Chicory leaf homogeneous polysaccharide, and preparation method and use thereof

Abstract

This invention provides a homogeneous polysaccharide from chicory leaves, derived from the leaves of chicory, a perennial herbaceous plant belonging to the genus *Cicadae* of the Asteraceae family. The invention also provides a method for preparing this homogeneous polysaccharide and its applications. This invention optimizes the extraction parameters of the homogeneous polysaccharide from chicory leaves using a high-pressure assisted eutectic solvent extraction process through single-factor multi-level experiments combined with response surface methodology. Furthermore, it achieves efficient extraction and separation of the homogeneous polysaccharide from chicory leaves by combining fractional alcohol precipitation and membrane separation. The homogeneous polysaccharide from chicory leaves prepared by this invention is a pectin-like acidic polysaccharide rich in galacturonic acid polysaccharides and type I rhamnogalacturonic acid polysaccharides, exhibiting good antioxidant activity, immunostimulatory activity, and anti-glycation activity.

Description

Technical Field

[0001] This invention relates to a homogeneous polysaccharide from chicory leaves, its preparation method, and its uses, belonging to the field of health foods or pharmaceuticals. Background Technology

[0002] Chicory (Cichorium intybus L.) is a perennial herb belonging to the genus Cichorium in the family Asteraceae. It has a clear dual value as both food and medicine in traditional ethnic medicine. According to the *Xinjiang Handbook of Traditional Chinese Medicine*, chicory has effects such as clearing heat, promoting diuresis, choleretic activity, and reducing inflammation. However, current enterprise technologies generate a large number of byproducts during the production and processing of chicory roots. For example, the cultivation of chicory roots produces a large amount of unutilized chicory leaf byproducts (approximately twice the yield of chicory roots). However, due to the lack of key technologies for the high-value utilization of chicory leaf byproducts, these byproducts are usually discarded directly, leading to significant resource waste and environmental pollution. In fact, research has confirmed that chicory leaf byproducts contain pectin-like acidic polysaccharides and polyphenolic active ingredients, both of which have various health benefits. Therefore, chicory leaf byproducts have extremely high development and utilization value.

[0003] Pectin-type acidic polysaccharides are mainly found in plant cell walls, playing a role in maintaining cell wall structure and strength. They are primarily composed of galacturonic acid polysaccharides, type I rhamnogalacturonic acid polysaccharides, type II rhamnogalacturonic acid polysaccharides, and xylose galacturonic acid polysaccharides, as well as multiple branched side chains. Studies have shown that pectin-type acidic polysaccharides possess various biological activities, such as antioxidant, immunomodulatory, anti-inflammatory, anti-tumor, lipid-lowering, and gut microbiota regulation. Therefore, pectin-type acidic polysaccharides have attracted increasing attention in the development of functional foods. However, current extraction techniques for pectin-type acidic polysaccharides still need improvement. Traditionally, hot water extraction is commonly used. Since polysaccharides are highly polar and easily soluble in water, hot water extraction is frequently employed. This method requires relatively simple equipment and procedures, but its disadvantages include long extraction time, high energy consumption, low extraction efficiency, and the lack of selectivity in hot water extraction, resulting in crude polysaccharides with many impurities and low purity (non-homogeneity).

[0004] Deep eutectic solvents (DES) are mixtures composed of hydrogen bond acceptors and hydrogen bond donors that, when mixed in specific proportions, form eutectic mixtures with melting points significantly lower than those of their individual components. These solvents possess unique physicochemical properties, such as high solubility, thermal stability, biodegradability, and ease of synthesis. DES can be used as solvents for polysaccharide extraction due to their non-toxicity or low toxicity, chemical and thermal stability, non-flammability, high solubility, and low melting point. They are also cost-effective, safe, and offer higher extraction rates than traditional extraction solvents. DES extraction exhibits high selectivity, high yield, and high purity of the final product for acidic polysaccharides. Some studies also utilize ultrasound, microwave, or high pressure to assist extraction. High pressure-assisted extraction offers advantages such as rapid heating, short extraction time, and high extraction efficiency. Therefore, high pressure-assisted DES extraction can be used to efficiently prepare highly active homogeneous polysaccharides from chicory leaves.

[0005] Currently, literature reports on chicory polysaccharides mainly focus on research on polysaccharides from chicory roots. Application number 202610147166.9, entitled "An Extraction Method for Chicory Aerial Parts Polysaccharides, Polysaccharides, and Related Applications and Evaluation Methods," discloses an extraction method for chicory aerial parts polysaccharides and related evaluation methods. In this invention, the aerial parts are mainly the stems and leaves of chicory, and the molecular weight of the polysaccharides is 50-800 kDa. Hot water extraction is used, but this method is energy-intensive, lacks selectivity, and takes 4 hours. The extraction method reported in this invention requires a long time, and the polysaccharide molecular weight varies greatly, lacking good homogeneity. Furthermore, the polysaccharides prepared by this method are mainly neutral glucuronidans. Therefore, it is necessary to provide an efficient and selective method for preparing homogeneous polysaccharides from chicory leaves, as well as for their structural characterization and activity evaluation. Summary of the Invention

[0006] This invention provides a homogeneous polysaccharide from chicory leaves, its preparation method, and its uses.

[0007] This invention provides a homogeneous polysaccharide from chicory leaves, which is a homogeneous polysaccharide derived from the leaves of chicory, a perennial herbaceous plant belonging to the genus Chicory in the family Asteraceae; wherein, the homogeneous polysaccharide from chicory leaves contains 91.44 ± 1.32 mg of total polysaccharides per 100 mg.

[0008] The homogeneous polysaccharide contains 44.59 ± 2.35 mg of total uronic acid, 1.53 ± 0.04 mg of total protein, and 1.37 ± 0.12 mg of total conjugated phenols (GAE).

