Novel inulin type oligosaccharide as well as preparation method and application thereof

Abstract

The invention discloses a novel inulin oligosaccharide and a preparation method and application thereof, the novel inulin oligosaccharide has a structure as shown in a formula 1, wherein m + n is more than or equal to 0 and less than or equal to 7. The novel inulin type oligosaccharide is composed of fructose and glucose, a glucosyl group is located in the middle of a molecule, two ends of the molecule are respectively connected with a plurality of fructosyl groups, and the fructosyl groups are mutually connected through beta (2-> 1) glucosidic bonds. The novel inulin oligosaccharide can obviously inhibit inflammatory mediators and proinflammatory cytokines secreted by cells in an in-vitro inflammation model, and has obvious anti-inflammatory activity; therefore, the novel inulin type oligosaccharide and the derivative thereof or the pharmaceutically acceptable salt thereof can be applied to functional health products or medicines to prevent and treat inflammatory diseases and / or improve intestinal functions, and can also be used as active ingredients of foods, drinks or health care products to improve immune functions and / or prevent inflammatory diseases.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and in particular relates to a novel inulin-type oligosaccharide and a preparation method and application thereof. Background Art

[0002] Inflammation is a defensive response of the human immune system to exogenous or endogenous stimuli (such as infection and trauma) and is a crucial physiological process for maintaining health. However, excessive or persistent inflammatory responses can lead to chronic inflammation, which in turn can cause a variety of diseases, including cardiovascular disease, chronic kidney disease, inflammatory bowel disease (IBD), and cancer. Inflammatory bowel disease (IBD), which includes ulcerative colitis (UC) and Crohn's disease (CD), has a global prevalence exceeding 10 million and is experiencing a significant increase in incidence in my country, making it a common clinical condition. The pathogenesis of IBD remains incompletely elucidated, but studies suggest that abnormal activation of the intestinal mucosal immune system and persistent inflammatory responses play a key role in its development and progression. Currently, clinical treatment for IBD relies primarily on anti-inflammatory drugs, such as salicylates (e.g., sulfasalazine), glucocorticoids (e.g., hydrocortisone), and immunosuppressants. However, long-term use of these drugs can cause serious side effects, such as gastrointestinal bleeding and abdominal pain, limiting their widespread use. Therefore, the development of new anti-inflammatory drugs with high efficiency and low toxicity, especially natural active ingredients with intestinal microenvironment regulation function, has become an important research direction in the current IBD treatment field.

[0003] Fructooligosaccharides (FOS) are a naturally occurring carbohydrate found in a wide variety of plants, such as Jerusalem artichokes, onions, and bananas. Their prebiotic properties have been extensively studied, with their ability to selectively promote the proliferation of beneficial intestinal bacteria (such as bifidobacteria and lactic acid bacteria), thereby regulating intestinal flora balance and demonstrating potential value in enhancing immune function. Existing research has largely focused on traditional inulin-type fructooligosaccharides, while systematic studies on novel inulin-type oligosaccharides with unique structures are lacking. In particular, research on novel inulin-type oligosaccharides in regulating intestinal immune responses and inhibiting inflammatory responses remains significant, significantly limiting their potential for development and application in the treatment of inflammatory diseases such as inflammatory bowel disease (IBD).

[0004] Therefore, there is an urgent need for a novel inulin-type oligosaccharide and a preparation method and application thereof to solve the deficiencies of the existing technical problems. Summary of the Invention

[0005] In view of the above problems, the purpose of the present invention is to provide a new inulin-type oligosaccharide and its preparation method and application. The new inulin-type oligosaccharide can significantly inhibit the inflammatory mediators and pro-inflammatory cytokines secreted by cells in an in vitro inflammation model and has obvious anti-inflammatory activity. Therefore, the new inulin-type oligosaccharide and its derivatives or pharmaceutically acceptable salts can be used in functional health products or medicines to prevent inflammatory diseases, treat inflammatory diseases and / or improve intestinal function, and can also be used as active ingredients in foods, beverages or health products to enhance immune function and / or prevent inflammatory diseases.

