A modified acidic polysaccharide from Ligusticum chuanxiong and its preparation method and use

Abstract

The present invention provides a modified acidic polysaccharide from the leaves of the elm, which is prepared by a chemical method or an enzymatic method from natural acidic polysaccharide from the leaves of the elm. The modified acidic polysaccharide contains, per 100 mg, 89.75 mg ± 1.04 mg to 90.67 mg ± 1.46 mg of total polysaccharide; 23.70 mg ± 1.58 mg to 71.74 mg ± 2.07 mg of total uronic acid; 2.06 mg GAE ± 0.06 mg GAE to 4.56 mg GAE ± 0.04 mg GAE (gallic acid equivalent); a degree of esterification of 3.72% ± 0.90% to 34.98% ± 0.62%; and a molecular weight of (0.562 ± 0.012) × 10 4 Da–(4.268±0.055)×10 4 Da; the proportion of galacturonic acid polysaccharide (HG): 15.28%–65.02%; the proportion of type I rhamnogalacturonic acid polysaccharide (RG-Ⅰ): 28.47%–72.37%; the side chain length: 0.44–10.31. The present invention also provides a preparation method and use of the modified acidic polysaccharide of the Artemisia selengensis. The modified acidic polysaccharide of the Artemisia selengensis prepared by the present invention has significant immunomodulatory activity, especially the modified acidic polysaccharide of the Artemisia selengensis with the arabinose side chain removed and the galactose side chain reduced, and the immune activity is better.

Description

Technical Field

[0001] The invention relates to a modified acidic polysaccharide from the leaves of Zingiber officinale, and belongs to the field of health-care foods or medicines. Background Art

[0002] Immunity is a physiological process that helps the body eliminate pathogens (bacteria, viruses, etc.) and tumor cells and maintains its health. It is of vital importance to human physiological health. Polysaccharides are considered natural immunomodulators or immunopotentiators due to their significant immunostimulatory activity. Polysaccharides can exert immunomodulatory effects by activating immune cell surface receptors, promoting the release of cytokines, enhancing the proliferation and differentiation of immune cells, strengthening the function of phagocytes, natural killer cells, and lymphocytes, promoting the growth of immune organs and the secretion of immunoglobulins, thereby enhancing the body's immunity.

[0003] Ligusticum chuanxiong (Ligusticum chuanxiong), also known as sweet tea, is an evergreen tree belonging to the genus Ligusticum in the family Fagaceae, Fagaceae, and is widely distributed in southern China. Ligusticum chuanxiong combines the functions of tea, sugar, and medicine and has long been used to prevent and treat diabetes. It will be listed as a new food ingredient in 2017, opening up its application in health foods and conventional foods. Ligusticum chuanxiong contains a variety of bioactive compounds, such as polysaccharides, phenolic acids, and flavonoids, with potential pharmacological effects. Ligusticum chuanxiong polysaccharides are one of the most important bioactive components of Ligusticum chuanxiong, exhibiting significant antioxidant, anti-inflammatory, anti-diabetic, and immunomodulatory effects, and hold great promise for development in the functional health food sector.

[0004] Fu Mengqian, Extraction optimization, structural characterization and in vitro bioactivity evaluation of polysaccharides from Ligusticum chuanxiong, Sichuan Agricultural University, June 2023. This article reports the conditions of pressurized hot water-assisted extraction technology and microwave-assisted low eutectic solvent extraction technology of polysaccharides from Ligusticum chuanxiong, and analyzes the structural characteristics of the purified polysaccharides from Ligusticum chuanxiong. The extracted polysaccharides from Ligusticum chuanxiong showed good in vitro antioxidant activity (ABTS free radical scavenging activity, DPPH free radical scavenging activity and NO free radical scavenging activity, as well as total reducing power), anti-glycation activity, hypoglycemic activity (α-glucosidase inhibitory activity), immunomodulatory activity and in vitro microbial beneficial activity. Patent application number 202110615589.6, invention name: Sweet tea polysaccharide extract with high antioxidant activity and extraction method thereof; the present invention discloses a sweet tea polysaccharide extract with high antioxidant activity and an extraction method thereof. The present invention adopts a low eutectic solvent-microwave assisted extraction method to extract polysaccharides from sweet tea, effectively improving the solvent's solubility for polysaccharides, achieving efficient extraction of polysaccharides and the prepared sweet tea polysaccharide has high antioxidant activity, and has a series of advantages such as high extraction efficiency, low extraction cost, simple extraction process, stable and reliable, and the extracted sweet tea polysaccharide has high antioxidant activity. Summary of the Invention

[0005] The present invention is based on the extraction of natural Zingiber officinale acidic polysaccharides with a low eutectic solvent, and the natural Zingiber officinale acidic polysaccharides are modified (chemically and enzymatically modified). The in vitro immunomodulatory activity of the Zingiber officinale acidic polysaccharides before and after modification is compared, and the key effector structures and potential mechanisms that affect the in vitro immunomodulatory effect of the Zingiber officinale acidic polysaccharides can be clearly defined, which is conducive to more effective utilization of Zingiber officinale resources, as well as providing a reference for the exploration of the immunomodulatory effect and mechanism of the Zingiber officinale polysaccharides, and providing theoretical and technical support for the development of Zingiber officinale polysaccharide-related functional foods. Compared with natural Zingiber officinale acidic polysaccharides, the modified Zingiber officinale acidic polysaccharides have better in vitro immune activity.

[0006] The present invention provides a modified acidic polysaccharide from the Artemisia argyi L. and also provides a preparation method and application of the modified acidic polysaccharide from the Artemisia argyi L.

[0007] The present invention provides a modified acidic polysaccharide of Zingiber officinale, which is a modified acidic polysaccharide prepared by chemical or enzymatic methods from natural Zingiber officinale acidic polysaccharide.

