Day lily polysaccharide with broad-spectrum antibacterial activity and application thereof

Abstract

The invention relates to the technical field of natural product extraction and purification and biological medicine, in particular to day lily polysaccharide with broad-spectrum antibacterial activity and application of the day lily polysaccharide. The molecular weight of the hemerocallis citrina polysaccharide is 1959 Da, monosaccharide components of the hemerocallis citrina polysaccharide mainly comprise fructose (88.87%) and glucose (11.13%), and the hemerocallis citrina polysaccharide is high-fructose neutral polysaccharide. The main chain is-> 1)-beta-D-Fruf-(2->), side chains are connected to O-6 sites of partial main chain residues, and the side chains comprise beta-D-Fruf-(2->) and-> 6)-beta-D-Fruf-(2->); the molar percentages of the sugar residues Fruf1, 2, Fruf2, Fruf1, 2, 6, Fruf2, 6 and Glc1, 6 are respectively 41.94%, 23.59%, 9.27%, 16.80% and 8.40%, and the molar percentages of the sugar residues Fruf1, 2, Fruf2, Fruf2, Fruf6 and Glc1, 6 are respectively 41.94%, 23.59%, 9.27%, 16.80% and 8.40%; the hemerocallis citrina baroni polysaccharide shows remarkable bacteriostatic activity on clinically separated multi-drug-resistant strains and standard strains, and shows dose dependence; in addition, the improvement effect of the hemerocallis citrina polysaccharide on the lung tissue injury is enhanced along with the increase of the dosage, the effect can be quantified and repeatable, and the requirements of drug development are met.

Description

Technical Field

[0001] This invention relates to the fields of natural product extraction and purification and biomedicine, specifically to a daylily polysaccharide with broad-spectrum antibacterial activity and its applications. Background Technology

[0002] With the widespread overuse of antibiotics, bacterial resistance has evolved into a global public health crisis. In particular, community-acquired methicillin-resistant Staphylococcus aureus (MRSA), represented by USA300 strain, poses a huge challenge to clinical treatment due to its high mortality rate and rapid spread. Developing novel antibacterial agents with new mechanisms of action that are less likely to induce resistance is urgently needed.

[0003] Natural plant polysaccharides have become an important direction for antibacterial drug research and development due to their safe source and good biocompatibility. As a traditional medicinal and edible plant, the research on polysaccharide components of daylily has focused on antioxidant and immunomodulatory aspects. More importantly, the daylily polysaccharides reported in the existing literature are mostly linear or low-branched fructans, and there are no reports of their direct antibacterial activity against multidrug-resistant bacteria (especially methicillin-resistant Staphylococcus aureus USA300), nor are there any publicly available technologies for verifying efficacy in animal models of Staphylococcus aureus pneumonia. Therefore, developing a daylily polysaccharide with efficient processing, well-defined structure, and effective resistance to drug-resistant bacterial infections has significant clinical translational value. Summary of the Invention

[0004] The primary objective of this invention is to provide a daylily polysaccharide with broad-spectrum antibacterial activity. This daylily polysaccharide is extracted, separated, and purified from dried daylilies. The polysaccharide has a molecular weight of 1959 Da, and its monosaccharide composition is mainly fructose (88.87%) and glucose (11.13%), classifying it as a high-fructose neutral polysaccharide. The main chain is →1)-β-D-Fru f -(2→,Some main chain residues have side chains attached to the O-6 positions, and the side chains contain β-D-Fru f -(2→and→6)-β-D-Fru f -(2→;sugar residue Fru f 1,2 Fru f 2. Fru f 1,2,6 Fru f 2,6 and Glc 1,6 The molar percentages are 41.94%, 23.59%, 9.27%, 16.80%, and 8.40%, respectively; its chemical structural formula is as follows: Another object of the present invention is to propose the application of the daylily polysaccharide in the following two aspects: (1) Application of the daylily polysaccharide in the preparation of drugs for treating bacterial pneumonia.

[0005] (2) Application of the daylily polysaccharide in the preparation of broad-spectrum antibacterial products.

