CN113402624A

CN113402624A - Extracellular polysaccharide and preparation method and application thereof

Info

Publication number: CN113402624A
Application number: CN202010185241.3A
Authority: CN
Inventors: 吴建勇; 黄永德; 李垄清; 宋昂芯
Original assignee: Shenzhen Research Institute HKPU
Current assignee: Shenzhen Research Institute HKPU
Priority date: 2020-03-17
Filing date: 2020-03-17
Publication date: 2021-09-17
Anticipated expiration: 2040-03-17
Also published as: CN113402624B

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to an extracellular polysaccharide and a preparation method and application thereof. The exopolysaccharide provided by the invention comprises: a main chain formed by glucose and mannose, and a side chain formed by galactose connecting the main chain; calculated according to molar ratio, the monosaccharide ratio of mannose, glucose and galactose is (3.5-4.5): (6.5-7.5): 0.8-1.5). The preparation method of the extracellular polysaccharide comprises the following steps: carrying out liquid fermentation on the strain Cs-HK1, and collecting fermentation liquor; carrying out alcohol precipitation treatment and deproteinization treatment on the fermentation liquor to prepare a polysaccharide crude extract; dissolving the crude polysaccharide extract with water, and performing gel column chromatography. The method is simple and convenient, is simple to operate and is easy for large-scale mass production. Tests show that the extracellular polysaccharide prepared by the method has good anti-inflammatory activity and shows good application prospect of the medicine.

Description

Extracellular polysaccharide and preparation method and application thereof

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to an extracellular polysaccharide, and a preparation method and application thereof.

Background

Inflammation is a defense response of the body to homeostasis and fine-tuning, including a complex pathoprotective response to tissue injury, infection, or other stimuli resulting from a tightly controlled inflammatory response. However, excessive inflammatory responses may cause abnormalities in the immune regulatory system of the human body, resulting in many harmful chronic inflammatory diseases in the host body. Since inflammation is considered to be closely related to various diseases, research on potential mechanisms of inflammation and anti-inflammatory active drugs has become a research hotspot of those skilled in the art

Disclosure of Invention

The invention mainly aims to screen anti-inflammatory active drugs with good drug application prospect, and the specific technical scheme is as follows:

to achieve the object, in a first aspect, the present invention provides an exopolysaccharide comprising: a main chain formed by glucose and mannose, and a side chain formed by connecting galactose with the main chain; calculated according to molar ratio, the monosaccharide ratio of mannose, glucose and galactose is (3.5-4.5): (6.5-7.5): 0.8-1.5).

In a second aspect, the invention provides a preparation method of exopolysaccharide, which comprises the following steps:

providing a strain Cs-HK1, wherein the strain Cs-HK1 belongs to the genus Tolypocladium china, is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, has the preservation address of No. 3 of Xilu No. 1 of Beijing Korean district, the preservation number of CGMCC No.6004 and the preservation time of 16 days at 4 months of 2012;

carrying out liquid fermentation on the strain Cs-HK1, and collecting fermentation liquor;

carrying out alcohol precipitation treatment and deproteinization treatment on the fermentation liquor to obtain a polysaccharide crude extract;

and dissolving the polysaccharide crude extract by using water, and performing gel column chromatography to obtain the exopolysaccharide.

In a third aspect, the invention provides the application of the exopolysaccharide or the exopolysaccharide prepared by the preparation method in preparing a functional preparation with anti-inflammatory activity.

The exopolysaccharide provided by the invention comprises glucose, mannose and galactose with a specific monosaccharide molar ratio, wherein a main chain is formed by the glucose and the mannose, and a side chain is formed by connecting the galactose with the main chain.

The preparation method of the extracellular polysaccharide provided by the invention is mainly obtained by carrying out liquid fermentation by taking the strain Cs-HK1 as a fermentation strain and then purifying and separating the fermentation liquor step by step. The method is simple and convenient, simple to operate and easy for large-scale mass production.

Based on the good anti-inflammatory activity of the exopolysaccharide, the strain, the exopolysaccharide or the exopolysaccharide prepared by the preparation method can be applied to preparing functional preparations with anti-inflammatory activity, including but not limited to medicines, health-care foods, cosmetics and the like.