[0006] To achieve the above objectives, the present invention provides a novel inulin-type oligosaccharide having a structure shown in Formula 1:

[0007] Formula 1 Among them, 0≤m+n≤7. Furthermore, the novel inulin-type oligosaccharide of the present invention is composed of fructose and glucose, and the specific characteristics of its structure are: the glucose group is located in the middle of the molecule, and several fructose groups are connected to both ends of the molecule, and the fructose groups are connected to each other through β (2→1) glycosidic bonds.

[0008] Furthermore, m and n are integers.

[0009] Furthermore, 0≤m+n≤2. Specifically, m=0, n=0; or m=1, n=0; or m=0, n=1; or m=1, n=1; or m=2, n=0; or m=0, n=2.

[0010] Correspondingly, the present invention also provides a method for preparing novel inulin-type oligosaccharides, wherein the traditional Chinese medicine Asparagus cochinchinensis is sequentially extracted, decolorized, separated and purified to obtain the novel inulin-type oligosaccharides.

[0011] Furthermore, the preparation method of the novel inulin-type oligosaccharide of the present invention comprises the following steps: (1) The Chinese herbal medicine Asparagus cochinchinensis is extracted by heating reflux extraction method, and then the extract is concentrated under reduced pressure to obtain crude oligosaccharide extract; (2) Decolorize the crude oligosaccharide extract using a macroporous resin column, then rinse with deionized water, collect the eluate, concentrate it by rotary evaporation, and freeze-dry it to obtain crude oligosaccharide powder; (3) Separate and purify the crude oligosaccharide powder by molecular exclusion chromatography, then concentrate the collected liquid by rotary evaporation and freeze-dry to obtain the oligosaccharide component; (4) Semi-preparative high performance liquid chromatography was used to separate and purify the oligosaccharide components, and then the organic solution in the collected liquid was removed by rotary evaporation and freeze-dried to obtain new inulin-type oligosaccharides.

[0012] Furthermore, in step (1) of the present invention, an ethanol aqueous solution is used as an extraction solvent to extract the Chinese herbal medicine Asparagus cochinchinensis by a heating reflux extraction method. Specifically, the concentration of the ethanol aqueous solution is 70% to 85% (v / v), and the heating reflux temperature is controlled at 70 to 90°C. The number of extractions is 3 to 5, and each extraction time is 1 to 3 hours.

[0013] Furthermore, the method further comprises step (5): separating and purifying the oligosaccharide isomer mixture in the novel inulin-type oligosaccharide by semi-preparative ion exchange chromatography, and then removing the organic solution in the collected liquid by rotary evaporation and freeze-drying.

[0014] Correspondingly, the present invention also provides the use of novel inulin-type oligosaccharides and their derivatives or pharmaceutically acceptable salts thereof in the preparation of products for preventing inflammatory diseases, treating inflammatory diseases and / or improving intestinal function. The products are functional health products or medicines, and the novel inulin-type oligosaccharides are the novel inulin-type oligosaccharides mentioned above or the novel inulin-type oligosaccharides obtained by the preparation method of the novel inulin-type oligosaccharides mentioned above.

[0015] Furthermore, the novel inulin-type oligosaccharide derivative is obtained by carboxylating, carbonylating, methylating, acetylating, phosphorylating, and esterifying the hydroxyl side chain groups of the novel inulin-type oligosaccharide.

[0016] Correspondingly, the present invention also provides the use of novel inulin-type oligosaccharides and their derivatives or pharmaceutically acceptable salts thereof in the preparation of products having the effect of improving immune function and / or preventing inflammatory diseases, the products being food, beverages or health products, and the novel inulin-type oligosaccharides being the novel inulin-type oligosaccharides mentioned above or the novel inulin-type oligosaccharides obtained by the preparation method of the novel inulin-type oligosaccharides mentioned above.

