Trametes gallica polysaccharide, its preparation method and application in preparing immunomodulatory drugs

Abstract

The present invention discloses a preparation method of Inonotus hispidus polysaccharide IHP-1aa: It is obtained by removing fat with ethanol, extracting with water and precipitating with ethanol, then removing proteins by the Sevage method, separating neutral polysaccharide components through a DEAE-52 cellulose column and a DEAE agarose gel column, and finally purifying with Sephadex G-100. The Inonotus hispidus polysaccharide has uniform purity and clear composition, and is a heteropolysaccharide composed of glucose, galactose, mannose, fucose and methylgalactose, with a molecular weight of 26.9 kDa. In vitro experiments show that it has no adverse effects on normal cells, can significantly stimulate macrophages RAW264.7 to secrete NO, IL-6 and TNF-α, has good immunomodulatory activity, and can be used for the development of immunomodulatory products.

Description

Technical Field

[0001] The present invention belongs to the technical field of polysaccharides, and particularly relates to a hispid polypore polysaccharide IHP-1aa with immunomodulatory activity, a preparation method thereof, and an application thereof in the preparation of immunomodulatory drugs. Background Art

[0002] Polysaccharides are another important type of macromolecules in organisms in addition to proteins and nucleic acids, and are widely present in animals, plants, and microorganisms. In recent years, polysaccharides have attracted wide attention due to their good physiological activities and the characteristics of being safe and non-toxic, and fungal polysaccharides, as broad-spectrum immune activators, have gradually become a research hotspot. The biological activities of polysaccharides are closely related to their structural characteristics, including molecular weight, monosaccharide and glycosidic bond composition, degree of branching, and chain conformation, etc.

[0003] Inonotus hispidus is a saprophytic fungus with high medicinal and edible value, and has been found parasitizing on various broad-leaved trees in China and Europe, including mulberry trees, ash trees, poplars, elm trees, mountain pears, and maple trees. In Aksu region of Xinjiang, China and Xiajin County of Shandong Province, the Inonotus hispidus on mulberry trees is also used as the traditional Chinese medicine "Sanghuang" for treating indigestion, cancer, diabetes, and stomach diseases, etc. Modern research has found that the extracts of Inonotus hispidus have good anti-tumor, antioxidant, antibacterial, antiviral, and neuroprotective activities, etc. Among them, polysaccharides are one of the main active components, but there has been no research on the structure and immunomodulatory activity of Inonotus hispidus polysaccharides. Therefore, separating, purifying, and preparing the active polysaccharides in Inonotus hispidus, characterizing their structures, and studying their efficacy and uses are of great significance for the development and utilization of Inonotus hispidus resources. Summary of the Invention

[0004] The purpose of the present invention is to overcome the defects of the prior art and provide a hispid polypore polysaccharide with immunomodulatory activity. The prepared polysaccharide HP-1aa has uniform purity and clear structure. In vitro experiments show that it can significantly stimulate macrophages RAW264.7 to secrete NO, IL-6, and TNF-α, and has good immunomodulatory activity, and can be used for the development and utilization of immune activity products.

[0005] The present invention also provides a preparation method of the above-mentioned hispid polypore polysaccharide IHP-1aa and its application in the preparation of immunomodulatory drugs.

[0006] To achieve the above purpose, the present invention adopts the following technical solutions:

[0007] A preparation method of a hispid polypore polysaccharide, which comprises the following steps:

[0008] 1) Defatting treatment of raw materials: Chopping the dried fruiting bodies of Inonotus hispidus, and then soaking them in an ethanol aqueous solution for defatting to obtain a crude extract of fruiting bodies;

[0009] 2) Water extraction and ethanol precipitation: Add distilled water to the crude extract of the fruiting body according to the solid-liquid ratio of 1 g: 8 - 30 ml, heat to 70 - 90 °C for extraction for 2 - 5 h, concentrate the extract, then add ethanol until the final ethanol concentration is 75%, let it stand for ethanol precipitation, collect the precipitate, and dry it in the shade;

[0010] 3) Protein removal: Use the Sevage method to remove protein, perform ethanol precipitation again, collect the precipitate, and freeze-dry to obtain the crude polysaccharide of Inonotus hispidus after removing protein;

[0011] 4) Isolation and purification: The crude polysaccharide of Inonotus hispidus after removing protein is separated and eluted successively through DEAE cellucose-52 and DEAE-Sepharose Fast Flow ion exchange chromatography columns, dialyzed, and freeze-dried. Finally, it is purified and eluted using a Sephadex G-100 chromatography column, the eluate is collected, concentrated under reduced pressure, dialyzed, and freeze-dried to obtain the polysaccharide of Inonotus hispidus, named IHP-1aa.