Drawings

FIG. 1 shows the results of GPC measurement of crude polysaccharide extract EPS80 in example 1;

FIG. 2 shows the elution procedure of DEAE-Sephadex A-25 column and the absorbance detection result of the eluate in example 1;

FIG. 3 shows the results of absorbance measurements of eluates from superdex 200pg columns in example 1;

FIG. 4 shows the inhibitory effect of EPS80-1 on the activation of inflammatory signals of NF-. kappa.B expressed by THO-1 cells induced by LPS;

FIG. 5 shows the inhibitory effect of EPS80-1 on the expression of Nitric Oxide (NO) by LPS-induced THP-1 cells;

FIG. 6 shows the inhibitory effect of EPS80-1 on the expression of IL-1. beta. by LPS-induced THP-1 cells;

FIG. 7 shows the inhibitory effect of EPS80-1 on IL-10 expression by LPS-induced THP-1 cells;

FIG. 8 shows the result of the cytotoxic activity test of EPS 80-1.

Detailed Description

In order to make the technical problems, technical solutions and advantageous effects to be solved by the present invention more clearly apparent, the present invention is further described in detail below with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

The mass of each component mentioned in the description of the embodiment of the present invention may not only refer to the specific content of each component, but also represent the proportional relationship of the mass between each component, therefore, it is within the scope of the disclosure of the description of the embodiment of the present invention to scale up or down the content of each component of the composition according to the description of the embodiment of the present invention. Specifically, the mass described in the description of the embodiment of the present invention may be a unit of weight known in the medical field such as μ g, mg, g, kg, etc.

In the description of the present invention, it is to be understood that the terms "first", "second" and "third" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implying any number of technical features indicated. Thus, features defined as "first", "second", "third" may explicitly or implicitly include one or more of the features. In the description of the present invention, "at least two" means two or more unless specifically limited otherwise.

An exopolysaccharide comprising: a main chain formed by glucose and mannose, and a side chain formed by galactose connecting the main chain; calculated according to molar ratio, the monosaccharide ratio of mannose, glucose and galactose is (3.5-4.5): (6.5-7.5): 0.8-1.5).

The exopolysaccharide provided by the embodiment of the invention comprises glucose, mannose and galactose with a specific monosaccharide molar ratio, wherein a main chain is formed by the glucose and the mannose, and a side chain is formed by connecting the galactose with the main chain.

As an embodiment, the exopolysaccharide is composed of at least two structural units comprising: a first glucose, a second glucose, a third glucose, a first mannose, a second mannose and a first galactose, the first glucose being linked to the first mannose by a 1, 5-glycosidic bond, the first mannose being linked to the second mannose by a 1, 5-glycosidic bond, the second mannose being linked to the second glucose by a 1, 6-glycosidic bond, the second glucose being linked to the third glucose by a 1, 6-glycosidic bond, and the first galactose being linked to the first glucose by a 1, 3-glycosidic bond. In some embodiments, the third glucose is linked to the first glucose by a 1, 6-glycosidic bond between two adjacent building blocks.

It is to be noted that the first glucose, the second glucose and the third glucose are only used for descriptive purposes to distinguish the connection relationship among the plurality of glucose, and the configurations of the first glucose, the second glucose and the third glucose may be the same or different, and the first mannose and the second mannose are the same.

As an embodiment, the molecular structure of the exopolysaccharide includes:

wherein n represents the number of the structural units and is a positive integer between 300 and 350;

beta-D-Galp is beta-D-galactose;

6) -beta-D-Glcp- (1 is C)₆And C₁beta-D-glucose which forms glycosidic linkages with adjacent monosaccharides, respectively;

[5)-β-D-Manf-(1]₂is C₅And C₁beta-D-mannose which forms glycosidic bonds with adjacent monosaccharides, respectively, and the number of repetitions of the beta-D-mannose is 2;

[6)-α-D-Glcp-(1]₂is C₆And C₁alpha-D-glucose which forms glycosidic bonds with adjacent monosaccharides, respectively, and the number of repetitions of the alpha-D-glucose is 2.

In a specific embodiment, the molecular structure of the exopolysaccharide is:

wherein n represents the number of the structural units and is a positive integer between 350 and 400; → indicates the monosaccharide attachment direction.

In a further embodiment, the extracellular polysaccharide has a weight average molecular weight of 300-400 kDa. In a specific embodiment, the weight average molecular weight of the exopolysaccharide is 360 kDa.

