An extraction method of Polygonatum cyrtonema polysaccharides and their applications

Abstract

The present invention relates to the technical field of natural product extraction and application, and particularly to an extraction method for Polygonatum cyrtonema polysaccharides and their applications. The extraction method comprises the following steps: Polygonatum cyrtonema is subjected to water extraction, alcohol precipitation, and drying to obtain a crude extract. The crude extract is then mixed with ethanol and centrifuged to collect the precipitate. This precipitate is subsequently subjected to water bath extraction to obtain a crude polysaccharide extract. The crude polysaccharide extract is then treated with enzymatic hydrolysis and impurity removal, followed by ion exchange purification and gel purification, to obtain purified Polygonatum cyrtonema polysaccharides. The Polygonatum cyrtonema polysaccharides obtained by this method can improve the condition, colon length, and histopathological features (HE staining) of DSS-induced colitis mice. In addition, they can inhibit the expression of inflammatory cytokines IL-6, IL-lß, and TNF-oc, as well as suppress the abnormal activation of the MAPK / NF-KB inflammatory signaling pathways.

Description

TECHNICAL FIELD This invention relates to the eld of natural product extraction and application, and more particularly, to . BACKGROUND Polygonatum cyrtonema Hua is one of the three source plants of Polygonatum recorded in the Chinese Pharmacopoeia. The polysaccharides of P. cyrtonema, being the major active components, are not only present in high content but also regarded as a quality control marker for Polygonatum in the Pharmacopoeia of China. Previous studies have reported that P. cyrtonema polysaccharides possess various biological functions, including anti-inammatory, anti-cancer, and anti-fatigue effects. In recent years, increasing attention has been paid to the correlation between the structural characteristics and biological activities of P. cyrtonema polysaccharides. However, in- depth investigations into their structural features remain limited, and studies focusing on the anti-inammatory effects of specic active fractions in the intestinal tract and the underlying mechanisms are still lacking. SUMMARY The present invention relates to the technical eld of natural product extraction and application, particularly to an extraction method of Polygonatum cyrtonema polysaccharides and the application of Polygonatum cyrtonema polysaccharides. Polygonatum cyrtonema Hua is one of the three original plants of Polygonatum Hua recorded in the Chinese Pharmacopoeia. Polygonatum cyrtonema polysaccharide, as the main active component in Polygonatum cyrtonema, has a relatively high content and is considered a quality control indicator of Polygonatum in the Chinese Pharmacopoeia. It has been reported to possess antiinammatory, anti-cancer, and anti-fatigue functions. In recent years, there have been many research reports on the relationship between the structural properties and functional activities of Polygonatum cyrtonema polysaccharides. However, in-depth studies on its structural characteristics are still limited, and there is a lack of research on the intestinal anti-inammatory effects of active sites of Polygonatum cyrtonema polysaccharides and their mechanisms of action. The purpose of the present invention is to provide an extraction method of Polygonatum cyrtonema polysaccharides and the application of Polygonatum cyrtonema polysaccharides. To achieve the above purpose, the present invention provides the following solutions: One of the technical solutions of the present invention, an extraction method of Polygonatum cyrtonema polysaccharides, comprising the following steps: Step 1, decocting Polygonatum cyrtonema with water twice, combining the two ltrates, and then concentrating, precipitating with ethanol, and drying to obtain a crude extract; Mixing the crude extract with ethanol and centrifuging, collecting the precipitate; Extracting the precipitate by water bath heating to obtain an extraction solution; concentrating the extraction solution and performing ethanol precipitation to obtain a crude polysaccharide extract; Step 2, mixing the crude polysaccharide extract with water and papain for enzymatic hydrolysis, then adding chloroform and n-butanol and mixing evenly, collecting the aqueous phase to obtain aqueous phase 1; adding petroleum ether to aqueous phase 1 and mixing, collecting the aqueous phase to obtain aqueous phase 2; adding macroporous resin AB8 to aqueous phase 2 and mixing for adsorption, dialyzing the obtained liquid and freezedrying to obtain crude polysaccharides; Step 3, dissolving the crude polysaccharides in water and centrifuging, subjecting the supernatant to ion exchange chromatography purication, selecting the elution solutions corresponding to the peaks eluted with puried water, 0.1M NaCl solution, 0.2M NaCl solution, and 0.3M NaCl solution, concentrating and dialyzing to obtain puried polysaccharides; Step 4, dissolving the puried polysaccharides in water and centrifuging, separating and purifying the supernatant with gel ltration chromatography, selecting the elution solution corresponding to the peak eluted with puried water, concentrating, dialyzing, and freeze-drying to obtain Polygonatum cyrtonema polysaccharides. Technical solution two of the present invention, Polygonatum cyrtonema polysaccharides prepared by the above extraction method. Technical solution three of the present invention, the above Polygonatum cyrtonema polysaccharides used in the preparation of medicine for treating colitis. Technical solution four of the present invention, the above Polygonatum cyrtonema polysaccharides used in the preparation of medicine for downregulating the content of inammatory cytokines IL-6, ILlB, and TNF-(x. The present invention discloses the following technical effects: The invention adopts hot water extraction, graded ethanol precipitation, and ion- exchange chromatography and gel chromatography to extract and purify Polygonatum polysaccharides, preparing Polygonatum cyrtonema polysaccharides. The Polygonatum cyrtonema polysaccharides prepared by the method of the invention