WO2014173058A1

WO2014173058A1 - Trametes robiniophila polysaccharide protein, preparation method therefor, and application thereof

Info

Publication number: WO2014173058A1
Application number: PCT/CN2013/082697
Authority: WO
Inventors: 徐无为; 陆正鑫
Original assignee: 启东盖天力药业有限公司
Priority date: 2013-04-24
Filing date: 2013-08-30
Publication date: 2014-10-30
Also published as: CN104119427B; CN104119427A

Abstract

The present invention relates to trametes robiniophila polysaccharide protein. Monosaccharide compositions of the polysaccharide protein are fucose, arabinose, galactose, glucose, xylose, and mannose, a mass ratio thereof is 0.1:1.0:5.4:4.4:2.1:7.0, and the weight-average molecular weight of the polysaccharide protein is 7.0x10⁵-2.0x10⁶Da and preferably is 1.69x10⁶Da. The polysaccharide protein of the present invention can be used to prepare medicine for curing tumour.

Description

Amaranth polysaccharide protein and preparation method and use thereof

The invention relates to a polysaccharide protein of cockroach and a preparation method and use thereof. Background technique

It is a fruiting body of Trametes robiniophila Murr., an important folk medicinal fungus with more than 1600 years of medication history in China. The Ainsworth fungal classification system attributed it to the fungus, the Basidiomycetes, the Phytophthora, the Polyporaceae, and the genus Trametes. It is medium to large, with cork, sterile handle, born on the trunk of broad-leaved trees such as alfalfa and locust, distributed in Hebei, Shaanxi, Liaoning, Hunan, Guangxi, Fujian, etc. Heartwood decayed wood rot fungus. This product is bitter, spicy, and non-toxic. It has the effects of "governing the wind", "breaking the blood", and "Yi Li,". It is clinically used to treat a variety of diseases. Due to the growing scarcity of old Chinese cockroaches, medicinal materials After exhaustion, it is difficult to meet the demand for clinical drugs, and it is unable to meet the needs of industrial production. In order to solve the problem of resources of Qi Er, Qidong Gaitian Pharmaceutical Co., Ltd. has solved the problem of large-scale fermentation of the mycelium after long-term research and practice. The industrial production of mycelium was realized, and the extracts of the ear fungus (the fermented material of the ear fungus) were used as raw materials to develop medicines such as the ear granules and the glutinous granules, which satisfied the clinical drug demand.

In recent years, a large number of chemical composition and pharmacological activity studies have confirmed that the effective component of the extract of the fungus is the polysaccharide protein of the ear, and its main function is to treat or assist in the treatment of neoplastic diseases. However, at present, there are few studies on the composition, structure and preparation methods of polysaccharides in the polysaccharides. In the chemical composition research, only the monosaccharide composition and ratio analysis of crude polysaccharides are reported in the literature, lacking the separation, purification and structure of the homopolysaccharides. Studies, such as the types and proportions of sugar residues contained in homogenous polysaccharide proteins, the order of arrangement in the main chain, etc., and the information on the mechanism and structure of the true medicinal components of the fungus, the polysaccharides of the ear polysaccharides Effective relationships are essential. Similarly, in the study of the biological activity of polysaccharides from the polysaccharides, the crude polysaccharides are used as research objects, and the in vivo and in vitro pharmacological research data obtained by applying uniform polysaccharide components are lacking, so that the polysaccharide protein and human immune cells or tumors cannot be further understood. The action process of cell surface-associated polysaccharide receptors cannot be used to clarify its principle of action. Summary of the invention

In order to overcome the deficiencies of the prior art, the present invention provides a polysaccharide protein of the ear and a preparation method and use thereof.

The above object of the present invention is achieved by the following technical solutions.

In a first aspect, the present invention provides a polysaccharide protein of the ear (also referred to as "the ear polysaccharide egg of the present invention" White"), the monosaccharide composition of the polysaccharide protein is fucose, arabinose, galactose, glucose, xylose and mannose, and the mass ratio thereof is 0.1: 1.0: 5.4: 4.4: 2.1: 7.0.

Preferably, the weight average molecular weight proteoglycans 7.0xl0 ⁵ -2.0xl0 ⁶ Da, preferably 1.69xl0 ⁶ Da.

Preferably, the method for preparing the polysaccharide protein comprises the following steps:

(1) removing the free protein from the aqueous solution of the fungus extract (such as scented ear cream) at a concentration of 2%-20% (mass/volume), preferably 8% (mass/volume), and collecting the free protein by the Sevage method. Class part

(2) adding the saccharide moiety obtained in the step (1) to anhydrous ethanol to form a sugar solution having an alcohol concentration of 55% to 70% by volume, preferably 60% by volume, and centrifuging to obtain a precipitate;

(3) dissolving the precipitate obtained in the step (2) with water, filtering, concentrating, and dialysis to collect the inner portion of the bag;

(4) Separating the inner portion of the bag collected in the step (3) by ion exchange column chromatography, wherein the eluent used is an aqueous solution of 0.85 Mol/L to 1.5 Mol/L of NaCl, preferably 1.0 Mol/L of NaCl. Aqueous solution.

In the present invention, the extract of the ear fungus includes an aqueous extract of the fungus, particularly a hot water (60-100 ° C) extract. The aqueous extract of the ear fungus can be used as it is or after concentration. The concentrated aqueous extract of the ear fungus (i.e., the ear cream) or the water extract of the unconcentrated ear fungus can be prepared or purchased by a conventional method.

Preferably, the method for preparing the polysaccharide protein further comprises the step of subjecting the product obtained by eluting the NaCl solution to dialysis and desalting.

Preferably, the method for preparing the polysaccharide protein further comprises the step of separating and purifying the product after dialysis and desalting using a Sepharose CL-6B agarose gel column.