[0017] Furthermore, the novel inulin-type oligosaccharide derivative is obtained by carboxylating, carbonylating, methylating, acetylating, phosphorylating, and esterifying the hydroxyl side chain groups of the novel inulin-type oligosaccharide.

[0018] Compared to the prior art, the present invention discloses a novel inulin-type oligosaccharide represented by the structure of Formula 1. It is prepared from the traditional Chinese medicine Asparagus cochinchinensis through extraction, decolorization, and separation and purification processes. This oligosaccharide has unique structural characteristics and significant biological activity. It can effectively inhibit the secretion of inflammatory mediators and proinflammatory cytokines in an in vitro inflammation model, demonstrating excellent anti-inflammatory effects. Based on this property, this oligosaccharide and its derivatives or pharmaceutically acceptable salts can be widely used in functional health products or medicines to prevent and treat inflammatory diseases and / or improve intestinal function. It can also be used as an active ingredient in foods, beverages, or health supplements to enhance immune function and / or prevent inflammatory diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1This is the high performance anion exchange chromatogram of the crude oligosaccharide powder in step (2) of Example 1.

[0020] Figure 2 The high performance anion exchange chromatograms of the six pure oligosaccharides obtained in Example 1 are shown.

[0021] Figure 3 This is the infrared spectrum of the pure DP3-1 oligosaccharide obtained in Example 1.

[0022] Figure 4 This is the infrared spectrum of the pure DP4-1 oligosaccharide obtained in Example 1.

[0023] Figure 5 This is the infrared spectrum of the pure DP4-3 oligosaccharide obtained in Example 1.

[0024] Figure 6 This is the infrared spectrum of the pure DP5-2 oligosaccharide obtained in Example 1.

[0025] Figure 7 This is the infrared spectrum of the pure DP5-3 oligosaccharide obtained in Example 1.

[0026] Figure 8 This is the infrared spectrum of the pure DP5-4 oligosaccharide obtained in Example 1.

[0027] Figure 9 This is the methylation spectrum of the pure DP3-1, DP4-1, and DP4-3 oligosaccharides obtained in Example 1.

[0028] Figure 10 This is the methylation spectrum of the pure DP5-2, DP5-3, and DP5-4 oligosaccharides obtained in Example 1.

[0029] Figure 11 This is the NMR spectrum of the pure DP3-1 oligosaccharide obtained in Example 1.

[0030] Figure 12 This is the NMR spectrum of the pure DP4-1 oligosaccharide obtained in Example 1.

[0031] Figure 13 This is the NMR spectrum of the pure DP4-3 oligosaccharide obtained in Example 1.

[0032] Figure 14 This is the NMR spectrum of the pure DP5-2 oligosaccharide obtained in Example 1.

[0033] Figure 15 This is the NMR spectrum of the pure DP5-3 oligosaccharide obtained in Example 1.

[0034] Figure 16This is the NMR spectrum of the pure DP5-4 oligosaccharide obtained in Example 1.

[0035] Figure 17 This is the ESI-QTOF-MS spectrum of the pure DP3-1, DP4-1, and DP4-3 oligosaccharides obtained in Example 1.

[0036] Figure 18 This is the ESI-QTOF-MS spectrum of the pure DP5-2, DP5-3, and DP5-4 oligosaccharides obtained in Example 1.

[0037] Figure 19 This is a graph showing the cytotoxicity test results of the six pure oligosaccharides obtained in Example 1.

[0038] Figure 20 This is a graph showing the effects of the six pure oligosaccharides obtained in Example 1 on the NO level in RAW 264.7 cells.

[0039] Figure 21 This is a graph showing the effects of the six pure oligosaccharides obtained in Example 1 on the IL-1β level in RAW 264.7 cells.

[0040] Figure 22 This is a graph showing the effects of the six pure oligosaccharides obtained in Example 1 on the IL-6 level in RAW 264.7 cells.