can improve the condition, colon length, and HE pathological features of colitis mice induced by DSS. It can also inhibit the content of inammatory cytokines IL-6, IL-lß, and TNF-a and the abnormal activation of the MAPK / NFKB inammatory pathway. BRIEF DESCRIPTION OF THE FIGURES In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings required for use in the embodiments will be briey introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this eld, other drawings can be obtained based on these drawings without creative work. In Figure l, A is a ow chart of extraction, separation and purication of Polygonatum sibiricum polysaccharide, B is the ion purication elution curve of Polygonatum sibiricum polysaccharide, and C is the gel purication elution curve of Polygonatum sibiricum polysaccharide PCPDl. Figure 2 is the physicochemical properties and spectral analysis of Polygonatum sibiricum polysaccharide PCPD-l; wherein A is the monosaccharide composition of the monosaccharide mixture sample and Polygonatum sibiricum polysaccharide; B is the molar mass distribution of Polygonatum sibiricum polysaccharide PCPD-l; C is the FT- IR chromatogram of Polygonatum sibiricum polysaccharide PCPD-l; D is the SEM image of Polygonatum sibiricum polysaccharide PCPD-l (from left to right, the scale bars are 20um, lOum, 4pm, and Zum, respectively). Figure 3 is the total ion ow chart of the sample. Figure 4 is a nuclear magnetic resonance spectrum of Polygonatum sibiricum polysaccharide; wherein A is a 1H NMR spectrum, B is a 13C NMR spectrum, C is a lH-lH COSY spectrum, D is a lH-lH NOESY spectrum, E is a lH-13C HSQC spectrum, and F is a lH-l3C HMBC spectrum. Figure 5 shows the chemical structure of PCPD-l, a polysaccharide from Polygonatum sibiricum. In Figure 6, A is the experimental ow chart, B is the weight of mice, C is the length of mouse colon, and D is the result of mouse colon tissue staining (X20). Figure 7 shows the anti-inammatory effect of PCPD-l on LPS-induced THP-1 macrophages; among them, A is the effect on the viability of THP-1 macrophages (24H), B is the effect on the viability of THP1 macrophages (48H), C is the expression level of IL6, D is the expression level of ILlß, E is the expression level of TNFa, and F is the relative protein expression of IKBOL, ERK, p3 8, NF-KB p65 and its phosphorylation. DETAILED DESCRIPTION OF THE INVENTION Various exemplary embodiments of the present invention are now described in detail. This detailed description should not be considered as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, features and embodiments of the present invention. It should be understood that the terms described in the present invention are only for describing specic embodiments and are not used to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the stated range are also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded in the range. Unless otherwise specied, all technical and scientic terms used herein have the same meanings as commonly understood by those skilled in the art in the eld to which the present invention relates. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specication are incorporated by reference to disclose and describe methods and / or materials related to the documents. In the event of a conict with any incorporated document, the content of this specication shall prevail. It is obvious to those skilled in the art that various modications and changes may be made to the specic embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments obtained from the present invention description are obvious to the technician. The present invention description and examples are only exemplary. The terms "include", "including", "have", "contain", etc. used in this article are open terms, which means including but not limited to. The "%" described in the present invention, unless otherwise specied, represents the mass percentage. The present invention adopts hot water extraction, graded alcohol precipitation, ion exchange chromatography, and gel column chromatography to extract and purify Polygonatum cyrtonema polysaccharides, preparing Polygonatum cyrtonema polysaccharides, and conducts preliminary characterization of their physicochemical properties, including monosaccharide composition, purity, and molecular weight characterization. Infrared spectroscopy, liquid chromatography, gas chromatography, mass spectrometry, nuclear magnetic resonance, and electron microscopy are employed to characterize the surface morphology and structural analysis of Polygonatum cyrtonema polysaccharides. On this basis, a DSS colitis mouse model and a THP-1 cell inammation evaluation system are established, and intervention with Polygonatum cyrtonema polysaccharides is carried out, laying a material foundation for elucidating the pharmacological mechanism of Polygonatum cyrtonema. A rst aspect of the present invention provides a method for extracting Polygonatum cyrtonema polysaccharides, comprising the following steps: Step 1: Polygonatum cyrtonema is decocted with water twice, the two ltrates are combined, then concentrated, precipitated with alcohol, and dried to obtain a crude extract; The crude extract is mixed with ethanol and centriiged, and the precipitate is collected; The precipitate is extracted by water bath to obtain an extraction solution; the extraction solution is concentrated and precipitated with alcohol to obtain a crude polysaccharide extract; Step 2: the crude polysaccharide extract is mixed with water and papain for enzymatic hydrolysis, then chloroform and nbutanol are added and mixed evenly, and the aqueous phase is collected to obtain aqueous phase 1; petroleum ether is added to aqueous phase 1 and mixed, the aqueous phase is collected to obtain aqueous phase 2, and macroporous resin AB8 is added to aqueous phase 2 and mixed for adsorption. The resulting liquid is dialyzed and freezedried to obtain crude polysaccharides; Step 3: the crude polysaccharides are dissolved in water and centrifuged, the resulting supernatant is puried through an ion exchange column, and the elution peaks corresponding to puried water, 0.1M NaCl solution, 0.2M NaCl solution, and 0.3M NaCl solution are selected. The eluted solutions are concentrated