[0008] The content of total sugars per 100 mg of modified acidic polysaccharide from Artemisia selengensis leaves is as follows: 89.75 mg ± 1.04 mg–90.67 mg ± 1.46 mg; total uronic acid: 23.70 mg ± 1.58 mg–71.74 mg ± 2.07 mg; total protein: 1.53 mg ± 0.02 mg–3.16 mg ± 0.07 mg; total bound phenols: 2.06 mg GAE ± 0.06 mg GAE–4.56 mg GAE ± 0.04 mg GAE (gallic acid equivalent); degree of esterification: 3.72% ± 0.90%–34.98% ± 0.62%;

[0009] Molecular weight M w :(0.562±0.012)×10 4 Da–(4.268±0.055)×10 4 Da; dispersion coefficient M w / M n : 1.163–1.729;

[0010] The sugar components of the modified acidic polysaccharide from the leaves of Artemisia selengensis mainly contain the following monosaccharides: galacturonic acid, galactose, arabinose, rhamnose, xylose, glucose, glucuronic acid, and mannose. The molar percentages of each monosaccharide are: (21.16%–70.56%): (17.26%–33.14%): (0.14%–27.48%): (5.53%–7.29%): ( 1.36%–6.85%):(1.71%–2.27%):(1.79%–2.89%):(0.83%–1.21%); among them, galacturonan (HG) accounted for 21.94%–65.02%, type I rhamnogalacturonan (RG-Ⅰ) accounted for 28.47%–72.37%, and the side chain length was 0.44–10.31.

[0011] More preferably,

[0012] The content of modified acidic polysaccharide from Artemisia selengensis leaf per 100 mg is as follows: total polysaccharide: 89.75 mg ± 1.04 mg–90.09 mg ± 1.07 mg; total uronic acid: 49.57 mg ± 0.94 mg–71.74 mg ± 2.07 mg; total protein: 3.03 mg ± 0.07 mg–3.16 mg ± 0.07 mg; total bound phenol: 2.06 mg GAE ± 0.06 mg GAE–2.69 mg GAE ± 0.10 mg GAE (gallic acid equivalent); degree of esterification: 7.74% ± 0.40%–14.54% ± 0.84%;

[0013] Molecular weight M w :(0.562±0.012)×10 4 Da–(1.317±0.018)×10 4 Da; dispersion coefficient M w / M n : 1.163–1.183;

[0014] The sugar components of the modified acidic polysaccharide from the leaves of Artemisia selengensis mainly contain the following monosaccharides: galacturonic acid, galactose, arabinose, rhamnose, xylose, glucose, glucuronic acid, and mannose. The molar percentages of each monosaccharide are: (55.89%–70.56%): (17.26%–28.99%): (0.14%–0.47%): (5.53%–6.46%): ( 1.36%–2.40%):(1.71%–1.78%):(2.22%–2.89%):(1.12%–1.21%); among them, galacturonan (HG) accounted for 49.44%–65.02%, type I rhamnogalacturonan (RG-Ⅰ) accounted for 28.47%–42.37%, and the side chain length was 0.44–0.86.

[0015] Wherein, the natural Zingiber officinale acidic polysaccharide is prepared by adopting a deep eutectic solvent assisted heating extraction method;

[0016] The chemically modified acidic polysaccharide is prepared by modifying with sodium hydroxide, hydrochloric acid and trifluoroacetic acid; the enzymatically modified acidic polysaccharide is prepared by modifying with pectinase derived from Aspergillus aculeatus.

[0017] The present invention also provides a method for preparing the modified acidic polysaccharide from the Artemisia selengensis, which comprises the following steps:

[0018] a. Using deep eutectic solvent to assist heating to extract natural acidic polysaccharides from Zingiber officinale;

[0019] b. The natural acidic polysaccharide of Zingiber officinale prepared in step a is modified by chemical or enzymatic methods.

[0020] Wherein, the method for modifying polysaccharide in step b is:

[0021] 0.5% (w / v) of natural acidic polysaccharide from the leaves of the tree of the elm was added to a sodium hydroxide solution with a pH value of 12.0, and stirred at 4°C for 30 minutes. Subsequently, hydrochloric acid (1M) was added until the pH value was 7.0. The solution was ultrafiltered through an ultrafiltration membrane with a molecular weight limit of 3.0 kDa and freeze-dried to obtain a modified acidic polysaccharide from the leaves of the tree of the elm (STP-AD).

[0022] 0.5% (w / v) of natural Zingiberis truncatula acidic polysaccharide was added to 0.5M trifluoroacetic acid solution to make the final concentration of trifluoroacetic acid in the system 0.25M, and the mixture was reacted at 95°C for 1.5h and 4h, respectively. Subsequently, sodium hydroxide solution (1M) was added until the pH value was 7.0, and the mixture was ultrafiltered through an ultrafiltration membrane with a molecular weight limit of 3.0kDa and freeze-dried to obtain Zingiberis truncatula modified acidic polysaccharide (STP-AH1) with arabinose side chains removed and Zingiberis truncatula modified acidic polysaccharide (STP-AH2) with arabinose side chains removed and galactose side chains reduced, respectively; wherein, under the condition of reaction for 1.5h, the Zingiberis truncatula modified acidic polysaccharide (STP-AH1) with arabinose side chains removed was prepared; under the condition of reaction for 4h, the Zingiberis truncatula modified acidic polysaccharide (STP-AH2) with arabinose side chains removed and galactose side chains reduced was prepared;

[0023] 0.5% (w / v) of natural acidic polysaccharide from the leaves of Artemisia selengensis was added to a 0.3U / mL pectinase solution, reacted at 40°C for 6 hours, inactivated, centrifuged, and the supernatant was taken; the supernatant was ultrafiltered through an ultrafiltration membrane with a molecular weight limit of 3.0kDa and freeze-dried to obtain modified acidic polysaccharide from the leaves of Artemisia selengensis (STP-E) with a reduced proportion of galacturonic acid polysaccharide.

[0024] The present invention also provides use of the modified acidic polysaccharide from the Artemisia selengensis leaf in preparing health-care food that helps to enhance immunity.

[0025] The present invention also provides use of the modified acidic polysaccharide of Zingiber officinale in preparing a medicine with immunomodulatory effect.

