Radix aconiti uniform polysaccharide with effect of improving ulcerative colitis, and preparation method and application thereof

Abstract

The application belongs to the technical field of aconite polysaccharide, and particularly relates to aconite uniform polysaccharide with the effect of improving ulcerative colitis, and a preparation method and application thereof. The aconite uniform polysaccharide is composed of glucose only, and has a molecular weight range of 1181-3063 kDa. Specifically, the number average molecular weight Mn is 1181 kDa, the peak molecular weight Mp is 3176 kDa, the weight average molecular weight Mw is 3063 kDa, the Z average molecular weight Mz is 6723 kDa, and the dispersion coefficient Mw / Mn is 2.595. The aconite uniform polysaccharide has a single molecular weight interval range and consistent molecular structure, can effectively relieve or treat ulcerative colitis-related symptoms, and can be used for preparing a drug for ulcerative colitis.

Description

Technical Field

[0001] The present application belongs to the technical field of aconite polysaccharides, and specifically relates to aconite uniform polysaccharide having the effect of improving ulcerative colitis, and its preparation method and application. Background Art

[0002] Ulcerative colitis (UC) is an inflammatory bowel disease characterized by persistent inflammation of the intestinal mucosa. Lesions begin in the rectum and progress retrogradely toward the proximal colon, potentially affecting the entire colon and even the terminal ileum. Clinical symptoms of UC include abdominal pain, diarrhea, rectal bleeding, and weight loss. The disease is protracted and difficult to cure, with a high recurrence rate. UC patients are also at increased risk of colorectal cancer, a serious life-threatening condition. In addition to causing significant pain for patients, UC also imposes a heavy financial burden. Therefore, the prevention and treatment of UC has become a major medical issue.

[0003] Aconite is a plant of the Ranunculaceae family (Aconitum Aconitum carmichaelii Debx. The root of the Chinese aconite root (Aconite Root) is pungent and sweet, highly caloric, and enters the heart, kidney, and spleen meridians. It has the effects of restoring yang, tonifying fire and supporting yang, and dispelling cold and relieving pain. The Shennong Bencao Jing first recorded the benefits of Aconite: "It is mainly used to treat wind-cold cough, evil spirits, warming the middle, treating metal wounds, breaking up hard masses, blood stasis, cold and heat, paralysis, cramps, knee pain, and inability to walk." Later, ancient Chinese medical texts documented the ability of aconite to treat UC-related symptoms. For example, the "Compendium of Materia Medica" records that aconite is primarily used to treat "…cholera, convulsions, and red and white diarrhea…" The "Compendium of Materia Medica" states that aconite "treats colds in the three yin meridians, yin-toxic cold hernias, stroke caused by middle cold…sudden diarrhea and loss of yang, chronic diarrhea and spleen diarrhea…" The "Shengji Zonglu" records that aconite pills can treat resting dysentery and red and white dysentery. The "Compendium of Materia Medica" states that aconite can "remove deep cold in the internal organs; replenish insufficient yang energy and warm the spleen and stomach." The "Changsha Yaojie" states that aconite "travels to the middle palace to warm the spleen and enters the lower jiao to warm the kidneys." In summary, aconite is effectively used to treat UC.

[0004] The active ingredient in Radix Aconiti Lateralis Preparata includes alkaloids, polysaccharides, glycosides etc., wherein, alkaloids are subject to extensive research at home and abroad owing to being the main active ingredient of Radix Aconiti Lateralis Preparata, and the research on Radix Aconiti Lateralis Preparata polysaccharide is then less. Prior art CN116672356A is directed to the application of Radix Aconiti Lateralis Preparata polysaccharide in treating inflammatory bowel disease and intestinal mucositis and has been studied, and successfully found that Radix Aconiti Lateralis Preparata polysaccharide has the effects such as regulating intestinal flora, suppressing the shortening of the colon and weight loss caused by ulcerative colitis, restoring damaged colon pathological tissue, improving diarrhea situation. However, the Radix Aconiti Lateralis Preparata polysaccharide found in prior art CN116672356A is a kind of homogeneous polysaccharide, and is relatively complicated on the composition of monosaccharide, and this causes it to need to consume certain resources to carry out quality control when carrying out standardized production, to ensure the stability of medicine and the repeatability of efficacy.

[0005] Therefore, a kind of aconite polysaccharide with relatively consistent molecular weight and structure is needed to fill the technical gap of aconite uniform polysaccharide. Summary of the Invention

[0006] The problem that the present application aims to solve is to provide a uniform polysaccharide of Aconite root with the effect of improving ulcerative colitis, as well as its preparation method and application. The uniform polysaccharide of Aconite root has a single molecular weight range and a consistent molecular structure, and can effectively alleviate or treat UC-related symptoms.

[0007] In order to solve the above technical problems, this application adopts the following technical solutions:

[0008] On the one hand, the present application provides a uniform polysaccharide of aconite root, which is composed only of glucose and has a molecular weight range of 1181~3063 kDa, wherein the number average molecular weight Mn is 1181 kDa, the peak molecular weight Mp is 3176 kDa, the weight average molecular weight Mw is 3063 kDa, the Z average molecular weight Mz is 6723 kDa, and the dispersion coefficient Mw / Mn is 2.595;

[0009] The structure of the Aconite root homogeneous polysaccharide is:

[0010] .

[0011] The molecular weight of the above-mentioned aconite uniform polysaccharide is within a single range. Functional groups such as pyranose rings and α-type glycosidic bonds exist in the molecule. The spatial configuration is a dense cross-linked macromolecule with a highly branched structure. The structure is relatively consistent, the morphology is smooth, and it has a large lamellar structure.

[0012] On the other hand, the present application provides a method for preparing the above-mentioned aconite uniform polysaccharide, comprising the following steps:

[0013] After the raw aconite root is reflux-extracted with anhydrous ethanol, the raw aconite root is filtered for the first time and the residue is collected;

[0014] leaching the filter residue with ultrapure water, collecting the leaching solution for a second filtration, performing a first centrifugation on the filtrate, and collecting a first supernatant;

[0015] The first supernatant is subjected to a first concentration, and when it is concentrated to 1 / 5 of the original volume, alcohol precipitation is performed to obtain a precipitate.

[0016] After washing, filtering and drying the precipitate, crude aconite polysaccharide is obtained;

[0017] After dissolving the crude aconite polysaccharide with a first solvent, performing a second centrifugation, and collecting a second supernatant to obtain a first sample solution;

[0018] The first sample solution is loaded onto a first chromatography column, and pure water is used as a mobile phase for a first elution. The first eluate obtained by the pure water separation is collected and subjected to a second concentration. When the eluate is concentrated to 1 / 5 of the original volume, the eluate is dialyzed in pure water using a dialysis bag. The liquid in the dialysis bag is collected and subjected to a first pre-freezing and a second drying to obtain a crude homogeneous polysaccharide product of Aconite root;

[0019] After dissolving the crude homogeneous polysaccharide of Aconite Radix with a second solvent, performing a fourth filtration to obtain a second sample solution;

[0020] The second sample solution is loaded onto a second chromatography column, and pure water is used as the mobile phase for a second elution. The second eluate is collected and subjected to a third concentration. When the eluate is concentrated to 1 / 5 of the original volume, a second pre-freezing and a third drying are performed to obtain the aconite uniform polysaccharide.

[0021] The uniform polysaccharide of aconite separated by the above method does not contain impurities such as nucleic acid and protein, has a uniform monosaccharide composition, and the molecular weight is within a single range.

[0022] Furthermore, the weight ratio of the raw aconite root to the anhydrous ethanol is 1:4.

[0023] Furthermore, the weight ratio of the filter residue to the ultrapure water is 1:10.

[0024] Furthermore, the solid-liquid ratio of the crude aconite polysaccharide to the first solvent is 20 mg:1 mL.

[0025] Furthermore, the solid-to-liquid ratio of the crude homogeneous polysaccharide of Aconite root to the second solvent is 5 mg:10 mL.

[0026] Furthermore, the first solvent and the second solvent include pure water.

[0027] Furthermore, the first chromatography column includes a DEAE-52 cellulose column.

[0028] Furthermore, the molecular weight cut-off of the dialysis bag is 3500 Da.

[0029] Furthermore, the second chromatography column includes a Sephadex G-100 gel column.

[0030] Furthermore, the temperature of the reflux extraction is 78° C., and the time of the reflux extraction is 4 h.

[0031] Furthermore, the extraction temperature is 95° C., the number of extractions is 2 times, and the extraction time is 2 hours per time.

[0032] Furthermore, the second filtration is performed using gauze.

[0033] Furthermore, the speed of the first centrifugation and the second centrifugation is 3000 r / min, and the time of the first centrifugation and the second centrifugation is 5 min.

[0034] Furthermore, the first concentration method includes rotary evaporation concentration.

[0035] Furthermore, the washing method includes washing with anhydrous ethanol.

[0036] Furthermore, the methods used for the first drying, the second drying and the third drying include freeze drying.

[0037] Furthermore, the flow rate of the first elution is 2.5 mL / min.

[0038] Furthermore, the dialysis time is 24 h, and the pure water in the dialysis bag is replaced 3 times during the dialysis period.

[0039] Furthermore, the temperature of the first pre-freezing and the second pre-freezing is -80°C.

