Mannan oligosaccharide with anti-inflammatory activity and application thereof

Abstract

The invention discloses anti-inflammatory activity of a group of mannodisaccharide and mannotriose containing beta-1, 2 glycosidic bonds, which are obtained by a microbial fermentation or enzymolysis method. Wherein the mannodisaccharide (M2-beta-1, 2) with the concentration of 1000 to 2000 [mu] g / mL and the mannotriose (M3-beta-1, 2) with the concentration of 250 to 500 [mu] g / mL can significantly inhibit LPS-induced RAW264.7 cells from secreting NO and inflammatory factors TNF-alpha, IL-6 and IL-1beta, and the inhibition effect is gradually enhanced along with the increase of the concentration. The mannan oligosaccharide containing the beta-1, 2 glucosidic bond is proved to have good anti-inflammatory activity, and can be used as an anti-inflammatory compound to be applied to health care product development, animal feed, anti-inflammatory drug additives and the like.

Description

Technical Field

[0001] The present invention belongs to the technical field of health products, and in particular relates to an anti-inflammatory activity evaluation method and application of manno-oligosaccharides containing beta-1,2 glycosidic bonds obtained by microbial fermentation. Background Art

[0002] Mannosyl-oligosaccharides (MOS) have the functions of enhancing animal immunity, regulating sugar and lipid metabolism, maintaining intestinal health, anti-inflammation, and anti-oxidation. It has been widely used as an additive in food and animal feed both at home and abroad. Masahisa et al. (Ibuki M, Fukui K, Kanatani H, Mine Y: Anti-inflammatory effects of mannanase-hydrolyzed copra meal in a porcine model of colitis. J Vet Med Sci 2014, 76 (5): 645-651.) studied the anti-inflammatory activity of mannobiose (purity 67.8%) containing β-1,4 glycosidic bonds obtained by mannanase hydrolysis of coconut meal. In the dextran sulfate sodium (DSS)-induced colitis pig model, MOS could attenuate the secretion of inflammatory cytokines TNF-α and IL-6, and also attenuated the expression levels of IL-1β, IL-6, IL-17 and TNF-α, proving that mannobiose exerts anti-inflammatory activity in pigs.

[0003] The study by Szilamer et al. (Ferenczi S, Szegi K, Winkler Z, Barna T, Kovacs KJ: Oligomannan prebiotic attenuates immunological, clinical and behavioral symptoms in mouse model of inflammatory bowel disease. Sci Rep 2016, 6: 34132.) showed that yeast cell wall MOS could significantly inhibit the production of IL-1α, IL-1β, IL-6 and granulocyte colony stimulating factor (G-SCF) in the in vitro model of mouse macrophages RAW264.7 induced by lipopolysaccharide (LPS), and had anti-inflammatory activity. Mannosyl oligosaccharides containing β-1,2 glycosidic bonds (β-1,2 glycosidic bond mannose disaccharide M2-β-1,2 and β-1,2 glycosidic bond mannose trisaccharide M3-β-1,2) have been reported in recent years to have significant immunoenhancing or immunomodulatory activities, but their anti-inflammatory effects have not been reported. Summary of the invention

[0004] In view of the above situation, it is critical to study the anti-inflammatory activity of β-1,2 glycosidic bond mannose oligosaccharides, expand their application in health care product development and pharmaceutical additives, and improve the quality of human life. The present invention mainly involves evaluating the anti-inflammatory activity of β-1,2 glycosidic bond mannose oligosaccharides obtained by microbial fermentation.

[0005] In this study, lipopolysaccharide (LPS) was added to the macrophage RAW264.7 cell culture system to construct an inflammatory model, and then M2-β-1,2 and M3-β-1,2 were added to the cell culture system to detect the changes in the secretion of NO and inflammatory factors TNF-α, IL-6, and IL-1β. The test results were then weighted to comprehensively evaluate the anti-inflammatory activity of manno-oligosaccharides containing β-1,2 glycosidic bonds.