In one embodiment, the extracellular polysaccharide is metabolically produced by a strain Cs-HK1, wherein the strain Cs-HK1 belongs to the genus Tolypocladium, Latin is named Tolypocladium sinense, and is preserved in the China general microbiological culture Collection center, the preservation address is No. 3 of Xilu No. 1 of Beijing Kogyo-sunward area, the preservation number is CGMCC No.6004, and the preservation time is 16 days 4 months 2012.

In some embodiments, the strain Cs-HK1 is selected from fruiting bodies of wild Cordyceps sinensis grown in the Tibet plateau, and the strain is selected by a process which is generally performed in the art, but is not particularly limited thereto.

Based on the technical scheme, the embodiment of the invention also provides a preparation method and application of the extracellular polysaccharide.

Correspondingly, the preparation method of the exopolysaccharide comprises the following steps:

s01, providing a strain Cs-HK1, wherein the strain Cs-HK1 belongs to the genus Tolypocladium china, is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, has the preservation address of No. 3 West Lu No. 1 of the Kyowa area of Beijing, has the preservation number of CGMCC No.6004, and has the preservation time of 16 days at 4 months and 16 days in 2012;

s02, carrying out liquid fermentation on the strain Cs-HK1, and collecting fermentation liquor;

s03, carrying out alcohol precipitation treatment and deproteinization treatment on the fermentation liquor to obtain a polysaccharide crude extract;

s04, dissolving the polysaccharide crude extract with water, and performing gel column chromatography to obtain the exopolysaccharide.

The preparation method of the extracellular polysaccharide provided by the embodiment of the invention takes the strain Cs-HK1 as a fermentation strain to carry out liquid fermentation, and the extracellular polysaccharide is prepared by purifying and separating the fermentation liquor step by step. The method is simple and convenient, is simple to operate and is easy for large-scale mass production.

Specifically, in step S01, the species and deposit information of strain Cs-HK1 are the same as those of strain Cs-HK1 described above, which are not described herein again.

In step S02, liquid fermentation is carried out on the strain Cs-HK1, so that the strain Cs-HK1 is metabolized to form the extracellular polysaccharide in the fermentation process, and the extracellular polysaccharide is enriched in the fermentation liquid.

In one embodiment, in the step of subjecting the strain Cs-HK1 to liquid fermentation, the strain Cs-HK1 is inoculated into a fermentation medium and fermented at 18 ℃ to 35 ℃ for 6 to 7 days. Under the fermentation temperature and the fermentation time, the strain Cs-HK1 is favorable for metabolizing the extracellular polysaccharide on a large scale, and the biosynthesis efficiency is improved.

The fermentation medium used in the fermentation process directly affects the synthesis of metabolites, and as an embodiment, comprises per 1 liter of said fermentation medium: 30-40g glucose, 5-10g peptone, 0.8-1.2g KH₂PO₄、0.4-0.6g MgSO₄·7H₂O and 5-15g of yeast extract, the solvent being water. In a specific embodiment, every 1 liter of the fermentation medium comprises: 40g glucose, 5g peptone, 1g KH₂PO₄、0.5g MgSO₄·7H₂O and 10g yeast extract in water. By using the optimized culture medium, the biomass of the cordyceps mycelium CS-HK1 is increased, and the extracellular polysaccharide synthesis efficiency of the strain Cs-HK1 is improved.

The specific process of fermentation can refer to the routine operation in the field, for example, the fermentation can be carried out in a large-scale fermentation tank, and the fermentation can also be carried out on a shaking table. In some embodiments, the fermentation is conducted on a shaker set at a speed of 200 rpm.

After the liquid fermentation of the strain Cs-HK1, the fermented product was centrifuged to collect the fermented liquid. In some embodiments, when the fermentation broth is centrifuged, the centrifugation is carried out at 12000rpm for 20 minutes, which is beneficial to efficiently enrich the extracellular polysaccharide formed by the metabolism of the strain Cs-HK1 in the fermentation broth.

In step S03, the fermentation broth is subjected to alcohol precipitation and deproteinization, so that the extracellular polysaccharide of the target metabolite in the fermentation broth is separated from other small molecular impurities and proteins, to obtain a crude polysaccharide extract with high extracellular polysaccharide content.