[0040] Furthermore, the third filtration method includes filtering using a 0.45 μm aqueous microporous filter membrane.

[0041] Furthermore, the flow rate of the second elution is 1.25 mL / min.

[0042] Furthermore, the second concentration and the third concentration are carried out by methods including reduced pressure evaporation concentration.

[0043] Furthermore, the alcohol precipitation comprises the following steps:

[0044] Under stirring, anhydrous ethanol was added to the concentrated first supernatant until the alcohol concentration reached 80%, and then the mixture was precipitated in a sealed state at 25° C. for 24 h. After precipitation twice, the precipitate was obtained.

[0045] On the other hand, the present application provides the use of the above-mentioned Aconite homogeneous polysaccharide or the Aconite homogeneous polysaccharide prepared by the above-mentioned method in the preparation of a drug for treating ulcerative colitis, wherein the drug for treating ulcerative colitis is a drug for alleviating or treating weight loss, bloody stools, diarrhea, colon pathological changes and / or colon inflammation.

[0046] Furthermore, the colon pathological changes include colon shortening, increased intestinal permeability, destruction of colonic crypt structure, destruction of colonic mucosa, increased inflammatory infiltration, decreased goblet cells and / or destruction of the mucus layer.

[0047] Furthermore, the method for alleviating or treating colon inflammation by the drug for treating ulcerative colitis includes reducing the content of inflammation-related factors in colon tissue.

[0048] Furthermore, the method for reducing the content of the inflammation-related factors by the drug for treating ulcerative colitis includes inhibiting the protein expression of IL-6 and TNF-α in colon tissue.

[0049] This application has the following beneficial effects:

[0050] 1. The monosaccharide composition of the homogeneous polysaccharide of Aconite root of the present application is only glucose, and the structure is relatively consistent, which facilitates quality control during industrial production;

[0051] 2. The method for preparing uniform polysaccharide from Aconite root of the present application involves simple operations and does not require complex instruments, thus reducing the cost of industrialization;

[0052] 3. The homogeneous polysaccharide of Aconite root of the present application can effectively alleviate the symptoms of weight loss, bloody stools, and diarrhea in UC model mice, has a significant therapeutic effect on colon pathology in UC model mice, and can also effectively alleviate colon inflammation in UC model mice. It is a very promising raw material for the preparation of UC therapeutic drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 The raw materials and products used in the extraction of crude polysaccharide from aconite root in this application (A. raw aconite root, B. crude polysaccharide from aconite root);

[0054] Figure 2 This is the DEAE-52 cellulose column elution curve of the crude polysaccharide of Aconite root of this application;

[0055] Figure 3 Sephadex G-100 gel column elution curves for APA, APB, APC, and APD (A.APA, B.APB, C.APC, D.APD);

[0056] Figure 4 UV scanning spectra of APA-1, APB-1, APC-1, and APD-1 of this application (A.APA-1, B.APB-1, C.APC-1, D.APD-1);

[0057] Figure 5 Light scattering spectrum curves of APA-1, APB-1, APC-1, and APD-1 of the present application (A. APA-1, B. APB-1, C. APC-1, D. APD-1, where the red line represents the multi-angle laser light scattering signal, the blue line represents the difference signal, and the black line represents the molecular weight fitted by the two signals);

[0058] Figure 6Molecular configuration analysis diagrams and slopes of APA-1, APB-1, APC-1, and APD-1 of the present application (A.APA-1, B.APB-1, C.APC-1, D.APD-1);

[0059] Figure 7 Ion chromatograms of APA-1, APB-1, APC-1, and APD-1 of the present application (A. ion chromatogram of monosaccharide standard, B. ion chromatogram of APA-1, C. ion chromatogram of APB-1, D. ion chromatogram of APC-1, E. ion chromatogram of APD-1, wherein Rha is rhamnose, Ara is arabinose, Gal is galactose, Glc is glucose, Xyl is xylose, Man is mannose, Fru is fructose, Rib is ribose, Gal-UA is galacturonic acid, Glc-UA is glucuronic acid, Man-UA is mannuronic acid, and Gul-UA is guluronic acid);

[0060] Figure 8 The infrared spectra of APA-1, APB-1, APC-1 and APD-1 of this application;

[0061] Figure 9 Scanning electron micrographs of APA-1, APB-1, APC-1, and APD-1 of this application (A.APA-1, B.APB-1, C.APC-1, D.APD-1);

[0062] Figure 10 This is the GC-MS ion chromatogram of the methylated sugar alcohol acetyl ester product of APA-1 of the present application;

[0063] Figure 11 The one-dimensional nuclear magnetic resonance spectrum of APA-1 of this application (A. 1H NMR spectrum, B. 13C NMR spectrum, C. DEPT135 spectrum);

[0064] Figure 12 The two-dimensional nuclear magnetic resonance spectrum of APA-1 in this application (AH 1 -H 1 COSY spectrum, B. HSQC spectrum, C. NOESY spectrum, D. HMBC spectrum);

[0065] Figure 13 This is a schematic diagram of the structure of APA-1 of this application;

[0066] Figure 14 Schematic diagram of the experimental design showing the animal grouping and treatment methods for the study of the pharmacological effects of APA-1 on UC mice in this application (wherein 5-ASA is 5-aminosalicylic acid, DSS is dextran sulfate sodium, and PSS is normal saline);

[0067] Figure 15 The results of the body weight, bloody stool and diarrhea test of each group of mice in this application (A. Body weight change trend, B. Body weight of each group between 4 and 7 days, C. Diarrhea score, D. Diarrhea score of each group between 3 and 7 days, E. Bloody stool score, F. Diarrhea score of each group between 4 and 7 days, G. DAI score, H. DAI score of each group between 4 and 7 days, among which, * P<0.05, ** P < 0.01 and *** P < 0.001);

[0068] Figure 16 The colon length and colon pathological changes of mice in each group of this application (A. Mouse colon length; B. Representative WB bands of Claudin and Occludin in mouse colon tissue and their relative protein expression levels; CH&E stained colon tissue sections, magnification 10x, scale bar 250 μm; D. Histopathological score; E. AB-PAS stained colon tissue sections, magnification 10x, scale bar 250 μm; F. AB-PAS staining positive area ratio; Among them, * P<0.05, ** P < 0.01 and *** P < 0.001);

[0069] Figure 17 The results of the detection of inflammatory factor concentrations and their relative protein expression levels in each group of mice in this application (A. IL-6 content; B. TNF-α content; C. IL-17 content; D. Representative WB bands of IL-6 and TNF-α in colon tissue; E. Relative protein expression level of IL-6 in mouse colon tissue; F. Relative protein expression level of TNF-α in mouse colon tissue; Among them, * P<0.05, ** P < 0.01 and *** P<0.001). DETAILED DESCRIPTION

[0070] The following is a clear and complete description of the technical solutions in some embodiments of the present application. Obviously, the embodiments described are only some of the embodiments of the present application, not all of them. Based on the embodiments provided in this application, all other embodiments obtained by ordinary technicians in this field are within the scope of protection of this application.

[0071] Unless the context requires otherwise, throughout the specification and claims, the term "comprising" is to be construed in an open, inclusive sense, that is, meaning "including, but not limited to."

[0072] When describing some embodiments, the expressions "at least one of A, B, and C" and "at least one of A, B, or C" may be used, both of which have the same meaning and include the following combinations of A, B, and C: only A, only B, only C, the combination of A and B, the combination of A and C, the combination of B and C, and the combination of A, B, and C.

[0073] In the specific implementation of the technical solution of this application, the main medicinal materials, medicines, reagents, instruments and animals used are as follows:

[0074] 1. Experimental medicinal materials

[0075] Raw aconite root was produced in Jiangyou, Sichuan, the authentic aconite production area, and purchased from Sichuan Yanchengtang Chinese Medicinal Materials.

[0076] 2. Experimental drugs and reagents

[0077] (1) Drugs and reagents involved in the preparation of uniform polysaccharide from Aconite

[0078] DEAE-52 cellulose (Shanghai Yuanye Biotechnology Co., Ltd.), Sephadex G-100 (Yangzhou Boruitang Biotechnology Co., Ltd.), rhamnose (Sigma-Aldrich, USA), arabinose, galactose, glucose, xylose, mannose and ribose (Sigma-Aldrich, USA), fructose (Sigma-Aldrich, USA), glucuronic acid (Sigma-Aldrich, USA), galacturonic acid (Sigma-Aldrich, USA), mannuronic acid (Beijing Solarbio Technology Co., Ltd.), guluronic acid (Shanghai Weiqiqi Biotechnology Co., Ltd.), 3500-5000 cellulose dialysis bag (Shanghai Yuanye Biotechnology Co., Ltd.).

