% D) in an NMR tube. NMR analysis, including 1 HNMR and 13 CNMR was monitored by a Bruker Avance 600MHz spectrometer (Germany). 1 In the HNMR spectrum, the δ4.8-5.3 ppm region is associated with heterotopic protons, with nine signals at 5.27, 5.24, 5.16, 5.11, 5.00, 4.97, 4.86, 4.81, and 4.69 ppm. The signals in the δ3.1-4.3 ppm region are believed to be associated with H2-H6 (Figure 5); 13 The δ90-110 ppm region in the CNMR spectrum is related to isomeric carbon, and nine isomeric carbon signals were observed, which can be attributed to 1The H NMR spectrum shows AIs of 102.30, 94.02, 99.39, 103.55, 106.93, 107.88, 100.14, 96.30, and 97.89 ppm, while chemical shifts at δ60-85 ppm are associated with non-isomeric carbons. The signal at 106.93 ppm is characteristic of the isomeric carbons of the β-galactofuranose molecule due to their extremely low field shift. The signal at 173.82 ppm is attributed to the presence of galacturonic acid in XEP-70 (Figure 6).

[0090] (2) The signals of each XEP-70 residue in the one-dimensional NMR spectrum were compared with the NMR data in the literature, and the results are shown in Table 2. Among them, the signals of C (5.16 / 99.39), H (4.86 / 96.30) and I (4.81 / 97.89) represent three different mannopyranose residues, the signals of A (5.27 / 102.30) and G (4.93 / 100.14) represent two different glucose pyranose residues, the signals of B (5.24 / 94.02) and E (5.00 / 106.93) represent two different galactose residues, and the remaining two signals D (5.11 / 103.55) is a galacturonic acid residue, and F (4.97 / 107.88) is a xylose residue.

[0091] Table 2 1 H and 13 CNMR chemical shift (δ): Residue AI of XEP-70

[0092] As shown in Table 2, XEP-70 is a pectin polysaccharide composed of (1→4)-linked and (1→4,6)-linked α-D-Glcp, (1→4)-linked α-D-GalpA, (1→2)-linked and (1→2,6)-linked α-D-Manp, (1→6)-linked β-D-Manp, (1→6)-linked α-D-Galp and (1→2,6)-linked β-D-Galf, and (1)-linked β-D-Xylp.

[0093] Test Example 2

[0094] Free radical scavenging ability test

[0095] Free radicals are unpaired groups in the human body with strong oxidizing properties. Excessive free radicals can induce protein denaturation and cell damage, ultimately leading to human diseases and aging. This test example uses the stone flower polysaccharides XEP-50, XEP-70 and XEP-90 obtained in Example 1, as well as vitamin C as the test samples to describe the antioxidant activity of different test samples at different concentrations, including the scavenging of free radicals (ABTS, DPPH, O2- and OH - ) and reducing power, the specific steps are as follows:

[0096] 1. DPPH free radical scavenging activity test

[0097] DPPH was dissolved in anhydrous ethanol to a final concentration of 0.2 mM to prepare a DPPH solution. 2 mL of the DPPH solution was then mixed with 2 mL of the test sample at varying concentrations. The mixture was incubated in the dark for 35 minutes, and the absorbance was recorded at 517 nm. This was repeated four times. The clearance rate was calculated according to the following formula, as shown in Figure 7A.

[0098] Clearance rate: DPPH = [1-(A1-A0) / A2]*100%

[0099] Wherein, A0 is the absorbance of the sample solution to be tested replaced by deionized water, A1 is the absorbance of the sample solution to be tested, and A2 is the absorbance of the sample solution to be tested replaced by deionized water instead of DPPH and other reagents.

[0100] DPPH is a relatively stable free radical commonly used to evaluate the free radical scavenging activity of natural compounds. As shown in Figure 7A, within the concentration range of 0.125-4 mg / ml, the three stone flower polysaccharides obtained in Example 1 exhibited excellent DPPH free radical scavenging ability, with scavenging activity increasing with increasing polysaccharide concentration. At the same concentration, the free radical scavenging ability of stone flower polysaccharide XEP-70 was superior to that of the other two polysaccharides, with a maximum scavenging rate of 70.38% ± 1.42%.

[0101] 2. ABTS free radical scavenging activity test

[0102] To prepare the ABTS assay reagent, Na2HPO4 and NaH2PO4 were dissolved in deionized water (50 mM PBS). Other chemical reagents were dissolved in PBS. Using Trolox as a reference, ABTS (5 mM), HRP (1 μM), H2O2 (0.018%), and PBS (50 mM) solutions were added to the test samples at varying concentrations. The mixtures were allowed to react in the dark for 10 minutes, and the absorbance was recorded at 730 nm. This was repeated four times. The clearance rate was calculated according to the following formula, as shown in Figure 7B.