[0006] The beneficial effects of this invention are: (1) The daylily polysaccharide DPH extracted in this invention showed significant antibacterial activity against clinically isolated multidrug-resistant strains and standard strains, and exhibited dose-dependent activity; the inhibition zone diameter of 50 mg / mL DPH against Staphylococcus aureus resistant strains USA300 and Mu50 reached 13 and 14 mm, respectively, and the inhibition zone diameter of 100 mg / mL DPH against Staphylococcus aureus resistant strains USA300, Mu50 and Mw2 reached 14-26 mm; The purified DPH-1 showed significant inhibitory effects on Salmonella pullorum CVCC519, Pseudomonas aeruginosa CMCC(B)10104, Escherichia coli MG1655, Lactobacillus plantarum ZJ316, and Lactobacillus amyloliquefaciens CICC6090, demonstrating its unique broad-spectrum inhibitory effect. Furthermore, the minimum bactericidal concentrations of the purified DPH-1 against Escherichia coli MG1655, Staphylococcus aureus USA300, and Salmonella pullorum CVCC519 were 12, 15, and 10 mg / mL, respectively.

[0007] (2) The effect of the daylily polysaccharide DPH extracted in this invention on lung tissue damage increases with increasing dose. The medium and high dose groups are significantly different from the model group. The effect is quantifiable and reproducible, which meets the requirements of drug development. Attached Figure Description

[0008] Figure 1 This is the elution curve of DPH in Example 1 of the present invention.

[0009] Figure 2 This is a structural analytical diagram of DPH in Embodiment 2 of the present invention, wherein... Figure 2 (a) is the high-performance gel permeation chromatogram of DPH-1. Figure 2 (b) is the ion chromatogram of DPH-1. Figure 2 (c) is the Fourier transform infrared spectrum of DPH-1.

[0010] Figure 3 This is a structural analytical diagram of DPH-1 in Embodiment 2 of the present invention; wherein, Figure 3 (a) A scanning electron microscope (SEM) image of the DPH-1 at 500x magnification. Figure 3 (b) A DPH-1 scanning electron microscope (SEM) image magnified 2000 times. Figure 3 (c) is a two-dimensional image of DPH-1 under an atomic force microscope. Figure 3(d) Three-dimensional image of DPH-1 under an atomic force microscope.

[0011] Figure 4 This is the nuclear magnetic resonance spectrum of DPH-1 in Embodiment 2 of the present invention; wherein Figure 4 (a) is the NMR spectrum of 1H; Figure 4 (b) is the NMR spectrum of 13C; Figure 4 (c) is the HSQC spectrum.

[0012] Figure 5 This is the nuclear magnetic resonance spectrum of DPH-1 in Embodiment 2 of the present invention; wherein Figure 5 (a) is the COSY spectrum; Figure 5 (b) is the HSQC-TOCSY spectrum; Figure 5 (c) is the HMBC spectrum.

[0013] Figure 6 This is the NMR NOESY spectrum of DPH-1 in Embodiment 2 of the present invention.

[0014] Figure 7 This is a structural model diagram of DPH-1 in Embodiment 2 of the present invention; wherein Figure 7 (a) is a schematic diagram of the primary structure of DPH-1. Figure 7 (b) is the chemical structural formula of DPH-1.