[0041] Figure 23 This is a graph showing the effects of the six pure oligosaccharides obtained in Example 1 on the TNF-α level in RAW 264.7 cells. DETAILED DESCRIPTION

[0042] To better illustrate the purpose, technical solutions and beneficial effects of the present invention, the present invention will be further described below with reference to specific embodiments and accompanying drawings. It should be noted that the following implementation method is a further explanation of the present invention and should not be used as a limitation of the present invention.

[0043] In the following examples, the high-performance anion exchange chromatography (HPLC) system used was a Thermo Scientific Dionex™ ICS-5000+ ion chromatography system. Protein purification was performed using a GE AKTA FPLC system coupled with a differential refractive index detector (DID), and semi-preparative HPLC was performed using a Shimadzu UFLC system coupled with a differential refractive index detector (DID). Unless otherwise noted, all reagents used in the following examples were commercially available.

[0044] Example 1 This embodiment provides a method for preparing a novel inulin-type oligosaccharide, comprising the following steps: (1) Add 200 g of Asparagus cochinchinensis powder to 4000 mL of 80% (v / v) ethanol aqueous solution, heat and perform reflux extraction three times at 85°C for 2 h each time, collect the supernatant by centrifugation, combine the supernatants and concentrate under reduced pressure to remove ethanol to obtain crude oligosaccharide extract; (2) The crude oligosaccharide extract was decolorized using an AB-8 macroporous resin column, washed with deionized water for 10 times the column volume, and the eluate was collected and concentrated by rotary evaporation, and then freeze-dried to obtain crude oligosaccharide powder with a yield of 81.69%; (3) The crude oligosaccharide components were separated and purified by size exclusion chromatography, specifically: using the AKTA protein purification system, Bio-Gel P-2 as the filler, isocratic elution with ultrapure water, collecting the eluate and determining the degree of polymerization using a high performance anion exchange chromatography-pulsed current detector, and concentrating the collected solution by rotary evaporation and freeze drying to obtain oligosaccharide components with a degree of polymerization of 3-5; (4) The oligosaccharide components with a degree of polymerization of 3-5 obtained in step (3) were further separated and purified by semi-preparative high performance liquid chromatography, specifically: a Shimadzu UFLC system was used, a Waters XBridge BEH Prep OBD Amide column was used, a differential refractive index detector was selected as the detector, eluted isocratically with a 70% acetonitrile aqueous solution, the eluate was collected, and the purity was determined by high performance anion exchange chromatography-pulsed current detector. After freeze drying, the pure oligosaccharides of DP3-1, DP4-3, DP5-3, and DP5-4 and the components mainly containing DP4-1 and DP5-2 were obtained; (5) The main DP4-1 component and DP5-2 component obtained in step (4) were further separated and purified by semi-preparative high performance anion exchange chromatography. Specifically, a Thermo ICS-5000+ ion chromatography system was used, a Dionex CarboPac PA 200 semi-prep column was used as the chromatographic column, and a pulsed current detector was used. The elution was isocratically eluted with a 100 mM sodium hydroxide aqueous solution containing 20 mM and 30 mM sodium acetate, respectively. The eluate was collected, and the sodium ions in the collected solution were removed by a cation exchange resin column. The solution was freeze-dried to obtain pure oligosaccharides of DP4-1 and DP5-2.

[0045] The crude oligosaccharide powder in step (2) was detected by high performance anion exchange chromatography-pulse current detector using a Dionex CarboPac PA 200 semi-prep column (3×250 mm) and a pulse current detector. The sample volume was 5 μL and the flow rate was 0.5 mL / min. The mobile phase was 100 mM NaOH containing 20 mM sodium acetate. The high performance anion exchange chromatogram of the crude oligosaccharide is shown in FIG. Figure 1 As shown, from Figure 1 It can be seen that the crude oligosaccharide powder contains oligosaccharides with different degrees of polymerization.

