Arabinan obtained from fructus lycii and application of araban

Abstract

The invention discloses araban obtained from fructus lycii and application of the araban. According to the molecular structure of the araban, a main chain is formed by repeated-> 5)-alpha-Araf-(1-> structural units, and the C-2 position of each sugar residue is substituted by T-alpha-Araf-(1-> structural units. According to the method, free protein is removed through a water extraction and alcohol precipitation method and an alkali solution method, graded alcohol precipitation and purification are combined, and the araban component only containing arabinose is extracted and separated from the fructus lycii for the first time. Compared with an arabinogalactan component, the arabinogalactan component has the advantages that the phagocytic ability of macrophages is remarkably improved, the macrophages are induced to secrete NO, the activity of acid phosphatase is improved, the phagocytic ability of the macrophages and the activity of the acid phosphatase can be activated in an LPS-induced inflammation model, NO secretion is inhibited, and the arabinogalactan component can be used for preparing an LPS-induced inflammation model. Therefore, an anti-inflammatory effect is generated, and a better bidirectional immunoregulation effect is achieved.

Description

Technical Field

[0001] This application relates to a polysaccharide extracted from wolfberry fruits and its applications. Background Art

[0002] The immune system is a key defense mechanism for maintaining host physiological homeostasis. When pathogens invade the host body, the activation of immune cells is crucial for defending against pathogens. However, the abnormal activation of immune cells and the excessive production of pro-inflammatory cytokines may lead to host tissue damage and trigger an inflammatory response. Under physiological conditions, macrophages play a central role in innate immunity. They eliminate pathogenic microorganisms, including fungal, bacterial, and virus-infected cells, through phagocytosis, and interact with T lymphocytes as antigen-presenting cells, thereby activating the adaptive immune response. However, under pathological conditions, the over-activation of macrophages may lead to the release of a large amount of pro-inflammatory cytokines, such as interleukin (ILs), tumor necrosis factor (TNF-α), etc. These cytokines can attract more immune cells to participate in the immune response. As immune cells are further activated, the amount of cytokines they produce increases, forming a negative feedback loop that exacerbates the inflammatory response. This excessive inflammatory response not only attacks pathogens but may also damage normal tissues and even induce autoimmune diseases, such as rheumatoid arthritis, systemic lupus erythematosus, etc. Therefore, regulating the balance between immune response and inflammation is crucial for maintaining host health, and the development of natural drugs with dual immunomodulatory effects is of great significance for the treatment of related diseases.

[0003] Wolfberry fruits are the dried ripe fruits of Lycium barbarum L. or Lycium chinense Mill. of the genus Lycium (Lycium L.) in the Solanaceae family. Wolfberry fruits have been used as food and medicine for more than 4,000 years, and among them, Lycium barbarum L. is the only variety included in the Chinese Pharmacopoeia (2010 Edition). "Shennong Ben Cao Jing" records that "long-term consumption of wolfberry can strengthen the bones and muscles, withstand cold and heat, and make the body light and not old, and it is the top grade among traditional Chinese medicines." In East Asia, Europe, and North America, wolfberry fruits have been widely used as functional foods or dietary supplements. More than 200 different active ingredients have been identified from wolfberry fruits, including polysaccharides, polyphenols, carotenoids (zeaxanthin and carotene), vitamins, alkaloids, etc. Polysaccharides are one of the most important active ingredients in wolfberry fruits, with immunomodulatory, hypoglycemic, hypolipidemic, neuroprotective, anti-tumor, antioxidant, and anti-aging biological activities, playing a key role in the functions of wolfberry, especially in terms of immunomodulatory ability. Nie Wei et al. found that the wolfberry polysaccharide LBP can reduce the levels of NA and 5-HT in the spleen and hypothalamus, and both NA and 5-HT can activate B cells to enhance the body's immune function. Zhang Xiaorui et al. showed that the arabinogalactan component LBPF4-OL in wolfberry fruits can significantly stimulate the proliferation of macrophages in BALB / c mice at a concentration of 10 μg / mL. Summary of the Invention

[0004] This application proposes an arabinan obtained from wolfberry fruits and its applications. For the first time, an arabinan component containing only arabinose was obtained from wolfberry fruits. The structure of its refined product was characterized, and through the RAW264.7 cell model and in vitro cell tests, it was found that it has effects such as two-way immunomodulation. The specific results are as follows:

[0005] An arabinan obtained from wolfberry fruits, whose molecular structure consists of a main chain composed of repeating →5)-α-Araf-(1→ structural units, and the C-2 position of each sugar residue is substituted by T-α-Araf-(1→. The molecular structure is:

[0006]

[0007] The average molecular weight of the above-mentioned arabinan is 22.78 kDa.

[0008] The preparation method of the above-mentioned arabinan includes:

[0009] Using wolfberry fruits as raw materials, extracting wolfberry polysaccharides by water extraction and alcohol precipitation, and combining with the alkali solution method to remove free proteins to obtain crude polysaccharides, denoted as ALBP;

[0010] Performing fractional alcohol precipitation of ALBP with different concentrations of ethanol. Among them, separating and purifying the supernatant collected by alcohol precipitation at an ethanol concentration of 70% to obtain an arabinogalactan component, denoted as ALBP-70; separating and purifying the supernatant collected by alcohol precipitation at an ethanol concentration of 80% to obtain an arabinan component, denoted as ALBP-80;

[0011] Further purifying ALBP-80 by molecular sieve gel chromatography to obtain a refined product, denoted as ALBGP-80.