[0012] Specifically, in step 1), the volume concentration of the ethanol aqueous solution is 70 - 80%, and the soaking time is 60 - 84 h.

[0013] Specifically, in step 2), let it stand for ethanol precipitation at 2 - 6 °C for 20 - 28 h.

[0014] Specifically, in step 4), the separation and elution through the DEAE cellucose-52 ion exchange chromatography column are as follows: Dissolve the crude polysaccharide of Inonotus hispidus after removing protein in distilled water to prepare a 15 - 25 mg / ml solution, centrifuge, take the supernatant, filter through a membrane, load the sample, elute with distilled water, and collect the eluate rich in polysaccharide (the eluate rich in polysaccharide refers to the tubes with color after color development by the phenol-sulfuric acid method), and concentrate under reduced pressure.

[0015] Furthermore, in step 4), the separation, elution, and dialysis through the DEAE-Sepharose Fast Flow ion exchange chromatography column are as follows: Load the polysaccharide solution purified by the DEAE cellucose-52 ion exchange chromatography column onto the DEAE-Sepharose Fast Flow ion exchange chromatography column, elute with distilled water, collect the eluate rich in polysaccharide, and concentrate under reduced pressure.

[0016] Specifically, in step 4), the purification and elution using the Sephadex G-100 chromatography column are as follows: Load the polysaccharide solution purified by the DEAE-Sepharose Fast Flow ion exchange chromatography column onto the Sephadex G-100 chromatography column, and elute with distilled water.

[0017] Further, in step 4), the dialysis bag used for dialysis has a molecular weight cut-off of 8000 - 12000 Da.

[0018] The present invention provides the Inonotus hispidus polysaccharide IHP-1aa prepared by the above preparation method. The Inonotus hispidus polysaccharide IHP-1aa is a heteropolysaccharide composed of glucose, galactose, mannose, fucose, and methyl galactose, with a molecular weight of 26.9 kDa, and its molecular structure is shown as follows.

[0019]

[0020] The present invention also provides the application of the above Inonotus hispidus polysaccharide in the preparation of immunomodulatory drugs.

[0021] The object of the present invention is to solve the gaps in existing research, and to propose an Inonotus hispidus polysaccharide IHP-1aa with immunomodulatory activity and its preparation method (obtained by degreasing with ethanol, extracting with water and precipitating with alcohol, then removing proteins by the Sevage method, separating neutral polysaccharide components through DEAE-52 cellulose column and DEAE agarose gel column, and finally purifying with Sephadex G-100). The prepared Inonotus hispidus polysaccharide HP-1aa has uniform purity and clear structure. In vitro experiments show that it can significantly stimulate macrophages RAW264.7 to secrete NO, IL-6, and TNF-α, has good immunomodulatory activity, and can be used for the development and utilization of immune activity enhancement products. Compared with the prior art, the beneficial effects of the present invention are as follows:

[0022] Through the extraction and separation of Inonotus hispidus, the present invention first obtains a biologically active macromolecular polysaccharide HP-1aa. It is detected that the neutral sugar content of this polysaccharide is over 95%. Through monosaccharide composition identification, it is found that this polysaccharide is a heteropolysaccharide composed of glucose, galactose, mannose, fucose, and methyl galactose. High performance gel permeation chromatography proves that it has uniform purity and the molecular weight distribution is 26.9 kDa. Nuclear magnetic resonance spectra determine its glycosidic bond linkage mode and its polysaccharide structure.

[0023] The preparation method of the present invention is simple in operation, has good repeatability, can obtain macromolecules with clear structures, and provides research value for further studying its structure-activity relationship. The polysaccharide prepared by the method of the present invention can better retain its natural structure, and this method has low requirements for equipment, which is conducive to large-scale popularization, development, and use in industrial production.