[0046] Purity testing: The purity of the six purified oligosaccharides (DP3-1, DP4-3, DP5-3, DP5-4, DP4-1, and DP5-2) obtained in Example 1 was determined using high-performance anion exchange chromatography coupled with a pulsed amperometric detector. Specifically, a Dionex CarboPac PA 200 semi-prep column (3 × 250 mm) was used with a pulsed amperometric detector. A sample volume of 5 μL was loaded at a flow rate of 0.5 mL / min. The mobile phase consisted of 100 mM NaOH containing 20 mM sodium acetate.

[0047] The high performance anion exchange chromatograms of 6 pure oligosaccharides are shown in Figure 2. Figure 2 As shown. Figure 2 It can be seen that the purity of the six oligosaccharides obtained by separation and purification is good. According to the calculation of peak area, the purity of the oligosaccharide samples is greater than 96%.

[0048] The structures of the six pure oligosaccharides (DP3-1, DP4-3, DP5-3, DP5-4, DP4-1, and DP5-2) obtained in Example 1 were analyzed using the following detection method: Infrared spectrum detection: Take about 1 mg of dry oligosaccharide sample and mix it with about 150 mg of KBr, press it into a sheet, and analyze it with an infrared spectrometer at 4000-400 cm -1 Infrared scanning is performed within the area with a resolution of 4 cm -1 .

[0049] The infrared spectra of DP3-1, DP4-1, DP4-3, DP5-2, DP5-3 and DP5-4 are shown in Figure 1. Figures 3 to 8 As shown in the figure, DP3-1, DP4-1, DP4-3, DP5-2, DP5-3, and DP5-4 all have characteristic absorption areas of sugars: at 3400~3200cm -1 OH stretching vibration (broad peak) appears near 2930 cm -1 There is a CH stretching vibration near 1650 cm -1 The HOH bending vibration of bound water appears near 1600~1400 cm -1 The CH symmetric vibration and C-OH deformation bands appeared nearby; the glycosidic bond characteristic region also appeared: 1150~1000 cm -1 COC and COH stretching vibrations (polysaccharide characteristic peaks) appeared, 900~700cm -1The characteristic region of anomeric carbon configuration appeared. Therefore, infrared spectroscopy confirmed that the pure oligosaccharides DP3-1, DP4-1, DP4-3, DP5-2, DP5-3, and DP5-4 were all typical inulin-type oligosaccharide structures.

[0050] Methylation experiment: Oligosaccharide samples were hydrolyzed with 1 mL of 1.5 M trifluoroacetic acid at 90°C for 2 hours and then dried with nitrogen. The hydrolyzate was reduced with NaBH4 to prepare alditol acetates. Partially methylated alditol acetates were analyzed by gas chromatography-mass spectrometry on an Agilent 6890 gas chromatograph system and an Agilent 5973 mass spectrometer. An HP-5MS capillary column (30 m × 0.25 mm) was used. The temperature program started at 120°C, then ramped to 200°C at 5°C / min, and then to 250°C at 8°C / min. The mass spectrometer was operated in electron impact mode with a scan range of 40–550 m / z. The ionization energy was 70 eV, and the scan rate was 2.89 s / cycle. The inlet and source temperatures were 250°C and 280°C, respectively.

[0051] The methylation patterns of DP3-1, DP4-1, DP4-3, DP5-2, DP5-3, and DP5-4 are shown in Figure 2. Figures 9 and 10 As shown in the figure, all samples show the following common peaks: t-Fru f (terminal fructose) (retention time ~14.5 min, abundance 1,200,000~1,800,000), this characteristic peak corresponds to 1,5,6-tri-O-acetyl-2,3,4-tri-O-methylfructitol, which indicates that the six oligosaccharide pure products all contain non-reducing terminal fructose (Fru f ), which is consistent with the typical structure of inulin-type oligosaccharides; all samples also contained significant 1,6-Glcp peaks, indicating the presence of 1,6-linked glucose units, which is a common bond type of inulin-type oligosaccharides. Except for DP3-1, 1,2-Fru was detected in all other samples (DP4-1, DP4-3, DP5-2, DP5-3, and DP5-4). f +1,6-Glc p peaks, indicating that these five oligosaccharide pure products may have more complex branched structures or the presence of 1,2-fructosidic bonds, while DP3-1 only contains a linear 1,6-bonded structure.

