A type of RG-I canarium album polysaccharide, and a preparation method and application thereof

Abstract

The application discloses a kind of RG-I type phragma polysaccharide and its preparation method and application, belong to novel polysaccharide development technical field.The method provided in the application is successfully obtained from phragma polysaccharide a novel structure novel, exact new polysaccharide-RG-I type phragma polysaccharide AMFPs-0-1, main chain is connected by→2)‑α‑L‑Rhap‑(1→ and→4)‑α‑D‑GalpA‑(1→ unit alternately, each Rhap of main chain is connected with the side chain consisting of arabinose or galactose;AMFPs-0-1 has no toxic side effects on cells, can be fermented and utilized by human intestinal flora, can significantly promote the growth of beneficial flora, significantly promote the production of short-chain fatty acids in intestinal tract, activate macrophages, effectively promote macrophage proliferation, has great application prospect in preventing and / or treating intestinal diseases, preparing immune enhancer drugs.

Description

Technical Field

[0001] This invention relates to the field of novel polysaccharide development technology, and in particular to an RG-I type olive polysaccharide, its preparation method, and its application. Background Technology

[0002] The human gut contains a complex community of microorganisms, collectively known as the gut microbiota. This community can influence almost every aspect of the host's physiology, including development, metabolism, immunity, and a range of behaviors. The gut microbiota has been shown to participate in the pathogenesis, development, and treatment of many chronic diseases, including cardiovascular disease, diabetes, and cancer.

[0003] A growing body of research indicates that many novel polysaccharides are non-toxic and possess the ability to selectively regulate the human gut microbiota, thus being recognized as an emerging prebiotic. In the gastrointestinal tract, human-produced enzymes cannot break down the glycosidic bonds of novel polysaccharides. However, gut microbiota in the colon carry polysaccharide utilization sites (PULs) and release carbohydrate-active enzymes. These enzymes work synergistically to break down polysaccharides, subsequently releasing the final metabolites SCFAs, thereby preventing and alleviating some common diseases and ultimately improving human health.

[0004] Activating macrophages is a crucial component of the immune defense system. Macrophages can directly kill pathogens through phagocytosis and indirectly kill them by releasing nitric oxide (NO) and cytokines, thereby regulating the immune system and helping the body fight infections and various diseases. Furthermore, research has shown that polysaccharides can initiate downstream signal transduction through receptors in macrophages, thus achieving therapeutic or preventative effects. Examples include widely used polysaccharides such as lentinan from shiitake mushrooms, maitake mushrooms, and Ganoderma lucidum.

[0005] With changing lifestyles and increasing health awareness, the demand for polysaccharide prebiotics is constantly growing. Commercially available prebiotics such as inulin, fructooligosaccharides (FOS), xylooligosaccharides (XOS), and galactomannans (GOS) possess excellent probiotic effects and are widely used in food additives and pharmaceuticals in various countries. Discovering novel, clearly defined, and naturally derived functional oligosaccharides and polysaccharides has become a hot topic in prebiotic research and development. Furthermore, identifying polysaccharide compounds from nature that possess good immunomodulatory activity and low toxicity has significant research and application value.

[0006] *Avicennia marina* (Forssk.) Vierh. is a highly salt-tolerant member of coastal mangrove forests, primarily growing in subtropical and tropical coastal areas. For centuries, its leaves, fruits, and bark have been used in traditional medicine. The fruit of *Avicennia marina*, also known as "olive money," yields 3-50 kg per plant, with annual production in my country exceeding 300,000 tons. It is the most widely used mangrove fruit for food and is also commonly used to treat constipation and dysentery.

[0007] Polysaccharides are among the most abundant bioactive substances in the female fruit of *Rhizophora stylosa*. Previous studies have found that its non-starch total polysaccharides have beneficial effects on regulating intestinal flora (Yuan, et al. Food Chemistry:X, 2022, 16, 100475), which is likely the main reason why *Rhizophora stylosa* exerts its therapeutic effects on constipation and dysentery. However, research reports on its homogeneous polysaccharides are scarce, especially those with well-defined structures, which have been reported as novel prebiotics. Furthermore, the immunomodulatory activity of well-defined, homogeneous *Rhizophora stylosa* polysaccharides requires further in-depth investigation. Summary of the Invention

[0008] The purpose of this invention is to provide an RG-I type eugenol polysaccharide, its preparation method and application, so as to provide a new polysaccharide with a novel and precise structure from natural plants and good application prospects.

[0009] To achieve the above objectives, the present invention provides an RG-I type eugenol polysaccharide, the structural formula of which is shown in formula (I), and is a mixture of homologous polysaccharides with the structure shown in formula (I).