[0103] Clearance rate: ABTS = (1-A1 / A0) * 100%

[0104] Wherein, A0 is the absorbance of the sample solution to be tested replaced by deionized water, and A1 is the absorbance of the sample solution to be tested.

[0105] As can be seen from Figure 7B, the increase in polysaccharide concentration and scavenging ability is synchronous, and the scavenging effect of stone flower polysaccharide XEP-70 is significantly higher than that of the other two polysaccharides.

[0106] 3. Superoxide radical scavenging activity test

[0107] NBT, NADH, and PMS were dissolved in deionized water. Then, 1 mL each of NBT (156 μmol / L), NADH (468 μmol / L), and PMS (60 μmol / L) were added sequentially to 1 mL of the test sample at various concentrations. The mixture was incubated in a 25°C water bath for 5 minutes. The absorbance was recorded at 560 nm. This reaction was repeated four times. The clearance rate was calculated according to the following formula, as shown in Figure 7, C.

[0108] Clearance rate: O 2- =[1-(A1-A2) / A0]*100%

[0109] Among them, A0 is the absorbance of the sample solution to be tested when deionized water replaces it, A1 is the absorbance of the sample solution to be tested, and A2 is the absorbance of the deionized water instead of NBT and other reagents.

[0110] As can be seen from Figure 7C, the three polysaccharides removed O 2- The free radical scavenging ability of the two polysaccharides was similar, and the scavenging ability increased with the increase of polysaccharide concentration. Among them, the stone flower polysaccharide XEP-70 had the best effect, reaching the maximum scavenging rate of 59.34±0.60% at 4mg / ml.

[0111] 4. Hydroxyl radical scavenging activity test

[0112] To 0.5 mL of three different concentrations of the test sample solution, 0.5 mL each of ferrous sulfate (7.5 mM), salicylic acid (5 mM), and hydrogen peroxide (10 mM) was added sequentially. Finally, 2 mL of deionized water was added to each mixed solution. The mixture was reacted in a 37°C water bath for 20 minutes. The absorbance was recorded at a wavelength of 510 nm. This was repeated four times. The clearance rate was calculated according to the following formula, as shown in Figure 7D.

[0113] Clearance: OH - =[1-(A1-A2) / A0]*100%

[0114] Among them, A0 is the absorbance of the sample solution to be tested replaced by deionized water, A1 is the absorbance of the sample solution to be tested, and A2 is the absorbance of the deionized water replaced by ferrous sulfate and other reagents.

[0115] According to Figure 7D, the three polysaccharides can remove OH -The free radical scavenging ability of the two polysaccharides was similar, and the scavenging ability increased with the increase of polysaccharide concentration. Among them, the stone flower polysaccharide XEP-70 had the best effect, reaching the maximum scavenging rate of 52.14±0.55% at 4mg / ml.

[0116] 5. Reduction test

[0117] Three different concentrations of the test sample were dissolved in 0.2 M phosphate buffer (pH 6.6). 2.5 mL of each sample solution was then mixed with 2.5 mL of potassium ferricyanide (1% w / v). After reacting in a 50°C water bath for 20 minutes, 2.5 mL of trichloroacetic acid (10% w / v) was added, the solutions were mixed, and centrifuged (3000 rpm for 20 minutes). Subsequently, 2.5 mL of the supernatant from each solution was added to 2.5 mL of deionized water and 250 μL of ferric chloride (0.1% w / v) and incubated for 10 minutes. The absorbance was recorded at a wavelength of 700 nm. This was repeated four times. The reducing power was calculated according to the following formula, as shown in Figure 7E.

[0118] Restoration power = A1

[0119] Where A1 is the absorbance of the sample solution to be tested.

[0120] According to Figure 7E, the changing trends of the reducing abilities of the three polysaccharides are similar to the changing trends of the free radical scavenging abilities mentioned above, but the reducing abilities they exhibit are not as significant as the free radical scavenging abilities.

[0121] From Figure 7A to E, it can be seen that the three polysaccharides all have certain free radical scavenging and reducing abilities, among which the stone flower polysaccharide XEP-70 has the strongest antioxidant activity.

[0122] Test Example 3

[0123] Antioxidant activity assay

[0124] 1. Cell Culture of LX-2 Cells

[0125] LX-2 cells were grown in high-glucose DMEM containing 20% ​​(v / v) FBS, 1% (v / v) streptomycin and penicillin at 37°C in an incubator containing 5% CO2.