[0026] Polysaccharides are a class of bioactive substances with complex and diverse structures. Polysaccharides with different structures exhibit different biological activities. Plant acidic polysaccharides are mainly pectin polysaccharides, and the main structural domains of pectin polysaccharides are galacturonan (HG) and type I rhamnogalacturonan (RG-I). A large number of studies have shown that the HG / RG I ratio, molecular weight, branch length, degree of esterification, glycosidic bonds and constituent monosaccharides of pectin polysaccharides are the key chemical structures of polysaccharides that have health-promoting effects, and these structures are closely related to their biological activities. Different modification methods will produce different degrees of substitution, and have different effects on activity, which can make the modified polysaccharides exhibit different physicochemical properties and biological activities. The present invention can clarify the key effector structure and potential mechanism of the in vitro immunomodulatory effect of the acidic polysaccharide of the leaf of the ginger. The natural acidic polysaccharide of the leaf of the ginger of the present invention has an immune-enhancing effect. The natural acidic polysaccharide of the leaf of the ginger is modified and the structural characteristics of the modified acidic polysaccharide of the leaf of the ginger and its immunostimulatory effect on RAW264.7 cells and its potential mechanism are analyzed. The modified acidic polysaccharide of Zingiber officinale prepared by the present invention has significant immunomodulatory activity, especially the modified acidic polysaccharide of Zingiber officinale with arabinose side chains removed and galactose side chains reduced has better immunomodulatory activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 High-performance gel exclusion chromatography of the acidic polysaccharide modified from the Artemisia selengensis (Note: STP, STP-AD, STP-AH1, STP-AH2, and STP-E represent the natural acidic polysaccharide from the Artemisia selengensis, the acidic polysaccharide modified from the Artemisia selengensis with esterification removed, the acidic polysaccharide modified from the Artemisia selengensis with arabinose side chains removed, the acidic polysaccharide modified from the Artemisia selengensis with arabinose side chains removed and galactose side chains reduced, and the acidic polysaccharide modified from the Artemisia selengensis with reduced galacturonic acid polysaccharide ratio, respectively);

[0028] Figure 2 Chromatogram of monosaccharide composition of modified acidic polysaccharide from Ligusticum chuanxiong (Note: STP, STP-AD, STP-AH1, STP-AH2 and STP-E represent natural acidic polysaccharide from Ligusticum chuanxiong, modified acidic polysaccharide from Ligusticum chuanxiong with esterification removed, modified acidic polysaccharide from Ligusticum chuanxiong with arabinose side chains removed, modified acidic polysaccharide from Ligusticum chuanxiong with arabinose side chains removed and galactose side chains reduced, and modified acidic polysaccharide from Ligusticum chuanxiong with reduced galacturonic acid polysaccharide ratio, respectively);

[0029] Figure 3Fourier transform infrared spectra of the acidic polysaccharide modified from the Artemisia selengensis (Note: STP, STP-AD, STP-AH1, STP-AH2 and STP-E represent the natural acidic polysaccharide from the Artemisia selengensis, the acidic polysaccharide modified from the Artemisia selengensis with esterification removed, the acidic polysaccharide modified from the Artemisia selengensis with arabinose side chains removed, the acidic polysaccharide modified from the Artemisia selengensis with arabinose side chains removed and galactose side chains reduced, and the acidic polysaccharide modified from the Artemisia selengensis with reduced galacturonic acid polysaccharide ratio, respectively);

[0030] Figure 4 Modified acidic polysaccharide from Artemisia selengensis 1 H NMR spectra (Note: STP, STP-AD, STP-AH1, STP-AH2, and STP-E represent natural acidic polysaccharide from Zingiber officinale, modified acidic polysaccharide from Zingiber officinale with esterification removed, modified acidic polysaccharide from Zingiber officinale with arabinose side chains removed, modified acidic polysaccharide from Zingiber officinale with arabinose side chains removed and galactose side chains reduced, and modified acidic polysaccharide from Zingiber officinale with reduced galacturonic acid polysaccharide ratio, respectively);

[0031] Figure 5 Modified acidic polysaccharide from Artemisia selengensis 13 C NMR spectra (Note: STP, STP-AD, STP-AH1, STP-AH2, and STP-E represent natural acidic polysaccharide from Zingiber officinale, modified acidic polysaccharide from Zingiber officinale with esterification removed, modified acidic polysaccharide from Zingiber officinale with arabinose side chains removed, modified acidic polysaccharide from Zingiber officinale with arabinose side chains removed and galactose side chains reduced, and modified acidic polysaccharide from Zingiber officinale with reduced galacturonic acid polysaccharide ratio, respectively);

[0032] Figure 6 Effects of modified acidic polysaccharides from Zingiber officinale on the cytotoxicity (A), production of nitric oxide (NO) (B), production of tumor necrosis factor-α (TNF-α) (C), and production of interleukin-6 (IL-6) (D) in RAW264.7 macrophages (Note: STP, STP-AD, STP-AH1, STP-AH2, and STP-E represent natural acidic polysaccharides from Zingiber officinale, modified acidic polysaccharides from Zingiber officinale with esterification removed, modified acidic polysaccharides from Zingiber officinale with arabinose side chains removed, modified acidic polysaccharides from Zingiber officinale with arabinose side chains removed and galactose side chains reduced, and modified acidic polysaccharides from Zingiber officinale with reduced galacturonic acid polysaccharides; error bars represent standard deviations; significant (p < 0.05) differences between different samples are indicated by data with different letters; significant differences between the tested samples and the blank control are indicated by *p < 0.05 and **p < 0.01);

[0033] Figure 7Effect of modified acidic polysaccharide from Ligusticum wallichii on RAW tissues treated with C29 or TAK-242 Effects of 264.7 on the production of nitric oxide (NO) (A), tumor necrosis factor-α (TNF-α) (B) and interleukin-6 (IL-6) (C) in macrophages, and the effect of modified acidic polysaccharides from Zingiber officinale on protein expression (D) (Note: STP, STP-AD, STP-AH1, STP-AH2 and STP-E represent natural acidic polysaccharides from Zingiber officinale, modified acidic polysaccharides from Zingiber officinale with esterification removed, modified acidic polysaccharides from Zingiber officinale with arabinose side chains removed, modified acidic polysaccharides from Zingiber officinale with arabinose side chains removed and galactose side chains reduced, and modified acidic polysaccharides from Zingiber officinale with reduced galacturonic acid polysaccharides, respectively; error bars represent standard deviations; significant (p < 0.05) differences between different samples are represented by data with different letters; significant differences between the tested samples and the blank control are represented by *p < 0.05 and **p < 0.01). DETAILED DESCRIPTION

[0034] Example 1 Preparation of the modified acidic polysaccharide of Zingiber officinale

[0035] 1. Preparation of natural acidic polysaccharide from Zingiber officinale:

[0036] a. Freeze-dry the harvested Litsea cubeba leaves, grind into powder and sieve:

[0037] b. Degreasing: Degrease the powder of Ligusticum wallichii with 80% ethanol, centrifuge and discard the supernatant to obtain the precipitate;

[0038] c. Deep eutectic solvent-assisted extraction: Use a deep eutectic solvent with a water content of 30% to assist in heating extraction, centrifuge, and reserve the supernatant;

[0039] d. Alcohol precipitation: Use 75% (v / v) ethanol for fractionation precipitation, then centrifuge and collect the precipitate;

[0040] e. Redissolve: Redissolve the precipitate from step d with ultrapure water;

[0041] f. Starch removal: Use α-amylase and saccharifying enzyme to remove starch in the reconstituted solution, centrifuge, and reserve the supernatant;

[0042] g. Ultrafiltration: ultrafiltration is performed on the supernatant, and fractions with molecular weights between 3 kDa and 100 kDa are collected by ultrafiltration membrane, and freeze-dried to obtain natural S. truncatula acidic polysaccharide (STP).