[0079] (2) Drugs and reagents involved in the pharmacodynamic study of Aconite homogeneous polysaccharide on UC

[0080] Colitis modeling: DSS (molecular weight: 36,000–50,000 Da, Shanghai Yisheng Biotechnology Co., Ltd.), 5-ASA (Shanghai Yuanye Biotechnology Co., Ltd.), RIPA lysis buffer (Wuhan Savier Biotechnology Co., Ltd.), GAPDH antibody (Wuhan Savier Biotechnology Co., Ltd.), Claudin1 antibody (Wuhan Sanying Biotechnology Co., Ltd.), Occludin antibody (Wuhan Sanying Biotechnology Co., Ltd.), horseradish peroxidase-conjugated goat anti-rabbit IgG (H+L) (Wuhan Sanying Biotechnology Co., Ltd.), IL-6 antibody (Wuhan Savier Biotechnology Co., Ltd.), TNF-α antibody (Shanghai Abimat Pharmaceutical Technology Co., Ltd.), femtogram-level ECL ultrasensitive luminescent solution (Shanghai Jinbeirui Biotechnology Co., Ltd.), bovine serum albumin (BSA) (Wuhan Savier Biotechnology Co., Ltd.), ready-to-use rapid blocking buffer (Guangzhou Boluteng Biotechnology Co., Ltd.), SDS-PAGE gel rapid preparation kit (Wuhan Savier Biotechnology Co., Ltd.), BCA protein quantification kit (Wuhan Savier Biotechnology Co., Ltd.), Tween 20 (Chengdu Kelong Chemical Reagent Co., Ltd.), skim milk powder (Wuhan Savier Biotechnology Co., Ltd.), 0.45 μm PVDF membrane (Merck, Germany), Omni-Easy™ instant protein loading buffer (Shanghai Yazyme Biopharmaceutical Technology Co., Ltd.), 10-180 kDa prestained protein marker (Wuhan Sevier Biotechnology Co., Ltd.), transfer buffer powder (Wuhan Sevier Biotechnology Co., Ltd.), TBS buffer powder (Wuhan Sevier Biotechnology Co., Ltd.), electrophoresis buffer powder (Wuhan Sevier Biotechnology Co., Ltd.), mouse Th1 / Th2 / Th17 flow cytometry microsphere array detection kit (BD Biosciences, USA).

[0081] 3. Experimental instruments

[0082] (1) Experimental instruments involved in the preparation of uniform polysaccharide from Aconite

[0083] YRE-5299 rotary evaporator (Gongyi Yuhua Instrument Co., Ltd.), digital display HL-2 constant flow pump experimental model (Shanghai Qingpu Huxi Instrument Factory), DBS-100N automatic fraction collector (Shanghai Qingpu Huxi Instrument Factory), vacuum freeze dryer (Tokyo Rikakigi Co., Ltd.), Nicolet iZ-10 Fourier transform infrared spectrometer (Thermo Fisher Scientific, USA), TANKPE030 water purifier (Sichuan Youpu Ultrapure Technology Co., Ltd.), N60 Touch ultra-micro spectrophotometer (Implen, Germany), ESJ30-5A electronic balance (Shenyang Shenyu Longteng Balance Co., Ltd.), Reacti-thermo nitrogen purge device (Thermo Fisher Scientific, USA), 7890A gas chromatograph (Agilent, USA), H1850 Xiangyi centrifuge (Hunan Xiangyi Laboratory Instrument Development Co., Ltd.), UltiMate 3000 high-performance liquid chromatograph (Thermo Fisher Scientific, USA), OPTILAB T-rex differential detector (Wyatt, USA), DAWN HELEOS-Ⅱ laser light scattering detector (Wyatt, USA), ICS 5000 + Ion chromatograph (Thermo Fisher Scientific, USA), 5977B mass spectrometer (Agilent Technologies, USA), Zeiss Merlin Compact high-resolution field emission scanning electron microscope (Zeiss, Germany), Bruker Avance Neo 600 MHZ nuclear magnetic resonance spectrometer (Bruker, Germany), BRT2.5 / 90 medium-pressure special glass chromatography column (Yangzhou Boruitang Biotechnology Co., Ltd.), 2.5*60 cm medium-pressure special glass chromatography column (Shanghai Qingpu Huxi Instrument Factory), SPECORD 250 PLUS UV-visible spectrophotometer (Analytik Jena, Germany).

[0084] (2) Experimental instruments involved in the pharmacodynamic study of Aconite homogeneous polysaccharide on UC

[0085] FA2004N electronic balance (Conan Xinling Instrument Equipment Co., Ltd.), JXLD-6K three-dimensional centrifugal cryo-grinder (Shanghai Jingxin Industrial Development Co., Ltd.), TGL-21M high-speed refrigerated centrifuge (Shanghai Luxiangyi Centrifuge Instrument Co., Ltd.), FlexStation-3 multifunctional microwell detector (Shanghai Meigu Molecular Instrument Co., Ltd.), Bio-Rad small vertical electrophoresis transfer system (Bio-Rad Corporation, USA), GYXH-265 floor-standing snowflake ice maker (Xiamen Guoyi Scientific Instrument Co., Ltd.), OS-03U small shaker (Jingqi Co., Ltd., USA), E-Blot contact chemiluminescence electronic slide imager (Shanghai e-BLOT Life Sciences Co., Ltd.), FACSVerse flow cytometer (BD Biosciences, USA), HCM100-Pro heating and shaking thermostat (Beijing Dalong Xingchuang Laboratory Instrument Co., Ltd.), Hamamatsu NanoZoomer digital pathology slide analyzer (Hamamatsu Photonics Trading Co., Ltd.).

[0086] 4. Experimental Animals

[0087] Specific pathogen-free grade C57BL / 6J male mice weighing 20.0 ± 2.0 g were purchased from Beijing Sibeifu Biotechnology Co., Ltd.

[0088] Example 1 Preparation of DEAE-52 cellulose column

[0089] (1) Pretreatment of DEAE-52 cellulose filler:

[0090] The DEAE-52 cellulose filler powder was taken out of the 4°C refrigerator and equilibrated at room temperature for 2 h. An appropriate amount of filler was added into 10 times the mass of pure water and fully swelled for 2 h. The mixture was then washed with pure water until neutral and finally ultrasonically degassed for 30 min.

[0091] (2) Wet column packing:

[0092] After cleaning the chromatography column with pure water, fix it vertically on an iron stand and connect the constant flow pump and collector in sequence with a hose. Add 1 / 2 column volume of pure water, turn on the pump switch, exhaust the air in the hose, adjust the pump flow rate and check the air tightness of the instrument. After a small amount of pure water flows to the bottom of the column, stir the pretreated DEAE-52 cellulose filler evenly, use a glass rod to slowly pour it into the chromatography column along the glass column wall until the filler is deposited to about 10 cm from the upper end of the chromatography column, stop loading the column, and then elute with pure water until the chromatography column is balanced. When the pure water liquid level is about 2-3 cm higher than the filler, turn off the pump to obtain a DEAE-52 cellulose column.

[0093] Example 2 Preparation of Sephadex G-100 gel column

[0094] (1) Pretreatment of Sephadex G-100 gel filler:

[0095] The Sephadex G-100 gel filler was taken out of the 4°C refrigerator and equilibrated at room temperature for 2 h. Then, 10 times the mass of pure water that had been ultrasonically degassed was slowly added and allowed to swell at room temperature for 24 h to allow it to fully swell. The pure water was replaced several times to ensure that the floating filler was completely removed. The ratio of gel microspheres to liquid was then adjusted to form a uniform 75% gel suspension, and vacuum degassed.

[0096] (2) Wet column packing:

[0097] After cleaning the chromatography column with pure water, fix it vertically on an iron stand and connect the constant flow pump and collector in sequence with a hose. Add 1 / 2 column volume of ultrasonically degassing pure water, turn on the pump switch, exhaust the air in the hose, adjust the pump flow rate and check the air tightness of the instrument. When a height of 5 cm of pure water is left at the bottom of the column, turn off the pump; slowly stir the degassed filler with a glass rod until it is suspended, turn on the pump, and carefully add the filler suspension to the chromatography column continuously along the glass rod until the filler is deposited to about 15 cm above the upper end of the chromatography column. Stop loading the column, then balance it with pure water at a low flow rate for 30 minutes. When the pure water level is about 5 cm higher than the filler, turn off the pump to obtain a Sephadex G-100 gel column.

[0098] Example 3 Extraction of crude polysaccharides from Aconite

[0099] 650 g of raw aconite root was extracted with 4 times its mass of anhydrous ethanol at 78°C for 4 hours under reflux, and the residue was collected by filtration. The collected residue was then added with 10 times the mass of heated ultrapure water and extracted twice with hot water, each time for 2 hours. The two extracts were combined and filtered through gauze. The filtered extract was centrifuged at 3000 rpm for 5 minutes, and the supernatant was collected. The supernatants were collected and combined, concentrated by rotary evaporation to 1 / 5 of the original volume, and the concentrate was collected and subsequently subjected to alcohol precipitation. The alcohol precipitation steps were as follows: the concentrate was stirred with a magnetic stirrer, then anhydrous ethanol was slowly added along a glass rod, monitoring the ethanol concentration with an alcoholometer until the alcohol concentration reached 80%. The addition of anhydrous ethanol was then stopped, and the container was sealed and cryogenically precipitated for 24 hours, repeating the alcohol precipitation twice. After alcohol precipitation, the extract was washed with anhydrous ethanol, filtered, and the ethanol solution was removed. Finally, the extract was freeze-dried to obtain crude aconite root polysaccharide.

[0100] This application adopts water extraction and alcohol precipitation method to extract raw aconite root ( Figure 1 The crude polysaccharide of Aconite extracted from A) is a light yellow coarse powder ( Figure 1 B), with a loose and light texture, stable properties, and good water solubility, 84.6 g of crude aconite polysaccharide was extracted from 650 g of raw aconite, with a yield of 13.01%.