[0009] The degree of esterification of the homogeneous polysaccharide from chicory leaves was 69.89% ± 0.89%, and the molecular weight M was [missing value]. w It is (3.24 ± 0.09) × 10 4 Da, polydispersion coefficient M w / M n It is 1.86 ± 0.14.

[0010] The homogeneous polysaccharide mainly consists of the following monosaccharides: mannose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose, and arabinose. The molar percentages of each monosaccharide are as follows: (0.93% ± 0.41%): (6.34% ± 0.67%): (1.92% ± 0.05%): (49.08% ± 1.81%): (2.58% ± 0.36%): (25.61% ± 1.37%): (13.54% ± 1.02%). Among these, the molar percentage of galacturonic acid polysaccharide is 42.75% ± 1.14%, and the molar percentage of type I rhamnol-galacturonic acid polysaccharide is 51.82% ± 1.86%.

[0011] The present invention also provides a method for preparing the homogeneous polysaccharide from chicory leaves, comprising the following steps:

[0012] a. Take fresh chicory leaves, freeze-dry them, grind them into powder, and sift them;

[0013] b. Add ethanol to the powder and remove the alcohol-soluble components by ultrasonication to obtain the extraction residue;

[0014] c. High-pressure assisted eutectic solvent extraction: Take the extraction residue prepared in step b, add the prepared extraction solvent, and extract under high pressure to obtain the extract;

[0015] d. Add α-amylase and saccharifying enzyme to the extract to remove starch;

[0016] e. Take the supernatant, add ethanol, precipitate with ethanol, and let stand overnight; after centrifugation, obtain the precipitate, and then wash the precipitate with ethanol;

[0017] f. Redissolve in water, separate the membrane, and dry to obtain homogeneous chicory leaf polysaccharide.

[0018] More preferably,

[0019] In step a, the freeze-drying conditions are -30 ℃ to -70 ℃ for 40 to 70 hours; the sieve mesh size is 60 to 80 mesh.

[0020] In step b, the final concentration of ethanol added is 20% to 90% ethanol; the weight-to-volume ratio of powder to ethanol is 1:(10-30) g / mL; the ultrasonic power is 300 W to 800 W; and the ultrasonic time is 20 min to 60 min.

[0021] In step c, high-pressure assisted eutectic solvent extraction is performed for 20-60 minutes, with a solvent-to-raw material ratio of 20 mL / g-60 mL / g. The eutectic solvent is composed of choline chloride, ethylene glycol, and ultrapure water, with a molar ratio of choline chloride to ethylene glycol of 1:3. The eutectic solvent has a water content of 35%-75%, an extraction pressure of 0.1 MPa, and an extraction temperature of 121 °C.

[0022] In step d, the conditions for removing starch by α-amylase and saccharifying enzyme are as follows: α-amylase with 5 U / mL - 20 U / mL, enzymatic hydrolysis temperature of 60℃ - 95℃, and enzymatic hydrolysis time of 3 h - 12 h; saccharifying enzyme with 5 U / mL - 20 U / mL, enzymatic hydrolysis temperature of 40℃ - 65℃, and enzymatic hydrolysis time of 10 h - 14 h.

[0023] In step e, the volume ratio of alcohol precipitation extract to ethanol is 1:(2-5), and the concentration of ethanol used is 50%-100% ethanol; the ethanol used to wash the precipitate is 50%-95% ethanol;

[0024] In step f, the membrane used for membrane separation has a molecular weight cutoff of 1000 Da - 3000 Da.

[0025] More preferably,

[0026] In step a, the product is freeze-dried at -40 °C for 48 h; the sieve is 80 mesh.

[0027] In step b, the ethanol added is 80% ethanol; the weight-to-volume ratio of powder to ethanol is 1:10 g / mL; the ultrasonic power is 480 W; and the ultrasonic time is 30 min.

[0028] In step c, high-pressure assisted eutectic solvent extraction is performed for 40 minutes, with a solvent-to-raw material ratio of 44 mL / g. The eutectic solvent is composed of choline chloride, ethylene glycol, and ultrapure water. The molar ratio of choline chloride to ethylene glycol is 1:3. The eutectic solvent has a water content of 43%, the extraction pressure is 0.1 MPa, and the extraction temperature is 121℃.

[0029] In step d, the amount of α-amylase added was 5 U / mL, the enzymatic hydrolysis temperature was 80 ℃, and the enzymatic hydrolysis time was 12 h; the amount of saccharifying enzyme added was 5 U / mL, the enzymatic hydrolysis temperature was 59 ℃, and the enzymatic hydrolysis time was 12 h.

[0030] In step e, the volume ratio of alcohol used for precipitation is 1:4; the ethanol used is 95% ethanol; the ethanol used for washing the precipitate is 76% ethanol.

[0031] The membrane used for membrane separation in step f has a molecular weight cutoff of 3000 Da.

[0032] This invention utilizes high-pressure assisted eutectic solvent extraction to extract homogeneous polysaccharides from chicory leaves. The extraction rate of homogeneous polysaccharides from chicory leaves using high-pressure assisted eutectic solvent extraction was 9.545% ± 0.210%. The extraction rate of chicory leaf polysaccharides using hot water extraction was 5.99% ± 0.15%. Compared to hot water extraction, the high-pressure assisted eutectic solvent extraction method increased the extraction rate by 59.35%. Furthermore, the optimal extraction time for the high-pressure assisted eutectic solvent method was 40 min, significantly shorter than the 2 h required for hot water extraction, thus improving extraction efficiency.

[0033] The present invention also provides the use of the chicory leaf homogeneous polysaccharide in the preparation of pharmaceuticals or health foods with antioxidant or immunostimulatory activities; or in the preparation of anti-glycation products.

[0034] The present invention also provides a health food or anti-glycation product that helps with anti-oxidation or helps enhance immunity, containing the aforementioned chicory leaf homogeneous polysaccharide.

[0035] The chicory leaf homogeneous polysaccharide described in this invention is a pectin-based acidic polysaccharide with a single symmetrical gel size exclusion chromatographic peak, mainly composed of galacturonic acid polysaccharide and type I rhamnogalacturonic acid polysaccharide.