[0010] Rhap is a pyranose rhamnosyl, GalpA is a pyranose galacturonic acid, Galp is a pyranose galactosyl, and Araf is a furanose arabinose.

[0011] R1 is a group with the structure shown in formula (II); R2 is a group with the structure shown in formulas (III), (IV), and (V).

[0012]

[0013] β-Galp-(1-[→4)-β-Galp] s -(V); where m is an integer with a mean of 1 to 6, n is an integer with a mean of 1 to 8, o is an integer with a mean of 6 to 10, p is an integer with a mean of 1 to 4, and q+r+s is an integer with a mean of 15 to 21.

[0014] Preferably, the monosaccharides comprising the RG-I type argan polysaccharide include rhamnose, galacturonic acid, galactose, and arabinose, with a molar ratio of rhamnose:galacturonic acid:galactose:arabinose of 1.0:(1.4±0.5):(2.8±0.5):(6.4±0.5).

[0015] Preferably, the average molecular weight (Mw) of the RG-I type eugenol polysaccharide is 700 kDa to 850 kDa, and the polydispersity index is between 1.0 and 2.0.

[0016] Preferably, the RG-I type olive polysaccharide is a non-starch polysaccharide obtained from the fruit of the sea olive female Avicennia marina (Forssk.) Vierh.

[0017] A method for preparing RG-I type eugenol polysaccharide as described above includes the following steps: using non-starch eugenol total polysaccharide as raw material, separation is performed using anion exchange chromatography column, the eluted fraction is collected by pure water, desalted by dialysis or ultrafiltration membrane filtration, concentrated, and then separated and purified by gel chromatography column, collecting the fractions of the main chromatographic peaks, desalting, and lyophilizing.

[0018] Preferably, when using anion exchange chromatography, a DEAE FF column with good water equilibration is selected, and gradient elution is performed using 0M, 0.1M, 0.3M, and 0.5M NaCl solutions as mobile phases; when using gel chromatography for separation and purification, a Sepharose Cl-6B gel chromatography column equilibrated with 0.2M NaCl is selected.

[0019] The application of the RG-I type argan polysaccharide as described above in the preparation of a drug to promote human gut health, wherein the application is to increase the relative abundance of beneficial bacteria and the level of acidic metabolites, including Bifidobacterium, Megalomonas, Macrococcus, and Gastrodia elata, and the acidic metabolites including short-chain fatty acids.

[0020] Preferably, the short-chain fatty acids are acetic acid and propionic acid.

[0021] The application of the RG-I type aralia polysaccharide described above in the preparation of a drug to enhance human immune stress response, wherein the drug can activate macrophages, promote the production of NO, TNF-α, and IL-6, and promote the expression of macrophage iNOS, IL-6, and TNF-α mRNA.

[0022] A health / functional food containing the above-mentioned RG-I type argan polysaccharide, the food being used to treat / alleviate intestinal flora imbalance and immunodeficiency.

[0023] Therefore, the present invention provides an RG-I type olive polysaccharide, its preparation method, and its application, the specific technical effects of which are as follows:

[0024] (1) Using the method provided by the present invention, a novel and precise new polysaccharide with a specific structure, namely RG-I type argan polysaccharide AMFPs-0-1, was successfully obtained. The main chain is composed of alternating →2)-α-L-Rhap-(1→ and →4)-α-D-GalpA-(1→ units. Each Rhap unit of the main chain is connected to a side chain composed of arabinose or galactose. The structure is unique and significantly different from RG-I type polysaccharides from other species.

[0025] (2) The RG-I type olive polysaccharide AMFPs-0-1 obtained in this invention can be fermented and utilized by human intestinal flora, and can significantly promote the growth of beneficial flora, especially significantly increase the relative abundance of beneficial flora such as Bifidobacterium, Gymnema sylvestris, Macrococcus and Megalosporium, and has great application prospects in the prevention and / or treatment of intestinal diseases.

[0026] (3) The RG-I type aralia polysaccharide AMFPs-0-1 obtained in this invention can significantly promote the production of short-chain fatty acids such as acetic acid, propionic acid, lactic acid and butyric acid in the intestine, which is of great significance for protecting the intestinal mucosa, maintaining intestinal function and promoting intestinal digestion and absorption.

[0027] (4) The RG-I type aralia polysaccharide AMFPs-0-1 obtained in this invention has no toxic side effects on cells, can activate macrophages, effectively promote macrophage proliferation, and improve human immunity. When the concentration of AMFPs-0-1 is 100 μg / mL, the macrophage proliferation rate is the largest, which is 230%, and it is expected to be used to prepare immune enhancer drugs.

[0028] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0029] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments of the present invention will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0030] Figure 1 The high-performance liquid chromatography gel chromatogram shows the molecular weight and distribution of AMFPs-0-1 in Example 1 of this invention.