[0015] Figure 8 This refers to the antibacterial activity of DPH against some drug-resistant bacteria in Example 3 of the present invention; wherein... Figure 8 (a) is Staphylococcus aureus USA300. DPH (50 mg / mL) was added to Oxford cup-1, DPH (100 mg / mL) was added to Oxford cup-2, oxacillin (8 μg / mL) was added to Oxford cup-3, and sterile enzyme-free water was added to Oxford cup-4. Figure 8 (b) Staphylococcus aureus Mw2, Oxford cup-1 contains DPH (100 mg / mL), Oxford cup-2 contains DPH (50 mg / mL), Oxford cup-3 contains ampicillin sodium (2 mg / mL), and Oxford cup-4 contains sterile enzyme-free water; Figure 8 (c) is Staphylococcus aureus Mu50. DPH (50 mg / mL) was added in Oxford cup-1, DPH (100 mg / mL) was added in Oxford cup-2, ampicillin sodium (2 mg / mL) was added in Oxford cup-3, and sterile enzyme-free water was added in Oxford cup-4. Figure 8(d) is Pseudomonas aeruginosa CMCC(B)10104. DPH (100 mg / mL) was added in Oxford Cup-1, DPH (50 mg / mL) was added in Oxford Cup-2, Ampicillin Sodium (2 mg / mL) was added in Oxford Cup-3, and sterile enzyme-free water was added in Oxford Cup-4. Figure 8 (e) is Salmonella pullorum CVCC519. In Oxford cup-1, DPH (100 mg / mL) was added; in Oxford cup-2, DPH (50 mg / mL) was added; in Oxford cup-3, sterile and enzyme-free water was added; and in Oxford cup-4, ampicillin (2 mg / mL) was added. Figure 8 (f) is Escherichia coli MG1655. DPH (100 mg / mL) was added in Oxford Cup-1, DPH (50 mg / mL) was added in Oxford Cup-2, sterile enzyme-free water was added in Oxford Cup-3, and ampicillin (2 mg / mL) was added in Oxford Cup-4. Figure 8 (g) is Lactobacillus plantarum ZJ316. DPH (50 mg / mL) was added to Oxford Cup-1, DPH (100 mg / mL) was added to Oxford Cup-2, ampicillin (2 mg / mL) was added to Oxford Cup-3, and sterile enzyme-free water was added to Oxford Cup-4. Figure 8 (h) is Lactobacillus amyloliquefaciens CICC6090. DPH (100 mg / mL) was added to Oxford Cup-1, DPH (50 mg / mL) was added to Oxford Cup-2, sterile enzyme-free water was added to Oxford Cup-3, and ampicillin (2 mg / mL) was added to Oxford Cup-4.

[0016] Figure 9 The images show the pathological changes in the lungs of mice treated under different conditions in Example 4 of this invention.

[0017] Figure 10 These are pathological sections of mouse lungs and CD177 immunohistochemistry from different treatment groups in Example 4 of this invention. Detailed Implementation

[0018] The present invention will be further described in detail below with reference to specific embodiments and accompanying drawings, but the scope of protection of the present invention is not limited to the content described; unless otherwise specified, all reagents used in the present invention are commercially available analytical grade reagents.

[0019] Example 1 Extraction of polysaccharides from daylily (1) Pretreatment: The dried daylily products were dried at 60℃ until the quality was constant, pulverized into powder by a pulverizer, and passed through a 400-mesh sieve; the powder was wrapped in filter paper and placed in a Soxhlet extractor, and defatted by reflux with petroleum ether (85℃, 3h); the filter paper package was removed and placed in a fume hood to air dry; the air-dried filter paper package was placed back into the Soxhlet extractor and refluxed with an 80% ethanol solution (85℃, 6h) to remove alcohol-soluble impurities such as monosaccharides and oligosaccharides in the sample, and finally the filter paper package was air-dried naturally.

[0020] (2) Hot extraction: The air-dried filter paper package was put into the Soxhlet extractor and 2000 mL of deionized water (solid-liquid ratio of 1:20) was added. The mixture was extracted at 90°C for 6 h. The filtrate was collected and filtered with filter paper. Then, 1000 mL of deionized water was added to the Soxhlet extractor and the mixture was extracted at 90°C for 3 h. The filtrate was collected and filtered with filter paper. The filtrates from the two filtrations were then combined.

[0021] (3) Rotary evaporation: The filtrate from step (2) is concentrated to 300 mL using a rotary evaporator to obtain a concentrated solution.

[0022] (4) Alcohol precipitation: Slowly add 4 times the volume of anhydrous ethanol to the concentrate, and slowly stir and mix with a glass rod until the ethanol volume fraction in the mixed solution is 80%. Let it stand at 4℃ for 24 h, centrifuge (6000 rpm, 5 min), and collect the precipitate, which is the crude polysaccharide. Hemerocallis citrina Baroni (CPH).

[0023] (5) Refining: Dissolve CPH in water at a solid-liquid ratio of 1:6, add 1.05% of the mass of CPH papain, dissolve, adjust the pH to 7 with hydrochloric acid, keep warm in a water bath (62℃, 100min), and centrifuge to collect the supernatant.