[0024] The Inonotus hispidus polysaccharide IHP-1aa prepared by the present invention has high purity, can significantly stimulate macrophages RAW264.7 to secrete NO, IL-6, and TNF-α, has good immunomodulatory activity, and can be used for the development of immune enhancer products, which is very helpful for improving the application value of Inonotus hispidus fruiting bodies. Description of the Drawings

[0025] Figure 1 Among them, A is the elution curve of a DEAE cellucose-52 ion exchange chromatography column; B is the elution curve of a DEAE-Sepharose Fast Flow ion exchange chromatography column; C is the elution curve of a Sephadex G-100 chromatography column. (Absorbance at 490nm is the absorbance value at 490nm, and Tubenumber is the tube number).

[0026] Figure 2 Among them, A is the HPGPC diagram and dextran standard curve for purity detection of IHP-1aa; B is the monosaccharide composition of IHP-1aa.

[0027] Figure 3 is the NMR spectrum of IHP-1aa, which are respectively 1 1H-NMR spectrum (A), 13 13C spectrum (B), 1 1H- 1 1H COSY(C), 1 1H- 13 13C HMBC(D), HSQC(E), TOSY(F);

[0028] Figure 4 is the effect of IHP-1aa on the viability of RAW264.7 cells (A); the effects of IHP-1aa on the production of NO (B), IL-6 (C), and TNF-α (D) in RAW264.7. Specific embodiments

[0029] The technical solutions of the present invention will be further introduced in detail below in conjunction with the embodiments, but the protection scope of the present invention is not limited thereto.

[0030] Inonotus hispidus ( Inonotus hispidus ) fruiting bodies were purchased from Nanyang Sanghuang Agricultural Development Co., Ltd. In the following embodiments, unless otherwise specified, the ethanol concentration refers to the volume percentage concentration.

[0031] Example 1:

[0032] A method for preparing the polysaccharide IHP-1aa from Inonotus hispidus includes the following steps:

[0033] 1) Defatting treatment of raw materials: After drying and chopping the Inonotus hispidus, it was soaked twice in a 75% ethanol aqueous solution (1 g of fruiting body was added with 10 ml of 75% ethanol aqueous solution, w / v) for defatting, each soaking for 72 h, and then filtered. The filter residue was the crude extract of the fruiting body.

[0034] 2) Water extraction and alcohol precipitation: The filter residue was extracted with distilled water (material-liquid ratio 1:20, g / ml) at 80 °C for 3 h, filtered, and the filtrate was collected. The operation was repeated three times. The extraction solutions were combined and concentrated under reduced pressure at 55 °C until the solution density reached 1.2 g / cm 3 , and 95% ethanol was added with stirring until the final ethanol concentration reached 75%. It was left to stand for alcohol precipitation at 4 °C for 24 h, filtered, and the precipitate was collected and air-dried.

[0035] 3) Protein removal: Sevage reagent (chloroform: n-butanol = 4:1, v / v) was used to remove proteins (this is a conventional technique in the art and not the innovation of this application, so it will not be elaborated here). Alcohol precipitation was performed again (i.e., 95% ethanol was added with stirring until the final ethanol concentration reached 75%, and it was left to stand for alcohol precipitation at 4 °C for 24 h), filtered, and the precipitate was collected. The precipitate was vacuum freeze-dried at -60 °C for 12 h until completely dry to obtain the crude Inonotus hispidus polysaccharide after protein removal, which was used for further purification.

[0036] 4) 300 mg of the crude Inonotus hispidus polysaccharide after protein removal was dissolved in 15 ml of distilled water to prepare a 20 mg / ml solution, centrifuged (8000 rpm, 5 min), the supernatant was taken, filtered through a 0.45 μm filter membrane, and 15 ml was loaded onto a DEAE cellucose-52 ion exchange chromatography column (2.6×40 cm) for purification. Gradient elution was carried out successively with distilled water, 0.05, 0.1, and 0.2 mol / L NaCl solutions (flow rate 1 mL / min, 8 min / tube). An automatic collector was used to collect the eluate, and the phenol-sulfuric acid method was used for color development detection. The absorbance (OD value) was measured at 490 nm. With the elution tube number as the abscissa and the absorbance as the ordinate, a polysaccharide elution curve was plotted (see Figure 1 A therein), the polysaccharide samples of the same elution peak were combined, concentrated under reduced pressure at 55 °C, dialyzed at 4 °C for 24 h (the molecular weight cut-off of the dialysis bag was 8000 - 12000 Da, and the water was changed 3 times), and vacuum freeze-dried at -60 °C for 12 h. Among them, the elution peak of distilled water was fraction (IHP-1) and the elution peak of 0.05 mol / L NaCl was fraction (IHP-2).