[0031] Figure 2 This is a high-performance liquid chromatogram of AMFPs-0-1 in Example 1 of the present invention for monosaccharide composition analysis;

[0032] Figure 3 This is the total ion chromatogram of the methylated sugar alcohol acetyl derivative of AMFPs-0-1 in Example 1 of the present invention;

[0033] Figure 4 This is the total ion chromatogram of the methylated sugar alcohol acetyl derivative of AMFPs-0-1-I, a partially acid-hydrolyzed sample of AMFPs-0-1 in Example 1 of the present invention;

[0034] Figure 5 For AMFPs-0-1 in Embodiment 1 of the present invention 1 H and 13 C NMR spectra and their assignments;

[0035] Figure 6 For AMFPs-0-1 in Embodiment 1 of the present invention 1 H- 13 C HSQC spectrum and its attribution;

[0036] Figure 7 This is the proton NMR spectrum of AMFPs-0-1-I, a partially acid-hydrolyzed sample of AMFPs-0-1 in Example 1 of the present invention;

[0037] Figure 8 The carbon spectrum and DEPT135 spectrum of AMFPs-0-1-I, a partially acid-hydrolyzed sample of AMFPs-0-1 in Example 1 of this invention;

[0038] Figure 9 The AMFPs-0-1-I partially acid-hydrolyzed sample of AMFPs-0-1 in Example 1 of this invention 1 H- 13 C HSQC spectrum and its attribution;

[0039] Figure 10 The HPGPC chromatogram shows the change in molecular weight distribution of AMFPs-0-1 before and after fermentation in Example 2 of this invention.

[0040] Figure 11 The images show the community structure distribution of intestinal flora at the phylum level (A) and genus level (B) and the community structure abundance heatmap at the genus level (C) after 24 hours of in vitro fermentation of AMFPs-0-1 in Example 2 of this invention.

[0041] Figure 12 The results of SCFAs content detection in the fermentation broth of AMFPs-0-1 at different fermentation times in Example 2 of the present invention are shown. A represents the acetic acid determination result, B represents the propionic acid determination result, C represents the lactic acid determination result, D represents the butyric acid determination result, E represents the isovaleric acid level determination result, F represents the valeric acid determination result, and G represents the total fatty acid level determination result.

[0042] Figure 13 This is the result of the effect of AMFPs-0-1 on the proliferation ability of RAW264.7 macrophage cells in Example 3 of the present invention;

[0043] Figure 14 This describes the effect of AMFPs-0-1 on NO release from RAW264.7 macrophage cells in Example 3 of the present invention.

[0044] Figure 15 The effect of AMFPs-0-1 on the release of IL-6 (A) and TNF-α (B) from RAW264.7 cells in Example 3 of this invention;

[0045] Figure 16 This invention relates to the effects of AMFPs-0-1 on the expression of iNOS mRNA (A), IL-6 mRNA (B), and TNF-α mRNA (C) in RAW264.7 cells in Example 3 of this invention. Detailed Implementation

[0046] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0047] To make the objectives, technical solutions, and advantages of this application clearer, more thorough, and more complete, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings and embodiments. The following detailed descriptions are all illustrations of embodiments, intended to provide further detailed explanation of the present invention. Unless otherwise specified, all technical terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0048] The instruments, equipment, reagents, and materials used in the examples were all obtained commercially.

[0049] The anion exchange resin DEAE Sepharose Fast Flow and cross-linked agarose gel Sepharose CL-6B were manufactured by GE Healthcare, USA; mouse macrophages RAW264.7 were provided by the Scientific Experiment Center of Guangxi University of Traditional Chinese Medicine; mouse IL-6 ELISA kits and mouse TNF-α ELISA kits were purchased from Xinbosheng Biotechnology Co., Ltd.; thiazolyl blue (MTT) was purchased from Maclean's Reagent Co., Ltd.; HiScript III RT SuperMix for qPCR (+gDNAwiper) and ChamQ Universal SYBR qPCRMasterMix were purchased from Novizan Biotechnology Co., Ltd.; penicillin-streptomycin antibiotics, trypsin-EDTA (0.25%, containing phenol red), imported Australian fetal bovine serum, and TRIzol were purchased from Thermo Fisher Scientific; PBS was purchased from Beijing Solarbio Science & Technology Co., Ltd.; lipopolysaccharide was purchased from Sigma-Aldrich, USA; and other reagents such as sulfonamides, dimethyl sulfoxide, and isopropanol were all commercially available analytical grade reagents.