In a second aspect, the present invention provides a method for preparing the above polysaccharide protein, which comprises the following steps:

(4) Separating the inner portion of the bag collected in the step (3) by ion exchange column chromatography, wherein the eluent used is an aqueous solution of 0.85 Mol/L to 1.5 Mol/L of NaCl, preferably 1.0. A solution of Mol/L in NaCl is obtained.

Preferably, the preparation method further comprises the step of dialysis and desalting of the product eluted with the aqueous solution of NaCl.

Preferably, the preparation method further comprises the step of separating and purifying the dialysis and desalted product using a Sepharose CL-6B agarose gel column.

In a specific embodiment, the preparation method comprises the following steps:

(1) Accurately weigh 300g of the ear fungus extract 槐 ear clear paste, dilute it to a dilute solution with a concentration of about 8% (mass/volume) by adding an appropriate amount of distilled water, then remove the protein by Sevage method, repeat Operate 5 times to collect the sugar part;

(2) slowly adding anhydrous ethanol to the saccharide portion of the sputum fungus obtained in the step (1) to obtain a sugar solution having an alcohol concentration of 60% by volume, and after standing at 4 ° C for 24 hours, 3000 Centrifuge at a speed of rpm for 10 minutes, and precipitate to obtain a precipitate;

(3) Weighing the appropriate amount of the precipitate obtained in the step (2), dissolving it in 150 ml of distilled water, filtering, concentrating under reduced pressure to a volume of 50 ml; dialysis collecting the inner portion of the bag, and

(4) The inner part of the bag collected in step (3) was separated by DEAE-52 ion exchange column chromatography, eluted with lO Mol/LNaCl solution, and the eluted part was concentrated and dialyzed to remove salt. The dialysis process was Dialysis dialysis for three days, dialysis for two days; and

(5) The product obtained in the step (4) was separated and purified using a Sepharose CL-6B agarose gel column until it was detected as a pure product by high performance liquid chromatography.

In a third aspect, the present invention provides the use of the above polysaccharide protein for the preparation of a medicament for treating a tumor. The polysaccharide protein of the present invention has remarkable antitumor activity and is expected to be an active ingredient of a novel antitumor drug.

In a fourth aspect, the present invention provides a pharmaceutical composition comprising an agaric acid protein and a pharmaceutically acceptable carrier, wherein the composition comprises 0.01% to 99.5% by weight of a polysaccharide protein as an active ingredient, and The auricular polysaccharide protein contains 90% by weight to 100% by weight of the polysaccharide protein of the first aspect of the present invention, based on the total weight of the polysaccharide protein of the ear.

The pharmaceutical composition preferably contains 0.1% to 99.9% by weight of the polysaccharide protein as an active ingredient, preferably 0.1% to 99.5% by weight of the polysaccharide protein as an active ingredient, more preferably a weight ratio. It is 0.5%-95% active ingredient.

The pharmaceutical composition, comprising a therapeutically effective amount of the polysaccharide protein of the present invention, has significant antitumor efficacy. The mixture of the polysaccharide protein of the ear and the pharmaceutically acceptable carrier, diluent and the like can be orally administered in the form of a tablet, a capsule, a granule, a powder or a syrup or in the form of an injection. Oral administration. The above formulations can be prepared by conventional pharmaceutical methods. Examples of useful pharmaceutically acceptable carriers include excipients (e.g., saccharide derivatives such as lactose, sucrose, Portuguese) Glucose, mannitol and sorbitol; starch derivatives such as corn starch, potato starch, dextrin and carboxymethyl starch; cellulose derivatives such as crystalline cellulose, hydroxypropyl cellulose, carboxymethyl cellulose, carboxy Methylcellulose calcium, sodium carboxymethylcellulose; gum arabic; dextran; silicate derivatives such as magnesium aluminum metasilicate; phosphate derivatives such as calcium phosphate; carbonate derivatives such as calcium carbonate; Such as calcium sulfate, etc.), binders (such as gelatin, polyvinylpyrrolidone and polyethylene glycol), disintegrants (such as cellulose derivatives such as sodium carboxymethylcellulose, polyvinylpyrrolidone), lubricants (for example Talc, calcium stearate, magnesium stearate, cetyl, boric acid, sodium benzoate, leucine), stabilizers (methyl p-hydroxybenzoate, propyl p-hydroxybenzoate, etc.), flavoring agents (eg commonly used) Sweeteners, sours, and flavors, etc.), diluents, and solvents for injections (such as water, ethanol, and glycerin, etc.).

When a pharmaceutical composition is used, a safe and effective amount of the polysaccharide protein of the invention is administered to a mammal, wherein the safe and effective amount is usually at least about 1 microgram per day, and in most cases no more than about 10 milligrams per kilogram of body weight. . Preferably, the dosage is from about 1 microgram per day to about 3 milligrams per kilogram of body weight. Of course, specific doses should also take into account factors such as the route of administration, the health of the patient, etc., which are within the skill of the skilled physician.

Further, the polysaccharide protein of the present invention can be used as a single drug or in combination with other drugs. Preferred combinations include: in combination with surgery, in combination with one or more western medicines, in combination with Chinese herbal medicines, in combination with radiation therapy.

The administration route of the pharmaceutical composition of the present invention is not particularly limited, and includes, but is not limited to, oral administration, injection administration, intratumoral administration, implantation administration, intraluminal administration, anal administration, transdermal administration. Administration, internal and external application; preferred injection administration includes: intravenous injection, intramuscular injection, subcutaneous injection, intraluminal injection, intratumoral administration.