[0024] (6) Decolorization: The pH of the supernatant obtained in step (5) is adjusted to 8 with ammonia water, and then 30% hydrogen peroxide is added dropwise until the polysaccharide solution is light yellow. The solution is then kept warm in a water bath (40℃, 50min).

[0025] (7) Purification: The obtained polysaccharide aqueous solution was placed at room temperature for 72 hours and then concentrated under reduced pressure to 300 mL. The precipitate was obtained by alcohol precipitation as in step (4). The precipitate was washed three times each with 95% ethanol, anhydrous ethanol, and acetone. The precipitate was then freeze-dried to obtain purified polysaccharides. Hemerocallis citrina Baroni (DPH).

[0026] (8) Purification of polysaccharides by chromatography: DEAE-52 cellulose anion exchange resin was swollen in water, and then the swollen resin was filled into the chromatography column. 300 mg of DPH from step (7) was dissolved in 10 mL of water, filtered through a 0.45 μm microporous membrane, and loaded onto the column. Gradient elution was performed using a constant flow pump (the elution components were water and 0.1 mol / L NaCl solution). The solution was then concentrated by a rotary evaporator, dialyzed for 24 h using a 1000 Da dialysis bag, and then freeze-dried to obtain homogeneous polysaccharides.

[0027] In this embodiment, the extraction of polysaccharides from daylily yielded a crude extraction rate of 23%, and the subsequent purification efficiency using a DEAE-52 cellulose column was 20%. During the column chromatography purification process, two DPH fractions were successfully eluted, such as... Figure 1 As shown, the yield of the component eluted by water was 20%, and the yield of the component eluted by 0.1 mol / L NaCl solution was 4.1%. The DPH component with the highest yield was named DPH-1 and used for subsequent studies.

[0028] Example 2 Structural analysis of daylily polysaccharide DPH-1 (1) Determination of the relative molecular weight of daylily polysaccharide DPH-1: 5 mg of DPH-1 was dissolved in 1 mL of NaCl solution (concentration of 0.01 mol / L) to prepare a DPH-1 solution with a concentration of 5 mg / mL. The relative molecular weight was determined by high performance gel permeation chromatography. Dextran T1 (1080 Da), maltotetraose (M4, 666 Da), and pullulan (6000 Da) were used as a series of standards to obtain the molecular weight calibration curve as follows: logM = 27 - 3.73V 1 +0.228V 2 -0.00504V 3 R 2 =0.9998.

[0029] In the formula, M is the molecular weight and V is the retention time of the sample.

[0030] according to Figure 2 (a) Its relative molecular weight is calculated to be 1959 Da.

[0031] (2) Determination of monosaccharide components of daylily polysaccharide DPH-1: The monosaccharide components of DPH-1 were analyzed by ion chromatography and determined by high performance anion exchange chromatography-pulse amperometric detection (HPAEC-PAD). The steps are as follows: ① Using fucose, rhamnose, arabinose, galactose, glucose, xylose, galactosamine, glucosamine, mannose, fructose, ribose, galacturonic acid, glucuronic acid, mannuronic acid, and guluronic acid as standards, prepare a single standard stock solution of 100 mg / mL, and then dilute it to a gradient mixed standard solution of 0.3~48 μg / mL for later use.

[0032] ② Weigh 2 mg of DPH-1 sample, add 1 mL of 2 mol / L trifluoroacetic acid, hydrolyze at 60℃ for 30 min, dry with nitrogen, wash 2-3 times with chromatographic grade methanol, and dissolve the residue in sterile water to obtain the test solution.

[0033] ③ An ICS 5000+ ion chromatography system was used, equipped with a Dionex CarboPac PA20 column (150×3.0mm, 10μm) and an electrochemical detector. Mobile phase A was water, mobile phase B was 0.1M NaOH solution, and mobile phase C was 0.1mol / L NaOH + 0.2mol / L NaAc. The flow rate was 0.5mL / min, the column temperature was 30℃, the injection volume was 5μL, and the elution gradient was shown in Table 1. Table 1 Elution gradient Qualitative analysis was performed using the retention time of standards. A standard curve was plotted using the external standard method. The content of each monosaccharide was calculated based on the peak area of ​​the samples to determine the monosaccharide composition and relative proportions of DPH-1. The ion chromatogram is shown below. Figure 2 As shown in (b), the monosaccharide composition of DPH-1 is mainly fructose (88.87%) and glucose (11.13%).