[0050] The biosafety cabinet (HR1500-ⅡB2) was purchased from Qingdao Haier Biomedical Co., Ltd.; the real-time fluorescence quantitative PCR instrument (LightCycler480) was purchased from Roche Applied Science; the carbon dioxide incubator (MCO-170AICDL-PC) and the autoclave (MLS-3781L-PC) were purchased from Puhuaxi Co., Ltd.; the full-wavelength multi-mode microplate reader (VICTORNivo) was purchased from PerkinElmer; the inverted phase contrast microscope (ICX41) was purchased from Ningbo Sunny Instruments Co., Ltd.; the nano-drop one nucleic acid and protein analyzer was purchased from Thermo Fisher Scientific; and the PCR amplification instrument (C1000Touch) was purchased from Bio-Rad Laboratories.

[0051] The basal culture medium used in the examples consisted of: 2g peptone, 2g yeast extract, 0.1g NaCl, 0.04g K2HPO4, 0.04g KH2PO4, 0.01g MgSO4·7H2O, 0.01g CaCl2·2H2O, 2.0g NaHCO3, 0.02g heme chloride, 0.5g L-cysteine ​​hydrochloride, 0.5g bile salts, 1mg resazurin, 2.0mL Tween 80, and 10μL vitamin K1 dissolved in 1L distilled water.

[0052] Example 1

[0053] The method for preparing RG-I type olive polysaccharide AMFPs-0-1 is as follows:

[0054] S1. Weigh 300 mg of total non-starch polysaccharides (AMFPs) from *Eriocaulon buergerianum* (preparation method see Yuan, et al. Food Chemistry: X, 2022, 16, 100475), dissolve in 30 mL of pure water, and load onto a DEAE-FF chromatography column (2.6 × 40 cm) equilibrated with pure water. Use different concentrations of NaCl solution (0 M, 0.1 M, 0.3 M, 0.5 M) as the mobile phase for gradient elution, and collect the elution using a collector. Detect the polysaccharides using the phenol-sulfuric acid method and plot the corresponding elution curves.

[0055] S2. According to the elution curve, the fractions of each elution peak were combined and collected, concentrated, dialyzed and desalted using a dialysis bag with a molecular weight cutoff of 3500 Da, concentrated, and lyophilized to obtain AMFPs-0. 120 mg was weighed and dissolved in 5 mL of distilled water, and loaded onto a Sepharose Cl-6B gel chromatography column (1.5 m × 2 cm) equilibrated with 0.2 M NaCl. The sample was collected using a collector. According to the elution curve results plotted by the phenol-sulfuric acid method, the fractions of each elution peak were combined and collected, concentrated, dialyzed and desalted using a dialysis bag with a molecular weight cutoff of 3500 Da, and lyophilized to obtain AMFPs-0-1.

[0056] S3. The molecular weight and distribution of AMFPs-0-1 were determined by high performance liquid chromatography-gel chromatography. Chromatographic conditions: Shimadzu high performance liquid chromatograph (LC-2030C 3D Plus), Shodex SB-804HQ (8.0 mm ID × 300 mm) gel column, refractive index detector (RID), flow rate 0.5 mL / min, and mobile phase 0.1 M sodium chloride solution.

[0057] S4. Pre-column derivatization high-performance liquid chromatography was used to detect the monosaccharide composition of AMFPs-0-1. The detector was a diode array detector (DAD) with a detection wavelength of 245 nm. The chromatographic column was an Eclipse Plus C18 (4.6 × 250 mm, 5 μm) with a column temperature of 30 °C. The mobile phase was a mixture of phosphate buffer (0.1 M, pH 6.7) and acetonitrile (volume ratio 83:17). The flow rate was 1.0 mL / min and the injection volume was 20 μL.

[0058] S5. The main chain structure of AMFPs-0-1 was determined by partial acid hydrolysis: 200 mg of AMFPs-0-1 sample obtained in step S2 was weighed and placed in a 50 mL round-bottom flask. 0.5 M trifluoroacetic acid (TFA) was added, the flask was sealed, and hydrolyzed in an oil bath with a magnetic stirrer at 100 °C for 1 h. After the reaction, the sample was cooled and repeatedly evaporated to dryness with methanol until no TFA was found. Then, deionized water was added and evaporated to dryness twice to remove methanol. The sample was dialyzed using a dialysis bag with a molecular cutoff of 3500 Da. The dialysate was collected, concentrated, and freeze-dried to obtain AMFPs-0-1-I.

[0059] S6. Methylation analysis of AMFPs-0-1 and AMFPs-0-1-I was performed according to the methylation method reported by Ciucanu et al. (Journal of the American Chemical Society, 2003, 125(52): 16213-16219).

[0060] Weigh 20 mg of AMFPs-0-1 obtained in step S2 and 30 mg of AMFPs-0-1-I obtained in step S5, dissolve them in 0.5 mL of D2O, centrifuge, collect the supernatant, freeze-dry, and repeat three times to obtain lyophilized samples. Dissolve the lyophilized samples in D2O containing internal standard.