[0036] The beneficial effects of this invention are:

[0037] 1. This invention employs a eutectic solvent for extraction. Eutectic solvents generally have lower toxicity and are more suitable for food and pharmaceutical extraction compared to traditional organic solvents. Furthermore, the composition and ratio of the eutectic solvent can be adjusted to optimize the extraction conditions for specific polysaccharides. Polysaccharides extracted using eutectic solvents may exhibit higher bioactivity, while water extraction may not maximize the preservation of polysaccharide bioactivity.

[0038] 2. This invention uses high-pressure assisted eutectic solvent extraction to extract homogeneous polysaccharides from chicory leaves. Compared with traditional extraction methods, the high-pressure extraction process is shorter, which can save extraction time and improve work efficiency.

[0039] 3. The molecular weight chromatogram of the chicory leaf homogeneous polysaccharide of the present invention shows a single symmetrical chromatographic peak, which means that the polysaccharide in the sample has high purity, that is, there is mainly a specific polysaccharide molecule, without interference from polysaccharides or impurities of other molecular weights. This helps to ensure its consistency in biological function and application.

[0040] The polysaccharide extract of this invention is a homogeneous polysaccharide with high purity. Furthermore, starch-like substances have been removed. Starch-like substances in polysaccharides can cross-react with other polysaccharides or biomolecules, interfering with the analysis and evaluation of the polysaccharides. Moreover, polysaccharides possess specific biological activities, such as immunomodulatory effects, and the presence of starch may reduce these activities or alter the bioavailability of the polysaccharides. Therefore, removing starch from polysaccharides yields a purer polysaccharide product with more specific functions. Attached Figure Description

[0041] Figure 1 Process flow diagram for high-pressure assisted eutectic solvent extraction of homogeneous polysaccharides from chicory leaves;

[0042] Figure 2 Effects of water content in eutectic solvent (A), extraction time (B), and solid-liquid ratio (C) on the extraction rate of homogeneous polysaccharides from chicory leaves using high-pressure assisted eutectic solvent extraction (Note: Significant differences between different levels are represented by the letter ad (p < 0.05)).

[0043] Figure 3 Three-dimensional stereographic plots (A, B, C) and two-dimensional contour plots (D, E, F) of the response surface to various factors on the extraction rate of homogeneous polysaccharides from chicory leaves.

[0044] Figure 4 Size exclusion chromatogram (A) of homogeneous polysaccharide (CLP) from chicory leaves, high performance liquid chromatogram (B) and Fourier transform infrared spectrum (C) of the constituent monosaccharides (Note: Man, mannose; Rha, rhamnose; GlcA, glucuronic acid; GalA, galacturonic acid; Glc, glucose; Gal, galactose; Xyl, xylose; Ara, arabinose).

[0045] Figure 5 Chicory leaf homogeneous polysaccharide (CLP) 1 H nuclear magnetic resonance spectroscopy and 13 C nuclear magnetic resonance spectrum;

[0046] Figure 6 Scavenging rates of chicory leaf homogeneous polysaccharide (CLP) against ABTS free radicals (A), DPPH free radicals (B), and total reducing power (C).

[0047] Figure 7 Anti-glycation ability of chicory leaf homogeneous polysaccharide (CLP);

[0048] Figure 8Effects of chicory leaf homogeneous polysaccharide (CLP) on RAW 264.7 macrophage cytotoxicity (A), nitric oxide (NO) production (B), tumor necrosis factor-α (TNF-α) production (C) and interleukin-6 (IL-6) production (D) (Note: *significant difference (p < 0.05) and **extremely significant difference (p < 0.01) between the sample and positive control and the blank control group). Detailed Implementation

[0049] Example 1: Extraction of homogeneous polysaccharide (hereinafter referred to as CLP) from chicory leaves using the high-pressure assisted eutectic solvent extraction method of the present invention.

[0050] (1) Ultrasonic removal of alcohol-soluble components: Fresh chicory leaves were freeze-dried, powdered, and passed through an 80-mesh sieve; alcohol-soluble compounds were removed by ultrasonic treatment at a ratio of chicory leaf powder to 80% ethanol of 1:10, with an ultrasonic power of 480 W and an ultrasonic time of 30 min. After ultrasonic treatment, the residue was retained after centrifugation at 5000 × g for 10 min.

[0051] (2) Extraction: Chicory leaf polysaccharides were extracted using a high-pressure assisted eutectic solvent at a material-to-liquid ratio of 1:44 (g / mL) for 40 min. The eutectic solvent consisted of choline chloride, ethylene glycol, and ultrapure water. The molar ratio of choline chloride to ethylene glycol was 1:3, the water content of the eutectic solvent was 43%, the extraction pressure was 0.1 MPa, and the extraction temperature was 121 ℃. After extraction, the mixture was centrifuged at 4000 × g for 10 min, and the supernatant was retained.

[0052] (3) Starch removal: Add 5 U / ml of α-amylase to the obtained chicory leaf polysaccharide extract, 80 ℃, 12 h; add 5 U / ml of saccharifying enzyme, 59 ℃, 12 h.

[0053] (4) Inactivation: 95 ℃ for 30 min; centrifuge at 4000 × g for 10 min and retain the supernatant.

[0054] (5) Alcohol precipitation: Add 4 times the volume of 95% ethanol to the supernatant, and let it stand overnight after alcohol precipitation.

[0055] (6) Reconstitution: Remove the supernatant, retain the precipitate, centrifuge at 4000 × g for 10 min to obtain the precipitate, then wash the precipitate with 76% ethanol; then reconstitute with ultrapure water, take the supernatant and remove small molecules (molecular weight cutoff, 3000 Da) using membrane separation. Freeze-dry to obtain chicory leaf homogeneous polysaccharide (extraction rate 9.545% ± 0.210%). The process flow chart for the preparation of chicory leaf homogeneous polysaccharide is shown below. Figure 1 .