[0037] 5) The main fraction (IHP-1) obtained in step 4) was dissolved in 10 ml of distilled water and loaded onto a DEAE-Sepharose Fast Flow chromatography column (2.6×40 cm) for purification. It was eluted with distilled water (flow rate 1 mL / min, 8 min / tube), and the phenol-sulfuric acid method was used for color development. A polysaccharide elution curve was plotted according to the absorbance (OD value) at 490 nm (see Figure 1In B), a single elution peak was obtained. The polysaccharide sample of this elution peak was concentrated under reduced pressure at 55 °C and dialyzed at 4 °C for 24 h (the molecular weight cut-off of the dialysis bag was 8000 - 12000 Da, and the water was changed 3 times). After vacuum freeze-drying at -60 °C for 12 h, the distilled water elution fraction (IHP-1a) was obtained.

[0038] 6) Dissolve the distilled water elution fraction (IHP-1a) obtained in step 5) in 5 ml of distilled water, load it onto a Sephadex G-100 chromatographic column for further purification, and elute with distilled water (flow rate 0.1 mL / min, 25 min / tube). The phenol-sulfuric acid method was used for color development, and the polysaccharide elution curve was plotted according to the absorbance (OD value) at 490 nm. The elution curve showed a group of uniform elution peaks (see Figure 1 In C). After merging, it was concentrated under reduced pressure at 55 °C and dialyzed at 4 °C for 24 h (the molecular weight cut-off of the dialysis bag was 8000 - 12000 Da, and the water was changed 3 times). After vacuum freeze-drying at -60 °C, high-purity refined Inonotus hispidus polysaccharide (IHP-1aa) was obtained.

[0039] The Inonotus hispidus polysaccharide IHP-1aa obtained by the above preparation was a white flocculent powder at room temperature and was stored in the dark and dry conditions at room temperature (below 25 °C).

[0040] The following uses the refined Inonotus hispidus polysaccharide (IHP-1aa) prepared in Example 1 for related detections and cell experiments.

[0041] Example 2 Purity Detection:

[0042] Figure 2 The HPGPC diagram of the purity detection of Inonotus hispidus polysaccharide IHP-1aa and the dextran standard curve (A) and the monosaccharide composition of IHP-1aa (B) are given.

[0043] As Figure 2 shown, the purity and molecular weight of the Inonotus hispidus polysaccharide IHP-1aa prepared in Example 1 were detected by the HPGPC method. Through the Shimadzu LC-20A HPLC system (Shimadzu Co., Ltd.), the chromatographic column was a Shodex OHpak SB-806 HQ column (10 μm, 8 mm × 300 mm), and it was detected with a 20A refractive index detector (RID). The result presented a single symmetric peak, proving that this Inonotus hispidus polysaccharide IHP-1aa was a homogeneous polysaccharide. The molecular weight of IHP-1aa was 26.9 KDa obtained from the dextran standard curve.

[0044] Example 3 Monosaccharide Composition Detection:

[0045] The crude Inonotus hispidus polysaccharide IHP-1aa prepared in Example 1 was determined by PMP-HPLC method. The mass percentage (%) of each monosaccharide was calculated by the internal standard method, and it was found that the crude Inonotus hispidus polysaccharide IHP-1aa was mainly composed of glucose (51.9%) and galactose (18.9%), containing a small amount of fucose (9.9%), mannose (5%), methyl galactose, and a trace amount of glucuronic acid (0.8%).