[0043] 2. Preparation of deesterified modified acidic polysaccharide from Ligusticum wallichii using low-temperature alkaline deesterification technology:

[0044] a. Take 0.5% (w / v) of the natural Zingiber officinale acidic polysaccharide described in 1 and add it to a sodium hydroxide solution with a pH value of 12.0, and stir at 4°C for 30 minutes;

[0045] b. Add hydrochloric acid (1 M) to the solution in step a until the pH value is 7.0, ultrafilter through an ultrafiltration membrane with a molecular weight limit of 3.0 kDa and freeze-dry to obtain a modified acidic polysaccharide of Artemisia selengensis (STP-AD) with esterification removed.

[0046] 3. Preparation of modified acidic polysaccharides from Ligusticum wallichii with arabinose side chains removed and galactose side chains reduced by controlled different acid hydrolysis technologies:

[0047] a. Take 0.5% (w / v) of the natural Zingiber officinale acidic polysaccharide described in 1 and add 0.5M trifluoroacetic acid solution to make the final concentration of trifluoroacetic acid in the system 0.25M, and react at 95°C for 1.5h and 4h respectively;

[0048] b. Sodium hydroxide solution (1M) was added to the solution of step a until the pH value was 7.0, and ultrafiltration and freeze-drying were performed with an ultrafiltration membrane having a molecular weight limit of 3.0 kDa to obtain a modified acidic polysaccharide (STP-AH1) of the leaf of the Chinese ginseng tree with arabinose side chains removed and a modified acidic polysaccharide (STP-AH2) of the leaf of the Chinese ginseng tree with arabinose side chains removed and galactose side chains removed. Wherein, under the condition of reaction for 1.5 h, the modified acidic polysaccharide (STP-AH1) of the leaf of the Chinese ginseng tree with arabinose side chains removed was prepared; under the condition of reaction for 4 h, the modified acidic polysaccharide (STP-AH2) of the leaf of the Chinese ginseng tree with arabinose side chains removed and galactose side chains reduced was prepared.

[0049] 4. Preparation of modified acidic polysaccharide of Zingiber officinale with reduced galacturonic acid polysaccharide ratio by directed enzymatic digestion technology a. Take 0.5% (w / v) of the natural Zingiber officinale acidic polysaccharide described in 1 and add it to 0.3U / mL pectinase solution, react at 40°C for 6h, inactivate, centrifuge, and take the supernatant;

[0050] b. The supernatant was ultrafiltered through an ultrafiltration membrane with a molecular weight limit of 3.0 kDa and freeze-dried to obtain a galacturonic acid polysaccharide modified from the leaves of the sedge (STP-E).

[0051] Example 2 Characterization of the physicochemical properties of the modified acidic polysaccharide from the leaves of Zingiber officinale of the present invention

[0052] Colorimetric methods were used to determine the total polysaccharide, total uronic acid, total bound phenolic, and total protein contents of the modified acidic polysaccharide from the leaves of the tree of the elm. SEC-MALLS-RID (Wyatt Technology Co., Santa Barbara, CA, USA) was used to determine the molecular weight and dispersity of the modified acidic polysaccharide from the leaves of the elm. The monosaccharide composition of the modified acidic polysaccharide from the leaves of the elm was determined by high-performance liquid chromatography (L-20A, Shimadzu, Japan) combined with pre-column derivatization with 1-phenyl-3-methyl-5-pyrazolone (PMP). The functional groups and degree of esterification of the modified acidic polysaccharide from the leaves of the elm were analyzed by Fourier transform infrared spectroscopy (PerkinElmer, Waltham, MA, USA). The glycosidic bonds of the modified acidic polysaccharide from the leaves of the elm were analyzed by nuclear magnetic resonance spectroscopy (Bruker, Rheinstetten, Germany). Specific method reference: Authors: Ding-Tao Wu, Meng-Xi Fu, Huan Guo, Yi-Chen Hu, Xiao-Qin Zheng, Ren-You Gan and Liang Zou; Title: Microwave-Assisted Deep Eutectic Solvent Extraction, Structural Characteristics, and Biological Functions of Polysaccharides from Sweet Tea (Lithocarpus litseifolius) Leaves; Journal Name: ANTIOXIDANTS, Volume 11, Issue 8; DOI: 10.3390 / antiox11081578.

[0053] 1. Chemical composition analysis of modified acidic polysaccharides from Ligusticum chuanxiong

[0054] Table 1 summarizes the chemical composition of natural S. truncatum acidic polysaccharide (STP) and modified S. truncatum acidic polysaccharides (STP-AD, STP-AH1, STP-AH2, and STP-E). The results showed no significant difference in the total polysaccharide content between natural S. truncatum acidic polysaccharide and modified S. truncatum acidic polysaccharide. The uronic acid content can influence the bioactivity of the polysaccharide to a certain extent; generally, higher uronic acid content indicates better bioactivity. The total uronic acid content of STP was 29.49 mg / 100 mg, while that of STP-AD decreased slightly to 26.71 mg / 100 mg and that of STP-E decreased significantly to 23.70 mg / 100 mg. The total uronic acid content of STP-AH1 and STP-AH2 was significantly increased to 49.57 mg / 100 mg and 71.74 mg / 100 mg, respectively. Furthermore, the total protein content of STP-E, STP-AH1, and STP-AH2 all increased significantly, ranging from 1.85 mg / 100 mg to 3.16 mg / 100 mg. The total bound phenol content of STP-E did not differ significantly from that of STP. The total bound phenol content of STP-AD, STP-AH1, and STP-AH2 all decreased, to 3.12 mg GAE / 100 mg, 2.69 mg GAE / 100 mg, and 2.06 mg GAE / 100 mg (gallic acid equivalents), respectively. The degree of esterification also affects polysaccharide activity; generally, the lower the degree of esterification, the better the bioactivity of the polysaccharide. As can be seen from Table 1, after mild alkaline deesterification, the esterification degree of STP (38.51%) decreased to 3.72% (STP-AD). After low-concentration acid hydrolysis for different times, the esterification degree decreased to 14.54% (STP-AH1) and 7.74% (STP-AH2). After pectinase degradation, the esterification degree decreased to 34.98% (STP-E).