[0056] Comparative Example 1: Preparation of chicory leaf polysaccharides using traditional hot water extraction method

[0057] (1) Ultrasonic removal of alcohol-soluble components: Fresh chicory leaves were freeze-dried, powdered, and passed through an 80-mesh sieve; alcohol-soluble compounds were removed by ultrasonic treatment at a ratio of chicory leaf powder to 80% ethanol of 1:10, with an ultrasonic power of 480 W and an ultrasonic time of 30 min. After ultrasonic treatment, the residue was retained after centrifugation at 5000 × g for 10 min.

[0058] (2) Extraction: The material-to-liquid ratio was 1:30 (w / v) for extraction residue to distilled water, the extraction temperature was 95 ℃, and the extraction time was 2 h. After extraction, the sample was centrifuged at 4000 × g for 10 min and the supernatant was retained.

[0059] (3) Starch removal: Add 5 U / ml of α-amylase to the obtained chicory leaf polysaccharide extract, 80 ℃, 12 h; add 5 U / ml of saccharifying enzyme, 59 ℃, 12 h.

[0060] (4) Inactivation: 95 ℃ for 30 min; centrifuge at 4000 × g for 10 min and retain the supernatant.

[0061] (5) Alcohol precipitation: Add 4 times the volume of 95% ethanol to the supernatant, and let it stand overnight after alcohol precipitation.

[0062] (6) Reconstitution: Remove the supernatant, retain the precipitate, centrifuge at 4000 × g for 10 min to obtain the precipitate, then wash the precipitate with 76% ethanol; then reconstitute with ultrapure water. Take the supernatant and remove small molecules (molecular weight cutoff, 3000 Da) by membrane separation. Freeze-dry to obtain chicory leaf polysaccharide (extraction rate 5.99% ± 0.15%).

[0063] Example 2: Screening experiment of condition parameters for high-pressure assisted eutectic solvent extraction method:

[0064] The prepared extraction solvent (choline chloride: ethylene glycol = 1:3) was added to the extraction residue after ultrasonic-assisted removal of alcohol-soluble components, and the mixture was extracted in a laboratory autoclave. A single-factor experimental design was used to optimize the extraction method, and the conditions are shown in Table 1.

[0065] Table 1 Single-factor experimental design table

[0066]

[0067] Secondly, a three-factor response surface methodology was used to further optimize the effects of various extraction parameters on the extraction rate of homogeneous polysaccharides from chicory leaves. Independent variables included eutectic solvent water content (X1, 35%, 45%, 55%), extraction time (X2, 30 min, 40 min, 50 min), and solid-liquid ratio (X3, 30 mL / g, 40 mL / g, 50 mL / g). Seventeen experiments were designed, and the response surface factor codes are shown in Table 2. The experimental data were then analyzed using Design-Expert. The obtained data were fitted using a second-order polynomial model, as shown in Table 3.

[0068] Table 2. Response Surface Experimental Design and Results

[0069]

[0070] Compared with traditional extraction methods, high-pressure assisted extraction of polysaccharides offers advantages such as high efficiency and energy saving. Therefore, this invention employs high-pressure assisted eutectic solvent extraction to extract homogeneous polysaccharides from chicory leaves. The water content of the eutectic solvent, extraction time, and solid-liquid ratio all significantly affect the extraction efficiency of high-pressure assisted eutectic solvent extraction of homogeneous polysaccharides from chicory leaves. Figure 2 As shown in Figure A, the extraction rate of homogeneous polysaccharides from chicory leaves was highest when the water content of the eutectic solvent increased from 35% to 45%. However, the extraction rate significantly decreased as the water content increased from 45% to 75%. Therefore, the optimal water content of the eutectic solvent obtained through single-factor experiments was 45%. The extraction rate of homogeneous polysaccharides from chicory leaves increased when the extraction time increased from 20 min to 50 min; however, the extraction rate decreased when the extraction time continued to increase from 50 min to 60 min. Therefore, the optimal extraction time obtained through single-factor experiments was 40 min. Figure 2 B). When the material-to-liquid ratio increased from 20 mL / g to 40 mL / g, the extraction rate of homogeneous polysaccharides from chicory leaves gradually increased; however, when the material-to-liquid ratio continued to increase from 40 mL / g to 60 mL / g, the extraction rate of homogeneous polysaccharides from chicory leaves gradually decreased. Figure 3 B). Therefore, the optimal material-to-liquid ratio determined through single-factor experiments is 40 mL / g.

[0071] Based on the results of the single-factor experiments, a Box-Behnken experimental design was further used to optimize the extraction rate of homogeneous polysaccharides from chicory leaves. The final BBD experimental data are shown in Table 3, and the derived second-order polynomial equation is as follows:

[0072] Y=-28.8522+0.83041X1+0.82752X2+0.12762X3-0.0006025X1X2-0.00107X1X3+0.00137X2X3-0.00833025X1 2 -0.0105702X2 2 -0.00160775X3 2

[0073] Where Y represents the extraction rate; X1, X2, and X3 are the water content (%) of the eutectic solvent, the extraction time (min), and the solid-liquid ratio (mL / g), respectively.

[0074] Table 3. Analysis of variance of the regression model for high-pressure assisted eutectic solvent extraction of homogeneous polysaccharides from chicory leaves.

[0075]

[0076] Note: X1: Water content of eutectic solvent (%); X2: Extraction time (min); X3: Solid-liquid ratio (mL / g); R 2 =0.9986, R 2 adj =0.9968, coefficient of variation (CV) =0.48%, adeq. precision =68.5917; *significant difference (p < 0.05), **extremely significant difference (p < 0.01).