[0101] Example 4 Separation and purification of crude polysaccharide from Aconite root

[0102] (1) Separation of polysaccharides using DEAE-52 cellulose column

[0103] Sample loading: Weigh 400 mg of crude aconite polysaccharide, dissolve it in 20 mL of pure water, centrifuge at 3000 r / min for 5 minutes, and collect the supernatant as the sample solution; turn on the pump to release the liquid in the column, and turn off the pump when the pure water level in the chromatography column is at the same height as the filler; use a rubber-tipped dropper to draw up the sample solution, and slowly rotate along the wall of the chromatography column to load the sample. After loading is completed, turn on the pump and start collecting the effluent.

[0104] Elution: After the sample solution has completely entered the packing, use pure water, 0.1 mol / L NaCl solution, 0.2 mol / L NaCl solution and 0.3 mol / L NaCl solution as the mobile phase for gradient elution. The flow rate of the mobile phase during elution is 2.5 mL / min. Collect the eluate with an automatic collector, and collect 10 mL in each collection tube.

[0105] Determination: The phenol-sulfuric acid method was used to detect the carbohydrate content of the eluate in each collection tube. The method was as follows: 200 μL of eluate from each collection tube was aspirated into a test tube, and then 100 μL of 6% phenol solution was added. After mixing evenly, 500 μL of concentrated sulfuric acid was added. After vortexing, the mixture was allowed to stand for 30 minutes to cool to room temperature, and the absorbance value was then measured at 490 nm using a UV spectrophotometer.

[0106] Analysis: With the tube number as the horizontal axis and the absorbance at 490 nm as the vertical axis, draw the elution curve of the DEAE-52 cellulose column ( Figure 2 ), according to the curve, it can be seen that elution peak 1 is from tubes 10 to 15, elution peak 2 is from tubes 41 to 43, elution peak 3 is from tube 68, and elution peak 4 is from tubes 100 to 102. The eluates in the collection tubes of different elution peaks are combined.

[0107] Collection: The combined eluate fractions were concentrated to 1 / 5 of their original volume by reduced pressure evaporation, and then dialyzed in pure water for 24 h using a dialysis bag with a molecular weight cutoff of 3500 Da. During this period, the pure water used for dialysis was replaced three times to remove low-molecular-weight substances in the mobile phase. The liquid in the dialysis bag was collected, pre-frozen at -80°C, and freeze-dried to obtain polysaccharides separated using pure water, 0.1 mol / L NaCl solution, 0.2 mol / L NaCl solution, and 0.3 mol / L NaCl solution as the mobile phase, respectively. They were labeled APA, APB, APC, and APD, respectively.

[0108] The final APA, APB, APC and APD obtained were 19.965 g, 2.047 g, 0.583 g and 0.609 g, respectively, with yields of 23.6%, 2.42%, 0.69% and 0.72% of the crude polysaccharides of Aconite root. Among them, the neutral APA separated using pure water as the mobile phase had the highest content, followed by APB, APC and APD. The four components of APA, APB, APC and APD separated were all white fluffy flocs with good water solubility.

[0109] (2) Purification of polysaccharides using Sephadex G-100 gel column

[0110] Sample loading: Weigh 50 mg of the above-mentioned APA, APB, APC, and APD respectively, dissolve them in 10 mL of pure water, and filter them through a 0.45 μm aqueous microporous filter membrane to obtain sample solutions; turn on the pump to release the liquid in the column, and when the pure water level in the chromatography column is at the same height as the Sephadex G-100 gel filler, turn off the pump; use a rubber-tipped dropper to draw up the sample solution, and slowly rotate along the wall of the chromatography column to load the sample; after loading is completed, turn on the pump and start collecting the effluent.

[0111] Elution: After the sample solution has completely entered the filler, use pure water as the mobile phase for elution. The flow rate of the mobile phase is 1.25 mL / min. Use an automatic collector to collect the eluate, and collect 5 mL in each collection tube.

[0112] Determination: The phenol-sulfuric acid method was used to detect the sugar content in each tube of eluate. The detection method was the same as that used for separating polysaccharides using a DEAE-52 cellulose column.

[0113] Analysis: With the tube number as the horizontal axis and the absorbance at 490 nm as the vertical axis, draw the elution curve of Sephadex G-100 gel column ( Figure 3 ), it can be seen from the curve that the molecular weights of the four components are all within a single range. The elution peak of APA is in tubes 22 to 24, the elution peak of APB is in tubes 25 to 28, the elution peak of APC is in tubes 25 to 29, and the elution peak of APD is in tubes 26 to 29. The eluates in the collection tubes with different elution peaks of each component were combined.

[0114] Collection: The combined eluate fractions were concentrated to 1 / 5 of their original volume by evaporation under reduced pressure, pre-frozen at -80°C, and freeze-dried to obtain purified polysaccharide fractions, which were designated as APA-1, APB-1, APC-1, and APD-1.

[0115] The final APA-1, APB-1, APC-1 and APD-1 obtained were 2.647 g, 0.2284 g, 0.076 g and 0.0423 g, respectively, with yields of 3.13%, 0.27%, 0.09% and 0.05% of the crude polysaccharides of Aconite root. Among them, the amount of APA-1 obtained from the separation and purification of APA was the highest, indicating that APA-1 is the main component of the crude polysaccharides of Aconite root.

[0116] Example 5 Structural Analysis of Purified Polysaccharide Fractions

[0117] (1) Ultraviolet spectrum analysis

[0118] APA-1, APB-1, APC-1 and APD-1 were prepared into 1 mg / mL solutions using pure water, and pure water was used as the blank control group. The full wavelength scanning was performed at 200-400 nm using a UV-visible spectrophotometer to obtain the following results: Figure 4 UV scanning spectrum shown.

[0119] The results showed that there were no obvious absorption peaks in the four components at 260 nm and 280 nm, indicating that there were no impurities such as nucleic acids and proteins in APA-1, APB-1, APC-1 and APD-1. It also showed that the purification effect of the crude polysaccharide of Aconitum carmichaelii was very good in this application, and the purified APA-1, APB-1, APC-1 and APD-1 components can be used for further research.

[0120] (2) Molecular weight determination

[0121] Sample pretreatment: APA-1, APB-1, APC-1, and APD-1 were dissolved in 0.1 M NaNO3 aqueous solution (containing 0.02% NaN3, w / w) to prepare a sample solution with a concentration of 1 mg / mL. The sample solution was filtered through a 0.45 μm filter and then tested on the instrument, as shown in the figure.

[0122] Chromatographic system: The chromatographic system was a gel exclusion chromatography differential display-multi-angle laser light scattering system. The gel exclusion liquid phase system was U3000 (Thermo, USA), the chromatographic columns were gel exclusion chromatography columns Ohpak SB-805 HQ (300×8 mm) and Ohpak SB-803 HQ (300×8 mm) connected in series, the differential detector was Optilab T-rEX (Wyatt technology, USA), and the laser light scattering detector was DAWN HELEOS Ⅱ (Wyatt technology, USA). The column temperature was 45°C, the injection volume was 100 μL, the mobile phase was 0.1 M NaNO3 (containing 0.02% NaN3), the flow rate was 0.6 mL / min, and the isocratic elution was 75 min.

[0123] The differential detector detects the concentration information of the sample, and the multi-angle laser light scattering instrument detects the light scattering information of the macromolecule. The molecular weight curve corresponding to each component is calculated based on the Mark-Houwink equation. The spectral curve of the sample is obtained with the retention time (Time, min) as the horizontal axis and the molar mass as the vertical axis. Figure 5 ), and the molecular weight and dispersion coefficient of the four polysaccharide components were calculated based on the chromatographic information (Table 1). In addition, the log (molar mass) was used as the horizontal axis and the log (root mean square radius) was used as the vertical axis for fitting. For the convenience of analysis, the scales of the horizontal and vertical axes were marked with molar mass and root mean square radius, respectively. The slope can be used as a reference for the molecular configuration of the polysaccharide components ( Figure 6 ).

[0124] Table 1 Molecular weight and dispersion coefficient of APA-1, APB-1, APC-1 and APD-1

[0125]

[0126] The results showed that APA-1 was a single broadly distributed peak, indicating that the molecular weight distribution of APA-1 was uniform and broad. The number average molecular weight (Mn) and weight average molecular weight (Mw) of APA-1 were 1181 kDa and 3063 kDa, respectively, and its polydispersity index was 2.595. APB-1 was a broadly distributed double peak, indicating that there were two molecular weight components in APB-1. The Mn and Mw of APB-1 were 86.024 kDa and 495.435 kDa, respectively, and its polydispersity index was 5.759. APC-1 was a single symmetrical single peak, indicating that the molecular weight distribution of APC-1 was uniform. The Mn and Mw of APC-1 were 134.118 kDa and 228.248 kDa, respectively. kDa, with a polydispersity index of 1.702; APD-1 has two peaks, one high and one low, indicating the presence of two molecular weight components in APD-1, with the high molecular weight component accounting for a higher proportion. The Mn and Mw of APD-1 are 140.545 kDa and 551.596 kDa, respectively, and its polydispersity index is 3.925. From the molecular configuration and its slope, it can be seen that the slope of APA-1 is 0.03±0.01, indicating that the spatial configuration of the APA-1 molecule is a dense cross-linked macromolecule with a highly branched structure, while the slopes of APB-1, APC-1, and APD-1 are 0.27±0.00, 0.20±0.01, and 0.56±0.01, respectively, indicating that APB-1 and APC-1 are branched clusters with nearly spherical structures, and APD-1 is a linear cluster with a Gaussian coil structure.