[0012] The specific steps can be:

[0013] (1) After water extraction of wolfberry fruits, the residue is re-extracted with water, the supernatant is combined, concentrated under reduced pressure, precipitated with alcohol, solid-liquid separated, and the precipitate is collected;

[0014] (2) After the precipitate is redissolved in water, NaOH is added, stirred, the pH value of the system is adjusted to 7.0 with hydrochloric acid, centrifuged, and the obtained supernatant is dialyzed and freeze-dried to obtain crude polysaccharides;

[0015] (3) Prepare the crude polysaccharide into a 1% aqueous solution of crude polysaccharide, add anhydrous ethanol until the volume percentage concentration of ethanol is 30%, perform alcohol precipitation, and centrifuge to obtain the first supernatant. Add anhydrous ethanol to the first supernatant until the volume percentage concentration of ethanol is 50%, perform alcohol precipitation, and centrifuge to obtain the second supernatant. Add anhydrous ethanol to the second supernatant until the volume percentage concentration of ethanol is 70%, perform alcohol precipitation, and centrifuge to obtain the third supernatant. Add anhydrous ethanol to the third supernatant until the volume percentage concentration of ethanol is 80%, perform alcohol precipitation, and centrifuge to obtain the fourth supernatant;

[0016] (4) Separate and purify the fourth supernatant using a polyacrylamide gel chromatography column to obtain arabinan ALBP-80 with an average molecular weight of about 15 kDa; then further purify it by Bio-gel-P100 gel column chromatography to obtain the refined product ALBGP-80 with a molecular weight of about 22.78 kDa.

[0017] Application of the above arabinan in the preparation of foods or drugs with dual immunomodulatory functions. In specific applications, as long as the food / drug contains substances with the above arabinan molecular structure, for example, ALBP-80 can be specifically used, or the purer ALBGP-80 can also be used.

[0018] Among them, the "food" mentioned here should be understood in a broad sense: the above arabinan can be made into capsules, tablets or other forms of dietary supplements for direct consumption by consumers; the above arabinan can be added to existing food products, such as yogurt, energy bars, fruit juices, etc., as a functional ingredient to enhance the nutritional value of the product; it can be used as a health food for specific populations (such as the elderly, athletes or those with low immunity); the above arabinan can also be used as a new food additive to improve the texture, stability or other properties of the food while providing additional health benefits. Of course, when applying the above arabinan to food, it is necessary to follow the food safety standards and regulations of the country or region.

[0019] An agent for inducing macrophages to secrete NO, whose active ingredient includes the above arabinan.

[0020] An agent for enhancing the activity of acid phosphatase, whose active ingredient includes the above arabinan.

[0021] An anti-inflammatory agent, whose active ingredient includes the above arabinan.

[0022] It should be noted that the above-mentioned arabinan can induce macrophages to secrete NO, enhance the activity of acid phosphatase, and has anti-inflammatory activity. These are not only the reasons for its ability to achieve bidirectional immune regulation, but also there are corresponding commercially available drugs for these three specific effects. Therefore, they also have patentable product uses respectively. For example:

[0023] Drugs for inducing macrophages to secrete NO already include LPS (lipopolysaccharide), Poly(I:C) (polyinosinic acid-polycytidylic acid), IFN-γ (interferon γ), etc.

[0024] Drugs for enhancing the activity of acid phosphatase already include LPS (lipopolysaccharide), PMA (phorbol ester), IFN-γ (interferon γ), etc.

[0025] Drugs with anti-inflammatory activity already include Dexamethasone, Aspirin, Ibuprofen, Resveratrol, etc.

[0026] This application has the following beneficial effects:

[0027] In this application, free proteins are removed by water extraction and alcohol precipitation, and the alkali solution method, and then combined with fractional alcohol precipitation for purification. For the first time, an arabinan component is extracted and isolated from wolfberries. The arabinan component (ALBP-80) and the arabinogalactan component (ALBP-70) obtained from the previous-stage alcohol precipitation are compared through the RAW264.7 cell model and in vitro cell experiments. It is found that compared with the arabinogalactan component, the arabinan component not only significantly improves the phagocytic ability of macrophages, induces macrophages to secrete NO, enhances the activity of acid phosphatase, but also can activate the phagocytic ability and acid phosphatase activity of macrophages and inhibit the secretion of NO in the inflammation model induced by LPS, thereby producing an anti-inflammatory effect. Therefore, the arabinan of this application has a better bidirectional immune regulation effect.

[0028] The preparation method of the arabinan component provided by this application is simple in operation, high in efficiency, and basically free of damage to the sugar component, and is suitable for large-scale production.

[0029] This application also provides a scientific basis for the in-depth development and utilization of wolfberry resources and health products. Description of the Drawings

[0030] Figure 1 It is the extraction and preparation flow chart of wolfberry arabinan ALBGP-80.

[0031] Figure 2 It is the molecular weight spectrum of crude polysaccharide ALBP, arabinogalactan ALBP-70, and arabinan ALBP-80.

[0032] Figure 3 They are the gas chromatograms of crude polysaccharide ALBP, arabinogalactan ALBP-70, and arabinan ALBP-80.

[0033] Figure 4 It is the elution diagram of ALBP-80 through the gel chromatography column Bio-gel-P100.

[0034] Figure 5 It is the molecular map of ALBGP-80.

[0035] Figure 6 It is the gas chromatogram of ALBGP-80.