[0077] As shown in Table 3, one-way ANOVA was used to evaluate the influence of extraction parameters on the extraction rate of chicory leaves and the validity of the fitted model. Based on the p-value (< 0.0001) and F-value (562.43), the fitted model was highly significant. Furthermore, the values ​​of the lack-of-fit term, coefficient of determination, and corrected coefficient of determination demonstrated that the model had a high good fit. Additionally, the values ​​of the coefficient of variation and moderate precision also demonstrated that the fitted model had good repeatability and reliability, and the p-values ​​of the linear coefficients, interaction coefficients, and quadratic coefficients of all extraction factors were less than 0.05, indicating that all extraction parameters significantly affected the extraction rate of homogeneous polysaccharides from chicory leaves. In fact, the three-dimensional response surface... Figure 3 A, 3B, 3C and two-dimensional contour lines Figure 3Figures D, 3E, and 3F further demonstrate that the interaction between extraction time and the solid-liquid ratio, as well as the interaction between the solid-liquid ratio and the water content of the eutectic solvent, is highly significant. All these data indicate that extraction time, solid-liquid ratio, and eutectic solvent water content are important parameters affecting the extraction of homogeneous polysaccharides from chicory leaves. Based on the analysis of the experimental results, the predicted optimal extraction conditions were determined as follows: extraction time of 40.35 min, solid-liquid ratio of 44.36 mL / g, and eutectic solvent water content of 42.65%. Considering the operability of the actual extraction process, the verification experiment was conducted under the conditions of an extraction time of 40 min, a solid-liquid ratio of 44 mL / g, and eutectic solvent water content of 43%. Under these conditions, the actual extraction rate was 9.545% ± 0.210%, very close to the predicted value of 9.559%.

[0078] Example 3: Chemical structure characterization of the homogeneous polysaccharide from chicory leaves of the present invention.

[0079] First, the total polysaccharide, total uronic acid, total bound phenols, and total protein content of the homogeneous polysaccharide from chicory leaves were determined using a colorimetric method. Subsequently, size exclusion gel chromatography coupled with multi-angle laser light scattering (MLS-RID, Wyatt Technology Co., Santa Barbara, CA, USA) was used to determine the molecular weight and dispersibility of the homogeneous polysaccharide from chicory leaves. High-performance liquid chromatography (HPLC) (L-20A, Shimadzu, Japan) combined with pre-column derivatization using 1-phenyl-3-methyl-5-pyrazolone (PMP) was used to determine the molar percentages of constituent sugars, galacturonic acid polysaccharides, and type I rhamnogalacturonic acid polysaccharides in the polysaccharide samples. Fourier transform infrared spectroscopy (PerkinElmer, Waltham, MA, USA) was used to analyze the functional groups and degree of esterification of the samples. Finally, nuclear magnetic resonance spectroscopy (NMR) was used to analyze the glycosidic bond types of the polysaccharides. For specific methodological references: Authors: Ding-Tao Wu, Meng-Xi Fu, Huan Guo, Yi-Chen Hu, Xiao-Qin Zheng, Ren-YouGan and Liang Zou; Title: Microwave-assisted deep eutectic solvent extraction, structural characteristics, and biological functions of polysaccharides from sweet tea (Lithocarpus litseifolius) leaves; Journal: ANTIOXIDANTS, Volume 11, Issue 8; DOI: 10.3390 / antiox11081578.

[0080] Table 4. Chemical composition, molecular weight, and monosaccharide composition of chicory leaf homogeneous polysaccharide (CLP)

[0081]

[0082] HG (%) = GalA (%) - Rha (%); RG-I (%) = 2 × Rha (%) + Gal (%) + Ara (%)

[0083] Chicory leaf homogeneous polysaccharide (CLP) was obtained by high-pressure assisted eutectic solvent extraction, combined with fractional alcohol precipitation and membrane separation. Its chemical composition was determined. Each 100 mg of CLP contained 91.44 ± 1.32 mg of total polysaccharides, 44.59 ± 2.35 mg of total uronic acids, 1.53 ± 0.04 mg of total protein, and 1.37 ± 0.12 mg of total conjugated phenols (GAE).

[0084] To further understand the structural characteristics of CLP, the molecular weight, functional groups, monosaccharide composition, and glycosidic bonds of CLP were studied using high-performance size exclusion chromatography, high-performance liquid chromatography, Fourier transform infrared spectroscopy, and nuclear magnetic resonance spectroscopy.

[0085] like Figure 4 As shown in Figure A, the molecular weight of CLP was determined by high-performance size exclusion chromatography (HPLC). The CLP molecular weight chromatogram shows a single symmetrical peak, indicating that the polysaccharide in the sample has high purity, meaning that a specific polysaccharide molecule is predominantly present, without interference from other polysaccharides or impurities. The molecular weight M of CLP is... w It is (3.24 ± 0.09) × 10 4 Da, polydispersion coefficient M w / M n It is 1.86 ± 0.14.

[0086] like Figure 4 As shown in B, the main sugars in CLP are the following monosaccharides: mannose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose, and arabinose. The molar percentages of each monosaccharide are: (0.93% ± 0.41%): (6.34% ± 0.67%): (1.92% ± 0.05%): (49.08% ± 1.81%): (2.58% ± 0.36%): (25.61% ± 1.37%): (13.54% ± 1.02%). Among them, the molar percentage of galacturonic acid polysaccharide is 42.75% ± 1.14%, and the molar percentage of type I rhamnol-galacturonic acid polysaccharide is 51.82% ± 1.86%. The above results indicate that the chicory leaf homogeneous polysaccharide CLP obtained by high-pressure assisted eutectic solvent extraction is a pectin-like acidic polysaccharide rich in galacturonic acid polysaccharide and type I rhamnogalacturonic acid polysaccharide.