[0055] Table 1 Chemical components of modified acidic polysaccharides from Zingiber officinale

[0056]

[0057] Note: STP, STP-AD, STP-AH1, STP-AH2, and STP-E represent native Zingiber officinale acidic polysaccharide, Zingiber officinale modified acidic polysaccharide with ester removal, Zingiber officinale modified acidic polysaccharide with arabinose side chains removed, Zingiber officinale modified acidic polysaccharide with arabinose side chains removed and galactose side chains reduced, and Zingiber officinale modified acidic polysaccharide with reduced galacturonic acid polysaccharide ratio, respectively. Results are expressed as (mean ± SD); superscript ae indicates significant difference (p < 0.05); statistical significance was assessed using ANOVA.

[0058] 2 Structural characteristics of modified acidic polysaccharides from Alpinia officinalis

[0059] (1) Molecular weight distribution

[0060] Molecular weight is often related to the activity of polysaccharides. Generally speaking, polysaccharides with lower molecular weight have better activity. Figure 1 The results show that STP, STP-AD, STP-AH1 and STP-AH2 all exhibit symmetrical elution curves. The molecular weights of STP and STP-AD are 4.472×10 4 Da and 4.268×10 4 Da, and the elution curves of STP and STP-AD were highly overlapped, and the retention times were almost the same. The retention times of STP-AH1, STP-AH2, and STP-E shifted later, and the molecular weights were significantly reduced to 1.317×10 4 Da, 0.562×10 4 Da and 1.458×10 4 In addition, the polydispersity (M w / M n ) is between 1.163 and 1.749, which is consistent with the trend of molecular weight change.

[0061] Table 2 Molecular weight of modified acidic polysaccharide from Ligusticum chuanxiong (M w ), dispersion coefficient (M w / M n ), monosaccharide composition molar ratio

[0062]

[0063] Note: STP, STP-AD, STP-AH1, STP-AH2 and STP-E represent natural acidic polysaccharide of Zingiberis chinensis, modified acidic polysaccharide of Zingiberis chinensis with esterification removed, modified acidic polysaccharide of Zingiberis chinensis with arabinose side chains removed, modified acidic polysaccharide of Zingiberis chinensis with arabinose side chains removed and galactose side chains reduced, and modified acidic polysaccharide of Zingiberis chinensis with reduced galacturonic acid polysaccharide ratio, respectively; HG(%) = GalA(%) - Rha(%); RG-I(%) = 2Rha(%) + Gal(%) + Ara(%); side chain length = (Ara(%) + Gal(%)) / Rha(%); superscript ae indicates significant difference (p < 0.05); statistical significance was tested by ANOVA test.

[0064] (2) Monosaccharide composition analysis

[0065] In order to reveal the effects of different modification methods on the chemical structure of natural acidic polysaccharides from the leaves of the tree, the monosaccharide composition, chemical groups and glycosidic bonds of the modified acidic polysaccharides from the leaves of the tree were systematically studied. Figure 2 As shown in Figures AF, STP, STP-AD, STP-AH1, STP-AH2, and STP-E all have the same monosaccharide composition, including rhamnose, mannose, glucuronic acid, galacturonic acid, glucose, galactose, xylose, and arabinose, indicating that the monosaccharide composition of the modified acidic polysaccharide from the leaves of the Artemisia selengensis plant is generally stable. As shown in Table 2, after alkaline deesterification, the molar percentages of the constituent monosaccharides in STP-AD and STP are very similar. Compared with STP, the galacturonic acid content of STP-E was significantly reduced to 21.26% by enzymatic hydrolysis, while the arabinose content of STP-AH1 was significantly reduced to 0.47% by acid hydrolysis, while the galacturonic acid content was significantly increased to 55.89%. With extended acid hydrolysis time, the arabinose and galactose contents of STP-AH2 further decreased to 0.14% and 17.26%, respectively, while the galacturonic acid content increased to 70.56%. The HG ratio, RG-I ratio, and RG-I side chain length can be estimated based on the galacturonic acid, rhamnose, arabinose, and galactose contents. Generally speaking, the HG ratio, RG-I ratio, and RG-I side chain length affect the activity of polysaccharides. Generally, a higher proportion of RG-I domains indicates greater activity, while a decrease in side chain length enhances activity. The HG ratio, RG-I ratio, and RG-I side chain length of modified acidic polysaccharides from the Artemisia selengensis plant are shown in Table 2. The HG ratio, RG-I ratio, and RG-I side chain length of STP are 25.23%, 64.86%, and 8.06, respectively. The HG ratio, RG-I ratio, and RG-I side chain length of STP-AD are 21.94%, 69.22%, and 7.49, respectively. These results indicate that mild alkali treatment has little effect on the HG ratio, RG-I ratio, and RG-I side chain length of the Artemisia selengensis plant acidic polysaccharides, maintaining overall stability. After enzymatic hydrolysis, the HG content of STP-E was significantly reduced to 15.28%, and the RG-I side chain length increased to 10.31. Pectinase significantly destroyed the HG domain of the natural Ligusticum chuanxiong acidic polysaccharide. This shows that pectinase treatment successfully prepared Ligusticum chuanxiong modified acidic polysaccharide with low HG content. After low-concentration acid hydrolysis, the HG content of Ligusticum chuanxiong modified acidic polysaccharide significantly increased to 49.44% (STP-AH1) and 65.02% (STP-AH2), and the RG-I side chain length was significantly reduced to 0.86 (STP-AH1) and 0.44 (STP-AH2), which significantly destroyed the side chain of Ligusticum chuanxiong acidic polysaccharide, indicating that acid hydrolysis successfully prepared Ligusticum chuanxiong modified acidic polysaccharide with reduced side chain length.