[0036] Figure 7 It is the GC-MS map of ALBGP-80.

[0037] Figure 8 It is the two-dimensional NMR map of ALBGP-80. In the figure, (A) 1 H- 1 H COSY map; (B) HSQC map; (C) HMBC map.

[0038] Figure 9 They are the effects of ALBP-70 and ALBP-80 components on the cell viability of RAW264.7 mouse macrophages. Compared with the blank group, *p < 0.05.

[0039] Figure 10 They are the statistical charts of the comparison of the immunological activities of ALBP-70 and ALBP-80; among them: (A) cell phagocytosis ability; (B) cell acid phosphatase activity; (C) cell NO secretion; the experimental results are expressed as mean ± standard deviation (n = 5); different letters indicate significant differences (P < 0.05), and the same letters indicate no significant differences (P < 0.05).

[0040] Figure 11 They are the statistical charts of the comparison of the in vitro anti-inflammatory activities of ALBP-70 and ALBP-80; among them: (A) cell phagocytosis ability; (B) cell acid phosphatase activity; (C) cell NO secretion; the experimental results are expressed as mean ± standard deviation (n = 5); different letters indicate significant differences (P < 0.05), and the same letters indicate no significant differences (P < 0.05).

[0041] Figure 12 Establishment of an inflammation model of mouse macrophages RAW264.7 induced by lipopolysaccharide; the experimental results are expressed as mean ± standard deviation (n = 5); compared with the blank group, *p < 0.05, **p < 0.01, ***p < 0.001. Specific implementation manners

[0042] The present application will be further described in detail below in conjunction with the accompanying drawings and embodiments.

[0043] Arabinan exists in the primary cell walls of plants, especially in seeds, fruits and roots, and has various biological activities, including gastrointestinal protection, immunomodulation, anticoagulation and anti-inflammatory effects. The main chain of arabinan is usually 1,5-α-Araf, and branching may occur at the C-3 or C-2 position, especially at the C-3 position. These branches are mainly composed of T-α-Araf and →3)-α-Araf-(1→. It has been found that arabinans from almond seeds, sugar beet root segments, Atractylodes macrocephala and Ephedra sinica have branches at the C-3 position, and the branches of the first three plants are only composed of α-L-Araf, and the branches of the latter also contain short side chains of →3)-α-Araf-(1→. The arabinan from Althaea officinalis has branches at both the C-3 and C-2 positions, and the degree of substitution of arabinan from Caesalpinia crista seeds at the C-2 position is higher than that at the C-3 position, but the degree of branching of these arabinans is generally less than 50%.

[0044] In the embodiment of the present application, Lycium barbarum is used as the raw material, and Lycium barbarum polysaccharide is extracted by water extraction and alcohol precipitation, and combined with an alkali method to remove proteins to obtain a crude polysaccharide component ALBP. Subsequently, the crude polysaccharide component ALBP is subjected to fractional alcohol precipitation, and an arabinan component containing only arabinose is obtained from Lycium barbarum for the first time. The structure of the arabinan component is analyzed by chemical methods (monosaccharide composition, methylation analysis) and spectroscopic techniques (GC, GC-MS, NMR). It is a highly branched arabinan structure, the main chain is composed of →5)-α-Araf-(1→, and the C-2 of each arabinose residue is replaced by α-Araf-(1→. The specific process is as follows:

[0045] I. Materials and Methods

[0046] 1. Materials

[0047] Wolfberry "Ningqi No. 1" is derived from the farm in Yinchuan City, Ningxia. Monosaccharide standards (L-rhamanose, L-fucose, D-arabinose, D-xylose, D-mannose, D-glucose, D-galactose, D-glucuronic acid, D-galacturonic acid), dextran standards with different molecular weights (5, 8, 12, 25, 50, 80, 150, 270, 670 kDa), heavy water (D2O), and DMSO are from Sigma Company (St. Louis, Mo, USA). Sephadex G-100 is purchased from Pharmacia, Sweden. MTT is purchased from Sangon Biotech (Shanghai, China). RAW263.7 cells are purchased from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences. BCA protein assay kit is purchased from Beyotime Institute of Biotechnology. Other reagents are all of analytical purity.

[0048] 2. Extraction, Isolation and Purification of Arabinan ALBP-80

[0049] In the previous stage of the laboratory, it was found that the main chain of the arabinogalactan complex extracted from wolfberry is composed of →6)-β-Galp-(1→ repeating units. Therefore, the released sugar chain will not undergo peeling degradation in alkaline solution. At the same time, the alkaline solution can also denature proteins and remove free proteins. We applied this idea to the extraction of wolfberry arabinan. Using wolfberry as the raw material, crude polysaccharide (ALBP) was obtained through hot water extraction, alcohol precipitation, removal of free proteins with alkaline solution, and dialysis. The crude polysaccharide (ALBP) was fractionated and precipitated with ethanol at different concentrations to obtain arabinogalactan fraction (ALBP-70) and arabinan fraction (ALBP-80). The arabinan fraction (ALBP-80) was further purified by molecular sieve gel chromatography to obtain refined arabinan (ALBGP-80). Specifically:

[0050] The preparation of ALBGP80 fraction is as Figure 1As shown below. First, weigh 1000 g of wolfberries and dry them in an oven for 2 hours. Mix them with double-distilled water at a ratio of 1 g:5 mL (w / v), homogenize and pulverize, and stir and extract at 80 °C for 2 hours. Subsequently, centrifuge (6000 rpm, 20 minutes), collect the supernatant, and re-extract the residue with water at a ratio of 1 g:2.5 mL (w / v), stir and extract at 80 °C for 2 hours, combine the supernatants from the two extractions, and concentrate under reduced pressure using a rotary evaporator. The volume after concentration under reduced pressure is 20% of the volume before concentration to obtain the water extract of wolfberries. Add 4 volumes of absolute ethanol to the concentrated solution, perform alcohol precipitation overnight at 4 °C, centrifuge again (6000 rpm, 20 minutes), and obtain the precipitate part. Dissolve the precipitate in water to 3 L, add 0.1 M NaOH solution, react at 55 °C for 10 hours, neutralize to pH 7 with HCl after the reaction, and then centrifuge (6000 rpm, 20 minutes) to collect the supernatant. Use a dialysis bag to desalt (the cut-off molecular weight of the dialysis bag is 3000 Da, dialysis time is 3 days), and finally freeze-dry the sample to obtain the crude polysaccharide ALBP of wolfberries.

[0051] Prepare a 1% solution of ALBP with water, slowly add 30% (v / v) ethanol, centrifuge (10000 rpm, 10 min) after precipitation for 12 h, and take the supernatant. Subsequently, slowly add ethanol to the supernatant to a concentration of 50% (v / v), precipitate again for 12 h and then centrifuge (10000 rpm, 10 min). For the collected supernatant, slowly add ethanol to a concentration of 70% (v / v), repeat the above steps to obtain the arabinogalactan component ALBP-70. Continue to slowly add ethanol to the collected supernatant to a concentration of 80% (v / v), perform the same operation to obtain the component ALBP-80.

[0052] Collect the component ALBP-80, separate and purify it through a Bio-gel P100 polyacrylamide gel chromatography column (2.0×1.5 m), and the elution solvent is 0.1 M NaCl to obtain the homogeneous arabinan component ALBGP-80.

[0053] 3. Physicochemical Property Analysis of Polysaccharide Components

[0054] 3.1 Molecular Weight Determination

[0055] According to the method of Gong et al., high-performance gel permeation chromatography (HPGPC) was used to determine the homogeneity and average molecular weight of the sample. The liquid chromatograph was a Shimadzu high-performance liquid chromatograph (Shimadzu2010A), equipped with a differential refractive index detector RID-20A and a gel chromatography column TSKGEL G4000PWXL (7.8 mm×30 cm). The determination process is as follows:

[0056] The molecular weight and homogeneity of each component were determined by high performance gel permeation chromatography (HPGPC). Preparation of dextran standards and samples. Weigh 2 mg of dextran standards (5 kDa, 12 kDa, 25 kDa, 50 kDa, 80 kDa, 150 kDa, 270 kDa, and 670 kDa series) and samples of each component of LBPs, dissolve them in 2 mL of 100 mM ammonium acetate buffer solution, and filter through a 0.22 μm aqueous filter membrane for further measurement. Chromatographic column: TSK-Gel G4000 PWxL dextran gel chromatography column (7.8 mm ID x 30.0 cm), detector: refractive index detector, mobile phase: 100 mM ammonium acetate buffer solution (pH = 6.0), flow rate: 0.3 mL / min, injection volume: 10 μL. Using the retention time of dextran standards as the abscissa and the logarithm of the standard molecular weight LgMw as the ordinate, a standard curve was plotted. Substitute the retention time of the sample to be measured into the standard regression equation, and the molecular weight of each component was obtained by calculation.

[0057] 3.2 Determination of sugar content and protein content

[0058] The total sugar content of each component was determined by the phenol-sulfuric acid method using glucose as the standard. The protein content of each component was determined by the BCA method using bovine serum albumin as the standard.

[0059] The determination process is as follows:

[0060] Since soluble sugars (such as hexoses and pentoses) are rapidly dehydrated under the action of concentrated sulfuric acid to form furfural or hydroxymethylfurfural, and then react with phenol to form an orange-red compound, which has the maximum absorption at 490 nm (or 480 nm), and the color depth is proportional to the sugar content within a certain range. Based on this principle, we used the phenol-sulfuric acid method to determine the sugar content of ALBGP-80 and each component. Accurately weigh 50 mg of glucose standard, and make up the volume to 500 ml in a volumetric flask with water. Respectively pipette 0, 0.2, 0.4, 0.6, 0.7, 0.8, 1.0, 1.2, 1.4, 1.6 m into glass test tubes, and make up to 2 mL with double-distilled water. Set 3 parallels for each concentration. Then add 50 μL of 80% (v / v) phenol solution and 2.5 mL of concentrated sulfuric acid to each test tube in turn, shake well, and react at room temperature for 20 min. Use a UV spectrophotometer to detect the absorbance at 490 nm, plot the standard curve, and get Y = 2.3075X + 0.0936, R 2 = 0.9919, and calculate the total sugar content of the sample. Accurately weigh 5 mg of LBGP-80 and each component sample, dissolve them in a 50 mL volumetric flask, and pipette 0.1 mL of each sample solution into a glass test tube, and make up to 2 mL with double-distilled water. Repeat the above steps, and substitute the absorbance value into the standard curve to calculate the sugar content of each component.