[0087] like Figure 4 As shown in Figure C, CLP exhibits typical signals of pectin-like acidic polysaccharides. In short, the FT-IR spectrum of CLP is in the range of 4000-400 cm⁻¹. -1 The wavelength range shows typical absorption peak characteristics of pectin-type acidic polysaccharides. At 3437 cm⁻¹... -1 and 2938 cm-1 The broad peaks at 1748 cm⁻¹ are caused by the stretching vibrations of the hydroxyl group and the asymmetric stretching vibrations of the CH bond, respectively. -1 and 1616 cm -1 The absorption band at 1427 cm⁻¹ is caused by the stretching vibrations of the esterified groups C=O and -COOR, confirming that CLP is mainly composed of acidic polysaccharides. Meanwhile, at 1427 cm⁻¹... -1 and 1245.5 cm -1 The absorption peaks at 1096 cm⁻¹ are attributed to CH / OH and -OCH₃, respectively. -1 and 1017 cm -1 The absorption peak at 1748 cm⁻¹ is attributed to the absorption of pyranoside. Furthermore, based on the absorption peak at 1748 cm⁻¹... -1 and 1616 cm -1 Based on the signal intensity, the degree of esterification of CLP was calculated to be 69.89% ± 0.89%.

[0088] To better understand the structural characteristics of CLP, 1D NMR analysis was performed. Figure 5As shown, both α and β configurations exist in CLP. The signals at 1.30 ppm, 5.25 ppm, and 16.67 ppm belong to H-6, H-1, and C-6 of 1,2,4-α-L-Rhap; the signals at 1.25 ppm and 5.33 ppm belong to H-6 and H-1 of 1,2-α-L-Rhap; the signals at 4.97 ppm, 100.35 ppm, and 170.68 ppm belong to H-1, C-1, and C-6 of 1,4-α-D-GalAMep; and the signals at 5.03 ppm, 99.49 ppm, and 173.32 ppm belong to H-1, C-1, and C-6 of 1,4-α-D-GalAp. The signals at 5.09 ppm and 107.36 ppm are correlated with the H-1 and C-1 of 1,5-α-L-Araf; the signals at 5.12 ppm and 109.18 ppm are correlated with the H-1 and C-1 of 1,3,5-α-L-Araf; the signals at 5.15 ppm and 109.18 ppm are correlated with the H-1 and C-1 of T-α-L-Araf; the signals at 5.18 ppm and 107.11 ppm are correlated with the H-1 and C-1 of 1,3-α-L-Araf; the signals at 4.47 ppm and 103.38 ppm belong to the H-1 and C-1 of 1,3,6-β-D-Galp; the signals at 4.54 ppm and 103.09 ppm belong to the H-1 and C-1 of 1,3-β-D-Galp; and the signals at 4.62 ppm... H-1 and C-1 signals of 1,4-β-D-Galp were observed at ppm and 104.22 ppm; signals at 3.81 ppm and 52.83 ppm originated from the methoxy group of GalA (GalA-OCH3); signals at 2.08 ppm, 2.18 ppm, 20.53 ppm, and 20.14 ppm represented the O-acetyl group of GalAp. In summary, the chemical structural characterization results confirm that the chicory leaf homogeneous polysaccharide CLP is mainly composed of galacturonic acid polysaccharide and type I rhamnogalacturonic acid polysaccharide, with galactan or arabinogalactan serving as side chains of type I rhamnogalacturonic acid polysaccharide.

[0089] The following efficacy tests demonstrate the beneficial effects of the present invention.

[0090] Experimental Example 1: Antioxidant Activity Test of the Homogeneous Polysaccharide CLP from Chicory Leaves of the Present Invention

[0091] 1. Determination of ABTS free radical scavenging ability:

[0092] Reagents: 7 mM ABTS solution, 2.45 mM potassium persulfate solution.

[0093] Methods: The ABTS radical cation solution was prepared by mixing 7 mM ABTS solution and 2.45 mM potassium persulfate aqueous solution at a 1:1 volume ratio and reacting in the dark for at least 16 hours at room temperature. The ABTS radical cation solution was further diluted with phosphate buffer (0.05 M, pH 6.6) to achieve an absorbance of 0.750 ± 0.02 at 734 nm. Then, in a 96-well microplate, 200 µL of the ABTS radical cation working solution was mixed with 20 µL of samples of different concentrations and shaken in the dark for 6 min, and the absorbance was measured at 734 nm. Vitamin C (Vc) was used as a positive control.

[0094] Results: Experiments were conducted with different concentrations of CLP (1.0 mg / mL, 2.0 mg / mL, 3.0 mg / mL, 4.0 mg / mL, and 5.0 mg / mL), and the IC50 of CLP for scavenging ABTS free radicals was calculated. 50 The value was calculated, with vitamin C used as a positive control. For example... Figure 6 As shown in Figure A, CLP exhibits significant ABTS radical scavenging activity, and its effect is dose-dependent. The IC50 of CLP's ABTS radical scavenging ability is... 50 The value was 2.03 ± 0.05 mg / mL.

[0095] 2. Determination of DPPH free radical scavenging capacity:

[0096] Reagents: 0.35 mM DPPH solution, anhydrous ethanol, 50% ethanol.

[0097] Methods: The DPPH working solution was prepared from 0.35 mM DPPH stock solution. Take 5 mL of DPPH stock solution and add 5 mL of water (the OD value after mixing should be around 0.8-0.9). Dilute with 50% ethanol, preparing fresh before use. Dilute 2-fold. In a 96-well microplate, add 200 μL of 0.35 mM DPPH directly to the plate, along with 25 μL of sample / water (blank). Incubate at 37 ℃ in the dark for 30 min, and measure the OD at 517 nm. The positive control is BHT (prepared with anhydrous ethanol).

[0098] Results: Experiments were conducted using different concentrations of CLP (1.0 mg / mL, 2.0 mg / mL, 3.0 mg / mL, 4.0 mg / mL, and 5.0 mg / mL), and the IC50 of CLP for scavenging DPPH free radicals was calculated. 50 The value was determined, with BHT used as a positive control. For example... Figure 6 As shown in Figure B, CLP exhibits some activity in scavenging DPPH free radicals, and its effect is dose-dependent. The IC50 of CLP's DPPH free radical scavenging ability is... 50The value was 7.85 ± 0.38 mg / mL.