[0066] (3) Infrared spectral characteristics

[0067] like Figure 3 As shown in the figure, compared with STP, the number of main characteristic absorption bands of modified acidic polysaccharides from the leaves of the tree (STP-AD, STP-AH1, STP-AH2 and STP-E) did not change significantly, including 3397.9 cm-1 (OH vibration), 2923.8cm -1 (CH vibration), 1738.8cm -1 (COO-CH3 vibration), 1626.7cm -1 (COO-H vibration), 1437.7cm -1 (C-OH vibration), 1239.1cm -1 (COC vibration), 1073.9cm -1 (CO vibration) and 1023.9cm -1 (CC vibration). Compared with STP, STP-AD, STP-AH1, STP-AH2 and STP-E have the -1 The peak intensity around 1626.7 cm -1 The peak intensity around 1738.8cm is usually used. -1 The peak area of ​​1738.8 cm -1 and 1626.7cm -1 The esterification degree of acidic polysaccharides from the leaves of Zingiber officinale was estimated by taking the average ratio of the sum of the peak areas. Figure 3 STP-AD at 1738.8cm -1 The peak almost disappeared, and the esterification degree decreased to 3.72%, indicating that the modified acidic polysaccharide of Ligusticum wallichii with low esterification degree was successfully prepared through low-concentration alkaline deesterification. The detailed results are shown in Table 1.

[0068] (4) Nuclear magnetic resonance spectroscopy

[0069] The effects of different modification methods on the chemical groups and glycosidic bonds of natural Zingiber officinale polysaccharides were evaluated by one-dimensional nuclear magnetic resonance analysis. Figure 4 、 5 As shown in Figure 2, the 1D NMR spectra of STP and modified acidic polysaccharides from the leaves of the tree (STP-AD, STP-AH1, STP-AH2 and STP-E) are similar. Typical signals of HG and RG-I domains were found in STP ( Figure 4 、 5For example, signals corresponding to residues of 1,4-α-D-GalAMep, 1,4-α-D-GalAp, 1,5-α-L-Araf, T-α-L-Araf, 1,3-α-L-Araf, 1,2,4-α-L-Rhap, and 1,2-α-L-Rhap were identified within the range of 4.96 to 5.30 ppm. Signals corresponding to residues of 1,3,6-β-D-Galp and 1,3-β-D-Galp were detected within the range of 4.46 and 4.54 ppm. Furthermore, C1 signals corresponding to residues of 1,4-α-D-GalAp, 1,4-α-D-GalAMep, 1,3,6-β-D-Galp / 1,3-β-D-Galp, 1,5-α-L-Araf, and T-α-L-Araf were also detected within the range of 99.50 to 109.15 ppm. In addition, at 3.81ppm ( 1 H) and 52.81 ppm ( 13 The typical signals under C) indicate the presence of methyl esterification on the carboxyl group of GalAp residue (GalA-OCH3), ranging from 2.07 to 2.18 ppm ( 1 H) range indicates the presence of acetylation of GalAp residues. The typical signal at 170.67ppm indicates the presence of α-1,4-D-GalAMep residues. However, compared with STP, the signal intensity of STP-AD at about 3.81ppm, 20.02ppm, 23.03ppm, 52.81ppm and 170.67ppm weakened or even disappeared, indicating that this study can successfully reduce or remove the methylation and acetylation degree of GalAp residues in natural Zingiber officinale acidic polysaccharides through mild alkali treatment. The signal intensity of STP-AH1 and STP-AH2 weakened or even disappeared at about 2.07ppm, 2.18ppm, 3.81ppm, 5.10ppm, 5.16ppm, 5.18ppm, 52.81ppm, 103.09ppm, 107.28ppm and 109.15ppm, and can be reduced or removed at 5.08ppm( 1 1,4-α-D-GalAMep residues of STP-AH1 were observed at 5.04 ppm and 52.81 ppm, indicating that low-concentration acid hydrolysis can successfully reduce or remove Araf and GalAp residues in natural Zingiber officinale acidic polysaccharides and partially reduce the methylation and acetylation levels of GalAp residues. The signal intensity of STP-E weakened or even disappeared at 5.04 ppm and 52.81 ppm, indicating that low-concentration pectin hydrolysis can be used to reduce GalAp residues in natural Zingiber officinale acidic polysaccharides.

[0070] The beneficial effects of the present invention are demonstrated by the following efficacy tests.

[0071] Experimental Example 1: Immunological structure-activity relationship and potential mechanism of action of modified acidic polysaccharide from Ligusticum chuanxiong

[0072] The present invention uses an in vitro model system to evaluate the influence of natural Zingiber officinale acidic polysaccharides and modified Zingiber officinale acidic polysaccharides on immune enhancement and their potential mechanisms of action, and clarifies the key effector structures and potential mechanisms that affect the in vitro immunomodulatory effects of Zingiber officinale acidic polysaccharides. The results of this invention will help promote its application in the food / functional food industry.

[0073] 1 Experimental materials and reagents

[0074] Table 3 Materials and reagents

[0075]

[0076] 2 Experimental methods

[0077] 2.1 Modification of acidic polysaccharides from Zingiber officinale

[0078] Extraction and modification of natural Zingiber officinale acidic polysaccharide prepared according to the method of Example 1

[0079] 2.2 In vitro immunological activity and structure-activity relationship analysis of modified acidic polysaccharides from Ligusticum chuanxiong

[0080] 2.2.1 Cytotoxicity assay

[0081] 5×10 4 100 μL of RAW 264.7 macrophages at a cell concentration of 100 μL / well were placed in a 96-well microplate and cultured in a 37°C incubator overnight. The supernatant was aspirated, and 100 μL of modified acidic polysaccharides (STP, STP-AD, STP-AH1, STP-AH2, and STP-E) of different concentrations (50, 100, 200 μg / mL) of Artemisia selengensis leaves were added to the wells. The culture medium blank was used as the blank control, and LPS (1 μg / mL) was used as the positive control. The cells were cultured for 24 hours. The supernatant was aspirated, and 100 μL of MTT solution (1 mg / mL) was added to the wells and cultured for 4 hours. The supernatant was aspirated, and 100 μL of DMSO was added to the wells. The OD was measured at 570 nm. The cell proliferation rate was calculated as follows:

[0082] Cell proliferation rate (%) = 1-(A1-A2) / A1×100% (2.1)

[0083] A1 is the absorbance value of the blank group, and A2 is the absorbance value of the sample.

[0084] 2.2.2 Determination of NO and cytokine (TNF-α, IL-6) content

[0085] 1×10 51 mL of RAW 264.7 macrophages (at a concentration of 1 cell / well) was plated into a 24-well microplate and cultured overnight at 37°C. The supernatant was aspirated, and 1 mL of STP, STP-AD, STP-AH1, STP-AH2, and STP-E at varying concentrations (50, 100, and 200 μg / mL) was added to the wells. A blank control was used, and LPS (1 μg / mL) was used as a positive control. The cells were incubated for 48 hours. The supernatant was aspirated, and NO and cytokine (TNF-α, IL-6) levels were determined according to the ELISA kit instructions.