[0061] AMFPs-0-1 and AMFPs-0-1-I were measured by 600MHz NMR. 1 H, 13 C 1 H- 1 H COSY / TOCSY / ROESY and 1 H- 13 C HSQC / HMBC spectra were used to further analyze the structures of AMFPs-0-1 and AMFPs-0-1-I, in order to deduce the chemical structural formula of the elm polysaccharide AMFPs-0-1.

[0062] Results Analysis

[0063] The high-performance liquid chromatography gel chromatograms of the molecular weight and distribution of AMFPs-0-1 are shown below. Figure 1 As shown, the purity of AMFPs-0-1 was >98% using the area normalization method. Based on the curve equation of the standard, the molecular weight of AMFPs-0-1 was calculated to be approximately 798 kDa, and the PDI was approximately 1.6.

[0064] The monosaccharide composition of AMFPs-0-1 was determined by pre-column derivatization high-performance liquid chromatography, and the results are as follows: Figure 2 As shown, by comparing the peak elution time with that of the standard monosaccharide and calculating the molar ratio of the monosaccharide composition by peak area, it was found that AMFPs-0-1 is composed of rhamnose (Rha), galacturonic acid (GalA), galactose (Gal), and arabinose (Ara), with a molar ratio of 1.0:1.4:2.8:6.4.

[0065] To resolve the main chain structure of AMFPs-0-1, AMFPs-0-1 was partially acid-hydrolyzed to obtain the hydrolyzed dialysis inner fluid sample AMFPs-0-1-I. Figure 2 Based on the obtained monosaccharide composition results, it can be inferred that Rhap and GalpA of AMFPs-0-1 are located in the main chain, while Araf and Galp are located in the side chain.

[0066] The total ion chromatogram of the methylated sugar alcohol acetyl derivative of AMFPs-0-1 is shown below. Figure 3AMFPs-0-1 contains T-linked-Araf, 1,3-linked-Araf, 1,5-linked-Araf, T-linked-Galp, 1,3,5-linked-Araf, 1,2,4-linked-Rhap, 1,4-linked-Galp, 1,3,4-linked-Galp, and 1,4,6-linked-Galp, with a molar ratio of 24.0:4.0:29.4:12.1:23.6:5.4:23.9:1.8:1.1.

[0067] The total ion chromatogram of the methylated sugar alcohol acetyl derivative of AMFPs-0-1-I, a partially acid-hydrolyzed sample of AMFPs-0-1, is shown in the figure. Figure 4 The connection methods are t-Rhap, 1,2-Rhap, t-linked-Galp, 1,2,4-Rhap, 1,4-Galp, 1,3,4-Galp, and 1,4,6-Galp, with a molar ratio of 3.1:21.2:18.7:11.9:37.9:2.6:1.6.

[0068] The structures of AMFPs-0-1 and AMFPs-0-1-I were further analyzed using nuclear magnetic resonance spectroscopy (1D NMR and 2D NMR). 1 H and 13 C NMR spectra and their attribution are as follows: Figure 5 As shown. 1 H- 13 C HSQC spectrum and its attribution as follows Figure 6 As shown. 1 H and 13 The CNMR signal attribution is shown in Table 1.

[0069] Table 1. AMFPs-0-1 1 H and 13 CNMR signal attribution

[0070]

[0071]

[0072] Note: a The bold numbers in the table represent glycosidic bond linkage sites; b Not detected.

[0073] The structure and connection mode of the main chain were further confirmed by 1D and 2D NMR spectra of AMFPs-0-1-I. 1 H and 13 C NMR spectra and their attribution are as follows: Figure 7 and8 As shown. 1 H- 13 C HSQC spectrum and its attribution as follows Figure 9 As shown. 1 H and 13 The C NMR signal assignments are shown in Table 2.

[0074] Table 2. AMFPs-0-1-I 1 H and 13 C NMR signal attribution

[0075]

[0076] Note: a The bolded numbers indicate the glycosylation position, with t being the terminal position.

[0077] Based on the monosaccharide composition, methylation analysis, and NMR spectrum resolution of AMFPs-0-1 and AMFPs-0-1-I, the structural formula of AMFPs-0-1 can be determined as follows:

[0078]

[0079] In formula (I): Rhap is a pyranose rhamnosyl group; GalpA is a pyranose galacturonic acid group; Galp is a pyranose galactosyl group; Araf is a furanose arabinose group.

[0080] R1 is a group with the structure shown in formula (II), and R2 is a group with the structure shown in formulas (III), (IV), and (V).