As an embodiment, the step of subjecting the fermentation broth to alcohol precipitation and deproteinization comprises:

s031, according to the proportion that the final volume percentage concentration of ethanol is 35% -40%, mixing the fermentation broth with ethanol, carrying out first alcohol precipitation treatment, centrifuging to obtain a first supernatant;

s032, mixing the first supernatant with ethanol according to the proportion that the final volume percentage concentration of the ethanol is 78% -84%, performing second alcohol precipitation treatment, and centrifuging to obtain a second supernatant;

s033, providing a Sevag reagent, mixing the second supernatant with the Sevag reagent, and performing deproteinization.

By the method, the content of the extracellular polysaccharide in the crude polysaccharide extract can be improved to the maximum extent, and the anti-inflammatory activity of the extracellular polysaccharide can be improved.

For the specific operations of the first alcohol precipitation process in step S031 and the second alcohol precipitation process in step S032, it can be referred to the conventional techniques in the art, such as mechanical stirring.

The Sevag reagent of step S033 and the specific procedure for deproteinization can be determined by conventional procedures in the art, such as, in some embodiments, the Sevag reagent is a 4:1, chloroform-n-butanol mixed solution; as some examples, the step of performing a deproteinization process comprises: and fully shaking the mixed solution of the second supernatant and the Sevag reagent, collecting the lower aqueous phase solution after the middle layer of the mixed solution is not turbid, and dialyzing the aqueous phase solution for 48 hours by adopting 3500mw of semipermeable membrane and distilled water.

In step S04, the crude polysaccharide extract is dissolved in water, and subjected to gel column chromatography, and further purification and separation of the target metabolite are achieved by using the molecular sieve effect of the gel column chromatography, so as to obtain the extracellular polysaccharide with high purity.

As an embodiment, the step of performing gel column chromatography comprises:

s041, preparing an aqueous solution of a polysaccharide crude extract, loading the aqueous solution of the polysaccharide crude extract on a DEAE-Sephadex column, eluting by using distilled water and an aqueous solution of sodium chloride, and collecting fractions;

s042, loading the fraction on a superdex 200pg column, and eluting by using ammonium bicarbonate aqueous solution.

In some embodiments, in step S041, the concentration of the crude polysaccharide extract in the aqueous solution of crude polysaccharide extract is 200-250mg/mL, the molar concentration of sodium chloride in the aqueous sodium chloride solution is 0.1-1M, and elution is performed at a flow rate of 0.3-0.5 mL/min.

In some embodiments, in step S042, when the elution is performed using an aqueous ammonium bicarbonate solution, the elution is performed at a flow rate of 0.3 to 0.5mL/min, and the molar concentration of ammonium bicarbonate of the aqueous ammonium bicarbonate solution is 0.1 to 0.3M.

The extracellular polysaccharide prepared by the preparation method has obvious inhibition effects on the activation of NF-kB inflammatory signals expressed by THO-1 cells induced by LPS, the expression of Nitric Oxide (NO) and the expression of IL-1 beta and IL-10 through in-vitro anti-inflammatory activity tests of cells, shows good application prospect of medicaments, and can be applied to the preparation of functional preparations with anti-inflammatory activity, including but not limited to medicaments, health-care foods, cosmetics and the like.

Correspondingly, the application of the strain, the exopolysaccharide or the exopolysaccharide prepared by the preparation method in preparing a functional preparation with anti-inflammatory activity.

Wherein, the functional preparation comprises but is not limited to drugs, health-care food, cosmetics and the like.

In order that the above implementation details and operation of the present invention can be clearly understood by those skilled in the art, and the advanced performance of the exopolysaccharide, the preparation method and the application thereof in the embodiment of the present invention can be obviously reflected, the implementation of the present invention is illustrated by the following examples.

Example 1

The embodiment provides an exopolysaccharide EPS80-1, and the preparation process specifically comprises the following steps:

s11, fermentation strain Cs-HK1

1) Taking a strain Cs-HK1, selecting a wild cordyceps sinensis fruiting body growing in Qinghai-Tibet plateau by a separation sieve, storing the wild cordyceps sinensis fruiting body in China general microbiological culture Collection center at 16 months 4 in 2012, wherein the storage address is No. 3 of Xilu No. 1 Bichen of the Korean district in Beijing, and the preservation number is CGMCC No. 6004;

2) according to the ratio of 40g/L glucose, 5g/L peptone and 1g/L KH₂PO₄、0.5g/L MgSO₄·7H₂O and yeast extract 10g/L, glucose, peptone, KH₂PO₄、MgSO₄·7H₂Dissolving O and yeast extract in water to obtain fermentation culture medium;

3) inoculating strain Cs-HK1 into fermentation medium, fermenting at 20 deg.C on shaker at rotation speed of 200rpm for 6-7 days, centrifuging fermentation product at 12000rpm for 20min, and collecting fermentation liquid.