[0034] (3) Characteristic analysis of daylily polysaccharide DPH-1: On a Fourier transform infrared spectrometer, at 4000~400cm -1 Infrared spectra were measured within the range, with 32 scans and a resolution of 4 cm⁻¹. -1 ,like Figure 2 As shown in (c), the analysis revealed that the daylily polysaccharide DPH-1 has polysaccharide characteristics and is a neutral polysaccharide; the purity of DPH-1 was determined by ultraviolet spectroscopy, and the results showed that the purity of DPH-1 was high; the amorphous and crystalline properties of DPH-1 were determined by X-ray diffraction, and the results showed that the polysaccharide has an amorphous structure; the structure of DPH-1 was analyzed by Congo red assay, and the results showed that DPH-1 does not have a triple helix structure.

[0035] (4) Morphological characteristics analysis of daylily polysaccharide DPH-1: The morphological characteristics of daylily polysaccharide DPH-1 were analyzed by scanning electron microscopy and atomic force microscopy, such as... Figure 3 (a) and Figure 3 As shown in (b), DPH-1 mainly has an irregular sheet-like structure with a smooth surface; Figure 3 (c) and Figure 3 As shown in (d), the structure of DPH-1 is circular or elliptical with an average height of about 200 nm, indicating that the polysaccharide sample exhibits a clear aggregate morphology on the mica surface. Moreover, the average height of DPH-1 is much higher than the typical height range (0.1-1 nm) of single-chain polysaccharides, indicating that the polysaccharide molecules in the sample do not exist in the form of single chains, but form large aggregates through intermolecular interactions. This result is consistent with the branched structure characteristics of polysaccharides, and the branched structure may promote interchain entanglement and aggregation.

[0036] (5) Methylation analysis of daylily polysaccharide DPH-1: 1 mg of daylily polysaccharide DPH-1 was dissolved in 1 mL of dimethyl sulfoxide (DMSO), 30 mg of NaOH solution was added and incubated for 30 min, and then iodomethane solution was added in two portions (250 μL each time) and the reaction was carried out in the dark for 2 h to complete methylation; the reaction solution was extracted with dichloromethane, washed with water to remove salt, dried with nitrogen, and then hydrolyzed with 0.5 M trifluoroacetic acid at 70 °C for 3 h. The hydrolysis product was reduced with sodium borodeuteride and acetylated with acetic anhydride to obtain methylated sugar alcohol acetate derivatives for GC-MS analysis.

[0037] ①Gas chromatography conditions: Chromatographic column: HP-5MS capillary column (30m×0.25mm×0.25μm).

[0038] Carrier gas: High-purity helium (≥99.999%).

[0039] Flow rate: 1.0 mL / min.

[0040] Column temperature: The initial temperature of the column oven is 50℃, held for 1.0 min; the temperature is increased to 130℃ at 50℃ / min; then the temperature is increased to 230℃ at 3℃ / min and held for 2 min.

[0041] ②Mass spectrometry conditions: Scan range: 30-600 mass-to-charge ratio.

[0042] The results of DPH-1 methylation by gas chromatography-mass spectrometry (GC-MS) are shown in Table 2.

[0043] Table 2. Gas chromatography-mass spectrometry (GC-MS) analysis results of DPH-1 methylated sugar alcohol acetate. (6) Nuclear magnetic resonance analysis of daylily polysaccharide DPH-1: The lyophilized DPH-1 sample was dissolved in 0.5 mL of deuterated water (D2O, 99.9%). After complete dissolution, it was analyzed using an Agilent 800MHz DD2 nuclear magnetic resonance spectrometer. The calibration was performed using HDO δ_H = 4.70 ppm and acetone methyl carbon δ_C = 29.84 ppm. The results are as follows: Figure 4-6 As shown, one-dimensional NMR is measured. 1 H-NMR ( Figure 4 (a) 13 C-NMR ( Figure 4 (b) and two-dimensional nuclear magnetic resonance HSQC ( Figure 4 (c)), COSY ( Figure 5 (a)), TOCSY ( Figure 5 (b) HMBC ( Figure 5 (c) NOESY spectrum ( Figure 6 ).