[0099] 3. Total reducing power determination:

[0100] Reagents: 1% potassium ferricyanide (prepared with 0.2M PBS solution, pH 6.6), 10% trichloroacetic acid, 0.1% ferric chloride

[0101] Methods: 100 µL of samples with different concentrations (1.0 mg / mL, 2.0 mg / mL, 3.0 mg / mL, 4.0 mg / mL, 5.0 mg / mL) were mixed with 100 µL of 1% potassium ferricyanide and shaken well. The mixture was then incubated in a water bath at 50 °C for 20 min. Then, 100 µL of 10% trichloroacetic acid, 300 µL of distilled water, and 60 µL of 0.1% ferric chloride were added sequentially, and the mixture was incubated in a water bath at 50 °C for 30 min. Finally, 200 µL of the reaction solution was placed in a 96-well microplate, and the absorbance was measured at 700 nm. Vitamin C (Vc) was used as a positive control.

[0102] Results: This experiment reflects the reducing power by measuring the degree of color change; the darker the color and the larger the OD value, the stronger the reducing power. Figure 6 As shown in C, the total reducing power of CLP increases with increasing concentration. When the CLP concentration is 5.0 mg / mL, its OD value reaches 1.42 ± 0.01.

[0103] Experimental Example 2: Anti-glycation activity test of the homogeneous polysaccharide CLP from chicory leaves of the present invention.

[0104] Reagents: Bovine serum albumin (BSA), anhydrous glucose, disodium hydrogen phosphate (Na2HPO4), sodium dihydrogen phosphate (NaH2PO4), sodium azide, aminoguanidine.

[0105] Methods: pH 7.4 PBS was prepared by mixing 100 mL of 7.164 g Na2HPO4 and 50 mL of 1.5605 g Na2HPO4 at a volume ratio of 81:19. A-PBS was prepared by dissolving 2.7024 g anhydrous glucose in 30 mL of pH 7.4, 0.2 mol / L PBS. B-PBS was prepared by dissolving 0.75 g BSA in 25 mL of PBS. A-PBS was prepared by dissolving 9.008 g glucose in 100 mL of water and then dissolving it in PBS. Then, in 96-well microplates, 200 μL of A-PBS and 200 μL of B-PBS were mixed, and different concentrations of polysaccharide samples or aminoguanidine positive controls were added. The mixture was incubated at 37 ℃ for 14 days. A blank control (CK) of 200 μL A-PBS + 200 μL B-PBS was used, with three CKs performed at the beginning and end of the reaction. After the reaction was completed, the fluorescence intensity was measured on a fluorescence microplate reader.

[0106] Results: Experiments were conducted using different concentrations of CLP (0.25 mg / mL, 0.50 mg / mL, 1.00 mg / mL, 2.00 mg / mL, and 4.00 mg / mL), and the IC50 was calculated. 50 The value was determined, with aminoguanidine (AG) used as a positive control. Figure 7 As shown, CLP exhibits certain anti-glycation activity, and the IC50 of CLP's anti-glycation activity is... 50 The value was 0.41 ± 0.02 mg / mL.

[0107] Experimental Example 3: Immunostimulatory Effect of the Homogeneous Polysaccharide CLP from Chicory Leaves of the Present Invention

[0108] Reagents: DMEM medium, thiazolyl blue (MTT), dimethyl sulfoxide (DMSO), lipopolysaccharide (LPS), ELISA kit

[0109] 1. Cytotoxicity assay:

[0110] Methods: RAW 264.7 macrophages were cultured at a concentration of 5 × 10⁻⁶ cells / mL. 3 Cells were cultured overnight in 96-well microplates at a concentration of [number] cells / well. The supernatant was aspirated, and 100 µL of CLP polysaccharide (12.5, 25, 50, 100 µg / mL) was added to each well. The plates were incubated for 24 h. A culture medium blank served as a control, and LPS (1 µg / mL) served as a positive control. The supernatant was aspirated, and 100 µL of LMTT solution (1 mg / mL) was added to each well. The plates were incubated for 4 h. The supernatant was aspirated, and 100 µL of DMSO was added to each well. OD was measured at 570 nm.

[0111] Result: As Figure 8 As shown in Figure A, CLP showed no toxicity to RAW 264.7 macrophages at concentrations ranging from 12.5 to 100 µg / mL.

[0112] 2. NO and cytokine content assay:

[0113] Methods: RAW264.7 cells were cultured at a rate of 1 × 10⁻⁶. 5 Cells were cultured overnight in 24-well plates at a concentration of [number] cells / well. The supernatant was aspirated, and 1 mL of CLP polysaccharide (12.5, 25, 50 µg / mL) was added to each well. The plates were incubated for 48 h. A culture medium blank served as a control, and LPS (1 µg / mL) served as a positive control. Subsequently, the supernatant from each well was collected, and the levels of NO and cytokines (TNF-α and IL-6) were measured according to the kit instructions.

[0114] Result: As Figure 8 As shown in B, 8C, and 8D, CLP significantly stimulated the release of NO, TNF-α, and IL-6 from RAW264.7 macrophages at concentrations ranging from 12.5 μg / mL to 50 μg / mL, with the increase being concentration-dependent, indicating that CLP has significant cellular immune-stimulating activity.

[0115] In summary, this invention optimizes the method for extracting homogeneous polysaccharide CLP from chicory leaves. The CLP extracted using this method exhibits good antioxidant activity, immunostimulatory activity, and anti-glycation ability, providing a theoretical basis for its development into functional foods.

Claims

1. A homogeneous polysaccharide from chicory leaves, characterized in that: It is a homogeneous polysaccharide derived from the leaves of Cichorium intybus L., a perennial herbaceous plant belonging to the genus Cichorium in the family Asteraceae; among which, the total polysaccharide in the homogeneous polysaccharide of chicory leaves contains 91.44 ± 1.32 mg of total polysaccharides per 100 mg.