and dialyzed to obtain puried polysaccharides; Step 4: the puried polysaccharides are added to water and centrifuged, the resulting supernatant is separated and puried through a gel ltration column, the elution peak corresponding to puried water is selected, the solution is concentrated, dialyzed, and freeze-dried to obtain Polygonatum cyrtonema polysaccharides. In a preferred embodiment of the present invention, in Step 1, the two decoction durations are independently 1.52 hours; the water bath extraction temperature is 60°C, and the duration is 4 hours. In a preferred embodiment of the present invention, in Step 2, the volume ratio of chloroform to n-butanol is 1:4; the amount of chloroform and n-butanol added is 1 / 4 of the enzymatic hydrolysis system volume. In a preferred embodiment of the present invention, in Step 3, the purication conditions for the supernatant through the ion exchange column are set as follows: ow rate of 4 mL / min; gradient elution is performed sequentially using puried water, 0.1M, 0.2M, and 0.3M NaCl solutions. In a preferred embodiment of the present invention, in Step 4, the separation and purication conditions for the supernatant through the gel ltration column are set as follows: ow rate of 1 mL / min; puried water is used to elute 1.5 times the column volume. In a preferred embodiment of the present invention, in Steps 3 and 4, the dialysis is independently performed using a 3000 Da dialysis bag for 4872 hours. A second aspect of the present invention provides Polygonatum cyrtonema polysaccharides prepared by the above extraction method. A third aspect of the present invention provides the application of the above Polygonatum cyrtonema polysaccharides in the preparation of a drug for treating colitis. A fourth aspect of the present invention provides the application of the above Polygonatum cyrtonema polysaccharides in the preparation of a drug for downregulating the contents of inammatory cytokines IL6, ILlB, and TNF-a. The technical solutions described in the present invention, unless otherwise specied, are all conventional methods in the eld. Reagents or raw materials used, unless otherwise specied, are all commercially available or already disclosed. The instruments used in the present invention are as follows: AKTA purication system (GE, USA), Allegra X12R refrigerated high-speed centrifuge (Beckman, USA), RE 5298A rotary evaporator (Yarong Biochemical, China), multifunctional microplate reader (Thermo Electron, USA), ESJ30-5A analytical balance (Shenyu Longteng), ICS 5000+ ion chromatograph (Thermo Electron, USA), gas chromatograph (Agilent, USA), mass spectrometer (Agilent, USA), AVANCE NEO SOOM nuclear magnetic resonance spectrometer (Bruker, Germany), Zeiss Merlin Compact high-resolution eld emission scanning electron microscope (Zeiss, Germany), Fourier transform infrared spectrometer (Thermo Electron, USA). The materials used in the present invention are as follows: The Polygonatum cyrtonema used in the present invention was purchased from Hunan Xinhui Pharmaceutical Co., Ltd., and is the dried mature fruit of Polygonatum cyrtonema Hua, a plant of the genus Polygonatum in the family Asparagaceae. Sephacryl S-400HR gel ltration column was purchased from GE. Dextran sulfate sodium (M0508D) was purchased from Meilun Bio. RPMIl640 culture medium and fetal bovine serum (FBS) were purchased from Hyclone, USA; phorbol lZ-myristate 13- acetate (PMA) was purchased from MCE, USA; LPS (Escherichia coli 01 1:B4), double antibiotics for cell culture, and Trizol were purchased from Sigma, USA; antibodies including IKBOL, p-IKBOL, ERK, pERK, pP38, P38, NF -KB, and p-NF-KB were purchased from Wuhan Sanying. The experimental animals used in the present invention are as follows: SPFgrade male C57BL / 6 mice aged 46 weeks (weighing 16l8g) were purchased from Hunan SJA Laboratory Animal Technology Co., Ltd. After purchase, the experimental animals were raised in the Animal Experiment Center of the Hunan Institute of Traditional Chinese Medicine (SPF level), with a 12-hour light / dark cycle, and provided with sufcient food and water. All animal experimental protocols were approved by the ethics committee of the respective institution. The experimental cells used in the present invention are as follows: Human acute monocytic leukemia cells (THP-l cell line) were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences. The test methods involved in the present invention are as follows: Infrared spectrum determination of Polygonatum cyrtonema polysaccharides An appropriate amount of the puried Polygonatum cyrtonema polysaccharide sample PCPD-l was mixed and ground with potassium bromide (KBr) at a mass ratio of 1:100, pressed into a pellet, and scanned on an infrared spectrometer. The Nicolet iZ-10 Fourier Transform Infrared Spectrometer was used for scanning analysis, with a resolution of 4.00 cm", a scanning range of 40004150 cm", and 32 scans. Determination of the molecular weight of Polygonatum cyrtonema polysaccharides The sample PCPD-l was dissolved in 0.1M NaN03 aqueous solution (containing 0.02% NaNOs, w / w) to a nal concentration of 1 mg / mL, ltered through a 0.45 um pore size lter, and then tested. The columns used were Ohpak SB-805 HQ (300><8 mm) and Ohpak SB803 HQ (300><8 mm) in series. Column temperature: 45°C; injection volume: 100 uL; mobile phase A (0.02% NaN03, 0.1M NaNOa); ow rate: 0.6 mL / min; elution gradient: isocratic for 75 min. Chromatographic data were processed using ASTRA 6.1 software. Monosaccharide composition analysis of Polygonatum cyrtonema polysaccharides The Thermo ICS 5000+ ion chromatography system was used with a DionexTM CarboPacTM PA20 (150><3.0 mm, 10 um) liquid chromatography column; injection volume: 5 L. Mobile phase A (H20), mobile phase B (0.1M NaOH), mobile phase C (0.1M NaOH, 0.2M NaAc); ow rate: 0.5 mL / min; column temperature: 30°C. Elution gradient: 0 min: A / B / C = 95:5:0 (V / V); 26 min: A / B / C = 85:5: 10 (V / V); 42 min: A / B / C = 85:5:10 (V / V); 42.1 min: A / B / C = 60:0:40 (V / V); 52 min: A / B / C = 60:40:0 (V / V); 52.1 min: A / B / C = 95:5:0 (V / V); 60 min: A / B / C = 95:5:0 (V / V). Preparation of mixed standards: 100 mg each of fucose, arabinose, galactose, glucose, xylose, mannose, fructose, ribose, galacturonic acid, and glucuronic acid were precisely weighed into a 10 mL volumetric ask, water was added to the mark to prepare a 10 mg / mL stock solution. The mixed standard stock solution was diluted 100 times to prepare a 100 ug / mL working solution, further diluted as needed, and placed into 1.5 mL EP tubes for use. An appropriate amount of PCPD-l polysaccharide sample was weighed, 1 mL of 2M TFA solution was added, and heated at 60°C for 1 hour. Nitrogen gas was used to dry the sample. 