[0044] Analysis revealed that: 1 In the H-NMR spectrum, there is no obvious strong signal in the anomeric hydrogen region δ4.2~5.5ppm, only a weak signal at δ5.30ppm, and the remaining hydrogen signals are concentrated in δ3.0~4.5ppm; 13 Multiple signals were observed in the δ90–110 ppm anodic carbon region of the C-NMR spectrum. Among them, the strong signal at δ103–105 ppm was a quaternary carbon without attached hydrogen. Combined with monosaccharide composition and methylation analysis, it was determined to be the anodic carbon (C-2) of a β-furan-type fructose residue. The anodic signals at δ5.30 ppm / δ92.16 ppm were assigned to the terminal groups of α-pyran-type glucose residues. Through joint analysis of multiple spectra, four β-furan-type fructose residues (→1)-β-D-Fru f -(2→;β-D-Fru f -(2→;→1,6)-β-D-Fru f -(2→and→6)-β-D-Fru f -(2→)and one α-pyranose glucose residue (→6)-α-D-Glc p - (1→) All 1 H, 13 Precise assignment of C chemical shifts.

[0045] By combining HMBC long-range correlation and NOESY spatial correlation signals, the linkage characteristics of each sugar residue were determined: ①→6)-α-D-Glc p -(1→H-1 and →1 in sugar residues)β-D-Fru f - (2→) The C-2 of the sugar residue has a β (2→1) linkage at the O-2 site.

[0046] ②→1)-β-D-Fru f -(2→between sugar residues,→1)-β-D-Fru f -(2→sugar residues and→1,6)-β-D-Fru f - (2→) sugar residues are all connected by β (2→1) links, with the site being O-2.

[0047] ③→1,6)-β-D-Fru f -(2→sugar residues and→6)-β-D-Fru f -(2→sugar residues have β(2→6) linkages at site O-6.

[0048] Carbon spectrum of DPH-1 sugar residues ( 13 C) and hydrogen spectrum ( 1 H) Signal attribution is shown in Table 3, Fru f 1,2 Fru f 2. Fru f 1,2,6 Fru f 2,6 and Glc 1,6 The molar percentages were 41.94%, 23.59%, 9.27%, 16.80%, and 8.40%, respectively, of which fructose residues were... f 1,2 Fru f 2. Fru f 1,2,6 Fru f 2,6 The molar ratio was 5:3:1:2, indicating that DPH-1 is a fructan.

[0049] Table 3 shows the carbon spectrum of DPH-1 ( 13 C) and hydrogen spectrum ( 1 H) Signal Attribution Based on the methylation analysis results of the polysaccharide sample and the one-dimensional and two-dimensional NMR information analysis, it is preliminarily inferred that the structure of the polysaccharide sample is a →1)-β-D-Fru f -(2→ main chain fructan, in part→1)-β-D-Fru f -(2→ has a side chain at position O-6, the side chain is β-D-Fru f -(2→、→6)-β-D-Fru f -(2→,its structural model is as follows Figure 7 As shown.

[0050] Example 3 Broad-spectrum antibacterial effect of daylily polysaccharides To verify the antibacterial effect of daylily polysaccharide, this invention purchased methicillin-resistant Staphylococcus aureus (USA300, Mu50, Mw2), Salmonella pullorum (CVCC519), Pseudomonas aeruginosa (CMCC(B)10104), Escherichia coli (MG1655), Lactobacillus plantarum ZJ316, and Lactobacillus amyloliquefaciens CICC6090 for antibacterial experiments.