[0006] The present invention provides a use of manno-oligosaccharides with β-1,2 glycosidic bonds in the preparation of anti-inflammatory products, wherein the manno-oligosaccharides with β-1,2 glycosidic bonds are preferably selected from β-1,2 glycosidic bond mannobiose or β-1,2 glycosidic bond mannotriose;

[0007] Wherein, the structural formula of the β-1,2 glycosidic bond mannobiose is:

[0008]

[0009] The structure of the β-1,2 glycosidic bond mannotriose is:

[0010]

[0011] Specifically, the anti-inflammatory compound is used in animal feed, food, or health products, or in additives for health products, animal feed, and anti-inflammatory drugs.

[0012] In a preferred embodiment, the concentration of the anti-inflammatory compound containing β-1,2 glycosidic bonds, mannobiose, is 1000-2000 μg / mL.

[0013] In a preferred embodiment, the concentration of the anti-inflammatory compound containing β-1,2 glycosidic bonds, mannotriose, is 250-500 μg / mL.

[0014] In a specific embodiment, the β-1,2 glycosidic bond mannobiose and mannotriose are obtained by microbial fermentation or enzymatic hydrolysis.

[0015] Additionally, the anti-inflammatory compounds are used for non-disease therapeutic purposes.

[0016] The present invention discloses the anti-inflammatory activity of β-1,2 glycosidic bond mannobiose and mannotriose. Among them, mannobiose (M2-β-1,2) with a concentration of 1000-2000μg / mL, especially mannotriose (M3-β-1,2) with a concentration as low as 250-500μg / mL can significantly inhibit the secretion of NO and inflammatory factors TNF-α, IL-6, and IL-1β by RAW264.7 cells induced by LPS, and the inhibitory effect gradually increases with the increase of concentration. It is proved that manno oligosaccharides containing β-1,2 glycosidic bonds have good anti-inflammatory activity and can be used as anti-inflammatory compounds in the development of health products, animal feed, anti-inflammatory drug additives, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Effects of M2-β-1,2 and M3-β-1,2 on the viability of RAW264.7 cells (*p<0.05, **p<0.01, indicating statistically significant differences between the experimental group and the control group).

[0018] Figure 2 Effects of M2-β-1,2 and M3-β-1,2 on LPS-induced NO release in RAW264.7 cells (*p<0.05, **p<0.01, ***p<0.001, indicating statistically significant differences between the experimental group and the control group).

[0019] Figure 3 Effects of M2-β-1,2 and M3-β-1,2 on LPS-induced TNF-α release in RAW264.7 cells (*p<0.05, **p<0.01, ***p<0.001, indicating statistically significant differences between the experimental group and the control group).

[0020] Figure 4 Effects of M2-β-1,2 and M3-β-1,2 on LPS-induced IL-6 release in RAW264.7 cells (*p<0.05, **p<0.01, indicating statistically significant differences between the experimental group and the control group)

[0021] Figure 5 Effects of M2-β-1,2 and M3-β-1,2 on LPS-induced IL-1β release in RAW264.7 cells (*p<0.05, ***p<0.001, indicating statistically significant differences between the experimental group and the control group). DETAILED DESCRIPTION

[0022] The technical scheme of the present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that the following embodiments are only exemplary descriptions and explanations of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are included in the scope that the present invention is intended to protect.

[0023] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

[0024] The following examples M2-β-1,2 and M3-β-1,2 were prepared by referring to the method in Artifcially designed routes for the conversion of starch to value-added mannosyl compounds through coupling in vitro and in vivo metabolic engineering strategies.

[0025] Example 1. Effects of M2-β-1,2 and M3-β-1,2 on RAW26.7 cell viability

[0026] The MTT method was used to detect the effects of M2-β-1,2 and M3-β-1,2 on the viability of macrophage RAW264.7 cells. RAW264.7 cells were revived in a 37°C, 5% CO2 incubator with DMEM containing 10% (v / v) fetal bovine serum and 1% (m / v) double antibodies (penicillin and streptomycin) and passaged every other day. Cells in the logarithmic growth phase were collected and the cell suspension concentration was 1.6×10 4 / well, inoculate 96-well plates, 200μL / well, and culture in a 37℃, 5% CO2 incubator for 12h. Discard the supernatant, add different concentrations of M2-β-1,2 (500, 1000, 1000μg / mL) and M3-β-1,2 (250, 500μg / mL) diluted with DMEM medium, and add only DMEM medium to the negative control group, 200μL / well, and culture in a 37℃, 5% CO2 incubator for 24h. Discard the supernatant, add 20μMTT solution (5mg / mL, i.e. 0.5% MTT (m / v)) to each well, and continue to culture for 4h. Discard the liquid in the well, add 200μL dimethyl sulfoxide to each well, and shake at 700rpm for 10min to fully dissolve the crystals. Measure the absorbance of each well at 490nm on a microplate reader. The formula for calculating the relative activity of cells is as follows:

[0027] Relative cell activity (%) = OD 样品 / OD 阴性对照 *100%

[0028] Test results such as Figure 1 As shown by Figure 1 A shows that after treating cells with 500-2000μg / mL M2-β-1,2 for 24h, there was no significant effect on cell activity compared with the negative control. This indicates that M2-β-1,2 has no toxicity to macrophages in vitro. Figure 1 B shows that after treating cells with 250-500μg / mL M3-β-1,2 for 24h, there was no significant effect on cell activity compared with the negative control and it promoted cell proliferation. Therefore, 500-2000μg / mL M2-β-1,2 and 250-500μg / mL M3-β-1,2 were selected as drug concentrations for the anti-inflammatory activity evaluation experiment.

[0029] Example 2 Effects of M2-β-1,2 and M3-β-1,2 on LPS-induced NO release in RAW264.7 cells

[0030] The Griess method was used to detect the effect of mannooligosaccharide on LPS-induced NO release in RAW264.7 cells to investigate its anti-inflammatory activity. RAW264.7 cells in the logarithmic growth phase were taken and the cell suspension concentration was 1×10 5 / well, inoculate 96-well plates, 200μL / well, culture in a 37℃, 5% CO2 incubator, and treat cells in groups after 12h. The control group did not receive any treatment, and the model group was added with a final concentration of 1μg / ml LPS (lipopolysaccharide) to induce inflammation; the treatment group was added with different concentrations of M2-β-1,2 (500, 1000, 1000μg / mL) and M3-β-1,2 (250, 500mg / mL) and LPS and incubated together. After 24h, the cell supernatant was collected, and Griess Reagent I and II were added according to the instructions of the kit. The absorbance was measured at 540nm by an enzyme reader, and a standard curve was prepared with the standard solution to calculate the concentration of NO in each sample. The results are shown in Figure 2 As shown in the results, 500-2000μg / mL of M2-β-1,2 and 250-500μg / mL of M3-β-1,2 can significantly inhibit the release of NO in RAW264.7 cells induced by LPS, and the inhibitory effect increases with the increase of the concentration, with the highest inhibition rates of 66.7% and 62%, respectively. This shows that M2-β-1,2 and M3-β-1,2 have good anti-inflammatory effects.

[0031] Example 3: Effect of Manno-oligosaccharide on the Release of Inflammatory Factors TNF-α, IL-6, and IL-1β from RAW264.7 Cells Induced by LPS

[0032] The ELISA method was used to detect the effects of M2-β-1,2 (500, 1000, 1000 μg / mL) and M3-β-1,2 (250, 500 μg / mL) on the release of inflammatory factors TNF-α, IL-6, and IL-1β in RAW264.7 cells induced by LPS to investigate their anti-inflammatory activity. Cells in logarithmic growth phase were taken and the cell suspension concentration was 4.8×10 5 pcs / well, inoculated in a 6-well plate, and cultured in a 37°C, 5% CO2 incubator for 12 hours. After 12 hours, the cells were divided into groups for treatment. The control group did not receive any treatment, and the model group was added with a final concentration of 1μg / ml LPS (lipopolysaccharide) to induce inflammation; the treatment group was added with different concentrations of M2-β-1,2 (500, 1000, 1000μg / mL) and M3-β-1,2 (250, 500μg / mL) and LPS for co-incubation. After 24 hours, the cell supernatant was collected, centrifuged at 4000rpm for 10min, and transferred to a sterile centrifuge tube for subsequent detection. According to the determination steps of the IL-6 ELISA kit, TNF-α ELISA kit, and IL-1β ELISA reagent, the contents of IL-6, TNF-α, and IL-1β in the cell culture supernatant were determined.