[0127] (3) Determination of monosaccharide composition

[0128] Preparation of monosaccharide standards: Weigh different monosaccharide standards and prepare 10 mg / mL single-standard stock solutions with pure water. Then, take appropriate amounts of the single-standard stock solutions and mix them to prepare standard mixed stock solutions with a concentration gradient. The concentration gradients of the monosaccharide standards in the standard mixed stock solutions are shown in Table 2.

[0129] Table 2 Concentration of monosaccharide standards in the standard mixture stock solution

[0130]

[0131] Sample pretreatment: Take a clean chromatographic bottle, weigh an appropriate amount of polysaccharide sample (2.02 mg APA-1, 1.91 mg APB-1, 2.00 mg APC-1 and 1.95 mg APD-1), dissolve them in 1 mL of 2 M trifluoroacetic acid solution, and then react at 121°C for 2 h. Then, blow dry with nitrogen, add 99.99% methanol to wash, blow dry again, repeat the washing three times, add 40 mL of pure water to dissolve, and transfer to a chromatographic bottle for testing.

[0132] Chromatographic method: Chromatographic system is Thermo ICS 5000 + Ion Chromatography System (ICS 5000 + , ThermoFisher Scientific, USA), the detector was an electrochemical detector, the chromatographic column was a Dionex™ CarboPac™ PA20 liquid chromatography column (150×3.0 mm, 10 μm), the mobile phases were A (H2O), B (0.1 M NaOH), and C (0.1 M NaOH, 0.2 M NaAc), the injection volume was 5 μL, the flow rate was 0.5 mL / min, the column temperature was 30°C, and the elution gradient was shown in Table 3.

[0133] Table 3 Ion chromatography elution gradient for monosaccharide composition analysis

[0134]

[0135] The ion chromatogram was drawn with the retention time (min) as the horizontal axis and the response value (nC) of the ion detection as the vertical axis ( Figure 7 ), and the equation for the relationship between the concentration and peak area of ​​each monosaccharide was calculated based on the peak area of ​​the monosaccharide standard curve at different concentrations (Table 4). The fit was good, indicating that the calculated monosaccharide content was reliable. At the same time, the monosaccharide composition and content of APA-1, APB-1, APC-1 and APD-1 were calculated (Table 5).

[0136] Table 4 Monosaccharide standard curve information

[0137]

[0138] Table 5 Monosaccharide composition and content (%) of APA-1, APB-1, APC-1, and APD-1

[0139]

[0140] The results showed that only glucose was detected in APA-1, and no other monosaccharides were detected, indicating that APA-1 is a polysaccharide composed only of glucose; APB-1 is mainly composed of glucose, with a glucose content as high as 96.56%, and only contains a small amount of arabinose and galactose; APC-1 is mainly composed of arabinose, with a content of 51.07%, followed by galactose, glucose, rhamnose, and also contains a small amount of galacturonic acid and glucuronic acid; the main monosaccharide of APD-1 is arabinose, with an arabinose content of 50.36%. However, compared with APC-1, the contents of galactose and glucose in APD-1 are decreased, while the contents of galacturonic acid and glucuronic acid are increased. The rhamnose content in APD-1 is higher than that in APC-1, and a small amount of xylose and mannose are detected in APD-1. Therefore, according to the results of monosaccharide composition detection, it can be seen that the monosaccharide types of the four purified AP components are APA-1 < APB-1 < APC-1 < APD-1, and the contents of uronic acid in the four components are APD-1 > APC-1 > APB-1 and APA-1. The content of uronic acid is consistent with the results of the DEAE cellulose elution curve.

[0141] (4)Infrared spectrum analysis

[0142] Weighed a small amount of samples of APA-1, APB-1, APC-1 and APD-1, mixed them with 200 mg of potassium bromide, pressed them into a sheet with a thickness of 1 mm, and then used a Nicolet iZ-10 Fourier transform infrared spectrometer to scan in the wavelength range of 450 to 4000 cm -1 to obtain the infrared spectrum as shown Figure 8 below.

[0143] The results showed that the infrared spectra of APA-1, APB-1, APC-1 and APD-1 all showed typical absorption peaks of carbohydrate polymers. The strong and broad peak at 3400~3200 cm −1 is the bending vibration absorption peak of the O-H bond, which is caused by the stretching vibration of the hydroxyl O-H bond related to intermolecular hydrogen bonds. APA-1, APB-1, APC-1 and APD-1 showed strong and broad absorption peaks at 3422.51 cm −1 , 3399.77 cm −1 , 3417.05 cm −1 and 3413.99 cm −1 respectively, indicating that APA-1, APB-1, APC-1 and APD-1 all contain a large number of O-H bonds; the broad peak at 3000~2800 cm −1 ​−1 、2929.15 cm −1 、2929.94 cm −1 and 2929.94 cm −1 There are absorption peaks at 1640 cm-1, indicating that these four components are polysaccharides; in addition, APA-1, APB-1, APC-1 and APD-1 all have absorption peaks at 1640 cm-1. −1 and 1395 cm −1 Absorption peaks appeared near 1640 cm-1 in APA-1 and APB-1, which were attributed to the ring stretching vibration of glucose. −1 and 1395 cm −1 The results of the absorption peak near 1740 cm are consistent with the results of Armillaria polysaccharide composed only of glucose; −1 There is no absorption peak near the 1740 cm −1 There are absorption peaks near the surface of the saccharide, and the absorption peak of APD-1 is more obvious, which is consistent with the results of monosaccharide. APA-1, APB-1, APC-1 and APD-1 are at 1079.18 cm −1 、1080.58 cm −1 、1040.09 cm −1 and 1042.62 cm −1 The absorption peak at 1153.92 cm indicates the presence of a pyranose ring; −1 and 1023.37 cm −1 The band at 1153.32 cm −1 and 1024.51 cm −1 The bands at 859.49 cm-1 and 861.77 cm-2 are attributed to the stretching vibrations of COH and COC, respectively; the bands at 859.49 cm-1 and 861.77 cm-2 of APA-1 and APB-1 are attributed to the stretching vibrations of COH and COC, respectively. −1 and 851.04 cm −1 A band with the characteristic α-pyranose configuration of glucose appeared at 47°C. In summary, the four components obtained all had characteristic absorption peaks of polysaccharides, indicating that these four components were all polysaccharides, and that APA-1 contained pyranose rings and α-type glycosidic bonds.

[0144] (5) Scanning electron microscopy

[0145] 5 mg of APA-1, APB-1, APC-1, and APD-1 samples were adhered to conductive carbon tape, sprayed with gold, and then photographed using a scanning electron microscope. The accelerating voltage of the scanning electron microscope was 0.02-30 kV, and the magnification was 500 times. The morphological characteristics of APA-1, APB-1, APC-1, and APD-1 were obtained ( Figure 9 ).

[0146] The image shows that there are obvious differences in the surface morphological structures of the four polysaccharide components, APA-1, APB-1, APC-1, and APD-1. APA-1 has a smooth morphology with large sheet-like structures. In contrast, part of APB-1 is still smooth with large sheet-like structures, but part is significantly fragmented. Compared with APA-1 and APB-1, APC-1 and APD-1 are no longer smooth, do not have large sheet-like structures, but instead show many fragments and scattered irregular filaments and fibers. It can be confirmed from the image that the ranking of the fragmentation and crosslinking degrees of the four components is APA-1 < APB-1 < APC-1 < APD-1, while the ranking of the smooth large lamellar structures is APA-1 > APB-1 > APC-1 > APD-1.

[0147] To sum up, since APA-1 is the main component of the crude polysaccharide of Aconitum carmichaeli Debx., has a uniform molecular weight distribution and a wide distribution, is a homogeneous polysaccharide of Aconitum carmichaeli Debx., has a dense crosslinked macromolecular spatial configuration, has a highly branched structure, and APA-1 is a polysaccharide composed only of glucose, does not contain uronic acid, there are pyranose rings and α-type glycosidic bonds between molecules, and at the same time APA-1 has a smooth morphology and large sheet-like structures, therefore, APA-1 is selected as the object for subsequent pharmacodynamic studies.

[0148] Example 6 Structure Analysis of APA-1

[0149] (1) Methylation Analysis

[0150] After methylation, hydrolysis, and acetylation of APA-1, gas chromatography-mass spectrometry (GC-MS) is used to determine and compare with the standard mass spectrometry library to judge the nucleoside linkage mode of APA-1.