[0051] (1) Analysis of the antibacterial effect of DPH on some drug-resistant strains The inhibitory effects of daylily polysaccharide DPH on Staphylococcus aureus (USA300, Mu50, Mw2), Pseudomonas aeruginosa (CMCC(B)10104), Salmonella pullorum (CVCC519), and Escherichia coli MG1655 were detected using the Oxford cup agar diffusion method. The specific steps were as follows: ① After activating the above-mentioned strains, prepare the original bacterial suspension, and dilute the bacterial suspension with culture medium to 1×10⁻⁶. 9 CFU / mL was prepared for use, with Staphylococcus aureus diluted with tryptone soybean broth, Pseudomonas aeruginosa diluted with Pseudomonas isolation broth, Salmonella cholerae diluted with hydrolyzed casein broth, and Escherichia coli diluted with LB broth.

[0052] ② Pour 8 mL of the melted corresponding agar medium into a petri dish and let it cool to form the bottom layer.

[0053] ③ Add 10 μL of diluted bacterial solution to 10 mL of melted corresponding agar medium, mix well, and pour it onto the bottom layer as the bacterial layer. When the bacterial layer is semi-solidified, place it into 4 Oxford cups.

[0054] ④ After cooling, add antibiotics (100 mg / mL) to each Oxford cup on each plate. -1 DPH, 50 mg / mL -1 The DPH and sterile enzyme-free water were added in a volume of 100 μL; the corresponding antibiotic for Staphylococcus aureus (USA300, Mu50, Mw2) was oxacillin (concentration of 8 μg / mL), and the corresponding antibiotic for the other strains was ampicillin (concentration of 2 mg / mL).

[0055] ⑤ Each plate was first diffused in a 4℃ refrigerator for 5 hours, then transferred to a 37℃ incubator and incubated upright for 24 hours; the diameter of the inhibition zone around the Oxford cup was measured, and the average value was taken to compare the antibacterial effects. The results are shown in Table 4 and . Figure 8As shown, DPH has antibacterial effects against eight Gram-positive and Gram-negative bacteria, including Staphylococcus aureus (USA300, Mu50, Mw2), Pseudomonas aeruginosa (CMCC(B)10104), Salmonella pullorum (CVCC519), Escherichia coli (MG1655), Lactobacillus plantarum (ZJ316), and Lactobacillus amyloliquefaciens (CICC6090). Among the Gram-positive bacteria, DPH has the most significant antibacterial effect against Lactobacillus plantarum ZJ316. When the DPH concentration is 100 mg / mL, the inhibition diameter of Lactobacillus plantarum ZJ316 reaches 32 mm.

[0056] Table 4 Comparison of the antibacterial effects of DPH on some bacterial strains Note: Inhibition zone <15mm indicates low sensitivity, 15mm≤inhibition zone<19mm indicates moderate sensitivity, and inhibition zone ≥19mm indicates high sensitivity.

[0057] Meanwhile, it was found that antibiotics had no antibacterial effect on Staphylococcus aureus Mu50, Mw2, Pseudomonas aeruginosa CMCC(B)10104 and Lactobacillus plantarum ZJ316 (inhibition zone diameter was 0 mm). However, the DPH prepared in this invention can effectively inhibit their growth. In particular, the DPH (100 mg / mL) component can form a stable inhibition zone for all tested strains, and the diameter of the inhibition zone is 14-32 mm.

[0058] (2) MIC / MBC determination of DPH against some drug-resistant strains The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of purified DPH-1 against Staphylococcus aureus USA300, Salmonella pullorum CVCC519, and Escherichia coli MG1655 strains were determined using the micro-broth dilution method and the agar plate spread method. The specific steps are as follows: ①MIC determination: After activating the strain, a raw bacterial culture was prepared, and the raw bacterial culture was diluted into a bacterial suspension using broth medium (OD200). 600=1), then dilute 1000 times with culture medium for later use; prepare 40 mg / mL DPH-1 solution, and dilute with culture medium to seven concentrations of 40, 30, 24, 20, 10, 4 and 2 mg / mL for later use; add 100 μL of the above concentrations of DPH-1 solution to each well of a 96-well plate, and perform three replicates for each concentration; set up control groups as follows: 200 μL bacterial suspension (growth control group), 200 μL pure water (negative control group), 100 μL pure water and 100 μL bacterial suspension (diluted growth control group); incubate at 37℃ for 24 h, and the concentration corresponding to the lowest concentration in the clear well is the MIC.