[0033] Inflammatory factors are a class of active protein molecules produced by immune cells when they are stimulated by external factors. In the body's immune response, inflammatory factors play an important role in enhancing the body's resistance, regulating the body's immune response and inflammatory response, such as Figure 3 As shown in the figure, after 12h of LPS stimulation, the inflammatory factor TNF-α released by the cells was significantly increased compared with the control group. Compared with the LPS model group, after 500-2000μg / mL M2-β-1,2 stimulation, the secretion of TNF-α by cells can be inhibited to varying degrees, and the inhibitory effect gradually increases with the increase of concentration. When the concentration of M2-β-1,2 is 2000μg / mL, the inhibition rate can reach 62.3%. Similarly, after 250-500μg / mL M3-β-1,2 stimulation, the inhibitory effect on the secretion of TNF-α by cells gradually increases with the increase of concentration. When the concentration of M3-β-1,2 is 500μg / mL, the inhibition rate can reach 62.0%.

[0034] like Figure 4As shown in the figure, after 12 hours of LPS stimulation, the release of inflammatory factor IL-6 increased significantly compared with the control group. Compared with the LPS model group, 1000-2000μg / mL M2-β-1,2 stimulation can inhibit the secretion of IL-6 in cells to varying degrees, and the inhibitory effect gradually increases with the increase of concentration. When the concentration of M2-β-1,2 is 2000μg / mL, the inhibition rate can reach 74.3%. Similarly, after 250-500μg / mL M3-β-1,2 stimulation, the inhibitory effect on cell IL-6 secretion gradually increases with the increase of concentration. When the concentration of M3-β-1,2 is 500μg / mL, the inhibition rate can reach 79.5%.

[0035] like Figure 5 As shown in the figure, after 12 hours of LPS stimulation, the inflammatory factor IL-1β released by the cells was significantly increased compared with the control group. Compared with the LPS model group, after stimulation with 1000-2000μg / mL M2-β-1,2, the secretion of IL-1β by cells can be inhibited to varying degrees, and the inhibitory effect gradually increases with the increase of concentration. When the concentration of M2-β-1,2 is 2000μg / mL, the inhibition rate can reach 55.6%. Similarly, after stimulation with 250-500μg / mL M3-β-1,2, the inhibitory effect on the secretion of IL-6 by cells gradually increases with the increase of concentration. When the concentration of M3-β-1,2 is 500μg / mL, the inhibition rate can reach 70.4%.

[0036] The above results show that M2-β-1,2 and M3-β-1,2 can inhibit the release of LPS-induced related inflammatory factors (TNF-α, IL-6, IL-1β), and M2-β-1,2 has good anti-inflammatory effect at 1000-2000μg / mL and M3-β-1,2 at 250-500μg / mL. Relatively speaking, M3-β-1,2 can achieve better anti-inflammatory effect at a lower concentration than M2-β-1,2.

[0037] The above is an explanation of the embodiments of the present invention. However, the present invention is not limited to the above embodiments. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. Use of manno-oligosaccharides with β-1,2 glycosidic bonds in the preparation of anti-inflammatory products.

2. The use according to claim 1, characterized in that The β-1,2 glycosidic bond manno-oligosaccharide is selected from β-1,2 glycosidic bond mannobiose or β-1,2 glycosidic bond mannotriose; Wherein, the structural formula of the β-1,2 glycosidic bond mannobiose is: The structure of the β-1,2 glycosidic bond mannotriose is:

3. The use according to claim 1, characterized in that The anti-inflammatory compound is used in animal feed, food, or health products.

4. The use according to claim 1, characterized in that The anti-inflammatory product is used as an additive for health products, animal feed, and anti-inflammatory medicines.

5. The use according to any one of claims 1 to 4, characterized in that The concentration of the anti-inflammatory compound containing β-1,2 glycosidic bond mannobiose is 1000-2000 μg / mL.

6. The use according to any one of claims 1 to 4, characterized in that The concentration of the anti-inflammatory compound containing β-1,2 glycosidic bond mannotriose is 250-500 μg / mL.

7. The use according to any one of claims 1 to 4, characterized in that β-1,2 glycosidic bond mannobiose and mannotriose are obtained by microbial fermentation or enzymatic hydrolysis.

8. The use according to any one of claims 1 to 4, characterized in that The anti-inflammatory compounds are used for non-disease therapeutic purposes.

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