[0151] Sample pretreatment: 2.5 mg of APA-1 sample was placed in a glass reactor, 500 μL of dimethyl sulfoxide was added to dissolve it, and then 1 mg of NaOH was quickly added. The solution was sealed and ultrasonicated for 30 min. After complete dissolution, 50 μL of iodomethane solution was added and the reaction was carried out in a water bath with a magnetic stirrer at 30°C for 1 h. Finally, 1 mL of ultrapure water and 2 mL of dichloromethane were added to terminate the methylation reaction. The solution was vortexed and centrifuged. The aqueous phase was discarded and washed with pure water three times. The lower dichloromethane phase was aspirated and dried with nitrogen to obtain the methylated polysaccharide sample. The methylated polysaccharide sample was taken and 100 μL of 2M trifluoroacetic acid was added. After hydrolysis at 121°C for 90 min, the product was rotary evaporated and dried. 50 μL of 2M ammonia and 50 μL of 1M sodium borodeuteride were added and mixed. After reacting at room temperature for 2.5 h, 20 μL of acetic acid was added to terminate the reaction and the product was dried with nitrogen. Subsequently, 250 μL of acetic anhydride was added and the product was reacted at 100°C for 2.5 h, and finally, 1 mL of pure water and 500 μL of dichloromethane were added, centrifuged, the aqueous phase was discarded, and the product was washed with pure water three times. The dichloromethane phase was aspirated and analyzed by GC-MS using an Agilent 7890A-5977B gas chromatograph-mass spectrometer to obtain the GC-MS ion chromatogram of the methylated sugar alcohol acetyl derivative of APA-1 ( Figure 10 ).

[0152] Chromatographic system: GC-MS analysis was performed using an Agilent 7890A-5977B gas spectrometer (Agilent Technologies, UAS) with a BPX70 column (30 m × 0.25 mm × 0.25 µm, Australia). The injection volume was 1 μL, and the carrier gas was high-purity helium at a flow rate of 1.5 mL / min. The initial temperature was 140°C, maintained for 2 min, and then programmed to 230°C at 3°C / min. The mass spectrometer system used an Agilent 5977B quadrupole mass spectrometer equipped with an electron impact ion source and a MassHunter workstation (Agilent Technologies, USA). Analytes were detected in full-scan mode with a mass scan range of 50–350 m / z.

[0153] Quantitative analysis: The ratio of the chromatographic peak area to the molecular weight of each sugar residue derivative was calculated to approximate the relative molar amount of each sugar residue derivative, and then the relative molar ratio of each sugar residue in the sample was calculated (Table 6). The calculation formula is as follows:

[0154] Relative molar amount = peak area / molecular weight;

[0155] Relative molar ratio (%) = relative molar amount / total relative molar amounts of each component.

[0156] Table 6 Main sugar residue types and relative molar ratios of APA-1

[0157]

[0158] The results showed that there were seven types of sugar residues in APA-1, namely Glc-(1→, →3)-Glc-(1→, →6)-Glc-(1→, →4)-Glc-(1→, →3,4)-Glc-(1→, →2,4)-Glc-(1→ and →4,6)-Glc-(1→), with relative molar proportions of 13.29%, 1.03%, 2.79%, 69.99%, 2.09%, 1.75% and 9.06%, respectively. Among them, →4)-Glc-(1→ was the main type of sugar residue, and all The majority of the residues are glucose residues, which is consistent with the monosaccharide composition of APA-1. In addition, the relative molar sum of the residues of →4)-Glc-(1→, →3,4)-Glc-(1→, →2,4)-Glc-(1→), and →4,6)-Glc-(1→ is 82.89%. Therefore, it can be determined that the connection mode of the sugar residues on the APA-1 main chain is glucose (1→4) connection, among which →3,4)-Glc-(1→, →2,4)-Glc-(1→), and →4,6)-Glc-(1→ are sugar residues connecting the side chains.

[0159] (2) Nuclear magnetic resonance analysis

[0160] 30 mg of APA-1 was weighed and dissolved in 1 mL of D2O. After complete dissolution, it was filtered through a 0.22 μm filter membrane and then subjected to 1H-NMR, 13C-NMR, 1H-1H COSY-HSQC-HMBC, NOESY, and DEPT135 spectra using a Bruker AVANCE NEO 600 MHz NMR spectrometer to further clarify the possible connection mode between the monosaccharide residues of APA-1. The one-dimensional NMR spectrum and two-dimensional NMR spectrum of APA-1 are shown as follows: Figure 11 and Figure 12 shown.

[0161] In the one-dimensional nuclear magnetic resonance spectrum of the polysaccharide, the 1H NMR spectrum distribution at δ4.5~4.80 ppm is usually the anomeric hydrogen signal of the β-glycosidic bond configuration, and the distribution above δ4.80 ppm is usually the anomeric hydrogen signal of the α-glycosidic bond configuration; the 13CNMR spectrum distribution at δ95.0~110 ppm is usually the signal of the anomeric carbon; the signal at δ60.36 ppm in the DEPT135 spectrum is attributed to the exomethylene C-6 of the glucose residual sugar ring. According to the 1H NMR spectrum of APA-1, there are multiple groups of anomeric hydrogen in APA-1, which are at δ4.87 ppm, δ5.29 ppm, δ5.23 ppm, δ5.36 ppm, δ4.83 ppm and δ5.03 ppm, respectively. This indicates that APA-1 mainly contains α-configuration sugar residues, and the 1H NMR spectrum in the range of δ3.0~4.0 ppm is the alkyl proton signal of the residual sugar ring except for the anomeric hydrogen. APA-1 has many signals in this region and the signals overlap seriously, which is a typical feature of the nuclear magnetic hydrogen spectrum of polysaccharides. According to the 13C NMR spectrum of APA-1, there are multiple anomeric carbons in APA-1, which are at δ97.63 ppm, δ99.46 ppm, δ99.70 ppm, δ99.41 ppm, δ99.01 ppm and δ97.02 ppm, respectively. In addition, in the range of 70~210 No obvious uronic acid carbonyl carbon signal was found in the ppm range, confirming that APA-1 is neutral. This result corresponds to the monosaccharide composition and methylation results. According to the DEPT135 spectrum of APA-1, APA-1 has a methylene C-6 outside the glucose residual sugar ring.

[0162] Further analysis by two-dimensional nuclear magnetic resonance (2D-NMR) spectra revealed that the H-1 / C-1 related signals of the sugar residues were δ4.87 / 97.63, δ5.29 / 99.46, δ5.23 / 99.70, δ5.36 / 99.41, δ4.83 / 99.01, and δ5.03 / 97.02, respectively. The methylation results showed that →4)-Glc-(1→ was the sugar residue with the highest proportion. According to 1H Based on the NMR spectrum, carbon spectrum 13C NMR spectrum and relevant research literature, δ5.29 / 99.46 ppm was assigned to the H-1 / C-1 of →4)-Glc-(1→, δ5.23 / 99.70 ppm was assigned to the H-1 / C-1 signal of →4,6)-Glc-(1→, and δ4.87 / 97.63 ppm was assigned to the H-1 / C-1 signal of Glc-(1→. At the same time, the attribution of multiple other peak signals and the remaining hydrogen and carbon of each sugar residue was determined based on the 1H-1H COSY, HSQC and NOESY of APA-1, and the final summary is shown in Table 7. For example, it was determined that δ5.29 ppm was assigned to the anomeric H-1 of →4)-Glc-(1→. By identifying the hydrogen-hydrogen related COSY and NOESY two-dimensional spectra, the signal with strong correlation was found to be δ3.51 ppm, confirming that the position of H-2 of →4)-Glc-(1→ is δ3.51 ppm. Combined with the HSQC spectrum, it is confirmed that the signal of C-2 of →4)-Glc-(1→ is δ71.47 ppm. Based on the information of COSY and NOESY spectra, the signal related to H-2 of →4)-Glc-(1→ is found to be δ3.84 ppm, confirming that H-3 of →4)-Glc-(1→ is at δ3.84 ppm. Combined with HSQC, the position of C-3 of →4)-Glc-(1→ is δ73.37 ppm. Using the same method, the position information of H-4 / C-4, H-5 / C-5 and H-6 / C-6 of →4)-Glc-(1→ are determined to be δ3.51 / 76.58 ppm, δ3.73 / 71.10 ppm and δ3.73 / 60.35 ppm, respectively.

[0163] Table 7 Signal assignments of 1H NMR and 13C NMR of APA-1

[0164]

[0165] Further analysis of the HMBC spectrum of APA-1 can obtain the connection information between sugar residues. In the HMBC spectrum, obvious correlation signals at δ5.29 ppm and δ76.58 ppm were found, confirming the presence of 1→4 glycosidic bonds of the same residual sugar. This glycosidic bond constitutes the majority of the residual sugar connections in the main chain. The information at this position also confirms the presence of glycosidic bonds between →4)-Glc-(1→ and →4,6)-Glc-(1→. Based on the ratio of methylation results, it was confirmed that →4)-Glc-(1→ and →4,6)-Glc-(1→ occupied the vast majority of the main chain, while a small amount of →3,4)-Glc-(1→ (2.09%) and →2,4)-Glc-(1→ (1.75%) residues existed. The signal response in NMR was very weak, making it difficult to find relevant information about the two-dimensional NMR signals at each site. Therefore, it was judged that these two residues were a small component of the main chain, forming the glycosidic bond of the main chain with a 1→4 connection, and forming the starting point of the branch chain at positions 2 and 3.

[0166] Based on the above analysis, it is judged that APA-1 is mainly a glucan with a 1→4 connection as the main chain, in which →3,4)-Glc-(1→, →2,4)-Glc-(1→ and →4)-Glc-(1→ are sugar residues connecting the side chains. The main structural repeating unit is as follows Figure 13 shown.