99.99% methanol was added for cleaning, followed by drying, and methanol cleaning was repeated 23 times. An appropriate amount of sterile water was added to dissolve the sample, which was then transferred into a chromatography vial for testing. Linkage structure analysis of Polygonatum cyrtonema polysaccharides An Agilent gas chromatography system (Agilent 7890A; Agilent Technologies, USA) was used, with column: BPX70 (30 m >< 0.25 mm >< 0.25 um, SGE, Australia). Injection volume: 1 uL; split ratio: 10: 1; carrier gas: high purity helium; initial oven temperature: 140°C held for 2.0 min, programmed temperature increase at 3°C / min to 230°C, held for 3 min. A quadrupole mass spectrometry system (Agilent 5977B; Agilent Technologies, USA) equipped with an electron ionization (EI) source and MassHunter workstation was used. Electron ionization (EI) mode was employed, analytes were detected in full scan (SCAN) mode, with a mass scan range (m / z): 50350. 5. Scanning electron microscopy (SEM) analysis of Polygonatum cyrtonema Polysaccharides The polysaccharide sample was passed through a lOOmesh sieve, a small amount was placed on conductive carbon adhesive tape, subjected to gold sputtering treatment, and scanned using an electron microscope at magnications ranging from 100X to 4000><. 6. Nuclear magnetic resonance (NMR) analysis of Polygonatum cyrtonema Polysaccharides A Bruker (Germany) 500 MHz nuclear magnetic resonance spectrometer was used for quantitative analysis at a scanning temperature of 25°C. An appropriate amount of puried polysaccharide was fully dissolved in D20 to prepare a polysaccharide solution at a concentration greater than or equal to 40 mg / mL. The dissolved solution was transferred into an NMR tube with an added volume of 0.5 mL. The NMR tube was then scanned using the spectrometer to acquire 1D 1H, 13C and 2D COSY, HSQC, HMBC, and NOESY spectra. 7. Animal grouping, administration, and processing After 7 days of acclimatization, 48 male C57BL / 6 mice were randomly divided into 6 groups: normal group (Ctrl), model group (DSS), positive control group (POS, sulfasalazine enteric-coated tablets), and low (PDl-L), medium (PDl-M), and high (PDl-H) dose groups of Polygonatum cyrtonema polysaccharide PCPD-l, with 8 mice per group. The POS group was administered 300 mg / kg of sulfasalazine suspension; the PDl-L, PDl-M, and PDl-H groups were administered 20 mg / kg, 40 mg / kg, and 60 mg / kg of Polygonatum cyrtonema polysaccharide PCPD-l solution, respectively. All groups received intragastric administration at 20 mL / kg of the corresponding drugs. The Ctrl and DSS groups received an equal volume of saline. Each group was pretreated for 3 days, and from day 4 to day 10, except for the normal and control groups, all other groups were given 2.5% DSS solution in their drinking water (note: DSS = dextran sulfate sodium solution, freshly prepared daily and protected from light). DSS water was replaced daily. After successful modeling, the mice were fasted but allowed water for 24 h. All animals were euthanized by intraperitoneal injection of sodium pentobarbital (60 mg / kg). After euthanasia, the colon segment from the anus to the end of the cecum was collected and its length measured. The tissue was ushed with precooled saline, dried using lter paper, weighed, and xed in 10% neutral formalin for HE staining. 8. Histopathological examination of colon tissue Colon tissue samples were xed in 10% neutral formalin for 24 h, embedded in parafn, and sliced into 6 um sections for HE staining. Under a light microscope, histopathological changes were observed at magnication, including crypt and goblet cell numbers, arrangement of glands and epithelial cells, mucosal layer damage, and the degree of inammatory cell inltration. 9. Cell culture THP-1 cells were cultured in suspension using RPMI-l640 medium (containing 10% fetal bovine serum, 50 M ß-mercaptoethanol, and 1% penicillin-streptomycin). After PMA-induced differentiation, cells were washed 3 times with PBS buffer, fresh 1640 medium was added, and LPS (nal concentration 1 L / mL) was added to stimulate THP- 1 cells, with different drug interventions added simultaneously. Blank control group: PBS; positive control group: LPS; PCPD-l low dose group: 50 ug / mL; medium dose group: 100 g / mL; high dose group: 200 g / mL. 10. MTT assay for cytotoxicity evaluation THP-1 cells were seeded into 96-well plates (cell density 7><105 cells / mL, 100 L per well), and treated with different concentrations of Polygonatum cyrtonema polysaccharides for 44 h. Then, 20 L of MTT (5 mg / mL) was added to each well, and the plates were incubated for an additional 4 h. After incubation, the supernatant was discarded, 150 L of DMSO was added, and the plates were shaken for 15 min to fully dissolve the formazan crystals. Absorbance at 490 nm was measured using a microplate reader. THP-1 cells were also seeded into 96-well plates at the same density, treated with LPS and different concentrations of polysaccharides for 44 h, and the same procedures were followed to measure absorbance at 490 nm. 1 1. Western blot analysis of inammation pathway proteins Cells were prepared as described in the cell culture section. After treatment with LPS and different concentrations of Polygonatum cyrtonema polysaccharides for 1 h, cellular proteins were extracted. Protein concentrations were determined using the BCA protein assay kit, and equal concentrations were prepared. Samples were denatured in a metal bath at 90°C for 10 min, separated via 10% SDSPAGE electrophoresis (15 g per lane), and transferred to PVDF membranes. The membranes were blocked with 5% skimmed milk at room temperature for 2 h, then incubated overnight at 