S12, preparation of polysaccharide crude extract EPS80

1) Adding ethanol into the fermentation liquid prepared in the step S11 according to the proportion that the final volume percentage concentration of the ethanol is 40%, carrying out alcohol precipitation, centrifuging, and collecting supernatant;

2) adding ethanol into the supernatant prepared in the step 1) according to the proportion that the final volume percentage concentration of the ethanol is 80%, carrying out alcohol precipitation, centrifuging, and collecting the supernatant;

3) preparing a chloroform-n-butanol mixed solution with a volume ratio of 4:1, adding the chloroform-n-butanol mixed solution into the supernatant prepared in the step 2), sufficiently shaking, centrifuging, removing an upper organic phase containing impurities such as protein and the like, and collecting a lower aqueous phase containing fungal polysaccharide; dialyzing the water phase for 48h, and freeze-drying to obtain polysaccharide crude extract EPS 80. The molecular weight distribution of the polysaccharide crude extract EPS80 was determined by GPC, and the results are shown in FIG. 1.

S13, separating and purifying extracellular polysaccharide EPS80-1

Re-dissolving the polysaccharide crude extract EPS80 in water to form a polysaccharide crude extract water solution with the concentration of 250 mg/mL;

loading the aqueous solution of polysaccharide crude extract on a DEAE-Sephadex A-25 column, sequentially eluting with distilled water and 0.1-1M sodium chloride aqueous solution at a flow rate of 0.5mL/min step by step, collecting eluate, detecting polysaccharide content in each fraction with anthrone reagent, dividing the eluate into 3 fractions according to polysaccharide content, which are respectively marked as EPS80-1, EPS80-2 and EPS80-3, as shown in FIG. 2;

loading fraction EPS80-1 onto superdex 200pg column, eluting with 0.3M ammonium bicarbonate water solution at flow rate of 0.3mL/min, collecting eluate, and analyzing sugar content in the eluate with anthrax method, the result is shown in FIG. 3; and (3) combining, concentrating, dialyzing and freeze-drying the main polysaccharide components to obtain the exopolysaccharide EPS 80-1.

Taking the exopolysaccharide EPS80-1 prepared in this example for structural characterization, the specific process is briefly described as follows:

(1) molecular weight analysis

The molecular weight distribution and uniformity of EPS80-1 were analyzed by High Performance Gel Permeation Chromatography (HPGPC). HPGPC was performed using a Waters-HPGPC instrument, a Waters-1515 isometric HPLC pump, a Waters-2414 Refractive Index (RI) detector, and a Waters-2998 UV detector. HPGPC (MW vs. elution time) was calibrated with dextran standards of 1-670kDa (Sigma-Aldrich, USA).

(2) Chemical composition analysis

The total carbohydrate content was determined by the anthrone assay (using glucose as standard) and the protein content by the Lowry method (using Bovine Serum Albumin (BSA) as standard). Monosaccharide composition was analyzed by 1-phenyl-3-methyl-5-pyrazolone (PMP) -high performance liquid chromatography as described previously. Briefly, EPS samples (1-2 mg) were completely hydrolyzed with 2M trifluoroacetic acid (TFA) at 110 ℃ for 4 h. The hydrolysate was dried under vacuum and then derivatised with 450. mu.l PMP solution (0.5M in methanol) and 450. mu.l 0.3M NaOH at 70 ℃ for 30 min. The reaction was stopped by neutralization with 450. mu.l of 0.3M HCl, followed by extraction with chloroform (1mL, 3 times), and the extract was subjected to HPLC analysis on an Agilent 1100 instrument using a G1312A cartridge pump and an ultraviolet detector using an Agilent ZORBAX Eclipse XDB-C18 column (5 μ M, 4.6X 150 mm).

(3) Partial acid hydrolysis and methylation analysis

Dried EPS80-1(5 mg) was pre-dissolved in 2.5ml of anhydrous dimethyl sulfoxide (DMSO) and stirring was continued for 3 hours. Anhydrous sodium hydroxide (30mg) was added under nitrogen and stirred for 30 minutes, and methyl iodide (0.8 ml) was added slowly in an ice bath under nitrogen and stirred in the dark. The mixture was left at room temperature for 1 hour.