[0052] MRI: The dried oligosaccharide sample (12-15 mg) was dissolved in 0.6 mL D2O at room temperature. 1D NMR spectrometer was used. 1 H. 13 C NMR, 2D 1 H-13 C HSQC, 1 H- 13 C HMBC, 1 H- 1 H COSY and 1 H- 1 H TOCSY NMR spectroscopy analysis.

[0053] The NMR spectra of DP3-1, DP4-1, DP4-3, DP5-2, DP5-3 and DP5-42 are as follows: Figures 11 to 16 As shown in the figure, the six oligosaccharide pure products 1 In HNMR, the anomeric proton (H-1) signal of the pyranose glucose residue can be observed around δ 5.4 ppm. The coupling constant of the anomeric proton (J = 3.9 Hz) indicates that glucose exists in the α configuration; the sugar ring proton (H-2 ~ H-6) signals are displayed in the range of δ 3.5 ~ 4.5 ppm, which are consistent with the chemical shift characteristics of typical oligosaccharides, including the doublet signal of δ 4.30 and 4.20 ppm assigned to H-3, and the triplet signal of δ 4.20 and 4.00 ppm assigned to H-4. 13 C NMR detected the fructose terminal carbon (C-2) signal in the range of δ 102.9~103.7 ppm, and the glucose terminal carbon (C-1) signal at around δ 92.0 ppm, further confirming the presence of glycosidic bonds. The oligosaccharide polymerization degree values ​​calculated by one-dimensional NMR spectroscopy were consistent with the results of methylation analysis and were consistent with the inference of two-dimensional NMR spectroscopy. 1 H- 13 The inter-residue correlation peaks in C HMBC NMR were used to determine the residue connection mode, Glc p -H-1 and Fru f- The cross peak between C-2 indicates that the connection between pyranose glucose and fructofuranose is Glc p -(1→2)-Fru f 6 pure oligosaccharides 1 H- 13 C HMBC NMR of Fru f -H-1 and Fru f -C-2 glycosidic bond connected via 1 H- 13 The CHMBC correlation peak is obtained, and 1 H- 1 H COSY and 1 H- 1 H TOCSY NMR spectroscopy further verified the connection mode, which was consistent with the structure of the new inulin-type oligosaccharide.

[0054] ESI-qTOF-MS analysis: The analysis was performed using a Bruker high-resolution impact HD quadrupole time-of-flight (qTOF) mass spectrometer equipped with an electrospray ionization source (ESI). The capillary voltage was set to 3000 V in negative ion mode, the mass range was set to m / z 100-1000 in full scan mode, the ion source temperature was set to 250°C, and the nitrogen flow rate was set to 4 L / min. The MS of the three highest intensity ion fragments was 2 Real-time intelligent analysis of data.

[0055] The ESI-QTOF-MS spectra of DP3-1, DP4-1, DP4-3, DP5-2, DP5-3 and DP5-4 are as follows: Figure 17-18 As shown in the figure, the fragmentation patterns of the six pure oligosaccharides include glycosidic bond cleavage and cross-ring fragmentation. The formation of fragment ions in glycosidic fragmentation is primarily due to the loss of a hexose unit (-162 Da) and subsequent water loss (-18 Da) from glycoside cleavage, a typical fragmentation pattern for inulin-type fructans. The mass reductions at m / z 545 and m / z 383 indicate that these ions can fragment into cross-ring products in two directions. The ion at m / z 383 is primarily derived from the terminal sucrose unit, consistent with the fragmentation pattern of novel inulin-type oligosaccharides.