[0061] Under alkaline conditions, the peptide bond in the protein can reduce Cu 2+ to Cu + . Cu + forms a purple-blue complex with the BCA reagent. This complex has a maximum absorption value at 562 nm and is proportional to the protein concentration within a certain range. Taking the standard protein concentration as the abscissa and the absorbance of bovine serum albumin (BSA) as the ordinate, a standard curve is plotted to obtain Y = 0.873X + 0.0879, R 2 = 0.9998, and the concentration of the protein to be measured is calculated.

[0062] 3.3 Monosaccharide composition analysis

[0063] The Jacob Lehrfeld method was used to determine the monosaccharide composition of each component. A Shimadzu GC2010 instrument equipped with a capillary column RTX-50 (30.0 m × 0.25 mm × 0.25 mm) was used, and the temperature program was referred to Qiang Peng et al.

[0064] 4. Methylation analysis

[0065] Based on the Needs method with modifications. Dissolve 5.0 mg of the polysaccharide sample in DMSO, add dry NaOH powder and CH3I, stir and react for 2 h, and terminate the reaction with distilled water. Repeat several times until methylation is complete. Each completely methylated sample is hydrolyzed with formic acid and TFA (2M), reduced with NaBH4, and acetylated to generate partially methylated alditol acetate derivatives. The resulting methylated alditol acetate derivatives were analyzed using a Shimadzu GCMS-QP2010 system equipped with an rtx-5ms capillary column (RTX-5MS, 30.0 m × 0.25 mm × 0.25 m).

[0066] 5. Nuclear magnetic resonance analysis

[0067] Dissolve 60 mg of the polysaccharide sample in 2 mL of D2O (99.9%), mix well, freeze-dry, and repeat the above steps 3 times to completely replace the hydrogen atoms with deuterium. Then dissolve the freeze-dried sample in 0.6 mL of D2O and place it in an NMR tube, and then use a Varian Unity Inova 600 MHz NMR spectrometer to collect 1 1H spectrum, 13 13C spectrum, 2D NMR ( 1 1H- 1 COSY, 1 1H- 1 1H TOCSY, 1 1H- 1 1HNOESY, 1 1H- 13 13C HSQC and1 H- 13 The HMBC spectrum was analyzed. The NMR data were processed by Mestrenove 14.0 software.

[0068] 6. ALBP-80 Immunoreactivity Study

[0069] 6.1 Cell Culture and Cell Viability Assay

[0070] RAW264.7 cells were seeded in 96-well plates at a density of 1×10 5 cells / mL. After culturing for 12 hours, the supernatant was removed, and samples (ALBP70 and ALBP80) at different concentrations (50 - 800 μg / mL) were added, with 6 parallel groups set for each well. A blank control group and a positive control group were set up. The blank control group was DMEM medium without serum, and the positive control group was LPS (10 μg / mL). After culturing at 37 °C for 24 hours, the supernatant was discarded, and the MTT method was used to determine cell viability to finally determine the optimal concentration of each component for subsequent experiments.

[0071] 6.2 Determination of Neutral Red Phagocytosis Ability

[0072] The method reported by Weeks et al. was used to detect the ability of the sample to phagocytose neutral red by macrophages. The cells were treated according to the above method (Section 6.1). A 0.1% neutral red solution was added to each well of the 96-well plate and incubated for 4 h. Then, 100 μL of cell lysate was added to each well, and the absorbance of each well was measured at 540 nm using an enzyme-linked immunosorbent assay (ELISA) reader. The phagocytosis ability of the cells was calculated as follows: phagocytosis index = Abs(sample) / Abs(control) × 100%.

[0073] 6.3 Determination of NO Secretion

[0074] The secretion of nitric oxide is related to immunity. The cells were treated according to the above method (Section 6.1). The Griess reagent method was used to detect the level of NO in the culture medium. After culturing the cells in 96-well plates for 24 h, the cells were treated with samples at different concentrations for 24 h. Then, the supernatant (100 μL) was transferred to a new 96-well plate and mixed with an equal volume of Griess reagent (50 μL of 1% sulfanilamide in 5% phosphoric acid and 50 μL of 0.1% N-1-naphthylethylenediamine dihydrochloride in distilled water) in the dark at room temperature for 10 min. The absorbance was measured at 540 nm using an ELISA reader. Using NaNO2 as the standard, the linear range was 0 - 100 μM. With the NO secretion (μmol / L) as the abscissa X and the absorbance value (A) as the ordinate Y, a standard curve of NO content was plotted, and the NO secretion was calculated based on the absorbance of the cell supernatant.

[0075] 6.4 Determination of Acid Phosphatase Activity

[0076] Acid phosphatase is a marker enzyme in lysosomes, and the level of it can indirectly reflect the immune regulation of the body. Cells were treated according to the above method (Section 6.1), and the acid phosphatase activity of the samples on RAW264.7 cells was detected by the method reported by Ke et al. 25 μL of 1% Triton X-100 and 150 μL of p-nitrophenyl phosphate solution (1 mg / mL) were added to each well to culture the cells for 1 h, and then 3.0 M NaOH solution was added to each well to terminate the reaction.

[0077] 7. Anti-inflammatory activity study of ALBP-80

[0078] RAW264.7 cells were seeded in 96-well plates at a density of 1×10 5 cells / mL. After culturing for 12 h, the supernatant was removed. After pretreating the cells with lipopolysaccharide (LPS) at different concentrations for 24 h, the cell viability was determined by the MTT method to determine the effective concentration that could cause an inflammatory response. Subsequently, after RAW264.7 cells were cultured for 12 h, the supernatant was removed, and the cells were cultured with samples (ALBP70 and ALBP80) at different concentrations (50 - 800 μg / mL) for 24 h, and then incubated with lipopolysaccharide culture solution for 24 h. The cell phagocytosis ability, NO secretion amount, and acid phosphatase activity were determined according to the method in Section 2.6.2 - 4.