The reaction was stopped by the addition of 2.5mL of distilled water and excess methyl iodide was removed by evaporation under vacuum at 40 ℃. A partially methylated sample was extracted with dichloromethane. The dichloromethane solution was washed three times with deionized water to remove impurities. The partially methylated EPS80-1 was evaporated to dryness in vacuo at room temperature in a rotary evaporator. The methylation process was repeated three times to completion. The partially methylated EPS80-1 was further hydrolyzed with 2M TFA (1mL) in a sealed tube at 110 ℃ for 6h, and excess TFA was removed with a stream of nitrogen in a boiling water bath. The dried hydrolysate was redissolved in 1mL of ammonia saturated water and reduced with excess NaBH4 for 12h at room temperature. Excess NaBH4 was reacted with acetic acid (until no bubbles appeared) and the boric acid formed was removed by co-distillation with methanol. The dried residue was acetylated for 2 hours with 1ml of acetic anhydride in a sealed tube at 110 ℃ to form partially methylated acetaldol (PMAA). PMAA was evaporated to dryness in vacuo, redissolved in chloroform and washed three times with water.

The product was analyzed by GC-MS on a fused silica capillary column (30 mm. times.0.25 mm ID, Agilent HP-5MS) using Agilent 6890N gas chromatography and 5975 vl-MSD. The column temperature was fixed at 100 ℃ for 3 minutes, then increased to 250 ℃ at 3 ℃/min and fixed at 250 ℃ for 10 minutes. The syringe and detector were fixed at 280 ℃ and 250 ℃ respectively.

(4) Nuclear magnetic resonance and infrared spectroscopy

The nmr was performed using a brook AV400 instrument. For NMR spectroscopy, a sample of EPS80-1 (30mg) was co-evaporated twice with heavy water by freeze-drying and then dissolved in 600. mu.l of heavy water. The infrared spectroscopy (IR) of EPS80-1 was performed at room temperature on an Avatar 360 FTIR (Thermo Nicolet, Cambridge, UK) with a resolution of 4cm-1 in the range of 4000-1 cm-1. All spectra were baseline corrected.

The molecular structure of the exopolysaccharide EPS80-1 is measured as follows:

the monosaccharide ratio (calculated by molar ratio) is shown in table 1, which indicates that the exopolysaccharide EPS80-1 prepared in this example is composed of two or more structural units, each structural unit includes: 3 first glucose, 2 mannose and 1 galactose; wherein, the head end of the structural unit is provided with 1 glucose, the glucose is connected with 1 mannose through a 1, 5-glycosidic bond, the mannose is continuously connected with the next mannose through a 1, 5-glycosidic bond, the next mannose is connected with 1 glucose through a 1, 6-glycosidic bond, the glucose is continuously connected with the next glucose through a 1, 6-glycosidic bond, and the galactose is connected with the first glucose through a 1, 3-glycosidic bond to form a side chain.

TABLE 1

The anti-inflammatory activity of the exopolysaccharide EPS80-1 prepared in example 1 was tested, wherein the anti-inflammatory effect was comprehensively evaluated by using LPS to induce THP-1 cells as inflammatory cells, and using NO kit and ELISA kit to test the expression level of inflammatory factors such as IL-10, 1L-1 beta, NO, etc. in the cells, and the inhibition rate of EPS80-1 against the inflammatory factors of the cells.

The inflammatory factor inhibition rate was the rate at which the expression level of inflammatory factors was reduced by inflammatory cells with EPS80-1 intervention compared to the group of inflammatory cells without drug intervention.

1. Test of the Effect of EPS80-1 on the activation of inflammatory signaling pathway NF-. kappa.B expression by LPS-induced THO-1 cells

Providing THP-1 cells loaded with NF-. kappa.B inflammatory signal activation indicator enzyme (which is an alkaline phosphatase SEAP), the activity of which was assessed using Quanti-Blue reagent (InvivoGen);

THP-1 cells were plated in 96-well plates (2X 10)⁵/well) for 48h, and Lipopolysaccharide (LPS) (Sigma-Aldrich, Shanghai, China) at a final concentration of 1. mu.g/mL was added to induce an inflammatory response in the cells; then, adding EPS80-1 with different concentrations into a part of culture wells added with LPS to induce cells to generate inflammatory response, wherein the part without any treatment on THP-1 cells is a control group, the part without intervention of EPS80-1 is an LPS group, the part with addition of EPS80-1 is an EPS80-1 group, and the EPS80-1 concentration in the EPS80-1 group is 1,5, 10 and 25 mug/mL respectively;

after the culture, the supernatant of the cells was added to a Quanti-Blue reagent at a ratio of 1:4, and the absorbance was measured at 625 nm.