[0126] 2. Toxicity test of XEP-70

[0127] LX-2 cells were seeded into 96-well cell plates at a density of 5000 cells / well. The agarwood polysaccharide XEP-70 obtained in Example 1 was added to each well to a final concentration of 12.5, 25, 50, 100, or 200 μg / mL. After 24 hours of culture, a cell counting kit (CCK-8) was added and the cells were cultured for another hour. The absorbance was measured and cell viability was calculated. The results showed that agarwood polysaccharide XEP-70 significantly promoted cell proliferation in the range of 25-100 μg / mL (P<0.05) (Figure 8). Therefore, 25, 50, and 100 μg / mL of agarwood polysaccharide XEP-70 were subsequently selected for subsequent experiments to evaluate its antioxidant activity on LX-2 cells.

[0128] 3. Screening of H2O2 concentration

[0129] LX-2 cells were seeded at a density of 5,000 cells / well in a 96-well plate. H₂O₂ was added to these wells to a final concentration of 20, 25, 30, 35, or 40 μg / mL and incubated for 24 hours. A cell counting kit (CCK-8) was added to each well, followed by an additional 1-hour incubation. Absorbance was measured and cell viability was calculated. The results showed that cell damage began at 20 μg / mL H₂O₂, and cell viability decreased with increasing concentrations (Figure 9). Therefore, the IC₅₀ concentration (30 μg / mL) was subsequently selected as the model concentration for oxidative damage in LX-2 cells.

[0130] 4. Antioxidant activity of stone flower polysaccharide

[0131] LX-2 cells were seeded into 96-well cell plates at a density of 5,000 cells / well. XEP-70, a polysaccharide obtained in Example 1, was added to these wells at final concentrations of 25, 50, or 100 μg / mL, with Vc used as a positive control (final concentration of 100 μg / mL). The cells were cultured for 24 hours. H₂O₂ was added to a final concentration of 30 μg / mL, and the cells were stimulated for 24 hours. CCK8 assays were then performed to measure cell viability. The results showed that, compared to the injury model group, different concentrations of XEP-70 protected the cells and increased their activity, with 100 μg / mL showing the greatest effect, approaching that of the positive control group (Figure 10).

[0132] 5. Antioxidant activity and metabolic content testing

[0133] 2×10 5LX-2 cells were seeded in 6-well plates at a density of 100 cells / well and randomly divided into a blank group (Control), a positive control group (Vc), a model group (H2O2), and an experimental group (XEP-70). The positive control group was treated with 100 μg / ml Vc, while the experimental groups were treated with 25, 50, and 100 μg / ml XEP-70, respectively, and cultured for 24 hours. The cells were then stimulated with H2O2 (30 μg / ml). After 24 hours, the proteins were washed three times and then lysed with RIPA lysis buffer (Bio-Ultrasound, Shanghai, China). The protein content in the cell lysate was determined using a BCA kit. The activities of T-AOC, ROS, LDH, CAT, GSH, and SOD, as well as the MDA content in each cell group were measured. The results showed that XEP-70 significantly reduced the MDA content and the activities of LDH and SOD in a dose-dependent manner (P < 0.05). The activities of T-AOC, GSH, SOD, and CAT in the model group were significantly lower than those in the blank group. However, in the experimental group pre-added with XEP-70, the activities of these enzymes were reversed and significantly enhanced in a dose-dependent manner (P < 0.05). Overall, these results indicate that XEP-70 effectively protects LX-2 cells from H2O2 oxidative damage by regulating the activity of antioxidant enzymes (Figure 11).

[0134] 6. Western Blotting

[0135] Cells were harvested and treated as described in step 5. The amount of protein used per well was then determined based on protein concentration. Protein lysates were separated by 10% SDS-PAGE electrophoresis and transferred to PVDF membranes, which were then blocked with blocking buffer for 1 hour at room temperature. Diluted secondary antibodies were added and incubated at room temperature for 30 minutes. Fluorescence imaging was performed using an ECL kit, followed by exposure, development, and fixation in the darkroom. Expression of key proteins in the Nrf2-Keap1-ARE signaling pathway was then assessed. Results showed that pretreatment with 50 and 100 μg / mL of XEP-70 significantly increased the expression of NQO1, HO-1, GCLC, and GCLM compared to the model group (p < 0.01). Therefore, it is suggested that XEP-70 activates Nrf2 in the Nrf2-Keap1-ARE signaling pathway and transmits it to the cell nucleus, further promoting the expression of NQO1, HO-1, GCLC, and GCLM, thereby reducing H2O2-induced oxidative damage and achieving an antioxidant effect (Figures 12-13).

[0136] Based on the above content, it can be seen that the stone flower polysaccharide XEP-70 provided in this application can enhance the cellular antioxidant capacity by activating the Nrf2-Keap1-ARE signaling pathway, thereby effectively protecting LX-2 cells from hydrogen peroxide-induced oxidative damage.

[0137] Although the above embodiment provides a detailed description of the present application, it is only a part of the embodiments of the present application, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present application.