[0046] Example 4 Nuclear magnetic resonance analysis:

[0047] 30 mg of the crude Inonotus hispidus polysaccharide IHP-1aa prepared in Example 1 was dissolved in 0.6 mL of 99.9% D2O and placed in a Bruker Avanced III 400 MHz NMR spectrometer (Bruker, Switzerland) to collect data, and the data was recorded at 298K 1 1H-NMR, 13 13C-NMR, 1 1H- 1 1H COSY, TOSY, HSQC and HMBC spectra. Through the results of monosaccharide composition, nuclear magnetic resonance analysis and comparison with relevant literature, the chemical shifts of the sugar residues of IHP-1aa were assigned in Table 1 below, and all the sugar residue signals are marked in Figure 3 (A-F), and the partial structure of this polysaccharide was predicted as:

[0048]

[0049] Table 1 Chemical shift assignment of sugar residues of IHP-1aa

[0050]

[0051] Example 5 In vitro cell experiment:

[0052] The crude Inonotus hispidus polysaccharide IHP-1aa prepared in Example 1 was subjected to an in vitro immune experiment

[0053] 1. Materials and reagents: The crude Inonotus hispidus polysaccharide IHP-1aa isolated and purified in Example 1; mouse macrophage RAW264.7 (purchased from the Cell Bank of the Chinese Academy of Sciences' Committee for the Preservation of Type Cultures); penicillin and streptomycin mixture (Beijing Solarbio Science & Technology Co., Ltd.); fetal bovine serum (FBS, Gibco BRL, USA), ELISA kit (Beijing Sznbio Biotechnology Co., Ltd.); NO kit (Shanghai Beyotime Biotechnology Co., Ltd.).

[0054] 2. Instruments: Carbon dioxide incubator (Thermo scientific, model 3111); FRESCO 21 centrifuge (Thermo); Multiskan GO microplate reader (Thermo); 96-well and 24-well cell culture plates (Corning); DMEM medium (Procell).

[0055] 3. Experimental methods

[0056] (1)MTT assay for cell viability

[0057] RAW 264.7 mouse macrophages were cultured in DMEM medium (hereinafter referred to as DMEM complete medium) containing penicillin (100 U / mL), streptomycin (100 μg / mL), and FBS (10%) at 37 °C and 5% CO2. The effect of IHP-1aa on the viability of RAW264.7 cells was detected by MTT assay. Briefly, cells were seeded in 96-well plates at a density of 1.5×10 4 cells / well and incubated in an incubator at 37 °C and 5% CO2 for 24 hours. Then, they were treated with different concentrations (50 - 800 μg / mL) of HP-1aa (100 μL) for 24 hours, with DMEM complete medium as the negative control group. Subsequently, 10 μL of MTT solution (5 mg / mL in PBS) was pipetted into each well, and the incubation continued for 4 hours. The supernatant was discarded, and then 100 μL of DMSO was used to dissolve the formazan crystals. The absorbance was measured at 490 nm using a microplate reader.

[0058] Cell viability was calculated as follows: Cell viability (%) = A / B × 100%, where A is the absorbance of the experimental group and B is the absorbance of the negative control group.

[0059] (2)Detection of the contents of NO, IL-6, and TNF-α released by RAW264.7 cells

[0060] RAW264.7 cells were seeded in 24-well plates at a density of 1.5×10 5 cells / well and incubated in an incubator at 37 °C and 5% CO2 for 24 hours. Then, they were treated with LPS (1 μg / mL, positive group), DMEM complete medium (control group), and different concentrations of IHP-1aa (200, 400, 800 μg / mL) at 37 °C and 5% CO2 for 24 hours. The culture supernatants of RAW264.7 cells were collected, and the production of NO and cytokines IL-6 and TNF-α was detected using Griess reagent kits and ELISA reagent kits, respectively.

[0061] 4. Data processing

[0062] The results were expressed as mean ± SD, and one-way ANOVA using Graphpad Prism 9.5 software was used for statistical analysis to compare their significant differences.