[0079] II. Results and analysis

[0080] 1. Preparation of arabinogalactan ALBGP-80

[0081] Using wolfberry as raw material, crude polysaccharide (ALBP) was obtained through hot water extraction, alcohol precipitation, removal of free protein with alkali solution, and dialysis, with a yield of 1.2%. The total sugar content of ALBP was determined to be 47.4%, and the protein content was determined to be 21.5%. The crude polysaccharide was fractionated by alcohol precipitation with different concentrations of ethanol to obtain arabinogalactan component (ALBP-70) and arabinan component (ALBP-80), with yields of 0.8% and 0.1% respectively, and the total sugar contents were determined to be 62.2% and 52.5% respectively; the protein contents were determined to be 10.3% and 8.6% respectively.

[0082] 2. Molecular weight determination

[0083] The average molecular weights of ALBP, ALBP-70, and ALBP-80 were determined by high performance gel permeation chromatography (HPGPC), and the results are as Figure 2As shown in the figure. By using the retention time of dextran standards on the chromatographic column and the logarithm of the molecular weight, a linear regression equation was plotted to calculate the molecular weights of ALBP, ALBP-70, and ALBP-80. The results showed that the average molecular weights of ALBP were 67 kDa and 15 kDa, the average molecular weights of ALBP-70 were 68 kDa and 21 kDa, and the average molecular weight of ALBP-80 was 15 kDa. The peak at 37.5 minutes was the solvent peak.

[0084] 3. Determination of monosaccharide composition

[0085] The results of monosaccharide analysis are as Figure 3 shown. The main monosaccharide components of the crude polysaccharide ALBP were Ara, Glc, and Gal, with molar percentages of 51.3:8.2:40.5, respectively. The crude polysaccharide was fractionated by alcohol precipitation to obtain ALBP-70 and ALBP-80 fractions. Further calculation of the relative molar percentages of the monosaccharide compositions of each fraction showed that ALBP-70 was mainly composed of arabinose (79.4%) and galactose (20.6%), while ALBP-80 was mainly composed of arabinose (93%).

[0086] 4. Purification of arabinan

[0087] The arabinan fraction ALBP-80 was purified by Bio-gel-P100 gel column chromatography, and the eluent was 0.1 M NaCl to further purify the fraction, and finally ALBGP-80 ( Figure 4 ) was obtained with a yield of 0.08%. The high-performance liquid chromatography (HPGPC) showed that ALBGP-80 was a single symmetric peak, indicating that ALBGP-80 was a homogeneous polysaccharide fraction ( Figure 5 ). Subsequently, monosaccharide analysis found that it was mainly composed of arabinose (97%).

[0088] The molecular structure is as follows:

[0089]

[0090] 5. Methylation analysis of ALBGP-80

[0091] To further analyze the glycosidic bond types and linkage modes of the polysaccharide, a complete methylation analysis of ALBGP-80 was performed. According to the retention times of the characteristic peaks in the total ion chromatogram and their characteristic fragment ion mass spectra in GC-MS, the types of methylated glycosyls could be determined. As Figure 7As shown, ALBGP-80 has two types of glycosidic bonds, namely T-Araf-(1→ and →2,5)-Araf-(1→. According to the GC peak areas corresponding to the glycosidic bonds and the corresponding response factors, their molar fractions were calculated, and the results are shown in Table 1. The number of branched sugar residues and terminal sugar residues in ALBGP-80 is basically equal, indicating that the methylation reaction is complete. The methylation analysis results show that ALBGP-80 contains a large number of T-Araf-(1→ terminal sugar residues, accounting for 49.75% of the arabinose residues, indicating that the structure of ALBGP-80 is highly branched.

[0092] Table 1 Methylation analysis of ALBGP-80

[0093]

[0094] 6. NMR analysis

[0095] Nuclear magnetic resonance spectroscopy is a method that can effectively determine the configuration of anomeric carbons in sugar residues and the type of glycosidic bonds in the structure of polysaccharides. The 1 chemical shift values in the 1H spectrum of ALBGP-80 are 5.20 ppm and 5.24 ppm, named A and B respectively. According to the chemical shifts of anomeric carbon hydrogens in the NMR spectra in the literature and the methylation analysis results, the relevant chemical shifts were assigned. Among them, both A and B belong to α sugar residues. Combining the types of sugar residues analyzed by methylation, the chemical shifts of A and B were respectively assigned to α-Ara, which are the signals generated by the sugar residues T-α-Araf-(1→ and →2,5)-α-Araf-(1→ respectively. 13 The anomeric carbon chemical shifts of sugar residues in the 13C spectrum generally concentrate between δ 90 - 110. According to the methylation analysis and the anomeric carbon hydrogen chemical shift table in relevant NMR literature, 109.37 ppm and 107.55 ppm can be assigned to α-Ara. The signal at 170.01 ppm may be due to the presence of acetyl groups in the sample.