[0167] Example 7 Pharmacodynamic Study of Aconite Homogeneous Polysaccharide APA-1 on UC

[0168] The experimental mice were housed in a standard specific pathogen-free environment in advance with a temperature of 22±2°C and a humidity of 50%±10%. During the adaptation period, the mice had free access to standard diet and drinking water. The animal experiments were approved by the Animal Experimentation Ethics Committee of Chengdu University of Traditional Chinese Medicine.

[0169] 1. Animal grouping and drug administration

[0170] according to Figure 14Schematic diagram of the experimental design. After 5 days of adaptive feeding, 30 mice were randomly divided into 5 groups, namely control (C), model (M), low-dose APA-1 (L), high-dose APA-1 (H) and positive (P) groups, with 6 mice in each group. The mice in group C received standard diet and water freely for 10 consecutive days. The mice in group M received normal saline treatment at a dose of 0.2 mL / 20 g (20 g mice were gavaged with 0.2 mL normal saline) for 10 consecutive days. The mice in group L received APA-1 treatment at a dose of 100 mg / kg (1 kg mice were gavaged with 100 mg APA-1) for 10 consecutive days. The mice in group H received APA-1 treatment at a dose of 200 mg / kg (1 kg mice were gavaged with 200 mg APA-1) for 10 consecutive days. The mice in group P received 5-ASA treatment at a dose of 100 mg / kg (1 kg mice were gavaged with 100 In addition, starting from the fourth day, the standard drinking water of groups M, L, H, and P was replaced with 3% DSS solution (3 g DSS dissolved in 100 mL of high-temperature sterilized pure water) to rapidly induce ulcerative colitis in mice, and the old DSS solution was replaced with fresh 3% DSS solution every two days until the end of the experiment.

[0171] 2. Record of weight, blood in stool and diarrhea

[0172] During the experiment, the weight of the mice was recorded at a fixed time every day, and the weight on the day when 3% DSS was started was used as the weight on day 0. The percentage of the weight of each day thereafter to the weight on day 0 was calculated. At the same time, a clean and sterilized mouse cage was taken, a clean white paper was placed on the cage, and the mice were placed in the cage. The fresh feces of the mice were waited and observed, and the stool consistency and intestinal bleeding were evaluated according to the rules described in the literature. The scores were scored according to the percentage of weight change, stool consistency and degree of intestinal bleeding according to the scoring rules in Table 8. The sum of the scores of the three indicators of weight loss, stool consistency and degree of intestinal bleeding was the disease activity index (DAI). The test results are shown as follows: Figure 15 As shown, in Figure 15 In the data, the results are displayed as the mean plus or minus the standard deviation.

[0173] Table 8 Scoring rules for weight loss, stool consistency, and intestinal bleeding

[0174]

[0175] Finally, from the perspective of body weight changes, starting from the fourth day after 3% DSS treatment, the body weight of mice in group M continued to decrease compared with that in group C, the body weight of mice in groups L and H began to decrease on the sixth day after DSS treatment, and the body weight of mice in group P began to decrease on the fifth day after DSS treatment; by the seventh day, compared with group C (98.25±2.79%), the body weight of mice in group M (78.50±5.09%) decreased by about 20%, and there was a significant difference between the two groups; compared with group M, the weight loss of mice in groups L (91.22±3.68%), group H (90.82±5.145%) and group P (87.50±7.211%) was significantly alleviated. In terms of diarrheal symptoms, mice in group M began to experience mild diarrhea starting on the third day after DSS treatment, and the diarrheal symptoms worsened as the experiment progressed. Compared with group M, groups L, H, and P developed diarrheal symptoms later. By the last day, mice in group M developed severe watery diarrhea, while groups L, H, and P had milder diarrhea. There was a significant difference in diarrhea scores between groups L and H and group M. In terms of bloody stools, mice in group M began to experience bloody stools starting on the third day after DSS treatment, and the symptoms became increasingly severe as the experiment progressed. Compared with group M, groups L, H, and P had milder bloody stools. On the seventh day, there was a significant difference in bloody stool scores between groups L and H and group M. In addition, based on the disease index (DAI), the DAI of mice in group M gradually increased starting on the third day after DSS treatment, while the disease index of groups L, H, and P was lower. On the last day, there was a significant difference in DAI between groups L and H and group M. In summary, after DSS treatment, mice in group M showed obvious weight loss, hematochezia, and diarrhea, indicating that the UC mouse model was successfully established, and APA-1 could significantly alleviate these symptoms.

[0176] 3. Colon length and colon pathological changes

[0177] Mice were dissected, and the intestinal segment from the cecum to the end of the colon was completely removed. The length of the colon was measured with a ruler and photographed. Then, a section of colon tissue was taken from mice in groups C, M, L, H, and P, and fixed in 4% paraformaldehyde. The fixed colon tissue was embedded in paraffin and 4 μm serial sections were prepared. Some sections were stained with Harris hematoxylin and 0.5% eosin to obtain colon H&E stained sections, and some sections were stained with Alcian blue dye and Schiff's reagent to obtain colon AB-PAS stained sections. All H&E and AB-PAS stained sections were observed and photographed using a digital pathology slicer. The H&E stained colon pathology scoring rules are as follows: inflammatory cell infiltration (0-3 points), crypt distortion (0-3 points), and colon tissue damage (0-3 points). The test results are shown in the figure below. Figure 16 shown.

[0178] The results showed that compared with group C, the colon length of mice in group M was significantly shortened, and compared with group M, the shortening of colon length of mice in groups L, H and P was significantly alleviated; Claudin and Occludin are two important intestinal tight junction proteins and are important indicators for evaluating the integrity of the intestinal epithelial barrier. Compared with group C, the relative expression of Claudin and Occludin proteins in the colon tissue of mice in group M was significantly decreased, while the relative expression of Claudin and Occludin proteins in the colon tissue of mice in groups L and H was significantly higher than that in group M, and the effect of APA-1 was dose-dependent; H&E staining results showed that DSS treatment caused obvious intestinal tissue damage, which was mainly manifested by the destruction of intestinal crypt structure. The results showed that compared with group C, group M had obvious tissue damage, while compared with group M, the intestinal damage in groups H and P was significantly alleviated. AB-PAS staining results showed that compared with group C, mice in group M had obvious goblet cell failure, mucus layer destruction and reduced mucus secretion, while these phenomena in groups L, H and P were alleviated. At the same time, the proportion of positive area of ​​AB-PAS staining showed that compared with group C, the proportion of positive area of ​​mice in group M was significantly reduced, while the proportion of positive area of ​​mice in groups L, H and P was significantly higher than that of group M, and the effect of APA-1 was dose-dependent. In summary, compared with group C, mice in group M showed colon pathological changes such as colon shortening, increased intestinal permeability, destruction of colonic crypt structure, colonic mucosal destruction, increased inflammatory infiltration, decreased goblet cells and mucus layer destruction, while APA-1 could effectively alleviate these pathological changes, indicating that APA-1 has a significant therapeutic effect on the colon pathology of UC mice.

[0179] 4. Detection of inflammatory factor concentration and relative protein expression level

[0180] (1) Colon tissue pretreatment

[0181] A section of colon tissue from mice in groups C, M, L, H, and P was quickly frozen in liquid nitrogen and then stored at -80°C. The colon tissue stored at -80°C was weighed, mixed with RIPA lysis buffer (lysis buffer volume / colon tissue weight = 9 μL / mg), and then intermittently ground and crushed using a tissue grinder six times, each time for 1 min. After crushing, the tissue was placed on ice for 30 min and then centrifuged at 4°C and 12,000 rpm for 10 min. The colon tissue supernatant was collected.

[0182] (2) Detection of inflammation-related factors

[0183] The mouse Th1 / Th2 / Th17 flow cytometric bead array detection kit (BD Biosciences, USA) was used to detect the levels of IL-6, TNF-α, and IL-17 in the colon tissue supernatant, and the concentrations of inflammatory factors in colon tissue cells were calculated using FCAP v3.0 software.

[0184] (3) Relative protein expression levels of inflammatory factors

[0185] After the total protein content in the colon tissue supernatant was quantified and leveled, instant protein loading buffer was added and denatured at 95°C for 10 min;

[0186] Gently inject 10% separating gel prepared according to the gel preparation kit instructions into the double-layer glass interlayer placed on the gel preparation rack. After adding the appropriate volume of separating gel, slowly add an appropriate amount of isopropyl alcohol to flatten it and eliminate bubbles. Let it solidify at room temperature for 45 minutes. After the separating gel has completely solidified, slowly pour out the isopropyl alcohol and absorb it with filter paper. Gently inject the prepared stacking gel into the glass interlayer, slowly insert the sample comb, and let it solidify at room temperature for 45 minutes.

[0187] After the stacking gel solidifies, place the gel glass plate into the electrophoresis tank and add the electrophoresis solution prepared with electrophoresis buffer powder. Carefully and slowly pull out the comb to remove the bubbles blocking the sample wells. Add 1-2 μL of pre-stained protein marker and an appropriate amount of the protein sample to be tested to the sample wells.

[0188] After loading, run electrophoresis at 80 V until the loading buffer runs out of the stacking gel. Then adjust the voltage to 120 V and continue running. Different proteins require different electrophoresis times. For Claudin1 and IL-6, run for 60 min, for TNF-α, run for 120 min, and for occludin, run until the 35 kDa marker runs to the end of the gel, a total of 180 min.