Compared with the prior art, the present invention has at least the following beneficial effects:

For the first time, the present invention uses a system of water extraction, alcohol precipitation, deproteinization, dialysis small molecule, ion exchange chromatography and gel molecular size exclusion chromatography to obtain a uniform polysaccharide protein component from the fungus of the fungus, and Comprehensive application of molecular weight analysis, monosaccharide composition and amino acid composition analysis, infrared spectrum analysis and methylation analysis, etc., confirmed the chemical structure characteristics, and defined its weight average molecular weight, monosaccharide composition and ratio, amino acid composition and The ratio, and the way in which the sugar residues are attached. Such a polysaccharide component with clear structure and uniform molecular weight is an ideal molecular model for studying the active constituents and biological activities of polysaccharides from Auricularia auricula, in order to elucidate the immunomodulatory and antitumor active components of Auricularia auricula polysaccharides and their effects. The mechanism lays a foundation, and the present invention will provide a scientific basis for the further development and utilization of the fungus and the quality control of related preparations. DRAWINGS

Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings, in which: Figure 1 is a flow chart of the alcohol precipitation of the ear extract of the ear fungus;

Fig. 2 is a flow chart of purification of TCP-60 column chromatography by 60% ethanol precipitation site of the ear fungus extract of the ear fungus;

Figure 3 is a standard curve for determining the molecular weight (Mw) of polysaccharide protein TP-3;

Figure 4 is an ion chromatogram of a reference solution in the analysis of the monosaccharide component of the polysaccharide protein TP-3; Figure 5 is a UPLC-GPC chromatogram of the polysaccharide protein TP-3;

Figure 6 is an ion chromatogram of the composition analysis of the polysaccharide of TP-3;

Fig. 7 shows the proliferation of mouse spleen cells by ³ H-TdR incorporation method, wherein: TCP: crude polysaccharide; TP-1: polysaccharide protein-1; TP-2: polysaccharide protein-2; TP-3: Polysaccharide protein-3; TP-4: polysaccharide protein-4; To-1: low molecular weight component-1; Το-2: low molecular weight component-2; Το-3: 槐 ears Low molecular weight component-3; Το-4: 低 ear low molecular weight component-4; TFP: 槐 ear coarse total protein fraction;

Figure 8 shows the proliferation of T and sputum lymphocytes in mice by ³ H-TdR incorporation, wherein Figure 为 is CD19+ cells; Figure B is CD3+ cells; Figure C is mouse T lymphocytes; Figure D is mouse B lymphocytes Cell; Dex: dextran; TCP: crude polysaccharide; TP-1: polysaccharide protein-1; TP-2: polysaccharide protein-2; TP-3: polysaccharide protein-3; TP-4: Polysaccharide protein-4; LPS: lipopolysaccharide;

Fig. 9 shows changes in the proportion of T and sputum lymphocytes in mice and humans after stimulation with polysaccharides from the ear, wherein Fig. B is mouse lymphocytes; Fig. B is mouse B lymphocytes; Fig. C is human T lymphocytes; For human B lymphocytes; Dex: Dextran; TCP: crude polysaccharide; TP-1: Polysaccharide protein-1; TP-2: Polysaccharide protein-2; TP-3: Polysaccharide protein -3; TP-4: polysaccharide protein-4; LPS: lipopolysaccharide;

Figure 10 shows the expression of CD69 on the surface of T and sputum lymphocytes after stimulation with polysaccharides from Auricularia auricula. Fig. B shows the expression of CD69 on the surface of T lymphocytes stimulated by polysaccharides from Auricularia auricula, and Figure B shows the expression of CD69 on the surface of B lymphocytes after stimulation with Auricularia auricula. Where Dex: Dextran; TCP: crude polysaccharide; TP-1: Polysaccharide protein-1; TP-2: Polysaccharide protein-2; TP-3: Polysaccharide protein-3; TP- 4: polysaccharide protein-4; LPS: lipopolysaccharide;

Figure 11 shows changes in NO release in mouse peritoneal macrophages after stimulation with polysaccharides from Lycium barbarum L., Dex: Dextran; TCP: crude polysaccharide; TP-1: Polysaccharide protein-1; TP-2: Polysaccharide protein-2; TP-3: polysaccharide protein-3; TP-4: polysaccharide protein-4; To-1: low molecular weight component-1; To-2: low molecular weight component -2; Το-3: 低 ear low molecular weight component-3; Το-4: 槐 ear low molecular weight component-4; TFP: 槐 ear coarse total protein fraction; LPS: lipopolysaccharide; Figure 12 槐 ear polysaccharide protein Inhibition of tumor angiogenesis endothelial cell proliferation, TP-3: Auricularia Polysaccharide-3. detailed description

The invention is illustrated by the following examples, which are intended to be illustrative of the invention and are not intended to limit the scope of the invention. Example 1: Method for separating and purifying polysaccharide protein of cockroach (ΤΡ-3)

Accurately weigh the ear extract of Quercus sinensis (provided by Qidong Gaitian Pharmaceutical Co., Ltd.) 300g, and dilute it to a dilute solution with a concentration of about 8% (mass/volume) by adding an appropriate amount of distilled water. Then, the Sevage method was used to remove the protein, and the operation was repeated 5 times to collect the sugar fraction (TCP) and the total free protein fraction (TFP) of the ear, and the total free protein of the ear was vacuum dried for later use. The substance fraction (TCP) was concentrated under reduced pressure to remove residual chloroform and n-butanol, followed by fractional alcohol precipitation. First, slowly add anhydrous ethanol to TCP to make a sugar solution with a volume concentration of 40% by volume of alcohol. After standing at 4 ° C for 24 hours, centrifuge at 3000 rpm for 10 minutes, and the precipitate number is TCP-40. Drying under reduced pressure for further separation and purification, and then adding an appropriate amount of absolute ethanol to the supernatant to make a 60% (volume) sugar solution containing alcohol, standing at 4 ° C for 24 hours, 3000 rpm, centrifugation 10 In minutes, the precipitate number is TCP-60, and dried under reduced pressure for further separation and purification. After that, the appropriate amount of absolute ethanol is added to the supernatant to 80% by volume of ethanol, and after standing at 4 ° C for 24 hours, Centrifuge at 3000 rpm for 10 minutes, the precipitate number is TCP-80, and dry under reduced pressure for further separation and purification. The specific fractionation alcohol precipitation flow chart is shown in Figure 1.