4°C with antibodies against IKBOL, p-IKBd, ERK, pERK, P38, pP38, NF-KB, p-NF-KB, and GAPDH (all diluted 1:1000). After washing with Tris buffer, the membranes were incubated with HRP-conjugated anti-mouse or anti-rabbit IgG antibodies (both diluted 1:10000) at room temperature for 2 h. After washing, bands were visualized using an ECL ultrasensitive chemiluminescence kit and a chemiluminescence imaging system (Tanon). The following embodiments are provided to describe the technical solutions of the present invention in detail, but they should not be construed as limiting the scope of protection of the present invention. Example 1 Step 1: Extraction of crude Polygonatum cyrtonema Polysaccharides Take 2 kg of Polygonatum cyrtonema, decoct twice with water. For the rst decoction, add 10 times the weight of P. cyrtonema in water and boil for 2 hours; for the second decoction, add 8 times the weight in water and boil for 1.5 hours. Combine the two ltrates, pass through a ZOO-mesh sieve, concentrate the ltrate to a density of 1.10 g / cm3, add anhydrous ethanol to a nal ethanol content of 70 wt%, stir evenly, let stand for 24 hours, lter, and collect the precipitate. Dry the precipitate in a vacuum oven (temperature 6570°C, vacuum degree 0.070.08 MPa) to obtain the crude extract of P. cyrtonema. Grind the crude extract and pass it through a 60-mesh sieve. Add anhydrous ethanol, stir to extract fat-soluble pigments and partial impurities, centrifuge for 10 min, and collect the precipitate. Add pure water to the precipitate (solid-liquid ratio 1:10 g / mL), extract in a 60°C water bath for 4 h, centrifuge for 10 min, and collect the supernatant. Repeat the extraction once with the residual sediment using the same procedure. Combine the two extracts, concentrate by rotary evaporation under vacuum to 1 / 10 of the original volume, and add four times the volume of anhydrous ethanol for overnight precipitation. Centrifuge for 10 min to collect the precipitate, which is the crude polysaccharide extract. Step 2: Deproteinization and impurity removal of crude P. cyrtonema Polysaccharides Add pure water to the crude polysaccharide extract to fully dissolve it, then add papain (papain volume fraction in the crude polysaccharide solution is 2%) for enzymatic hydrolysis overnight. Add 1 / 4 volume of chloroform and n-butanol (volume ratio of chloroform to n-butanol is 1:4) to the enzymatic system, mix thoroughly, and collect the upper aqueous phase to obtain aqueous phase 1. Add petroleum ether to aqueous phase 1, mix thoroughly, and collect the lower aqueous phase to obtain aqueous phase 2. Add macroporous resin AB-8 to aqueous phase 2, mix thoroughly, and adsorb overnight. Collect the solution, dialyze using a 3000 Da dialysis bag for 48 h to remove small molecular components, and freeze-dry the polysaccharide solution to obtain crude polysaccharides. Step 3: Ion-exchange purication of crude P. cyrtonema polysaccharides Dissolve an appropriate amount of crude polysaccharides from Step 2 in pure water to prepare a polysaccharide stock solution at a concentration of 50 mg / mL. Centrifuge for 10 min, and use the supernatant for ion-exchange column purication at a ow rate of 4 mL / min. Use gradient elution with pure water, 0.1 M, 0.2 M, and 0.3 M NaCl solutions. Collect one tube every 15 mL, and collect all eluates. Use the phenol-sulfuric acid method to determine the total sugar content in each tube, and plot the ion-purication elution curve. Combine eluates corresponding to the same elution peak (pure water, 0.1 M, 0.2 M, and 0.3 M NaCl solutions), concentrate via rotary evaporation to 1 / 5 of the original volume. Dialyze with a 3000 Da dialysis bag for 60 h (4872 h) to remove salts and obtain the ion-exchange puried polysaccharides. Measure the content and purity of the polysaccharides. Step 4: Gel ltration purication of P. cyrtonema Polysaccharides Take the ionpuried polysaccharide sample from Step 3, dissolve in pure water to prepare a polysaccharide stock solution at a concentration of 20 mg / mL. Centrifuge for 10 min, and use the supernatant for gel ltration chromatography at a ow rate of 1 mL / min. Use pure water to elute 1.5 times the column volume. Collect one tube every 12 mL and collect all eluates. Determine the total sugar content in each tube using the phenolsulfuric acid method, and plot the gelpurication elution curve. Combine eluates corresponding to the same elution peak (pure water), concentrate via rotary evaporation to 1 / 5 of the original volume. Dialyze using a 3000 Da dialysis bag for 48 h and freeze-dry to obtain Polygonatum cyrtonema polysaccharides (denoted as PCPD-l). Determine the polysaccharide content and purity. Detection of the Polygonatum cyrtonema Polysaccharide prepared in Example 1: Isolation and purication analysis results of P. cyrtonema Polysaccharide In the present invention, P. cyrtonema polysaccharides were extracted and puried through hot water extraction, graded alcohol precipitation, deproteinization, ion exchange column, and gel ltration column, as shown in the owchart in Figure 1A. The extraction process of P. cyrtonema polysaccharides is often accompanied by coexisting proteins, forming high-molecular-weight polymers. To improve the purity of the polysaccharides and facilitate pharmacological effects, papain was used for protein removal. DEAE cellulose was used for crude separation and preliminary purication based on differences in charge properties of the polysaccharides. Different concentrations of elution buffers (e.g., NaCl) were applied to achieve separation of polysaccharides. Taking the tube number of eluate as the X-axis and the total sugar content (blue) and NaCl concentration in the eluate (green) as the Y-axis, the ion-purication elution curve of the sample was plotted, as shown in Figure 1B. Step 3 yielded three ion elution peaks: ion elution peak 1: PCP-D1; ion elution peak 2: PCP-D2; ion elution peak 3: PCP-D3. According to the total sugar content determination, the polysaccharide purities were: PCP-D1: 60.1%, PCP-D2: 75.4%, and PCP-D3: 43.3%. The overall recovery rate was 46.9%. Gel ltration chromatography, also known as sizeexclusion chromatography, separates polysaccharides based on molecular weight from large to