[0056] Therefore, through the above-mentioned infrared spectroscopy detection results, methylation experiment results, nuclear magnetic resonance experiment results and ESI-QOF-MS analysis results, comprehensive analysis can determine that DP3-1 (m=0, n=0), DP4-1 (m=0, n=1), DP4-3 (m=1, n=0), DP5-2 (m=0, n=2), DP5-3 (m=n=1), and DP5-4 (m=2, n=0) are new inulin-type oligosaccharides.

[0057] The pharmacological activities of six novel inulin-type oligosaccharides (numbered 1-6, 1=DP3-1; 2=DP4-1; 3=DP4-3; 4=DP5-2; 5=DP5-3; 6=DP5-4) were tested using an in vitro cell model. The specific methods are as follows.

[0058] Cytotoxicity assay: RAW 264.7 cells (5 × 10 3 100 μL of 10% CCK8 solution was added to each well, and the plates were kept in the dark for 4 h. The absorbance was measured at a wavelength of 450 nm using a microplate reader.

[0059] Cytotoxicity test results Figure 19 shown.

[0060] NO level test: RAW 264.7 cells (5 × 10 4 Cells were seeded overnight in 96-well plates (100 μg / well). Different concentrations of novel inulin-based oligosaccharides were then added to the experimental groups. After a 2-hour preincubation at 37°C, 5% CO₂, cells were incubated in the presence or absence of LPS (1 μg / mL) for 24 hours. The supernatant was then mixed with an equal volume of Griess reagent at room temperature in the dark for 15 minutes. The absorbance was measured at 540 nm using a microplate reader. NO production was calculated as the ratio of the absorbance values ​​between the sample and LPS-treated groups.

[0061] NO level test results are as follows Figure 20 shown.

[0062] Cytokine level test: RAW 264.7 cells (5 × 10 4 Cells were seeded overnight in 96-well plates (100 μg / well). Different concentrations of novel inulin-based oligosaccharides were then added to the experimental groups. After a 2-hour preincubation at 37°C, 5% CO₂, cells were cultured in the presence or absence of LPS (1 μg / mL) for 24 hours. Supernatants were collected for cytokine analysis, using enzyme-linked immunosorbent assay kits to assess levels of IL-1β, IL-6, and TNF-α.

[0063] IL-1β level test results Figure 21 As shown, the IL-6 level test results are as follows Figure 22 As shown, the TNF-α level test results are as follows Figure 23 shown.

[0064] from Figure 19 It can be seen that the cell survival rate treated with the six novel inulin-type oligosaccharides had no significant difference from that of the blank group at the experimental concentration, indicating that the six novel inulin-type oligosaccharides had no toxic effect on RAW 264.7 cells at this concentration.

[0065] from Figure 20 The results showed that in the LPS-induced macrophage inflammation model, all six novel inulin-type oligosaccharides reduced the NO content in the LPS-induced macrophage supernatant in a concentration-dependent manner. Specifically, compared with the LPS group, the six novel inulin-type oligosaccharides at a concentration of 1000 μg / mL reduced the NO concentration in the macrophage inflammation model by approximately 80%.

[0066] from Figures 21 to 23It can be seen that in the LPS-induced macrophage inflammation model, pre-incubation of 6 novel inulin-type oligosaccharides can reduce the content of cytokines IL-1β, IL-6 and TNF-α in the cell supernatant to varying degrees, and at the highest concentration, the concentration of cytokines can be reduced by about 50%.

[0067] In summary, the novel inulin-type oligosaccharide provided by the present invention shows its non-toxicity in an in vitro cell model, and can reduce the secretion of related pro-inflammatory cytokines when the cells are inflammatory, thereby reducing the occurrence of inflammation and having an anti-inflammatory effect. In terms of clinical treatment, compounds or drugs with anti-inflammatory effects are often used in the treatment of inflammatory bowel disease. Therefore, the novel inulin-type oligosaccharide provided by the present invention can be used to prepare drugs for preventing and treating inflammatory diseases, improving intestinal function, and treating inflammatory bowel disease. It can also be used to prepare foods, beverages, and health care that have the effects of improving immune function and preventing inflammatory diseases, and is used for quality control of oligosaccharide components. It also helps to better utilize plant resources containing novel inulin-type oligosaccharides.