[0086] 2.3 Potential cellular immune mechanisms of modified acidic polysaccharides from Zingiber officinale

[0087] 1×10 5 1 mL of RAW 264.7 macrophages at a concentration of 1 μg / well was plated in a 24-well microplate and cultured overnight at 37°C. The supernatant was aspirated, and 1 mL of culture medium with or without TAK-242 (1 μM) or C29 (30 μM) was added to each well and incubated for 4 hours. The supernatant was aspirated, and 1 mL of STP, STP-AD, STP-AH1, STP-AH2, and STP-E at varying concentrations (50, 100, and 200 μg / mL) was added to each well. A blank medium control was used, and LPS (1 μg / mL) was added as a positive control. The cells were incubated for 48 hours. The supernatant was aspirated, and NO and cytokine (TNF-α, IL-6) levels were determined according to the ELISA kit instructions. In addition, a modified acidic polysaccharide from the plant Zingiber officinale (STP-AH1) with reduced galactose and arabinose side chains was selected. RAW 264.7 cells were incubated with 200 μg / mL of STP-AH1 in six-well plates for 48 hours. Blank medium and LPS (1 μg / mL) were used as blank and positive controls, respectively. The supernatant was removed and the plates were rinsed twice with PBS. 1 mL of PBS was added to each well, and the cells were gently pipetted to homogenize and transferred to a 1.5 mL centrifuge tube. The cells were collected after centrifugation. RIPA cell lysis buffer (containing 0.1% PMSF and protein phosphatase inhibitors) was added and cells were lysed on ice for 30 minutes. Western blot analysis of capillary proteins was performed on a Protein Simple Wes system using a 12–230 kDa separation module (SM-W004) and an anti-rabbit detection module (DM-001) according to the manufacturer's instructions. Data were analyzed using Compass software, with appropriate exposure times set to ensure maximum signal.

[0088] 3 Experimental results

[0089] 3.1 In vitro immunological activity and structure-activity relationship of modified acidic polysaccharides from Ligusticum chuanxiong

[0090] Figure 6A shows the toxic effects of STP, STP-AD, STP-AH1, STP-AH2 and STP-E on RAW264.7 cells. The results showed that the acidic polysaccharide modified from the leaves of Alpinia officinalis had no obvious toxic effect on cell viability at the experimental concentration. Figure 6 As shown in BD, different concentrations (50-200 μg / mL) of STP, STP-AD, STP-AH1, STP-AH2 and STP-E can stimulate the release of NO, TNF-α and IL-6 by RAW264.7 macrophages, and the increase is concentration-dependent, indicating that the modified acidic polysaccharide of Ligusticum wallichii has significant immunostimulatory effects in vitro. Figure 6 BD showed that STP had the weakest immunostimulatory effect, while the immunostimulatory effects of various modified acidic polysaccharides from the leaves of the genus Pinellia were enhanced, indicating that modification can enhance the in vitro immunostimulatory activity of native acidic polysaccharides from the leaves of the genus Pinellia. Among them, STP-AD showed a significantly enhanced in vitro immunostimulatory effect, suggesting that mild alkaline deesterification can reduce the degree of esterification of acidic polysaccharides from the leaves of the genus Pinellia, thereby enhancing their immunostimulatory activity. STP-E, despite a slight decrease in degree of esterification, molecular weight, and HG fraction, exhibited enhanced immunostimulatory effects, suggesting that enzymatic hydrolysis can enhance the immunostimulatory activity of acidic polysaccharides from the leaves of the genus Pinellia by reducing the HG fraction and increasing the RG-I fraction. However, STP-E had a lower activity than STP-AD, likely due to a significantly lower uronic acid content in STP-E compared to STP-AD, while having a higher degree of esterification. These results further indicate that lower esterification is associated with a stronger immunostimulatory effect of acidic polysaccharides from the leaves of the genus Pinellia. Figure 6 BD showed that the immunostimulatory effects of STP-AH1 and STP-AH2 were significantly enhanced, significantly higher than those of the other groups, and the immunostimulatory effect of STP-AH2 was higher than that of STP-AH1. STP-AH1 removed the arabinose side chain, while STP-AH2 further reduced the length of the galactose side chain on the basis of removing the arabinose side chain. This suggests that acid hydrolysis can reduce the side chain length of the acidic polysaccharide RG-Ⅰ of the leaves of the Artemisia selengensis, exposing more of the main chain and enhancing the immunostimulatory effect. The molecular weight and esterification degree of STP-AH1 and STP-AH2 were significantly reduced, and the uronic acid content was significantly increased, which may also lead to the further enhancement of the immunostimulatory effects of STP-AH1 and STP-AH2. The results showed that the esterification degree, HG and RG-Ⅰ ratio, RG-Ⅰ side chain length, uronic acid and molecular weight of the acidic polysaccharide from the leaves of the ginger tree all affected the in vitro immune effect of the acidic polysaccharide from the leaves of the ginger tree. Among them, the esterification degree, RG-Ⅰ side chain length and HG ratio of the acidic polysaccharide from the leaves of the ginger tree were negatively correlated with its immunostimulatory effect, while the exposure degree of the main chain, uronic acid content and RG-Ⅰ ratio were positively correlated with its immunostimulatory effect. In addition, the side chain length of RG-Ⅰ, i.e. the exposure degree of the main chain and uronic acid content, had a more significant effect on the in vitro immune activity of the acidic polysaccharide from the leaves of the ginger tree.

[0091] 3.2 Potential cellular immune mechanisms of modified acidic polysaccharides from Zingiber officinale

[0092] To further reveal the potential mechanism of macrophage activation mediated by natural and modified S. zingiberensis acidic polysaccharides (STP) (STP-AD, STP-AH1, STP-AH2, and STP-E), we investigated the effects of C29 (a selective intracellular TLR2 signaling inhibitor) and TAK-242 (a selective intracellular TLR4 signaling inhibitor) on the production of NO, TNF-α, and IL-6 in RAW 264.7 cells. Figure 7 As shown in Figures AC, STP, STP-AD, STP-AH1, STP-AH2, and STP-E, untreated with C29 or TAK-242, all significantly promoted the production of NO, TNF-α, and IL-6 by RAW 264.7 cells. However, the addition of C29 (30 μM) or TAK-242 (1 μM) to STP, STP-AD, STP-AH1, STP-AH2, and STP-E significantly inhibited NO, TNF-α, and IL-6 secretion from RAW 264.7 cells. The inhibitory rate with TAK-242 was significantly higher than that with C29. These results confirm that STP, STP-AD, STP-AH1, STP-AH2, and STP-E can activate RAW 264.7 cells and exert immunostimulatory effects by interacting with TLR2 or TLR4 receptors on the surface of macrophages, with TLR4 receptors having a greater impact on the immunogenic effects of modified acidic polysaccharides from the leaves of Artemisia selengensis. In addition, the present invention also determined that the acidic polysaccharide of Zingiber officinale activated RAW264.7 cells to increase the expression levels of NF-κBp65 and NF-κBp-p65. STP-AH1 was selected for determination. Figure 7 As shown in D, STP-AH1 can significantly upregulate the protein expression level of NF-κB p-p65, indicating that STP-AH1 can activate the TLR4 / NF-κB signaling pathway, thereby exerting an immunostimulatory effect.