Weigh an appropriate amount of 40% (volume) ethanol precipitation site TCP-40, dissolve it in 150ml distilled water, filter, concentrate to a volume of 50ml under reduced pressure, dialysis collection bag inside and outside the bag, outside the bag Part of the low molecular weight component-1 (ΤΟ-1) of the ear, the inner part of the bag was separated by DEAE-52 ion exchange column chromatography, respectively, using distilled water and 0.1M NaCl solution, 0.5M NaCl, and collected in each part. 3.0L, the water-eluting fraction was directly concentrated to dryness, and the sodium chloride eluting fraction was concentrated and dialyzed to remove salt. The dialysis process was dialysis dialysis for three days, and the distilled water was dialyzed for two days. Then, each section was separated and purified by Sepharose CL-6B agarose gel column until it was pure by high performance liquid chromatography. After the above column chromatography, it was separated from TCP-40 ion exchange column by 0.1 Mol/L. Purification of the eluted fraction of NaCl obtained UPLC (Ultra High Performance Liquid Chromatography) as a homogeneous component of the polysaccharide protein TP-1 (397 mg) and a homogeneous polysaccharide protein TP-2 (1212 mg) from the 0.5 Mol/L NaCl elution site. ), the separation flow chart is shown in Figure 2.

Weigh an appropriate amount of 60% (volume) ethanol precipitation site TCP-60, dissolve it in 150ml of distilled water, filter, concentrate to a volume of 50ml under reduced pressure, and dialysis collection bag part and The outer part of the bag is divided into the low molecular weight component-2 (TO-2) of the ear, and the inner part of the bag is separated by DEAE-52 ion exchange column chromatography, respectively, using distilled water and 1.0 Mol/L NaCl solution, respectively. 3.0L was collected in each part, and the water-eluting fraction was directly concentrated to dryness. The eluted part of sodium chloride was concentrated and dialyzed and desalted. The dialysis process was dialysis dialysis for three days, and the dialysis water was dialyzed for two days. Then, each section was separated and purified by Sepharose CL-6B agarose gel column until it was pure by high performance liquid chromatography. After the above column chromatography, it was separated from TCP-60 ion exchange column by 1.0 Mol/L. Purification of the eluted fraction of NaCl resulted in a UPLC (Ultra High Performance Liquid Chromatography) verification of the homogeneous component of the polysaccharide protein TP-3 (1. 21 g).

Weigh an appropriate amount of 80% (volume) ethanol precipitation site TCP-80, dissolve it in 150ml of distilled water, filter, concentrate to a volume of 50ml under reduced pressure, dialysis collection bag inside and outside the bag, outside the bag The part is the low molecular weight component -3 ( TO-3 ) of the ear, and the inner part of the bag is separated by DEAE -52 ion exchange column chromatography, and is respectively eluted with distilled water and 1.0 Mol/L NaCl water, and 3.0L is collected in each part. The water-eluting fraction was directly concentrated to dryness, and the sodium chloride eluting fraction was concentrated and dialyzed to remove salt. The dialysis process was dialysis for three days, and the dialyzed water was dialyzed for two days. Then, each section was separated and purified by Sepharose CL-6B agarose gel column until it was pure by high performance liquid chromatography. After the above column chromatography, it was separated from TCP-80 ion exchange column by 1.0 Mol/L NaCl. The eluted site was purified by UPLC (Ultra High Performance Liquid Chromatography) to verify the homogenous component of the polysaccharide protein TP-4 (10.0g), and the 80% (volume) ethanol precipitate supernatant of the ear cream was collected. Molecular weight component -4 (TO-4) (Figure 2). Example 2: Study on the chemical structure of polysaccharide protein (TP-3)

This example investigates the chemical structure of the polysaccharide protein TP-3 prepared in Example 1.

I. Research method for chemical structure of polysaccharide protein (TP-3)

1. Determination of purity and molecular weight

UPLC-GPC-ELSD instrument configuration and chromatographic conditions: US Waters UPLC, TSK-3000 GPC column, autosampler, Millipore ultrapure water ion exchanger Preparation of high purity water (0.45 μιη cellulose acetate membrane filtration); flow rate 0.3 ml / min „

Preparation of the standard curve: Weigh the appropriate amount of dextran standard, add deionized water, configure it as a reference solution with a concentration of 0.5 mg/ml, and then perform UPLC test one by one. The results are shown in Figure 3.

Sample solution preparation: Weigh a certain amount of the polysaccharide protein TP-3 prepared in Example 1, add appropriate amount of deionized water, prepare it into a solution with a concentration of lmg/ml, Millipore 0.22μιη water filter to filter, injection detection . 2, monosaccharide composition analysis

Complete acid hydrolysis: Weigh accurately the polysaccharide protein TP-3 (10 mg) prepared in Example 1 into a thick-walled pressure bottle, add 4 ml of 2 Mol/L trifluoroacetic acid (TFA), fill with nitrogen, and seal the tube. It was hydrolyzed at 100 °C for 2 hours and evaporated to dryness under reduced pressure.