[0081]

[0082] β-Araf-(1-[→3)-α-Araf] n - (III)

[0083]

[0084] β-Galp-(1-[→4)-β-Galp] s - (V)

[0085] m is an integer with a mean of 1 to 6; n is an integer with a mean of 1 to 8; o is an integer with a mean of 6 to 10; p is an integer with a mean of 1 to 4; q+r+s is an integer with a mean of 15 to 21.

[0086] Example 2

[0087] The intestinal flora fermentation and probiotic function of RG-I type olive polysaccharide AMFPs-0-1 prepared in Example 1 were investigated using the following methods:

[0088] Stool samples were provided by four volunteers (aged 20–25, 2 males and 2 females) who had no gastrointestinal disease and had not received antibiotic treatment for at least three months. After sampling, the stool samples were placed in sterile centrifuge tubes, and the time from sampling to experimental operation did not exceed 2 hours.

[0089] (1) Prepare 1L of basic culture medium, autoclave at 121℃ for 20min, cool to room temperature in a clean bench, and adjust the pH to 7.0 with 0.1M HCl solution filtered through a membrane.

[0090] Equal amounts of fecal samples were weighed from four fecal samples and placed in 50 mL centrifuge tubes. Sterile modified physiological saline was added, vortexed, and centrifuged (500 rpm × 5 min) to obtain a fecal suspension. 0.5 mL of the fecal suspension was added to 4.5 mL of a solution containing 50 mg AMFPs-0-1 (solvent: sterile modified physiological saline) and mixed thoroughly to create the AMFPs-0-1 group. Another 0.5 mL of the fecal suspension was added to 4.5 mL of a solution containing 50 mg FOS (solvent: sterile modified physiological saline) and mixed thoroughly to create a positive control. 4.5 mL of sterile modified physiological saline was used as a blank control solution. The tubes were sealed with sealing film and placed in an anaerobic fermentation chamber. The chamber was then placed in an anaerobic gas generator and sealed quickly. Fermentation was carried out in an incubator at 37°C. Three replicates were set for each group.

[0091] (2) The anaerobic box was opened at 0, 6, 12 and 24 h after the start of fermentation to terminate the reaction. The fermentation broth was centrifuged (10000 rpm × 5 min) and the supernatant was collected.

[0092] Bacterial precipitates were collected at 24-hour fermentation time points, stored at -80℃ and placed on dry ice before being transported to Guangzhou Gediao Biotechnology Co., Ltd. for genomic DNA extraction and 16S rRNA V3-V4 hypervariable region sequencing. The amplification primers were 341F (sequence shown in SEQ ID NO.1) and 806R (sequence shown in SEQ ID NO.2). The purified amplification products were ligated into sequencing adapters to construct sequencing libraries, which were then sequenced on the Illumina platform.

[0093] SEQ ID NO.1: 5′-CCTACGGGNGGCWGCAG-3′;

[0094] SEQ ID NO. 2: 5'-GGACTACHVGGGTATCTAAT-3'.

[0095] (3) Take the collected fermentation supernatant and use gas chromatography (GC) to determine the content of short-chain fatty acids (SCFAs) at different fermentation time points.

[0096] Results Analysis

[0097] Utilization of AMFPs-0-1 by gut microbiota: Changes in molecular weight distribution of AMFPs-0-1 samples at different fermentation times, as shown in the figure. Figure 10 As shown, the response value of the sample at around 12.117 min decreased with the extension of fermentation time, and the molecular weight distribution became more and more widespread, indicating that AMFPs-0-1 was gradually degraded into small fragments by intestinal microorganisms during the fermentation stage.

[0098] Analysis of community structure distribution after 24 hours of fermentation is as follows: Figure 11 As shown, at the phylum level, Firmicutes (Bacillota), Desulfobacteria, Proteobacteria, Bacteroidetes, and Actinobacteria were the dominant bacterial groups in each sample group (AMFPs-0-1 group, positive control group, and blank control group), but their proportions varied among the sample groups. AMFPs-0-1 fermentation had a positive effect on the growth of Firmicutes (Bacillota) and Actinobacteria.

[0099] At the genus level, the dominant genera in the AMFPs-0-1 group were Bifidobacterium, Megamonas, Megasphaera, and Mitsuokella. Compared with the BLK group, the relative abundance of Phascolarctobacillus was significantly decreased in the FOS-positive control group and the AMFPs-0-1 group, while the relative abundance of Escherichia coli-Shigella in the FOS-positive control group and Bifidobacterium was significantly increased in the AMFPs-0-1 group.

[0100] To further compare the differences in gut microbiota among the sample groups, heatmap analysis at the genus level was performed, as shown in the attached figure. Figure 11 As shown in Figure C, Bifidobacterium, Mitsuokella, Megasphaera, and Megamonas are unique bacteria in the AMFPs-0-1 group. Bifidobacterium is the most abundant genus in the AMFPs-0-1 group, accounting for 46.43% of the bacterial community. Bifidobacterium is representative of actinomycetes, which are Gram-positive bacteria and can help prevent intestinal diseases.