2. The chicory leaf homogeneous polysaccharide according to claim 1, characterized in that: The homogeneous polysaccharide contains, per 100 mg, total uronic acid: 44.59 ± 2.35 mg; total protein: 1.53 ± 0.04 mg; and total conjugated phenols: 1.37 ± 0.12 mg GAE (gallic acid equivalent).

3. The chicory leaf homogeneous polysaccharide according to claim 1 or 2, characterized in that: The degree of esterification of the homogeneous polysaccharide was 69.89% ± 0.89%, and the molecular weight M was [missing value]. w It is (3.24 ± 0.09) × 10 4 Da, polydispersion coefficient M w / M n It is 1.86 ± 0.

14.

4. The chicory leaf homogeneous polysaccharide according to any one of claims 1-3, characterized in that: The homogeneous polysaccharide mainly comprises the following monosaccharides: mannose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose, and arabinose. The molar percentages of each monosaccharide are: (0.93% ± 0.41%): (6.34% ± 0.67%): (1.92% ± 0.05%): (49.08% ± 1.81%): (2.58% ± 0.36%): (25.61% ± 1.37%): (13.54% ± 1.02%). Among these, the molar percentage of galacturonic acid polysaccharide is 42.75% ± 1.14%, and the molar percentage of type I rhamnol-galacturonic acid polysaccharide is 51.82% ± 1.86%.

5. The chicory leaf homogeneous polysaccharide according to any one of claims 1-4, characterized in that: High-pressure assisted eutectic solvent extraction was used to extract homogeneous polysaccharides from chicory leaves.

6. A method for preparing a homogeneous polysaccharide from chicory leaves according to any one of claims 1-5, characterized in that: It includes the following steps: a. Take fresh chicory leaves, freeze-dry them, grind them into powder, and sift them; b. Add ethanol to the powder and remove the alcohol-soluble components by ultrasonication to obtain the extraction residue; c. High-pressure assisted eutectic solvent extraction: Take the extraction residue prepared in step b, add the prepared extraction solvent, and extract under high pressure to obtain the extract; d. Add α-amylase and saccharifying enzyme to the extract to remove starch; e. Take the supernatant, add ethanol, precipitate with ethanol, and let stand overnight; after centrifugation, obtain the precipitate, and then wash the precipitate with ethanol; f. Redissolve in water, separate the membrane, and dry to obtain homogeneous chicory leaf polysaccharide.

7. The method for preparing homogeneous polysaccharide from chicory leaves according to claim 6, characterized in that: In step a, the freeze-drying conditions are -30℃ to -70℃ for 40 to 70 hours; the sieve mesh size is 60 to 80 mesh. In step b, the final concentration of ethanol added is 20% to 90% ethanol; the weight-to-volume ratio of powder to ethanol is 1:(10-30) g / mL; the ultrasonic power is 300 W to 800 W; and the ultrasonic time is 20 min to 60 min. In step c, high-pressure assisted eutectic solvent extraction is performed for 20 min to 60 min, with the extraction solvent to raw material ratio being 20 mL / g to 60 mL / g. The eutectic solvent is composed of choline chloride, ethylene glycol, and ultrapure water. The molar ratio of choline chloride to ethylene glycol is 1:3, the water content of the eutectic solvent is 35% to 75%, the extraction pressure is 0.1 MPa, and the extraction temperature is 121 ℃. In step d, the conditions for removing starch by α-amylase and saccharifying enzyme are as follows: α-amylase with 5 U / mL - 20 U / mL, enzymatic hydrolysis temperature of 60℃ - 95℃, and enzymatic hydrolysis time of 3 h - 12 h; saccharifying enzyme with 5 U / mL - 20 U / mL, enzymatic hydrolysis temperature of 40℃ - 65℃, and enzymatic hydrolysis time of 10 h - 14 h. In step e, the volume ratio of alcohol precipitation extract to ethanol is 1:(2-5), and the concentration of ethanol used is 50%-100% ethanol; the ethanol used to wash the precipitate is 50%-95% ethanol; In step f, the membrane used for membrane separation has a molecular weight cutoff of 1000 Da - 3000 Da.

8. The method for preparing homogeneous polysaccharide from chicory leaves according to claim 7, characterized in that: In step a, the product is freeze-dried at -40 °C for 48 h; the sieve is 80 mesh. In step b, the ethanol added is 80% ethanol; the weight-to-volume ratio of powder to ethanol is 1:10 g / mL; the ultrasonic power is 480 W; and the ultrasonic time is 30 min. In step c, high-pressure assisted eutectic solvent extraction is performed for 40 minutes, with a solvent-to-raw material ratio of 44 mL / g. The eutectic solvent consists of choline chloride, ethylene glycol, and ultrapure water. The molar ratio of choline chloride to ethylene glycol is 1:

3. The eutectic solvent has a water content of 43%, the extraction pressure is 0.1 MPa, and the extraction temperature is 121℃. In step d, the amount of α-amylase added was 5 U / mL, the enzymatic hydrolysis temperature was 80 ℃, and the enzymatic hydrolysis time was 12 h; the amount of saccharifying enzyme added was 5 U / mL, the enzymatic hydrolysis temperature was 59 ℃, and the enzymatic hydrolysis time was 12 h. In step e, the volume ratio of alcohol used for precipitation is 1:4; the ethanol used is 95% ethanol; the ethanol used for washing the precipitate is 76% ethanol. The membrane used for membrane separation in step f has a molecular weight cutoff of 3000 Da.

9. Use of the chicory leaf homogeneous polysaccharide according to any one of claims 1-5 in pharmaceuticals or health foods having antioxidant or immunostimulatory activity; or in the preparation of anti-glycation products.

10. A health food or anti-glycation product that helps with antioxidation or boosts immunity, characterized in that: It contains the chicory leaf homogeneous polysaccharide as described in any one of claims 1-5.

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