Sample ion chromatographic analysis

Instrument configuration and chromatographic conditions: Dionex ICS 3000 ion color language, CarboPac PA20 analytical column, 150 x 3mm, S/N 002823, CarboPac PA20 guard column, 50*3mm, S/N 002652, eluent composition and flow rate l- 25 min, Im Mol/L KOH; 25.1-32 min, 30 m Mol/L KOH,; 32.1-35 min, Im Mol/L KOH; 0.45 mL/min, 10 L injection.

Preparation of the reference solution: Take the appropriate amount of fucose, arabinose, galactose, glucose, xylose, mannose reference substance, dissolve it into 10.0 mg/L of the reference solution with deionized water, shake it, that is, .

Standard curve preparation: Accurately draw the appropriate amount of reference stock solution, and dilute it with deionized water to 0.5 mg/L, lmg/L, 5 mg/L, 10 mg/L, 15 mg/L standard solution, 0.45 After filtration through μιη microporous membrane, the chromatographic conditions were determined by ion chromatography. Dionex ICS 3000 ion chromatography, CarboPac PA20 analytical column, 150 x 3mm, S/N 002823, CarboPac PA20 guard column, 50*3mm, S /N 002652 , eluent composition and flow rate l-25min, lm Mol / L KOH; 25.1-32 min, 30m Mol / L KOH, ; 32.1-35 min, lm Mol / L KOH; 0.45mL / min, injection 10 L. The peak area integral value is plotted on the ordinate (Y) and each standard concentration is plotted on the abscissa (X). The standard curve of each reference is plotted and the regression equation is calculated. The results are shown in Table 1 and Figure 4.

Table 1 Linear relationship study results Correlation product name Relative retention time Regression equation Correlation coefficient (R) Fucose 0.20 Υ=1.282Χ+0.055 99. .9724 Arabinose 0.67 Υ=1.848χ-0.100 99. .9973 Galactose 0.85 Υ =2.138χ-0.170 99. .9765 Glucose 1.00 Υ=2.297χ-0.164 99. .9630 Xylose 1.20 Υ=2.629χ-0.176 99. .9620 Mannose 1.30 Υ=1.911χ-0.147 99. .9826 Test article Solution preparation: The polysaccharide peptone-3 completely acid hydrolyzate prepared in Example 1 was redissolved in 50 ml of deionized water, sonicated for 10 minutes to dissolve, and the solution was taken in an appropriate amount. The pore size of 0.22 μιη water filtration membrane and DIONEX RP II solid phase extraction were performed. Small column. Separately draw the reference solution and the test solution for each ΙΟμ, and inject them into the ion chromatograph.

3, methylation analysis

Complete methylation of polysaccharide protein: Weigh the appropriate amount of the polysaccharide peptone-3 prepared in Example 1 in a reaction flask, dry it in a vacuum oven for 5 h (50 °C), add 2 ml of DMSO treated with molecular sieve, and ultrasonically oscillate. 5 minutes, after the sample is completely dissolved, add 20 mg of powdered NaOH, while exhausting the air in the bottle with nitrogen, sonicating at room temperature for 10 minutes, let stand for 90 minutes, after the DMSO in the reaction bottle is completely frozen, add dropwise 0.1 ml of methyl iodide (this process takes about 15 to 20 minutes), and the reactants slowly thaw and gradually clarify until they become bright yellow. It was then sonicated for 10 minutes and allowed to stand for 30 minutes. The mixture was distilled under reduced pressure at room temperature, and excess methyl iodide was removed, and it was dialyzed against water for one day, and steamed to about 2 ml under reduced pressure. And then freeze-dried in a desiccator for 5 hours, after the above operation is repeated 2 times, and a small sample an infrared detector, if infrared spectroscopy at 3300cm- ¹ Central strong and broad hydroxyl peak disappeared, and a peak at 2900cm- ¹ methyl Significantly enhanced, indicating that the sample's permethylation reaction has been completed.

Partially methylated Aldiol acetyl ester preparation: The sample which has been completely methylated is dissolved in 3 mL of 90% by volume of formic acid solution, sealed, depolymerized at 100 °C for 6 h, and added to the reaction flask 2~ 3 mL of methanol, concentrated and evaporated to dryness under reduced pressure at 40 ° C. The above operation was repeated three times to remove excess formic acid, and then 4 mL of 2 Mol/LTFA solution was added to the depolymerized sample, sealed and hydrolyzed at 110 ° C for 2 h. The solution in the bottle was evaporated to dryness under reduced pressure at 40 ° C, and then 2 to 3 mL of methanol was added thereto, and evaporated to dryness. The above operation was repeated several times to remove excess TFA. After the hydrolyzed sample is dissolved in 3~4mL of distilled water, add about 20mg NaBH ₄ to reduce at room temperature for 3h, then adjust the pH value to about 5 with glacial acetic acid, add 1~2mL methanol and a drop of glacial acetic acid, then decompress. Evaporate and repeat the above procedure several times to remove excess acetic acid. The sample treated above was placed in a P ₂ 0 ₅ vacuum desiccator and dried under reduced pressure for one day. After drying at 110 ° C for 10-15 min, 3 mL of acetic anhydride was added, and the reaction was carried out at 110 ° C for 1 h, and 2 mL of toluene was added to the reaction solution. After shaking, unreacted acetic anhydride was distilled off under reduced pressure at 40 ° C, and this was repeated several times to remove acetic anhydride. Then, the acetylated sample was dissolved in chloroform, and the chloroform layer was washed three times with an equal volume of distilled water, and the aqueous layer was removed. The chloroform layer was dried over anhydrous sodium sulfate for 10 min, filtered, and the chloroform solution was concentrated at room temperature under reduced pressure. After about 0.7 mL, GC/MS analysis was performed. Temperament conditions are: starting temperature 50 ° C, temperature program 40 ° C / min, to 215 ° C, hold 40 min, detector temperature 250 ° C, DB-5 capillary GC-MS column detection.