Claims

1. A polysaccharide XEP-70, wherein the sugar residues and glycosidic bond types of the polysaccharide XEP-70 include: →4)-α-D-Glcp-(1→, →4,6)-α-D-Glcp-(1→, →4)-α-D-GlcpA-(1→, →2)α-D-Manp(1→, →2,6)-α- D-Manp-(1→,→6)-β-D-Manp-(1→,→6)-α-D-Galp-(1→,→2,6)-β-D-Galf-(1→,β-D-Xylp-(1→; The weight average molecular weight of the Shihua polysaccharide XEP-70 is 430.98 kDa.

2. The stone flower polysaccharide XEP-70 according to claim 1, characterized in that The total sugar content of the stone flower polysaccharide XEP-70 is 99.05 wt.%, and the uronic acid content is 13.09 wt.%.

3. The stone polysaccharide XEP-70 according to claim 1 or 2, characterized in that: The stone flower polysaccharide XEP-70 consists of mannose, galacturonic acid, glucose, galactose and xylose.

4. The stone flower polysaccharide XEP-70 according to claim 3, characterized in that The molar ratio of mannose, galacturonic acid, glucose, galactose and xylose is 39.28:16.09:17.56:23.20:3.

87.

5. The method for preparing the stone polysaccharide XEP-70 according to any one of claims 1 to 4, characterized in that: The steps include: After hot extraction of the stone flower with water, the solid-liquid separation is carried out, and the obtained aqueous solution is concentrated to obtain a crude stone flower polysaccharide solution; subjecting the crude polysaccharide solution of the stone flower to alcohol precipitation, collecting the precipitate and freeze-drying it to obtain crude polysaccharide of the stone flower; The crude polysaccharide of the stone flower is separated and purified by using a DEAE anion exchange column; The separation and purification comprises: redissolving the crude polysaccharide of stone flower in water to prepare an aqueous solution, applying the solution to a DEAE anion exchange column, eluting with water and a 0.5 mol / L NaCl salt solution in sequence, collecting the eluted fractions of the 0.5 mol / L NaCl salt solution, dialyzing, and freeze-drying to obtain the polysaccharide XEP-1 of stone flower; The agarwood polysaccharide XEP-1 was redissolved in water, and anhydrous ethanol was added to make the final concentration of ethanol 70% v / v. After standing at 0-4° C. for 8-48 hours, the precipitate was collected by centrifugation and dried to obtain the agarwood polysaccharide XEP-70.

6. The preparation method according to claim 5, characterized in that The number of times of adding water for hot extraction is 3 times, and the mass volume ratio of stone flower and water is 1kg:10L each time; The temperature of the water-adding thermal extraction is 90-100°C.

7. The preparation method according to claim 5, characterized in that The concentration temperature is 95°C; The final concentration of ethanol in the alcohol precipitation is 80% v / v.

8. The preparation method according to claim 5, characterized in that The dialysis includes distilled water dialysis and running water dialysis; the molecular weight cutoff of the distilled water dialysis is 3500Da, and the dialysis time is 24h; the molecular weight cutoff of the running water dialysis is 1000-10000Da, and the dialysis time is 24h.

9. The preparation method according to claim 8, characterized in that The running water dialysis is performed using tap water at a flow rate of 300 mL / min.

10. The preparation method according to claim 5, characterized in that During the separation and purification, the volume ratio of the aqueous solution, the elution water and the 0.5 mol / L NaCl salt solution for elution is 1:40:

40.

11. The preparation method according to claim 5, characterized in that The freeze-drying conditions are: pressure of 10 to 30 MPa and temperature of -60 to -80°C.

12. Use of the Shihua polysaccharide XEP-70 according to any one of claims 1 to 4 or the Shihua polysaccharide XEP-70 obtained by the preparation method according to any one of claims 5 to 11 in the preparation of antioxidant products.

13. The use according to claim 12, characterized in that The antioxidant products include drugs for preventing and / or treating oxidative damage.

14. The use according to claim 12, characterized in that The drug for preventing and / or treating oxidative damage includes a drug for preventing and / or treating oxidative damage to liver cells.

15. The use according to claim 14, characterized in that The hepatocytes include LX-2 cells.

16. The use according to claim 14, characterized in that The oxidative damage includes H2O2-induced oxidative damage.

17. The use according to any one of claims 13 to 16, characterized in that: The effective concentration of XEP-70 in the medicine is 25-100 μg / mL.

18. A drug for preventing and / or treating oxidative damage, characterized in that: The active ingredient of the medicine includes the stonecrop polysaccharide XEP-70 according to any one of claims 1 to 4 or the stonecrop polysaccharide XEP-70 obtained by the preparation method according to any one of claims 5 to 11.

19. The drug according to claim 18, characterized in that The effective concentration of XEP-70 in the medicine is 25-100 μg / mL.

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