small. The major component PCPD1 was further puried by gel ltration using Sephacryl S400HR dextran gel. The elution curve, plotted with tube number vs. absorbance value, is shown in Figure 1C. A single symmetric peak was collected, and after vacuum concentration and freezedrying, the puried PCPDl polysaccharide was obtained, with a purity of 79.0%. Physicochemical Properties and Spectral Analysis Results of PCPD-l Since fructose is highly sensitive to strong hydrolysis conditions, appropriate optimization was performed in the preliminary phase. The hydrolysis conditions selected were 0.5 mol / L sulfuric acid for 2 hours. The hydrolyzed PCPD-l was analyzed by high- performance anion exchange chromatography to determine monosaccharide composition. Fructose was the main monosaccharide in PCPD- 1 , accounting for as much as 96.96%, with the remaining 3.04% being glucose (as shown in Figure 2A). As shown in Figure 2B, the molecular weights were as follows: number-average molecular weight (Mn) = 6.037 kDa, peak molecular weight (Mp) = 6.736 kDa, weight-average molecular weight (Mw) = 6.175 kDa, and Z-average molecular weight (Mz) = 6.326 kDa. Infrared spectroscopy provided a deeper understanding of the chemical structure of PCPD-l. The IR spectrum of the sample is shown in Figure 2C. The absorption band at 36003200 cm1 corresponds to the stretching vibration of 40H, a characteristic peak of polysaccharides. Specically: 3420.73 cm1 is the stretching vibration peak of OH, characteristic of sugars. 2940.27 cm1 corresponds to the stretching vibration of CH. 1026.09 cm1 is an absorption peak attributed to the C0 stretching vibration. To macroscopically study the physicochemical properties of PCPD-l, scanning electron microscopy (SEM) was used to analyze its microstructure. As shown in Figure 2D, under a magnication of 10K, the surface of the polysaccharide sample appeared dense with many pores and a clearly visible network structure. Methylation analysis is one of the most powerful methods for structural analysis of polysaccharides and oligosaccharides. It produces partially methylated alditol acetates, which are analyzed qualitatively and quantitatively by gas chromatographymass spectrometry (GCMS). By calculating the ratio of chromatographic peak area to the molecular weight of the corresponding derivative, the relative molar content of the compound can be approximated. This allows for determining the relative molar ratio of different glycosidic linkages in the sample, thereby revealing the linkage patterns of the sugar residues. The methylation analysis results are shown in Figure 3 and Table 1. Sample Derivative Name Derivative Relative Connec molecular molar tion weight (MW) ratio (%) 2,5-di-O-acetyl-(2-deuterio)- t-Fru(f) 323 6.39 1 ,3 ,4,6-tetraO-methyl mannitol 2,5-di-O-acetyl-(2-deuterio)- t-Fru(f) 323 11.09 1 ,3 ,4,6-tetra-O-methyl mannitol 1,2,5-tri-O-acetyl-(2-deuterio)- 1-Fru(f) 351 32.82 3 ,4,6-tri-O-methyl mannitol 1,2,5-tri-O-acetyl-(2-deuterio)- D1N1 1-Fru(f) 351 25.68 3 ,4,6-tri-O-methyl mannitol 6- 1,5 ,6-tri-O-acetyl-2,3 ,4-tri-O- 351 3.12 Glc(p) methyl glucitol 1,6- 1,2,5,6-tetraO-acetyl-(2-deuterio)- 379 7.65 Fru(f) 3,4di-O-methyl mannitol 1,6- 1,2,5,6-tetraO-acetyl-(2-deuterio)- 379 13.26 F ru(f) 3 ,4-di-O-methyl mannitol A detailed nuclear magnetic resonance (NMR) analysis of PCP-D1 was performed to obtain the main chemical shift distribution in the sample (as shown in Table 2) for further structural elucidation. As shown in Figure 4A, the 85.35 ppm 1H signal was assigned to the (itconguration of a pyranose sugar. The absence of anomeric proton signals in the ö4.45.2 ppm range indicates the presence of a furanose sugar in the structure, presumed to be fructose. Compared with 1H NMR, the chemical shift distribution of PCPD1 in the 13C NMR spectrum is broader, with anomeric carbon signals concentrated in the range of 6951 10 ppm (Figure 4B). At chemical shifts 5103.66, 103.8, and 103.19 ppm, three distinct peaks were identied, designated as monosaccharide residues A, B, and C, respectively. However, due to the high molecular weight and ordered structure of PCP-D1, there is signicant signal overlap in the 1D NMR spectrum, necessitating 2D NMR for a deeper structural analysis of PCP. By combining 1D and 2D NMR, residue D, which has non-overlapping NMR signals, was conrmed to have a unique signal at 6535 / 9218 ppm (Figure 4E). Based on the monosaccharide composition results (Section 3.X), residue D may be (it-glucose. Notably, based on the H1 signal of residue D (85.35 ppm), AlH-AlH NMR enabled the sequential assignment of H2 to H6 signals, with chemical shifts at 83.49 ppm, 63.88 ppm, 63.71 ppm, 63.8 ppm, and 63.47 ppm, respectively (Figures 4C and 4D). Simultaneously, the chemical shifts of C2 to C6 on the pyranose ring were determined by HSQC signals, which were 671.09 ppm, 675.14 ppm, 672.42 ppm, 674.44 ppm, and 669.19 ppm, respectively (Figure 4E). The downeld shifts of C1 and C6 suggest substitutions at the O-1 and 0-6 positions of the pyranose ring. Combined with the methylation analysis results (Section 3.X), it is inferred that residue D may be >6)-0t- D-glcp-(1>. On the other hand, in the anomeric region of the HSQC spectrum, no cross peaks were observed for residues A, B, and C, which is consistent with the structural characteristics of fructose. At 8103.66 ppm, the terminal carbon signal of residue A corresponds to the characteristic of a B-glycosidic linkage. Based on the linkage information and anomeric signals, residue A may be fructofuranose: (>1)-[3-D-fruf-(2>). Similarly, residues B and C were also assigned as fructose residues, indicating that residue B may be >1,6)- BDfruf(2> and residue C as BDfruf(2>. According to the 1H and 13C chemical shifts of each residue, the HMBC spectrum showed cross peaks between residue AC2 and residue A-H1 at 6103.66 / 3.76 ppm (3.64 ppm), and between residue AHl and residue BHl at 8103.66 / 3.71 ppm (Figure 4F). Cross peaks were also observed between residue BC2 and residue AH1 at 8103.8 / 3.76 ppm (3.64 ppm), and between residue CC2 and residue BH6 at 8103.19 / 3.88 ppm. However, due to the low content of residue D (<5 %), no relevant correlations were found in either the HMBC or NOESY spectra. In