4. Amino acid analysis:

An appropriate amount of the polysaccharide protein TP-3 prepared in Example 1 was weighed into a test tube, and 4 ml of 6 M HCl was added thereto to carry out hydrolysis at 100 ° C for 6 hours, and HC1 was distilled off, centrifuged, and filtered, and the filtrate was analyzed by an amino acid analyzer. 5, IR spectrum analysis:

1 mg of the polysaccharide protein TP-3 prepared in Example 1 was weighed and dried under vacuum overnight, and the next day, IR detection was carried out by potassium bromide tableting.

2. Results of chemical structure study of polysaccharide protein (TP-3)

The chemical structure of the polysaccharide protein (TP-3) prepared in Example 1 was studied according to the research method of the first part. The results are as follows:

1, purity and molecular weight

TP-3 is a dark brown powdery substance, soluble in water, DMSO, insoluble in high concentrations of organic solvents such as methanol and ethanol. The UPLC-GPC-ESLD pattern of the polysaccharide protein (see Figure 5) presents a symmetrical narrow The chromatographic peak indicates that it is a pure polysaccharide protein material compared with the molecular weight standard. The molecular weight of the polysaccharide protein is 1.69×10 ⁶ Da (see Figure 3), the Lowry reaction is positive, and the UV scan has a weak absorption at 280 nm, indicating This substance contains protein.

2, monosaccharide composition analysis

After complete acid hydrolysis of TP-3, a part of the hydrolyzed product was directly subjected to ion chromatography analysis, and compared with the standard monosaccharide, the analysis showed that the polysaccharide consisted of fucose, arabinose, galactose, glucose, xylose and mannose. No signal related to uronic acid and nitroacetyl glucosamine was found during the test, suggesting that it is a neutral polysaccharide (Table 2, Figure 6).

Table 2 Polysaccharide protein of scorpion polysaccharide TP-3 monosaccharide composition and ratio (mass ratio) Ion chromatographic analysis sample name fucose arabinose galactose glucose xylose mannose

TP-3 0.1 1.0 5.4 4.4 2.1 7.0 It can be seen from the monosaccharide composition analysis that the polysaccharide protein TP-3 contains six monosaccharides, namely fucose, arabinose, galactose, glucose, xylose and mannose. Among them, the content of mannose and galactose is the highest, followed by glucose, xylose, arabinose and fucose.

3, methylation analysis

In order to confirm the attachment of sugar residues in TP-3, TP-3 was methylated by Needs method, and after three methylation, it was depolymerized with 90% formic acid to 2 Mol/LTFA complete acid. Hydrolysis, reduction of NaBH ₄ and acetylation of acetic anhydride to prepare an alcohol derivative of alditol, and GC-MS analysis was carried out, and the results are shown in Table 3.

Table 3 TP-3 methylation analysis results

Connection method Peak area percentage

Non-reducing end-linked arabinose 16% non-reducing end-linked xylose 8% 1,5 connections to arabinose 4%

1,3 connections to arabinose 5%

1,3-position linked fucose 5% non-reducing end-linked glucose 13% non-reducing end-linked galactose 2%

1,4 connections to xylose 6%

1,6-position linked galactose 2%

1,4 mannose 5%

1,4 connections to glucose 26%

1,3 connections to mannose 6%

1,3,6 linked mannose 1%

The 1,3,6-linked galactose methylation analysis showed that the structure of the polysaccharide protein-3 was very complicated. Containing 14 different linked sugar residues, wherein the arabinose is linked to other sugar residues by the 1st, 1st, 3rd, and 1,5th hydroxyl groups, and the xylose is at the 1st and 1st and 4th hydroxyl groups. Other sugar residues form a glycosidic linkage, and fucose is only linked to other sugar residues by the 1,3 hydroxyl group. The mannose is 1,4, 1, 3, 1, 3, and 6 The hydroxyl group is linked to other sugar residues to form a glycosidic bond. The glucose is linked to the other sugar residue by a hydroxyl group at the 1st, 1st, 4th positions. The galactose passes through the 1st position, 1, 6th position, 1, 3, and 6 positions. The hydroxyl group is linked to other sugar residues to form a glycosidic bond. Note: The ratio of the five-carbon sugar to the six-carbon sugar residue in the above results differs from the monosaccharide composition analysis, which is related to the difference in the degree of loss of different types of sugar residues during the reaction.

4. Amino acid analysis:

In order to analyze the amino acid composition and ratio of the polysaccharide protein TP-3 in the ear, the amino acid composition and ratio analysis were carried out by hydrolyzing hydrochloric acid combined with Hitachi automatic amino acid analyzer. The results are shown in Table 4.

Table 4 Results of amino acid composition analysis of polysaccharide protein TP-3

Amino acid name content μ g/mg

Aspartic acid 13.10

Threonine 7.15

Serine 5.82

Glutamate 18.20

Glycine 11.06 Alanine 6. 65

Proline 6. 33

Methionine 0. 84

Isoleucine 2. 64

Leucine 6. 12

Tyrosine 2. 27

Phenylalanine 3. 32

Lysine 5. 60

Histidine 2. 63

Arginine 6. 53

Proline 9. 10

5, IR spectrum analysis:

The infrared light of TP-3 was not absorbed at 1735 cm ^-1 , suggesting that TP-3 does not contain uronic acid. Example 3 Study on the immunological activity of the ear cream and its chemical components in vivo

1, sample source

槐耳清膏 is provided by Qidong Gaitian Pharmaceutical Co., Ltd. Crude polysaccharide TCP, homopolysaccharide TP-1, TP-2, TP-3, TP-4, low molecular weight components ΤΟ-1, ΤΟ-2, ΤΟ-3, ΤΟ-4 were prepared by Example 1.