[0101] Changes in the main metabolic products of gut microbiota, SCFAs, such as Figure 12As shown, the fermentation products of AMFPs-0-1 were mainly acetic acid, propionic acid, and lactic acid. The concentrations of acetic acid, propionic acid, lactic acid, and butyric acid in the AMFPs-0-1 group all increased significantly during the 24-hour fermentation process. After fermentation by fecal microbiota, the intestinal microbiota structure of the AMFPs-0-1 group changed, significantly promoting the production of intestinal SCFAs. The increase in SCFAs can have beneficial effects on human health.

[0102] Example 3

[0103] The immunomodulatory activity of RG-I type eugenol polysaccharide AMFPs-0-1 prepared in Example 1 was investigated using the following method:

[0104] Cell culture, including cell resuscitation, cell passage, and cell cryopreservation, was performed according to standard experimental methods. The effect of AMFPs-0-1 on the proliferation of RAW264.7 cells was investigated according to the method reported by Sun et al. (International Journal of Biological Macromolecules, 2018, 108:314-323.). The effect of different concentrations of AMFPs-0-1 on the proliferation of RAW264.7 macrophages was determined using the MTT assay, and the absorbance (OD490) of each well was measured using a microplate reader to calculate cell viability.

[0105] The effect of AMFPs-0-1 on NO secretion in RAW264.7 cells was determined using the Griess method.

[0106] The effects of AMFPs-0-1 on the secretion of TNF-α and IL-6 in RAW264.7 cells were investigated using a kit, following the instructions provided.

[0107] The effects of AMFPs-0-1 on the expression of iNOS, IL-6, and TNF-α mRNA in RAW264.7 cells were investigated using a kit method. 2 mL of 0 μg / mL, 25 μg / mL, 50 μg / mL, 100 μg / mL, and 200 μg / mL of AMFPs-0-1 polysaccharide and 1 μg / mL LPS were added to six-well plates containing cells cultured for 12 h, and incubated at 37°C for 24 h. The supernatant was discarded, and the cells were washed twice with PBS to remove the culture medium. Then, 250 μL of TRIzol reagent was added, and the cells were gently shaken and repeatedly pipetted to ensure complete cell lysis. The lysis buffer was transferred to a 1.5 mL RNase-free centrifuge tube. After total RNA extraction, the total RNA was reverse transcribed into cDNA according to the RNA reverse transcription kit instructions, and then amplified using the cDNA as a template. GAPDH was used as an internal control. The method is used to quantitatively analyze the target gene in the sample.

[0108] Results Analysis

[0109] The effect of AMFPs-0-1 on the proliferation of RAW264.7 cells (macrophages) is as follows: Figure 13 As shown in the figure. Compared with the control group, AMFPs-0-1 significantly promoted cell proliferation within the measured concentration range (p<0.05). The cell proliferation rate was highest at a concentration of 100 μg / mL (230%), and AMFPs-0-1 still promoted cell proliferation at the lowest concentration of 25 μg / mL without cytotoxicity.

[0110] The effect of AMFPs-0-1 on NO secretion in RAW264.7 cells is as follows: Figure 14 As shown in the figure. Compared with the blank control group, AMFPs-0-1 activated macrophages to secrete NO, and the NO content was significantly increased in a dose-dependent manner (p<0.05), especially in the high-dose group (200 μg / mL), where the NO secretion was 31.3 μmol / L, which was slightly lower than that in the lipopolysaccharide (LPS) positive control group.

[0111] The effect of AMFPs-0-1 on macrophage secretion of cytokines, such as Figure 15 As shown, compared with the control group, AMFPs-0-1 significantly promoted the secretion of IL-6 by macrophages (p<0.05), and the increase was dose-dependent, reaching a maximum of 605 pg / mL at a concentration of 200 μg / mL. When the concentration of AMFPs-0-1 was 12.5-100 μg / mL, it significantly promoted the secretion of TNF-α by macrophages in a dose-dependent manner (p<0.05), reaching a maximum of 36067 pg / mL at a concentration of 100 μg / mL. At a concentration of 200 μg / mL, the secretion of TNF-α was lower than that at 100 μg / mL.

[0112] The effects of AMFPs-0-1 on the expression levels of iNOS, IL-6, and TNF-α mRNA secreted by macrophages are as follows: Figure 16 As shown, compared with the blank control group, treatment of RAW264.7 macrophages with AMFPs-0-1 significantly increased iNOS expression levels in a dose-dependent manner (p<0.05), and iNOS mRNA expression induced NO production in RAW264.7 cells. Simultaneously, AMFPs-0-1 also significantly promoted the expression of IL-6 and TNF-α mRNA, with mRNA expression increasing by up to 310-fold and 7-fold, respectively, compared with the blank control group. These results indicate that AMFPs-0-1 promotes cytokine secretion by upregulating the expression of iNOS, IL-6, and TNF-α mRNA.