As shown in the results of FIG. 4, the EPS80-1 group can effectively inhibit the activation of NF-kB inflammatory signal pathways of THP-1 cells induced by LPS, and the EPS80-1 group with the concentration of 25 mug/mL has the most obvious inhibition effect on the activation of NF-kB inflammatory pathways of THP-1 cells induced by LPS.

2. Test of the Effect of EPS80-1 on the expression of Nitric Oxide (NO) by LPS-induced THP-1 cells

THP-1 cells were plated in 96-well plates (1X 10)⁵Per well), wherein the culture medium of the control group is RPMI1640 culture medium, the culture medium of the EPS80-1 group is RPMI1640 culture medium containing LPS and EPS80-1 with different concentrations, the concentration of the EPS80-1 group is 1,5, 10 and 25 mu g/mL respectively, and the culture medium of the LPS group is standard culture medium containing LPS;

after 48h of culture, 100. mu.L of cell culture supernatant was taken and added with NaNO₂The supernatant was analyzed for nitrite content using a colorimetric method as a standard. The method specifically comprises the following steps: the supernatant was mixed with Griess reagent in equal volume and after 20min, absorbance was measured at 540 nm. Wherein, the Griess reagent is formed by dissolving a mixed solution of N- (1-naphthyl) ethylenediamine (the mass volume percentage is 0.1%) and sulfanilamide (the mass volume percentage is 1%) in phosphoric acid with the mass percentage of 5%.

As shown in the results of FIG. 5, the EPS80-1 group can effectively inhibit the expression of NO by the THP-1 cells induced by LPS, and the expression level of NO in the EPS80-1 group with the concentration of 25 mug/mL is 76.7% lower than that in the LPS group, which indicates that the inhibition effect of the EPS80-1 group with the concentration of 25 mug/mL on the expression of NO by the cells is most significant.

3. Test of the Effect of EPS80-1 on IL-1 β and IL-10 expression by LPS-induced THP-1 cells

THP-1 cells were plated in 96-well plates (1X 10)⁵/well), the cells were washed with Phosphate Buffered Saline (PBS) to the remaining culture wells except for the control group, the medium was changed to RPMI1640 medium (without FBS), LPS was then added to stimulate the inflammatory reaction of THP-1, and EPS80-1 was added to a part of the culture wells at final concentrations of 1,5, 10, and 25. mu.g/mL, respectively, wherein the LPS group without the intervention of EPS80-1 was used as the LPS group, and EPS80-1 group was used as the case with EPS 80-1.

After 48h incubation, cytokine concentrations in the supernatants were determined using a polysite enzyme-linked immunosorbent assay (ELISA) kit (R & D Systems) (Sigma-Aldrich, Shanghai, China).

As shown in the results of FIG. 6 and FIG. 7, both EPS80-1 group were able to effectively inhibit the expression of IL-1 β and IL-10 by LPS-induced THP-1 cells, wherein the expression level of IL-1 β was 92.4% lower in EPS80-1 group at a concentration of 25 μ g/mL compared to LPS group, and the expression level of IL-10 was 74.3% lower in LPS group, indicating that the inhibition effect of IL-1 β and IL-10 expression by cells was most significant in EPS80-1 group at a concentration of 25 μ g/mL.

In conclusion, the exopolysaccharide EPS80-1 provided by the embodiment of the invention can effectively inhibit the THP-1 cell inflammatory reaction induced by LPS, and the exopolysaccharide EPS80-1 has good anti-inflammatory activity.

To evaluate the toxic activity effect of EPS80-1 on THP-1 cells, the extracellular polysaccharide EPS80-1 prepared in example 1 was subjected to a cytotoxic activity test using MTT method as follows, which comprises:

cells were first cultured in 96-well flat-bottom tissue culture plates (105 cells/well) for 24 hours to obtain stable growth, and then stimulated with different concentrations of EPS80-1 for 48 h. After 48h incubation, analysis was performed by MTT method (methylthiazolyl tetrazolium salt, Sigma). Succinate dehydrogenase in the mitochondria of living cells reduces exogenous MTT to water-insoluble blue and purple crystals deposited inside the cells, whereas dead cells do not. After 4h incubation of MTT, dimethyl sulfoxide (DMSO) was added to dissolve MTT and the supernatant was shaken up in a plate shaker. The Optical Density (OD) at 490nm was then determined using a microplate reader.