2. Promote the proliferation of mouse spleen cells

1) Testing whether polysaccharides from the ear can stimulate the proliferation of mouse spleen cells

Method: The components of the ear cream (the crude polysaccharide TCP; the polysaccharides TP-1, TP-2, TP-3, TP-4; low molecular weight components ΤΟ-1, ΤΟ-2, ΤΟ-3, ΤΟ -4; total free protein fraction (TFP) and negative control drug dextran (Dextran) were co-cultured with mouse spleen cells. After 42 h, ³ H-TdR was spiked, and 48 h later, the cell harvester Filtermate harvester was used to detect it. Proliferation index. The result is shown in Figure 7.

Conclusion: From the results of ³ H-TdR, the polysaccharide components extracted from the ear cream (including crude polysaccharide TCP, polysaccharide components TP-1, TP-2, TP-3, TP-4) can stimulate mice. Splenocyte proliferation; low molecular weight components can not stimulate spleen cell proliferation except TO-3.

2) Auricular polysaccharide can stimulate mouse B cell proliferation, but not T cells

Method: Crude polysaccharide TCP, TP-1, TP-2, TP-3, TP-4 and negative control dextran (Dextran) (concentration gradient 100 μg/ml, 30 μg/ml, 10 μ g/ml ), the positive control lipopolysaccharide (LPS) was co-cultured with purified T and sputum lymphocytes, and ³ H-TdR was incorporated at 42 h. After 48 h, the proliferation index was measured using a cell harvester Filtermate harvester. The results are shown in Figure 8. Conclusion: Magnetic beads were sorted to obtain high-purity 1\ B cells. After incubation with polysaccharides of Auricularia auricula for 48h, the results of ³ H-TdR showed that polysaccharides from scorpion TP-1, TP-2, TP-3 and TP- 4. TCP stimulates B cells, not T cells. And this stimulation is dose dependent.

3) After auricular polysaccharide stimulates mouse spleen cells and human peripheral blood mononuclear cells (PBMC), T,

Β fine proportion change 4匕

Methods: Crude polysaccharide TCP, TP-1, ΤΡ-2, ΤΡ-3, ΤΡ-4 and dextran (Dextran) (concentration of 50 μg/ml), lipopolysaccharide (LPS) and mouse spleen The cells and human peripheral blood PBMC were co-cultured, and after 24 hours, the proportion of T and Β fine packets was detected by flow cytometry. The results are shown in Figure 9.

Conclusion: After the polysaccharides of 槐-1, ΤΡ-2, ΤΡ-3, ΤΡ-4 stimulated mouse spleen cells or human peripheral blood PBMC, the proportion of B cells was significantly up-regulated compared with the control drug dextran (Dextran). There was no significant change in the proportion of T cells.

4) Expression of CD69 on sputum and B cells after spleen cells stimulated by spleen cells

Method: Crude polysaccharide TCP, TP-1, TP-2, ΤΡ-3, ΤΡ-4 and control polysaccharide dextran (Dextran) (concentration 50 μg/ml), lipopolysaccharide (LPS) and small The mouse spleen cells were co-cultured, and after 6 hours, the expression of CD69 on the surface of T and sputum cells was detected by flow cytometry. The results are shown in Figure 10.

Conclusion: After 6 hours of stimulation of mouse spleen cells by polysaccharides, the B cells began to express CD69, but T cells did not change significantly.

3. Promote macrophage release NO

METHODS: Mouse peritoneal macrophages were cultured in vitro, and crude polysaccharide TCP, ear polysaccharide TP-1,

TP-2, ΤΡ-3, ΤΡ-4, low molecular weight components ΤΟ-1, ΤΟ-2, ΤΟ-3, ΤΟ-4, 槐粗 coarse total free protein part TFP and control polysaccharide dextran (Dextran), Lipopolysaccharide (LPS) was co-cultured, and after 48 hours, the content of NO in the cell culture supernatant was measured using a Griess Reagent kit, and the results are shown in Fig. 11.

Conclusion: The results of NO test showed that polysaccharides from Auricularia auricula could stimulate the secretion of NO by mouse macrophages, and the secretion amount was dose-dependent with the concentration of polysaccharide. There was no significant change in NO secretion after stimulation with low molecular weight components. Example 4 Inhibition of the growth of tumor angiogenesis endothelial cells by the polysaccharide protein of Odonata I. Experimental methods and steps:

1. Experimental materials

Cells: human tumor-derived vascular endothelial cells

Reagents: M199 medium, double antibody, fetal bovine serum (FBS), basic fibroblast growth factor (bFGF), gelatin (gelatin). Sample to be tested: TP-3: Ear polysaccharide protein-3

2. Experimental steps:

(1) Add 0.2% gelatin to a 96-well plate with a lance, 40 μl per well, and shake the hook. Incubate in the incubator for more than 30 minutes.

(2) Take out the 96-well plate, use a lance to suck out the gelatin, wash it with PBS, and put it on the ultra-clean bench.

(3) Prepare 20% FBS and add double-resistance M199 medium.

(4) Take the resuscitated human tumor-derived vascular endothelial cells, aspirate the culture solution, wash once with PBS, centrifuge, and resuspend with 10% serum M199 to adjust the cell concentration to 40000-50000/ml.

(5) Add the cells to a 96-well plate with a lance, 100 μl per well, and add an equal amount of PBS to the marginal wells. Put in the incubator.

(6) After 12 hours, a M199 medium containing 1%-5% FBS and 10-100 ng/ml bFGF was prepared.