[0068] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the scope of protection of the present invention. Although the present invention is described in detail with reference to the preferred embodiments, it is not limited to those listed in the embodiments. Those skilled in the art should understand that the technical solutions of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A novel inulin-type oligosaccharide, characterized in that: It has the structure shown in formula 1: Formula 1 Among them, 0≤m+n≤7.

2. The novel inulin-type oligosaccharide according to claim 1, characterized in that 0≤m+n≤2.

3. The method for preparing the novel inulin-type oligosaccharide according to claim 1, wherein: The Chinese herbal medicine Asparagus cochinchinensis was sequentially extracted, decolorized, separated and purified to obtain novel inulin-type oligosaccharides.

4. The method for preparing the novel inulin-type oligosaccharide according to claim 3, wherein the steps include: (1) extracting the Chinese herbal medicine Asparagus cochinchinensis by a heating reflux extraction method, and then concentrating the extract under reduced pressure to obtain a crude oligosaccharide extract; (2) Decolorizing the crude oligosaccharide extract using a macroporous resin column, then washing with deionized water, collecting the eluate, concentrating it by rotary evaporation, and freeze-drying it to obtain crude oligosaccharide powder; (3) Separating and purifying the crude oligosaccharide powder by molecular exclusion chromatography, and then concentrating the collected liquid by rotary evaporation and freeze-drying to obtain the oligosaccharide component; (4) The oligosaccharide components were separated and purified by semi-preparative high performance liquid chromatography, and then the organic solution in the collected liquid was removed by rotary evaporation and freeze-dried to obtain novel inulin-type oligosaccharides.

5. The method for preparing the novel inulin-type oligosaccharide according to claim 4, characterized in that: The method further comprises step (5): separating and purifying the oligosaccharide isomer mixture in the novel inulin-type oligosaccharide by semi-preparative ion exchange chromatography, and then removing the organic solution in the collected liquid by rotary evaporation and freeze-drying.

6. Use of a novel inulin-type oligosaccharide and its derivatives or pharmaceutically acceptable salts thereof in the preparation of a product for preventing or treating inflammatory diseases and / or improving intestinal function, wherein the product is a functional health product or medicine, characterized in that: The novel inulin-type oligosaccharide is the novel inulin-type oligosaccharide according to any one of claims 1 to 2 or the novel inulin-type oligosaccharide prepared by the preparation method of the novel inulin-type oligosaccharide according to any one of claims 3 to 5.

7. The use according to claim 6, characterized in that The novel inulin-type oligosaccharide derivative is The novel inulin-type oligosaccharide is obtained by subjecting the hydroxyl side chain groups of the novel inulin-type oligosaccharide to carboxylation, carbonylation, methylation, acetylation, phosphorylation and esterification.

8. Use of a novel inulin-type oligosaccharide and its derivatives or pharmaceutically acceptable salts thereof in the preparation of a product having the effect of improving immune function and / or preventing inflammatory diseases, wherein the product is a food, beverage or health product, characterized in that: The novel inulin-type oligosaccharide is the novel inulin-type oligosaccharide according to any one of claims 1 to 2 or the novel inulin-type oligosaccharide prepared by the preparation method of the novel inulin-type oligosaccharide according to any one of claims 3 to 5.

9. The use according to claim 8, characterized in that The novel inulin-type oligosaccharide derivative is The novel inulin-type oligosaccharide is obtained by subjecting the hydroxyl side chain groups of the novel inulin-type oligosaccharide to carboxylation, carbonylation, methylation, acetylation, phosphorylation and esterification.

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