[0063] 5. Experimental results

[0064] NO is a non-specific molecule secreted by activated macrophages that can kill or inhibit the growth of a variety of pathogenic microorganisms. Activated macrophages can produce a variety of cytokines (such as TNF-α and IL-6) to regulate cellular and humoral immune responses. Figure 4 In A, it was demonstrated that IHP-1aa had no cytotoxicity. Figure 4 (B-D) showed the effects of IHP-1aa on the production of immune active factors. Figure 4 In B, it was shown that when the concentration of IHP-1aa was 200, 400, and 800 μg / mL, the production of NO was significantly increased compared with the control group. After stimulating the cells with 800 μg / mL IHP-1aa for 24 h, the NO secretion reached 23.47 ± 0.52 μM, an increase of 10.9 μM compared with the blank control group and 56.24% of the LPS group, and the differences were all significant (P<0.001). At the same time, compared with the blank control group, IHP-1aa at different concentrations (200-800 μg / mL) could significantly promote the secretion of IL-6 by RAW264.7 cells and showed a dose-response effect ( Figure 4 In C). As Figure 4 shown in D, TNF-α was significantly increased at 400 μg / mL compared with the blank group (p<0.001). In summary, the obvious increase in NO production indicated that the polysaccharide IHP-1aa from Inonotus hispidus might have good immune-stimulating effects and the ability to inhibit tumor cell proliferation. The release of cytokines TNF-α and IL-6 indicated that IHP-1aa had good immune-enhancing effects, suggesting that it could be used to prepare immune-enhancing drugs.

Claims

1. Use of Inonotus hispidus polysaccharide in the preparation of immunomodulatory drugs, characterized in that, The Inonotus hispidus polysaccharide is prepared through the following steps: 1) Defatting treatment of raw materials: The dried fruiting bodies of Inonotus hispidus are chopped and then soaked in an ethanol aqueous solution for defatting to obtain a crude extract of fruiting bodies; 2) Water extraction and ethanol precipitation: Distilled water is added to the crude extract of fruiting bodies, and the mixture is heated to 70 - 90 °C for extraction. After concentrating the extract, ethanol is added until the final ethanol concentration is 75%, and it is left to stand for ethanol precipitation. The precipitate is collected and air-dried; 3) Protein removal: The Sevage method is used to remove proteins, followed by ethanol precipitation again. The precipitate is collected and freeze-dried to obtain the crude polysaccharide of Inonotus hispidus after protein removal; 4) Isolation and purification: The crude polysaccharide of Inonotus hispidus after protein removal is successively separated, eluted, dialyzed through DEAE cellulose-52 and DEAE-Sepharose Fast Flow ion exchange chromatography columns, and finally purified and eluted using a Sephadex G-100 chromatography column. The eluate is collected, concentrated under reduced pressure, dialyzed, and freeze-dried to obtain the product; In step 4), for separation and elution through the DEAE cellulose-52 ion exchange chromatography column, specifically: The crude polysaccharide of Inonotus hispidus after protein removal is dissolved in distilled water, centrifuged, the supernatant is taken, filtered through a membrane, loaded onto the column, and eluted with distilled water. The eluate is collected and concentrated under reduced pressure.

2. Use of the Inonotus hispidus polysaccharide according to claim 1 in the preparation of an immunomodulatory drug, characterized in that, In step 1), the volume concentration of the ethanol aqueous solution is 70 - 80%, and the soaking time is 60 - 84 h.

3. Use of the Inonotus hispidus polysaccharide according to claim 1 in the preparation of an immunomodulatory drug, characterized in that, In step 2), it is left to stand for ethanol precipitation at 2 - 6 °C for 20 - 28 h.

4. Use of Inonotus hispidus polysaccharide according to claim 1 in the preparation of an immunomodulatory drug, characterized in that, In step 4), for separation and elution through the DEAE-Sepharose Fast Flow ion exchange chromatography column, specifically: The polysaccharide solution purified through the DEAE cellulose-52 ion exchange chromatography column is loaded onto the DEAE-Sepharose Fast Flow ion exchange chromatography column, and eluted with distilled water. The eluate is collected and concentrated under reduced pressure.

5. Use of the polysaccharide of Inonotus hispidus as claimed in claim 1 in the preparation of an immunomodulatory drug, characterized in that, In step 4), for purification and elution using the Sephadex G-100 chromatography column, specifically: The polysaccharide solution purified through the DEAE-Sepharose Fast Flow ion exchange chromatography column is loaded onto the Sephadex G-100 chromatography column, and eluted with distilled water.

6. The application of the Inonotus hispidus polysaccharide according to claim 1 in the preparation of an immunomodulatory drug, characterized in that, In step 4), the molecular weight cut-off of the dialysis bag selected during dialysis is 8000 - 12000 Da.

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