[0113] Therefore, the method provided by this invention successfully yielded a novel and precisely structured new polysaccharide—RG-I type AMFPs-0-1—from argan polysaccharide. The main chain is composed of alternating →2)-α-L-Rhap-(1→ and →4)-α-D-GalpA-(1→ units, with each Rhap unit linked to a side chain composed of arabinose or galactose. AMFPs-0-1 has no toxic side effects on cells, can be fermented and utilized by human intestinal flora, significantly promotes the growth of beneficial bacteria, significantly promotes the production of short-chain fatty acids in the intestine, activates macrophages, and effectively promotes macrophage proliferation. It has great application potential in the prevention and / or treatment of intestinal diseases and in the preparation of immune-enhancing drugs.

[0114] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. An RG-I type eugenol polysaccharide, characterized in that: The structural formula of the RG-I type aralia polysaccharide is shown in formula (I), and it is a mixture of homologous polysaccharides with the structure shown in formula (I). Rhap is a pyranose rhamnosyl, GalpA is a pyranose galacturonic acid, Galp is a pyranose galactosyl, and Araf is a furanose arabinose. R1 is a group with the structure shown in formula (II), and R2 is a group with the structure shown in formulas (III), (IV), and (V). β-Araf-(1-[→3)-α-Araf] n - (III) β-Galp-(1-[→4)-β-Galp] s - (V); where m is an integer with a mean of 1 to 6, n is an integer with a mean of 1 to 8, o is an integer with a mean of 6 to 10, p is an integer with a mean of 1 to 4, and q+r+s is an integer with a mean of 15 to 21.

2. The RG-I type olive polysaccharide according to claim 1, characterized in that: The monosaccharides that make up the RG-I type argan polysaccharide include rhamnose, galacturonic acid, galactose, and arabinose, with a molar ratio of rhamnose:galacturonic acid:galactose:arabinose of 1.0:(1.4±0.5):(2.8±0.5):(6.4±0.5).

3. The RG-I type olive polysaccharide according to claim 1 or 2, characterized in that: The average molecular weight (Mw) of the RG-I type aralia polysaccharide is 700kDa to 850kDa, and the polydispersity index is between 1.0 and 2.

0.

4. The RG-I type olive polysaccharide according to claim 1 or 2, characterized in that: The RG-I type olive polysaccharide is a non-starch polysaccharide obtained from the fruit of *Avicennia marina* (Forssk.) Vierh.

5. A method for preparing RG-I type olive polysaccharide as described in any one of claims 1 to 4, characterized in that, The steps are as follows: using non-starch total polysaccharide of olive as raw material, separation is carried out using anion exchange chromatography column, the elution fraction is collected by pure water, desalted by dialysis or ultrafiltration membrane filtration, concentrated, and then separated and purified by gel chromatography column. The fractions of the main chromatographic peaks are collected, desalted, and lyophilized.

6. The method for preparing RG-I type olive polysaccharide according to claim 5, characterized in that: When using anion exchange chromatography, a DEAE FF column with good water equilibration was selected, and gradient elution was performed using 0M, 0.1M, 0.3M, and 0.5M NaCl solutions as mobile phases. When using gel chromatography for separation and purification, a Sepharose Cl-6B gel chromatography column equilibrated with 0.2M NaCl was selected.

7. The use of RG-I type eugenol polysaccharide as described in any one of claims 1 to 4 in the preparation of a drug for promoting human intestinal health, characterized in that: The application aims to increase the relative abundance of beneficial bacteria and the level of acidic metabolites. The beneficial bacteria include Bifidobacteria, Megalosporins, Coccidia, and Pleurotus ostreatus, and the acidic metabolites include short-chain fatty acids.

8. The application of the RG-I type aralia polysaccharide according to claim 7 in the preparation of drugs that promote human intestinal health, characterized in that: The short-chain fatty acids are acetic acid and propionic acid.

9. The use of RG-I type eugenol polysaccharide as described in any one of claims 1 to 4 in the preparation of drugs to enhance human immune stress response, characterized in that: The drug can activate macrophages, promote the production of NO, TNF-α, and IL-6, and promote the expression of iNOS, IL-6, and TNF-α mRNA in macrophages.

10. A health / functional food comprising the RG-I type eugenol polysaccharide according to any one of claims 1 to 4, characterized in that: The food is used to treat / alleviate intestinal flora imbalance and weakened immunity.

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