[0189] Cut a piece of PVDF membrane that matches the size of the sample gel. Activate the cut PVDF membrane in methanol for 30 seconds, then take it out and quickly immerse it in the pre-prepared transfer buffer. Equilibrate at room temperature for 10 minutes to ensure that the membrane is fully wetted and active. The transfer buffer is prepared by dissolving the ice-free rapid transfer buffer powder in an appropriate amount of deionized water. Stir thoroughly during the preparation process to ensure complete dissolution. Then assemble the transfer clamp in the order of cathode (black) → sponge → 3 layers of filter paper → gel → PVDF membrane → 3 layers of filter paper → sponge → anode (white). During the assembly process, use tweezers to gently press each layer to eliminate possible bubbles. Place the assembled transfer clamp in the transfer tank and add pre-cooled transfer buffer to the tank to ensure that the liquid level of the buffer can completely cover the transfer device. Then. Perform the transfer operation in an ice bath, set the constant current to 250 mA, and the transfer time to 90 minutes;

[0190] After the transfer is completed, the membrane was placed in a rapid blocking solution and blocked on a shaker at room temperature for 15 minutes. After the blocking is completed, the membrane was washed with TBST buffer: 10 minutes each time under shaking conditions, for a total of 3 washes, replacing fresh washing solution each time;

[0191] Dilute the primary antibodies (GAPDH, TNF-α, Occludin, Claudin1, and IL-6) with 5% BSA solution. The specific dilution ratios are: 1:5000 for GAPDH and Claudin1, 1:2000 for TNF-α, 1:1000 for IL-6, and 1:15000 for Occludin. Use a pipette to aspirate the diluted primary antibodies into an incubation box of appropriate size. Transfer the blocked and washed transfer membrane to the corresponding incubation box and incubate on a shaker at 4°C overnight to complete the primary antibody incubation.

[0192] Remove the transfer membrane after primary antibody incubation and wash it three times with TBST buffer for 10 minutes each time. After washing, transfer the transfer membrane to an incubation box containing secondary antibody diluent (dilute the secondary antibody with 5% milk at a ratio of 1:5000) and incubate it on a shaker at room temperature for 90 minutes to complete the secondary antibody incubation.

[0193] After incubation with the secondary antibody, the transfer membrane was washed three times with TBST buffer for 10 minutes each time. During the washing period, equal volumes of ECL supersensitive luminescent solution A and solution B were mixed to obtain the luminescent working solution, and the luminescent working solution was transferred to a light-proof incubation box of appropriate size. During development, the washed imprinted membrane was removed and dried with filter paper, then placed in the luminescent working solution for incubation for 1-3 seconds. The membrane was then placed on an E-Blot chemiluminescent electronic sheet imager for exposure and photography. Finally, ImageJ software was used to calculate the optical density of the protein and analyze the relative expression levels of IL-6 and TNF-α proteins in mouse colon tissue.

[0194] The results of inflammatory factor concentration and protein relative expression level detection are as follows Figure 17 As shown, the results showed that compared with group C, the inflammatory factors IL-6, TNF-α, and IL-17 in the colon tissue of mice in group M were significantly increased. Compared with group M, the levels of inflammatory factors IL-6, TNF-α, and IL-17 in the colon tissue of mice treated with APA-1 and 5-ASA were all reduced. Among them, the levels of TNF-α and IL-17 in group P were significantly reduced, the level of IL-6 in group L was significantly reduced, and the level of IL-17 in group H was significantly reduced. In addition, compared with group C, the relative protein expression of IL-6 and TNF-α in the colon tissue of mice in group M was significantly increased, while compared with group M, APA-1 treatment significantly reduced the relative protein expression of IL-6 and TNF-α in the colon tissue. In summary, DSS-treated UC mice developed obvious colon inflammation, and APA-1 treatment could effectively alleviate colon inflammation.

[0195] The foregoing description is merely a preferred embodiment of the present disclosure. It should be understood that the present disclosure is not limited to the form disclosed herein and should not be construed as excluding other embodiments. Rather, the present disclosure is applicable to various other combinations, modifications, and environments and can be modified within the scope of the concepts described herein through the above teachings or techniques or knowledge in the relevant fields. Modifications and variations made by those skilled in the art that do not depart from the spirit and scope of the present disclosure are intended to be protected by the claims appended hereto.

Claims

1. Aconite root homogeneous polysaccharide, characterized in that The homogeneous polysaccharide of aconite root is composed only of glucose, wherein the number average molecular weight Mn is 1181 kDa, the peak molecular weight Mp is 3176 kDa, the weight average molecular weight Mw is 3063 kDa, the Z average molecular weight Mz is 6723 kDa, and the dispersion coefficient Mw / Mn is 2.595; The structure of the Aconite root homogeneous polysaccharide is:

2. The method for preparing the uniform polysaccharide of Radix Aconiti Lateralis Preparata as claimed in claim 1, wherein: The following steps are involved: After reflux extraction of raw aconite root with anhydrous ethanol, the raw aconite root is first filtered and the filter residue is collected; leaching the filter residue with ultrapure water, collecting the leaching solution for a second filtration, performing a first centrifugation on the filtrate, and collecting a first supernatant; The first supernatant is subjected to a first concentration, and when it is concentrated to 1 / 5 of the original volume, alcohol precipitation is performed to obtain a precipitate. After washing, filtering and drying the precipitate, crude aconite polysaccharide is obtained; After dissolving the crude aconite polysaccharide with a first solvent, performing a second centrifugation, and collecting a second supernatant to obtain a first sample solution; The first sample solution is loaded onto a first chromatography column, and pure water is used as a mobile phase for a first elution. The first eluate obtained by the pure water separation is collected and subjected to a second concentration. When the eluate is concentrated to 1 / 5 of the original volume, the eluate is dialyzed in pure water using a dialysis bag. The liquid in the dialysis bag is collected and subjected to a first pre-freezing and a second drying to obtain a crude homogeneous polysaccharide product of Aconite root; After dissolving the crude homogeneous polysaccharide of Aconite Radix with a second solvent, performing a fourth filtration to obtain a second sample solution; The second sample solution is loaded onto a second chromatography column, pure water is used as a mobile phase for a second elution, the second eluate is collected and subjected to a third concentration, and when the eluate is concentrated to 1 / 5 of the original volume, a second pre-freezing and a third drying are performed to obtain the aconite uniform polysaccharide; wherein the first solvent and the second solvent include pure water; The first chromatography column comprises a DEAE-52 cellulose column; The molecular weight cut-off of the dialysis bag is 3500 Da; The second chromatography column includes a Sephadex G-100 gel column.

3. The method according to claim 2, characterized in that The weight ratio of the raw aconite root to the anhydrous ethanol is 1:4; And / or, the weight ratio of the filter residue to the ultrapure water is 1:10; and / or, the solid-to-liquid ratio of the crude aconite polysaccharide to the first solvent is 20 mg:1 mL; And / or, the solid-to-liquid ratio of the crude uniform polysaccharide of Aconite root to the second solvent is 5 mg:10 mL.

4. The method according to claim 2, characterized in that The reflux extraction temperature is 78°C, and the reflux extraction time is 4 h; And / or, the leaching temperature is 95° C., the leaching times are 2 times, and the leaching time is 2 hours per time; And / or, the second filtration is performed using gauze; And / or, the speed of the first centrifugation and the second centrifugation is 3000 r / min, and the time of the first centrifugation and the second centrifugation is 5 min; And / or, the first concentration method includes rotary evaporation concentration; And / or, the washing method comprises washing with anhydrous ethanol; And / or, the methods adopted for the first drying, the second drying and the third drying include freeze drying; and / or, the flow rate of the first elution is 2.5 mL / min; And / or, the dialysis time is 24 hours, and the pure water in the dialysis bag is replaced 3 times during the dialysis period; And / or, the temperature of the first pre-freezing and the second pre-freezing is -80°C; And / or, the third filtration method includes filtering using a 0.45 μm aqueous microporous membrane; and / or, the flow rate of the second elution is 1.25 mL / min; And / or, the second concentration and the third concentration are carried out by methods including reduced pressure evaporation concentration.

5. The method according to claim 2, characterized in that The alcohol precipitation comprises the following steps: Under stirring, anhydrous ethanol was added to the concentrated first supernatant until the alcohol concentration reached 80%, and then the mixture was precipitated in a sealed state at 25° C. for 24 h. After precipitation twice, the precipitate was obtained.

6. Use of the Aconite root homogeneous polysaccharide as claimed in claim 1 or the Aconite root homogeneous polysaccharide prepared by the method according to any one of claims 2 to 5 in preparing a medicament for treating ulcerative colitis, characterized in that, The drug for treating ulcerative colitis is a drug for alleviating or treating weight loss, blood in the stool, diarrhea, colon pathological changes and / or colon inflammation.

7. The use according to claim 6, characterized in that The colon pathological changes include colon shortening, increased intestinal permeability, destruction of colonic crypt structure, destruction of colonic mucosa, increased inflammatory infiltration, decreased goblet cells and / or destruction of the mucus layer.

8. The use according to claim 6, characterized in that The method for alleviating or treating colon inflammation by the drug for treating ulcerative colitis includes reducing the content of inflammation-related factors in colon tissue.

9. The use according to claim 8, characterized in that The method for reducing the content of the inflammation-related factors by the drug for treating ulcerative colitis comprises inhibiting the protein expression of IL-6 and TNF-α in colon tissue.

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