[0059] ②MBC determination: Take a sample from the clear well obtained in step ①, spot it onto a drug-free agar medium, and observe after incubation. The lowest concentration at which no colonies grow is the MBC.

[0060] The results are shown in Table 5. The purified DPH-1 fraction had a MIC of 10 mg / mL and an MBC of 12 mg / mL against the Gram-negative bacterium Escherichia coli MG1655; a MIC of 12 mg / mL and an MBC of 15 mg / mL against the Gram-positive bacterium Staphylococcus aureus USA300; and a MIC and MBC of 10 mg / mL against Salmonella pullorum CVCC519, demonstrating good broad-spectrum antibacterial properties.

[0061] Table 5 Comparison of the antibacterial effects of DPH-1 on some bacterial strains In summary, DPH and its purified component DPH-1 exhibit antibacterial activity against both Gram-positive and Gram-negative bacteria, demonstrating their broad-spectrum antibacterial properties and potential application value in the prevention and control of bacterial diseases in livestock and poultry.

[0062] Example 4 The therapeutic effect of daylily polysaccharide DPH on a mouse model of Staphylococcus aureus pneumonia. (1) Thirty-six four-week-old SPF-grade female C57 mice were randomly divided into six groups of six mice each. The mice were challenged by nasal drops and divided into control group, Staphylococcus aureus model group, positive control group, low-dose DPH group, medium-dose DPH group and high-dose DPH group. Before the experiment, the mice's head, nostrils and oral cavity were disinfected with 75% alcohol by swabs, and then the alcohol was dripped into the mouth and nose. The treatment of each group of mice is shown in Table 6.

[0063] Table 6. Treatment of different groups in the mouse pneumonia model Mice in each group were euthanized one day and three days after treatment, and lung tissue was collected. Figure 9 As shown, the pathological changes in the lungs of mice indicate that the therapeutic effect of daylily polysaccharide DPH on the mouse pneumonia model is dose-dependent, and after 3 days of treatment, the medium- and high-dose daylily polysaccharide DPH group showed significant improvement compared with the model group.

[0064] The pathological results of lung tissue sections of mice in each group are as follows: Figure 10 As shown, after HE staining, except for the high-dose DPH group and the vancomycin group, increased red blood cells were observed in the lung tissue of each infected group, accompanied by a large number of inflammatory cell infiltrations, which led to partial alveolar collapse or disappearance of consolidation. This phenomenon was more significant on the first day after modeling. In the high-dose DPH treatment group, no obvious tissue damage was observed, and the treatment effect was better compared with the VAN (vancomycin) group.

[0065] CD177 + CD177 is an important marker of neutrophils. + Cells can mediate intense inflammatory responses by secreting pro-inflammatory cytokines; such as Figure 7 As shown, CD177 in the lung tissue of mice infected with USA300 + The area and intensity of positive staining in cells were significantly increased; however, after DPH intervention, CD177... + Cellular expression was significantly downregulated; these results suggest that DPH may inhibit CD177. + The expression level of cells, thereby exerting an anti-inflammatory effect.

[0066] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A daylily polysaccharide with broad-spectrum antibacterial activity, characterized in that: Derived from daylily, the polysaccharide has a molecular weight of 1959 Da, and its monosaccharides are composed of fructose and glucose by mass percentage, with fructose accounting for 88.87% and glucose for 11.13%. The sugar residues in the daylily polysaccharide are... f 1,2 Fru f 2. Fru f 1,2,6 Fru f 2,6 and Glc 1,6 The molar percentages were 41.94%, 23.59%, 9.27%, 16.80%, and 8.40%, respectively.

2. The daylily polysaccharide according to claim 1, characterized in that, The chemical structural formula of daylily polysaccharide is as follows.

3. The use of the daylily polysaccharide according to claim 1 in the preparation of a drug for treating bacterial pneumonia.

4. The application of the daylily polysaccharide according to claim 1 in the preparation of broad-spectrum antibacterial products.

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