(7) Take out the 96-well plate, aspirate the medium with a lance, add M199 and drug containing 1%-5% FBS and bFGF, 100 μl per well, and set the control group and the zero group. And add the samples to be tested, which are TCP, TP-1, TP-2, TP-3 and ΤΡ-4. Set 4 concentrations for each sample, respectively, l (^g / L, 10 (^g / L, 50 (^g / L, and 100 (^g / L. Then at 37 ° C, 5% C02) Continuous incubation.

(8) After 72 hours, the medium was aspirated, MTT (concentration 0.5 mg/ml, filter sterilization) was added, and incubation was carried out for 4-6 hours.

(9) Aspirate MTT, add 100 μl of DMSO, and measure OD by 570.

Second, the experimental conclusion:

Polysaccharide protein-3 (TP-3) has a significant inhibitory effect on the proliferation of tumor vascular endothelial cells. The results are shown in Figure 12. The values in the figure are shown as mean ± standard deviation. Each drug-administered group was compared with the control group, and analyzed by analysis of variance and post hoc T test. *P<0.05, indicating a significant difference between the two groups. **P<0.01, indicating a significant difference between the two groups. ***P< 0.001, indicating a significant difference between the two groups.

Claims

Rights request

1. A sophora polysaccharide protein. The monosaccharide composition of the polysaccharide protein is fucose, arabinose, galactose, glucose, xylose and mannose, and its mass ratio is 0.1: 1.0: 5.4: 4.4: 2.1: 7.0 .

2. The polysaccharide protein according to claim 1, characterized in that the weight average molecular weight of the polysaccharide protein is 7.0xl0 ⁵ -2.0xl0 ⁶ Da, preferably 1.69<10 ⁶ Da.

3. The polysaccharide protein according to claim 1 or 2, characterized in that the preparation method of the polysaccharide protein includes the following steps:

(1) Use the Sevage method to remove free proteins from an aqueous solution of Sophora odorifera bacterial plasm extract with a concentration of 2%-20% (mass/volume), preferably 8% (mass/volume), and collect the sugar portion;

(2) Add the sugar part obtained in step (1) to absolute ethanol to form a sugar solution with an alcohol concentration of 55%-70% (volume), preferably 60% (volume), and centrifuge to obtain a precipitate. ;

(3) Dissolve the precipitate obtained in step (2) with water, filter, concentrate, and dialyze the inner part of the collection bag; and

(4) Use ion exchange column chromatography to separate the portion in the bag collected in step (3), where the eluent used is a NaCl aqueous solution of 0.85 mol/L-1.5 mol/L, preferably 1.0 mol/L NaCl. aqueous solution.

4. The polysaccharide protein according to claim 3, characterized in that the preparation method of the polysaccharide protein further includes the step of dialyzing the product eluted from the NaCl aqueous solution to remove salt.

5. The polysaccharide protein according to claim 4, characterized in that the preparation method of the polysaccharide protein further includes the step of using a Sepharose CL-6B agarose gel column to separate and purify the product after dialysis and desalination.

6. A method for preparing the polysaccharide protein according to any one of claims 1 to 5, which method includes the following steps:

(1) Use the Sevage method to remove free proteins from the aqueous solution of Sophora odorifera bacterial plasm extract with a concentration of 2%-20% (mass/volume), preferably 8% (mass/volume), and collect the sugar portion;

(2) Add the sugar part obtained in step (1) to absolute ethanol to form a solution with an alcohol concentration of 55%-70% (volume), preferably 60% (volume), and centrifuge to obtain a precipitate;

7. The preparation method according to claim 6, characterized in that, the preparation method further It includes the step of dialyzing the product eluted from the NaCl aqueous solution to remove salt.

8. The preparation method according to claim 7, characterized in that the preparation method further includes the step of using a Sepharose CL-6B agarose gel column to separate and purify the product after dialysis and desalination.

9. The preparation method according to any one of claims 6 to 8, characterized in that the preparation method includes the following steps:

(1) Accurately weigh 300g of Sophora japonica fungus extract Sophora odorifera clear paste, add an appropriate amount of distilled water to dilute it to a dilute solution with a concentration of about 8% (mass/volume), and then use the Sevage method to remove free protein, repeat Operate 5 times to collect the sugar part;

(2) Slowly add absolute ethanol to the sugar part of Sophora fungus obtained in step (1) to form a sugar solution with an alcohol concentration of 60% (volume). After standing at 4°C for 24 hours, add it at 3000 Centrifuge at a speed of 10 rpm for 10 minutes to precipitate and obtain the precipitate;

(3) Weigh an appropriate amount of the precipitate obtained in step (2), dissolve it in 150 ml of distilled water, filter, and concentrate under reduced pressure to a volume of 50 ml; the inner part of the dialysis collection bag, and

(4) Use DEAE-52 ion exchange column chromatography to separate the part in the bag collected in step (3), and use 1.0 mol/L NaCl solution for elution. The elution part is dialyzed to remove salt after concentration. The dialysis process Three days for countercurrent water dialysis and two days for distilled water dialysis; and

(5) Use Sepharose CL-6B Sepharose column to separate and purify the product obtained in step (4) until it is detected as pure product by high performance liquid chromatography.

10. Use of the polysaccharide protein according to any one of claims 1 to 5 in the preparation of drugs for treating tumors.

11. A pharmaceutical composition, characterized in that the pharmaceutical composition contains Sophora polysaccharide protein and a pharmaceutically acceptable carrier, wherein the composition contains Sophora polysaccharide in a weight ratio of 0.01% to 99.95% Protein is used as the active ingredient, and based on the total weight of the Sophora polysaccharide protein, the Sophora polysaccharide protein contains 90wt%-100wt% of the Sophora polysaccharide protein according to any one of claims 1 to 5.