FIG. 8 shows the result of the cytotoxicity test of EPS80-1, and the result shows that when the concentration of EPS80-1 is as high as 100 μ g/mL, the survival rate of THP-1 cells is always close to 90%, which indicates that under the condition of high dose, EPS80-1 has no obvious toxicological effect on cells, has good safety performance and potential drug development value, and can be applied to the preparation of anti-inflammatory drugs.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.

Claims

1. An exopolysaccharide, comprising: a main chain formed by glucose and mannose, and a side chain formed by galactose connecting the main chain; calculated according to molar ratio, the monosaccharide ratio of mannose, glucose and galactose is (3.5-4.5): (6.5-7.5): 0.8-1.5).

2. Exopolysaccharide according to claim 1, characterized in that it is composed of at least two structural units comprising: a first glucose, a second glucose, a third glucose, a first mannose, a second mannose and a first galactose, the first glucose being linked to the first mannose by a 1, 5-glycosidic bond, the first mannose being linked to the second mannose by a 1, 5-glycosidic bond, the second mannose being linked to the second glucose by a 1, 6-glycosidic bond, the second glucose being linked to the third glucose by a 1, 6-glycosidic bond, and the first galactose being linked to the first glucose by a 1, 3-glycosidic bond.

3. Exopolysaccharide according to claim 2, characterized in that its molecular structure comprises:

wherein n represents the number of the structural units and is a positive integer between 350 and 400;

beta-D-Galp is beta-D-galactose; /

4. Exopolysaccharide according to any one of claims 1 to 3, which is metabolised by the strain Cs-HK1, wherein the strain Cs-HK1 belongs to the genus Campylobacter, China, and is preserved in the China general microbiological culture Collection center with the preservation number of CGMCC No.6004 and the preservation time of 16/4/2012; and/or

The weight average molecular weight of the extracellular polysaccharide is 300-400 kDa.

5. The preparation method of the exopolysaccharide is characterized by comprising the following steps:

providing a strain Cs-HK1, wherein the strain Cs-HK1 belongs to the genus Tolypocladium, is preserved in China general microbiological culture collection center with the preservation number of CGMCC No.6004 and the preservation time of 2012, 4 months and 16 days;

6. The method of claim 5, wherein the step of subjecting the strain Cs-HK1 to liquid fermentation comprises inoculating the strain Cs-HK1 into a fermentation medium and fermenting at 18 ℃ to 35 ℃ for 6 to 7 days.

7. The method according to claim 6, wherein the fermentation medium comprises, per 1 liter: 30-40g glucose, 5-10g peptone, 0.8-1.2g KH₂PO₄、0.4-0.6g MgSO₄·7H₂O and 5-15g of yeast extract, the solvent being water.

8. The method according to any one of claims 5 to 7, wherein the step of subjecting the fermentation broth to alcohol precipitation and deproteinization comprises:

mixing the fermentation liquor with ethanol according to the proportion that the final volume percentage concentration of the ethanol is 35-40%, carrying out first alcohol precipitation treatment, and centrifuging to obtain first supernatant;

mixing the first supernatant with ethanol according to the proportion that the final volume percentage concentration of the ethanol is 78-84%, carrying out second alcohol precipitation treatment, and centrifuging to obtain a second supernatant;

providing a Sevag reagent, mixing the second supernatant with the Sevag reagent, and performing deproteinization treatment.

9. The method according to any one of claims 5 to 7, wherein the step of performing gel column chromatography comprises:

preparing a polysaccharide crude extract aqueous solution, loading the polysaccharide crude extract aqueous solution on a DEAE-Sephadex column, eluting by using distilled water and a sodium chloride aqueous solution, and collecting fractions;

the fractions were loaded onto a superdex 200pg column and eluted with aqueous ammonium bicarbonate.

10. Use of exopolysaccharide according to any one of claims 1 to 4 or exopolysaccharide obtained by the process according to any one of claims 5 to 9 for the preparation of a functional preparation having anti-inflammatory activity.