conclusion, based on the combined results of 1D NMR, 2D NMR, and methylation analysis, PCP was inferred to consist mainly of a backbone composed of >1)-B-D-fruf- (2> and >1,6)-B-D-fruf-(2>, with side chains primarily composed of B-D-fruf-(2> linked to the O-6 position of >1,6)-B-D-fruf-(2> as terminal sugars. Therefore, the proposed structure of PCP is shown in Figure 5. Additionally, due to the low content of residue D, the corresponding connectivity relationships are not shown in the gure. Table 2 1H and 13C chemical shifts of sugar residues Chemical shifts (ppm) Cod Glycosyl residues H3 / C H4 / C e H1 / C1 H2 / C2 H5 / C5 H6 / C6 3 4 >1)B-D-Fruf- A 3.76,3.64 n.d 4.16 4.03 3.8 3.64 (24 103.6 62.25 76.74 74.57 81.07 60.45 6 >1,6)B-D-Fruf- B 3.71 n.d 4.18 4.13 4.04 3.88 (2> 62.52 103.8 74.35 74.64 81.17 63.26 C B-D-Fruf-(2> 3.8 n.d 4.12 4.01 3.87 3.79 103.1 60.78 77.03 75.08 80.22 59.95 9 >6)0tDGlcp D 5.35 3.49 3.88 3.71 3.8 3.47 (1> 92.18 71.09 75.14 72.42 74.44 69.19 n.d: Abbreviation for not detected, meaning not identied. 3. Effects of Polygonatum cyrtonema polysaccharides PCPDl on DSSInduced Colitis in Mice As shown in the workow diagram in Figure 6A, after three days of pretreatment with Polygonatum cyrtonema polysaccharides, 2.5% DSS was added to the drinking water to induce colitis. During the 10day experimental period, mice in the Ctrl group showed a slow increase in body weight. By day 7 of modeling, body weight in the DSS groups had decreased. Compared with the Ctrl group, body weight in the DSS group was signicantly reduced (p < 0.001). However, compared with the DSS group, body weight differences in the PCPD-l low-, medium-, and high-dose groups as well as the positive drug control group were not statistically signicant (Figure 6B). As shown in Figure 6C, colon length in DSS group mice was signicantly shorter than in the Ctrl group (p < 0.001). Compared with the DSS group, colon lengths were signicantly increased in the POS group (p < 0.01), PDl-L group (p < 0.01), PDl-M group (p < 0.01), and PDl-H group (p < 0.05). These results indicate that Polygonatum cyrtonema polysaccharides PCPD-l can alleviate DSS-induced shortening of colon length. Figure 6D shows the results of H&E staining of mouse colon tissue. In the Ctrl group, colon structure was intact and clear, with neatly arranged glands and epithelial cells. In the DSS group, colon structure was severely damaged, with extensive loss of crypts and goblet cells, disorganized gland arrangement, and massive inltration of inammatory cells. Compared with the DSS group, colon damage was less severe in the POS and all PCPD-l dose groups, with only slight reduction in goblet cells and mild inammatory inltration. These results indicate that Polygonatum cyrtonema polysaccharides alleviated DSS-induced pathological changes in the colon, including reduction of crypts and goblet cells and inammatory cell inltration, thus exerting a therapeutic effect on colitis. The intestine is not only an organ of digestion and absorption but also the bodys largest immune organ. When externally stimulated or infected by pathogenic microorganisms, innate immune cells in the intestinal mucosa and submucosa initiate effective immune responses. Colitis, as one of the main forms of inammatory bowel disease, is a specic recurrent inammatory disease affecting the rectum and colon. Its main features include immune dysregulation, cytokine imbalance, and epithelial barrier dysfunction. Dextran sulfate sodium (DSS) is a polyanionic derivative that induces colitis models primarily through mechanisms such as macrophage dysfunction, gut microbiota imbalance, epithelial toxicity, and cytokine signaling. In this study, a 2.5% DSS-induced colitis model was successfully established, characterized by signicant weight loss, shortened colon length, disappearance of crypts and goblet cells, disorganized glands, and strong inammatory inltration. The polysaccharide backbone of Polygonatum cyrtonema is mainly composed of fructooligosaccharides, which exhibit signicant anti-colitis activity. During ulcerative colitis, activated macrophages produce excessive pro-inammatory cytokines (IL-113, IL-6, TNF-a), triggering a so-called inammatory cascade that disrupts epithelial secretory function and increases permeability, causing pathological damage to intestinal mucosa and exacerbating UC. Therefore, subsequent experiments explored the anti- inammatory effects of Polygonatum cyrtonema polysaccharides on LPS-induced THP- 1 macrophages. 4. Anti-Inammatory effect of Polygonatum cyrtonema polysaccharides PCPD-l on LPS-Induced THP-1 Macrophages LPS-stimulated THP-l-derived macrophages were treated with various concentrations (50, 100, 200, 400 g / mL) of Polygonatum cyrtonema polysaccharides PCPD-l and cultured in vitro for 24 and 48 hours. Cell viability was assessed via MTT assay. As shown in Figures 7A and 7B, OD values in all treatment groups (0200 g / mL) showed no signicant differences compared to the untreated group, indicating no cytotoxicity of PCPD-l at concentrations up to 200 g / mL. Based on previous MTT results conrming lack of toxicity, three concentrations (50, 100, 200 g / mL) were selected for further experiments. As shown in Figures 7C7E, compared with the blank group, ILlß, TNFa, and IL6 levels were signicantly elevated in the LPSstimulated group (p < 0.01 or 0.001). However, treatment with PCPDl signicantly reduced TNFa levels across all doses (p < 0.001). At 200 g / mL, PCPDl also signicantly decreased IL6 (p < 0.01) and ILlß (p < 0.05) levels. Western blot analysis was used to detect the effect of PCPDl on inammatory signaling pathway proteins in LPSstimulated macrophages (Figure 7F). Compared with the PBS control group, the LPS group showed no signicant changes in total expression levels of IKBOL, ERK, p38, and NF-KB p65. However, their phosphorylated forms (p-IKBa, p- ERK, p-p38, and p-NF-KB p65) were signicantly upregulated. Treatment with 200 g / mL PCPD-l reduced the expression of these phosphorylated proteins compared with the LPS group. The MAPK signaling pathway plays a central regulatory role in cellular inammatory responses, proliferation, and apoptosis, with key hubs including p38 and ERK. Phosphorylated ERK (p-ERK) is essential in