Claims

1. A modified acidic polysaccharide from Ligusticum chuanxiong, characterized in that: It is a modified acidic polysaccharide prepared from natural Zingiber officinale acidic polysaccharide by trifluoroacetic acid method or pectinase method; The modified acidic polysaccharide from the leaves of Artemisia selengensis contains 89.75 mg – 90.67 mg of total polysaccharides per 100 mg; 23.70 mg – 71.74 mg of total uronic acid; 1.85 mg – 3.16 mg of total protein; 2.06 mg of total bound phenols (GAE) – 4.56 mg of GAE gallic acid equivalents; and esterification degree: 7.74% – 34.98%. Molecular weight M w :0.562× 10 4 Da –1.458× 10 4 Da; dispersion coefficient M w / M n : 1.163 – 1.729; The sugar components of the modified acidic polysaccharide from the leaves of Artemisia selengensis mainly include the following monosaccharides: galacturonic acid, galactose, arabinose, rhamnose, xylose, glucose, glucuronic acid, and mannose. The molar percentages of each monosaccharide are: (21.16% – 70.56%): (17.26% – 33.14%): (0.14% – 27.48%): (5.53% – 6.46%): (1.36% – 6.85%): (1.71% – 2.27%): (2.22% – 2.89%): (0.93% – 1.21%); among them, galacturonic acid polysaccharide HG accounts for 21.94% – 65.02%, type I rhamnogalacturonic acid polysaccharide RG-Ⅰ accounts for 28.47% – 72.37%, and the side chain length is 0.44–10.

31.

2. The modified acidic polysaccharide of Artemisia selengensis according to claim 1, characterized in that: The modified acidic polysaccharide from the leaves of Artemisia selengensis contains 89.75 mg – 90.09 mg of total polysaccharides per 100 mg; 49.57 mg – 71.74 mg of total uronic acid; 3.03 mg – 3.16 mg of total protein; 2.06 mg of total bound phenolics – 2.69 mg of GAE gallic acid equivalents; and esterification degree: 7.74% – 14.54%. Molecular weight M w :0.562× 10 4 Da–1.317× 10 4 Da; dispersion coefficient M w / M n : 1.163 – 1.183; The sugar components of the modified acidic polysaccharide from Artemisia selengensis mainly include the following monosaccharides: galacturonic acid, galactose, arabinose, rhamnose, xylose, glucose, glucuronic acid, and mannose. The molar percentages of each monosaccharide are: (55.89% – 70.56%): (17.26% – 28.99%): (0.14% – 0.47%): (5.53% – 6.46%): (1.36% – 2.40%): (1.71% – 1.78%): (2.22% – 2.89%): (1.12% – 1.21%); among them, galacturonic acid polysaccharide HG accounts for 49.44% – 65.02%, type I rhamnogalacturonic acid polysaccharide RG-Ⅰ accounts for 28.47% – 42.37%, and the side chain length is 0.44–0.

86.

3. The modified acidic polysaccharide of Ligusticum chuanxiong according to claim 1 or 2, characterized in that: The natural Zingiber officinale acidic polysaccharide is prepared by adopting a deep eutectic solvent-assisted heating extraction method; The pectinase is derived from Aspergillus aculeatus.

4. A method for preparing the modified acidic polysaccharide of Zingiber officinale according to any one of claims 1 to 3, characterized in that: It includes the following steps: a. Using deep eutectic solvent to assist in the extraction of natural acidic polysaccharides from the leaves of Zingiber officinale; b. The natural acidic polysaccharide of Zingiber officinale prepared in step a is modified by trifluoroacetic acid method or pectinase method.

5. The method for preparing the modified acidic polysaccharide of Artemisia selengensis according to claim 4, characterized in that: The methods for modifying the acidic polysaccharide in step b are: a. Take 0.5% natural Zingiber officinale acidic polysaccharide w / v 0.5 M trifluoroacetic acid solution was added to make the final concentration of trifluoroacetic acid in the system 0.25 M, and the reaction was carried out at 95 °C for 1.5 h and 4 h, respectively. Subsequently, 1 M sodium hydroxide was added until the pH value was 7.

0. The products were ultrafiltered through an ultrafiltration membrane with a molecular weight limit of 3.0 kDa and freeze-dried to obtain the modified acidic polysaccharide STP-AH1 from the leaves of the Artemisia selengensis with the arabinose side chains removed and the modified acidic polysaccharide STP-AH2 from the leaves of the Artemisia selengensis with the arabinose side chains removed and the galactose side chains reduced, respectively. Among them, the modified acidic polysaccharide STP-AH1 from the leaves of the Artemisia selengensis with the arabinose side chains removed was prepared under the reaction condition of 1.5 h, and the modified acidic polysaccharide STP-AH2 from the leaves of the Artemisia selengensis with the arabinose side chains removed and the galactose side chains reduced was prepared under the reaction condition of 4 h. b. Take 0.5% natural Zingiber officinale acidic polysaccharide w / v 0.3 U / mL pectinase solution was added, reacted at 40°C for 6 h, inactivated, centrifuged, and the supernatant was collected. The supernatant was ultrafiltered through an ultrafiltration membrane with a molecular weight limit of 3.0 kDa and freeze-dried to obtain the modified acidic polysaccharide STP-E from the Artemisia selengensis leaf with a reduced proportion of galacturonic acid polysaccharide.

6. Use of the modified acidic polysaccharide from Ligusticum chuanxiong according to any one of claims 1 to 3 in the preparation of health-care foods that help enhance immunity.

7. Use of the modified acidic polysaccharide of Zingiber officinale according to any one of claims 1 to 3 in the preparation of a medicament having an immunomodulatory effect.

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