[0096] All 1 1H and 13 13C signal chemical shifts were assigned by homonuclear correlation spectroscopy (COSY), total correlation spectroscopy (TOCSY), TNNOESY spectrum, and (HMBC) spectrum respectively. Since the chemical shifts of non-anomeric protons in the sugar ring generally overlap, it is necessary to analyze with the proton shift correlation maps of each sugar residue. The chemical shift values of non-anomeric proton signals can be assigned by two-dimensional COSY ( Figure 8 A). The anomeric signals of the six residues A and B in the two-dimensional HSQC spectrum are δ 5.20 / 109.37 ppm and δ 5.24 / 107.55 ppm respectively ( Figure 8B). Based on the methylation analysis and the relevant literature on the NMR of the arabinan structure, it can be inferred that these two sugar residues are T-α-L-Araf(A), →2,5)-α-L-Araf-(1→(B) respectively. The chemical shift values of the H and C spectra of all sugar residues are shown in Table 2. Two-dimensional HMBC and NOESY can infer the connection modes and sequences of different sugar residues. Through the cross-peaks of the HMBC spectrum, information on the anomeric carbon of the sugar residue and the proton of the sugar residue connected to it, as well as the anomeric proton of the sugar residue and the carbon of the sugar residue connected to it, can be obtained. For example, (δ84.05 / δ5.20) indicates that C-1 of residue A is connected to O-2 of residue B, and (δ4.65 / δ84.03) indicates that C-5 of residue B is connected to O-1 of the adjacent residue B. Figure 8 C).

[0097] In summary, based on the monosaccharide composition analysis, methylation analysis, and one-dimensional and two-dimensional NMR spectra, the possible repeating structure of ALBGP-80 can be speculated. The main chain is mainly composed of →5)-α-Araf-(1→, and it is a multi-branched arabinan structure in which α-Araf-(1→ replaces at C-2.

[0098] Table 2 13 C NMR and 1 HNMR chemical shifts of ALBGP-80

[0099]

[0100] Since ALBGP-80 is obtained by purifying ALBP-80, from the determination results of the monosaccharide composition, both ALBP-80 and ALBGP-80 are mainly composed of arabinan ( Figure 3 and Figure 6 ). Arabinan accounts for 93% in ALBP-80 and 97% in ALBGP-80. In the determination of molecular weight, it is found that their molecular weights are also similar, about 15 - 30 kDa. Therefore, it can be determined that their physicochemical properties are nearly the same. If ALBP80 has biological activity, it is mainly ALBGP80 that plays the main activity.

[0101] 7. Immunological activity studies of ALBP-70 and ALBP-80

[0102] 7.1 Effects on the viability of RAW264.7 cells

[0103] Natural plant polysaccharides can act as immunomodulators. After binding to immune regulation-related receptors, they activate immune cells to secrete cytokines, thereby playing an immunomodulatory role. RAW264.7 cells are a type of mononuclear macrophage extracted from mice, which participate in the body's immune regulation and are a commonly used model for studying cellular immunity. In previous laboratory studies, it was found that the arabinogalactan component extracted from wolfberry has activities such as immunomodulation, alleviating colitis, anti-aging, and inhibiting cancer cell proliferation, and it is the main active ingredient of wolfberry. Therefore, using wolfberry arabinogalactan as a positive control, this study investigated the immunomodulatory activity of the arabinogalactan component first extracted from wolfberry for the first time.

[0104] To study the toxic and side effects of each component on RAW264.7 cells, the MTT method was used to determine the effect of each component on cell viability. As Figure 9 shown, it was found that compared with the blank control group, when the sample concentration was above 400 μg / mL, it significantly promoted cell proliferation, while when the concentration was below 200 μg / mL, the sample had no effect on the number of RAW264.7 cells. Therefore, polysaccharides with a concentration lower than 200 μg / mL were used for further research.

[0105] 7.2 Effect on the phagocytic function of RAW264.7 cells

[0106] The phagocytosis of macrophages is one of the most important non-specific immune responses of the body. When macrophages are biologically stimulated, they phagocytose and digest apoptotic cells, cell debris, or pathogens, and the strength of phagocytic ability can reflect their immunomodulatory ability. As Figure 10 shown in A, compared with the blank control, both arabinogalactan (ALBP-70) and arabinan (ALBP-80) promoted the phagocytic ability of RAW264.7 cells. When the concentration was 200 μg / mL, ALBP-70 had a significant effect on promoting its viability, while ALBP-80 had a significant effect at a lower concentration of 50 μg / mL, and the viability indices were 1.12 and 1.40 (p < 0.05), respectively, and ALBP-80 was close to the positive control group at 1.44 (p < 0.05).

[0107] 7.3 Effect on the activity of acid phosphatase

[0108] Acid phosphatase is a signal enzyme that activates macrophages, and the strength of acid phosphatase activity can reflect the activation strength of macrophages. As Figure 10As shown in Figure B, compared with the blank control group, each component could increase the acid phosphatase activity. Compared with ALBP-70, which had a significant effect at 50 μg / mL and an extremely significant promoting effect between 100 - 200 μg / mL, and the concentration had a dose-dependence. While ALBP-80 had an extremely significant effect at a low concentration of 50 μg / mL, approaching that of the positive control group at 1.99 (p < 0.05). This indicated that arabinan was superior to arabinogalactan in promoting acid phosphatase activity.