multiple inammatory processes, while phosphorylated p38 (p-p3 8) is involved in both inammation and apoptosis. Therefore, transcription factors p-ERK and p-p38 serve as important therapeutic targets for colitis. Studies have reported that the nuclear transcription factor NF-KB, a downstream effector of MAPK, regulates the expression of various pro-inammatory cytokines and is involved in inammatory bowel disease (IBD). Upon stimulation, macrophages promote the phosphorylation and degradation of IKB by IKK, releasing NF-KB, which translocates to the nucleus and activates the transcription of downstream inammatory cytokines, triggering inammation. NF-KB and IKB are involved in a wide range of pathophysiological processes, including acute and chronic inammation, tissue brosis, and apoptosis. They also regulate the transcriptional levels of pro-inflammatory cytokines such as TNF-(x, IL-lß, and IL-6. Treatment with Polygonatum cyrtonema polysaccharides signicantly inhibited LPS- induced phosphorylation in the MAPK pathway, as well as the degradation of IKBu and phosphorylation of NF-KB p65, indicating that the polysaccharide suppresses the activation of the MAPK / NFKB signaling pathway. In summary, this invention describes the preparation of three Polygonatum cyrtonema polysaccharides fractionsPCPDl, PCPD2, and PCPD3through a purication and isolation strategy. The physicochemical properties of PCPDl were characterized. Structural analysis of the homogeneous component PCPDl was performed using ion chromatography, FTIR, SEM, GCMS, and NMR techniques. The results showed that PCPD-l possesses characteristic functional groups of polysaccharides and contains both pyranose and furanose rings. Monosaccharide composition analysis revealed that PCPD- 1 is composed primarily of fructose (96.96%) and glucose. SEM imaging showed that PCPD-l exhibits a dense, porous, and clearly networked surface morphology. Further structure elucidation through methylation and NMR analysis indicated that the main chain of PCPD-l is composed of >1)-B-D-Fruf-(2> and >1,6)-B-D-Fruf-(2> linkages. These ndings provide a material basis for subsequent activity screening. In vivo experiments using the chemical inducer DSS to establish a colitis mouse model demonstrated that Polygonatum cyrtonema polysaccharide PCPD-l improved clinical conditions in colitis mice, including body weight maintenance, colon length, and pathological features observed in H&E staining. To further explore the effect of Polygonatum cyrtonema polysaccharide on macrophages, an LPS-induced inammation model using THP-1 macrophages was employed. Results showed that PCPD-l inhibited the secretion of inammatory cytokines IL-6, IL-lß, and TNF-a, and suppressed the aberrant activation of the MAPK / NF-KB signaling pathways. The above-described embodiments represent preferred implementations of the invention and are not intended to limit its scope. Modications and improvements made by those skilled in the art, without departing from the spirit of the invention, shall fall within the scope of the claims of this invention.

Claims

1. An extraction method for Polygonatum cyrtonema polysaccharides, characterized in that this includes the following steps: step 1: Polygonatum cyrtonema is washed twice with water decocted; the ltrates from both extractions are combined, concentrated, subjected to alcohol precipitation and dried to obtain a crude extract; the crude extract is then mixed with ethanol and centrifuged to remove the precipitate to collect; the precipitate is extracted in a water bath to obtain an extraction solution obtain, which is then concentrated and subjected to alcohol precipitation to obtain a to obtain crude polysaccharide extract; step 2: the crude polysaccharide- extract is enzymatically hydrolyzed with water and papain, chloroform and n-butanol are added and mixed, and the aqueous phase is collected as aqueous phase 1, Petroleum ether is added and mixed into aqueous phase 1, and the resulting aqueous phase is collected as aqueous phase 2, AB-8 macroporous resin is added for adsorption; the resulting liquid is dialyzed and freeze-dried to obtain a crude to obtain polysaccharide; step 3: the crude polysaccharide is dissolved in water and centrifuged; the supernatant is purified by means of ion exchange chromatography with deionized water, 0.1 M NaCl, 0.2 M NaCl and 0.3 M NaCl as eluents; the eluate fractions are collected, concentrated and dialyzed to to obtain purified polysaccharides; step 4: the purified polysaccharides are dissolved in water and centrifuged; the supernatant is further purified by Gel chromatography with deionized water as eluent. The eluate fractions are collected, concentrated, dialyzed and freeze-dried to obtain Polygonatum cyrtonema- to obtain polysaccharides.

2. The extraction method according to claim 1, wherein in step 1 the two decoctions are independently are carried out for 1.52 hours; the water bath extraction is carried out for 4 hours performed at 60 C.

3. The extraction method according to claim 1, wherein in step 2 the volume ratio of chloroform to n-butanol is 1:4; the volume of the chloroform / n-butanol mixture is 1 / 4 of the volume of the enzymatic hydrolysis system.

4. The extraction method according to claim 1, wherein step 3 comprises ion exchange purification is performed at a flow rate of 4 ml / min, using a gradient elution with deionized water, 0.1 M, 0.2 M and 0.3 M NaCl solutions.

5. The extraction method according to claim ], wherein step 4 comprises gel chromatography performed at a flow rate of 1 ml / min, and the elution is performed at 1.5 times the column volume of deionized water.

6. Extraction method according to any one of claims 1 to 5, wherein the dialysis in steps 3 and 4 is performed independently using 3000 Da dialysis membranes for 48-72 hours.

7. Polygonatum cyrtonema polysaccharides prepared by the extraction method according to any of claims 1 to 6.

8. Use of the Polygonatum cyrtonema polysaccharides according to claim 7 in the preparation of a drug for the treatment of colitis.

9. Use of the Polygonatum cyrtonema polysaccharides according to claim 7 in the preparation of a drug to lower the levels of the inammatory cytokine IL-6, lLlb and TNFa. 1 / 6 Figure1