[0109] 7.4 Effects on the production of NO by RAW264.7 cells

[0110] The Griess reagent method can be used to determine the content of NO secreted by RAW264.7 cells, which can indirectly reflect the immunomodulatory activity of macrophages. As Figure 10 shown in Figure C, compared with the blank control, both arabinogalactan (ALBP-70) and arabinan (ALBP-80) could stimulate RAW264.7 cells to secrete NO. Among them, the amount of NO secreted by cells in the ALBP-80 group showed a decreasing trend, while the amount of NO secreted by cells in the ALBP-70 group gradually increased with the increase of concentration (p < 0.05). When the concentration was 200 μg / mL, the NO secretion of ALBP-70 was the highest, at 1.16 μmol / L, while when the concentration was 50 μg / mL, the NO secretion of ALBP-80 was the highest, at 1.22 μmol / L, approaching the positive control (1.22 μmol / L). Therefore, the ability of ALBP-80 to promote NO secretion was significantly stronger than that of ALBP-70 (p < 0.05).

[0111] 8. In vitro anti-inflammatory activity study of ALBP-70 and ALBP-80

[0112] Inflammation is the initial response of the body's immune system to stimuli such as injury, infection, and stress, and belongs to a complex biological process.

[0113] First, lipopolysaccharide (LPS) was used to induce the activation of mouse RAW264.7 macrophages to establish an in vitro inflammation model. As Figure 12 shown, when the concentration of LPS was 20 μg / mL, the cell inhibition rate reached 43%, so 20 μg / mL was selected to induce its inflammatory response. In Figure 10 Figure A, both ALBP-70 and ALBP-80 significantly enhanced the phagocytic ability of macrophages. However, in the LPS-induced macrophage inflammatory response, compared with the ALBP-70 component, all concentrations of ALBP-80 could significantly increase the activity of acid phosphatase with strong phagocytic ability, and the effect was the best when the concentration was 50 μg / mL and was close to the blank group, indicating that ALBP-80 could resist the inflammatory response by activating the function of macrophages ( Figure 11 Figure A - B).

[0114] NO is a biomarker of inflammation and inflammatory diseases and plays a key role in the inflammatory response of macrophages. LPS stimulation can significantly increase the secretion of NO in macrophages, and excessive NO can lead to the programmed death of macrophages. Figure 11 In group C, the NO secretion in the model group reached more than twice that of normal cells (P < 0.05). After treatment with ALBP-70 and ALBP-80, it was found that the NO secretion decreased significantly (P > 0.05), indicating that ALBP-70 and ALBP-80 have an inhibitory effect on LPS-induced NO production. Among them, ALBP-80 at a concentration of 50 μg / mL had the best inhibitory effect, approaching that of the blank control group. In summary, at low concentrations, arabinan (ALBP-80) not only has significant immunomodulatory activity but also has good anti-inflammatory activity, and its anti-inflammatory activity is stronger than that of arabinogalactan (ALBP-70) (p < 0.05), indicating that arabinan (ALBP-80) has a dual immunomodulatory effect.

[0115] In the above study, we found that Lycium barbarum arabinan also has an enhanced immunomodulatory effect. Its optimal concentration is 50 μg / mL, and its phagocytosis index (1.4), acid phosphatase activity (1.99), and nitric oxide secretion (1.22 μmol / L) are all significantly higher than those at other concentrations. Moreover, as the concentration increases, its enhanced immune activity decreases, which may be related to multiple binding sites or multiple targets of polysaccharides on cells.

[0116] Based on the above research, it can be considered that arabinan can be applied to the preparation of foods or drugs with dual immunomodulatory functions. In specific applications, as long as the food / drug contains substances with the above arabinan molecular structure, for example, ALBP-80 can be specifically used, or purer ALBGP-80 can also be used.

[0117] The ability of this arabinan to induce macrophages to secrete NO, enhance the activity of acid phosphatase, and have anti-inflammatory activity is not only the reason for its dual immunomodulatory effect but also, based on these three specific effects themselves, corresponding medicaments for inducing macrophages to secrete NO, medicaments for enhancing the activity of acid phosphatase, and anti-inflammatory medicaments can be prepared.

Claims

1. An arabinan obtained from wolfberry, characterized in that: Its molecular structure consists of repeated →5)-α-Araf-(1→ structural units forming the main chain, and the C-2 position of each sugar residue is replaced by T-α-Araf-(1→. The molecular structure is as follows:

2. The arabinan according to claim 1, characterized in that The molecular weight of the arabinan is 15 kDa to 30 kDa.

3. The arabinan according to claim 2, characterized in that The preparation method of the arabinan comprises: Lycium barbarum was used as raw material, and Lycium barbarum polysaccharide was extracted by water extraction and alcohol precipitation, and free protein was removed by alkaline solution method to obtain crude polysaccharide, which was recorded as ALBP. ALBP was subjected to graded alcohol precipitation with ethanol of different concentrations, wherein the supernatant collected from the alcohol precipitation at an ethanol concentration of 70% was separated and purified to obtain an arabinogalactan component, which was recorded as ALBP-70; the supernatant collected from the alcohol precipitation at an ethanol concentration of 80% was separated and purified to obtain an arabinogalactan component, which was recorded as ALBP-80; ALBP-80 was further purified by molecular sieve gel chromatography to obtain a refined product, which was designated as ALBGP-80.

4. Use of the arabinan according to claim 1 in the preparation of food or medicine with bidirectional immunomodulatory function.

5. A medicament for inducing macrophages to secrete NO, wherein the active ingredient of the medicament comprises the arabinan according to claim 1.

6. A medicament for enhancing the activity of acid phosphatase, wherein the active ingredient of the medicament comprises the arabinan according to claim 1.

7. An anti-inflammatory agent, the active ingredient of which comprises the arabinan according to claim 1.

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