Polygonum cuspidatum homogeneous polysaccharide PCPs as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of traditional Chinese medicine, and discloses polygonum cuspidatum homogeneous polysaccharide PCPs and a preparation method and application thereof.The polygonum cuspidatum homogeneous polysaccharide PCPs is mainly composed of fucose, rhamnose, arabinose, galactose and glucose according to the molar ratio of 1.17: 3.94: 1.58: 6.72: 86.59, and the weight-average molecular weight of the polygonum cuspidatum homogeneous polysaccharide PCPs is 93.245 kDa. The Polygonum cuspidatum homogeneous polysaccharide PCPs is a novel homogeneous polysaccharide compound, has fixed components and high yield, and has good effects of preventing and relieving colitis.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine technology, and relates to a homogeneous polysaccharide PCPs of Polygonum cuspidatum, its preparation method and application. Background Technology

[0002] Polygonum cuspidatum (scientific name: Polygonum cuspidatum Sieb. et Zucc Polygonum cuspidatum is a perennial herb belonging to the genus Polygonum in the family Polygonaceae. It has a bitter taste and cold properties, and possesses the effects of clearing heat, dampness, reducing jaundice, and dispersing blood stasis. It has clinical functions in treating inflammation, gout, diabetes, viral infections, and other diseases. Previous studies have found that Polygonum cuspidatum polysaccharides, as the main active ingredient of Polygonum cuspidatum, have various active functions, such as lowering blood sugar, lowering blood lipids, anti-tumor, antioxidant, and enhancing the body's immunity. Research has found that Polygonum cuspidatum polysaccharides, as a prebiotic with the function of regulating intestinal microecology, can not only reshape the intestinal flora structure but also effectively protect the intestinal barrier and promote the healthy recovery of the intestinal mucosa.

[0003] Inflammatory bowel disease (IBD), including ulcerative colitis (UC) and Crohn's disease (CD), is primarily caused by unhealthy lifestyle habits such as diets high in sugar and fat, irregular work schedules, restricted physical activity, and heavy work stress. IBD is a chronic, complex inflammatory disease characterized by gastrointestinal symptoms such as abdominal pain, diarrhea, and rectal bleeding. Epidemiological surveys show that the incidence of IBD in China is on the rise, with an estimated 1.5 million cases by 2025. However, its etiology remains unclear, weakening the effectiveness of current treatments. Current mainstream treatment strategies include drug intervention and surgery, with the main drug formulations being 5-aminosalicylic acid (5-ASA) and sulfadiazine (SASP). However, these options can lead to adverse reactions or financial burdens. Therefore, the pursuit of low-cost, high-efficiency, and low-toxicity drug formulations or functional foods has become a development trend.

[0004] Numerous studies have demonstrated that natural polysaccharides can effectively protect against and alleviate IBD. Natural polysaccharides reduce IBD symptoms by regulating the gut microbiota and promoting the recovery of the intestinal mucosal barrier (IMB) and immune system. The gut microbiota participates in the absorption and metabolism of nutrients, maintains intestinal homeostasis and physiological function, and effectively regulates the immune, nervous, and endocrine systems. The gut microbiota significantly influences the development and treatment of UC. Gut microbiota-based treatments for UC, such as fecal transplantation and prebiotic blends, are increasingly being used clinically.

[0005] Currently, there is limited research on the preparation and pharmacological activity of Polygonum cuspidatum polysaccharides, and the composition and activity of Polygonum cuspidatum polysaccharides obtained by different extraction methods vary significantly. Existing Polygonum cuspidatum polysaccharide preparation processes result in low extraction rates, limiting its application in practical production. Therefore, developing a method for producing Polygonum cuspidatum polysaccharides with a fixed composition and high yield would greatly promote the industrialization of Polygonum cuspidatum and expand its application scope. Summary of the Invention

[0006] This invention addresses the technical problem of limited applications of Polygonum cuspidatum polysaccharide by providing a homogeneous Polygonum cuspidatum polysaccharide PCPs, its preparation method, and its applications. This homogeneous Polygonum cuspidatum polysaccharide PCPs is a novel homogeneous polysaccharide compound with fixed components and a high yield, exhibiting good preventive and alleviating effects on colitis.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: On one hand, this invention provides a homogeneous polysaccharide PCPs from Polygonum cuspidatum, which is mainly composed of fucose, rhamnose, arabinose, galactose, and glucose in a molar ratio of 1.17:3.94:1.58:6.72:86.59, with a weight-average molecular weight of 93.245 kDa. Its structural unit has the following structural formula: .

[0008] On the other hand, the present invention provides a method for preparing the above-mentioned homogeneous polysaccharide PCPs from Polygonum cuspidatum, comprising the following steps: 1) Boil the chopped Japanese knotweed in water, cool, filter, and obtain Japanese knotweed polysaccharide extract; 2) The extract of Polygonum cuspidatum polysaccharide was subjected to alcohol precipitation to obtain crude polysaccharide precipitated with alcohol; 3) The crude polysaccharide precipitated with alcohol was deproteinized using the Sevag method to obtain total polysaccharide of Polygonum cuspidatum; 4) The total polysaccharides of Polygonum cuspidatum were separated and purified by ion exchange chromatography and gel column chromatography, then concentrated and freeze-dried to obtain homogeneous polysaccharides PCPs of Polygonum cuspidatum.

[0009] Furthermore, the boiling time described in step 1) is 1-2 hours.

[0010] Furthermore, in step 2), the alcohol precipitation uses ethanol with a concentration of 80-95%, the precipitation temperature is 4-25℃, and the time is 2-12 h.

[0011] Furthermore, in step 3), when removing protein using the Sevag method, a mixture of chloroform and n-butanol is used for extraction; the volume ratio of chloroform to n-butanol is 3~5:1.

[0012] Further, in step 4), the ion exchange chromatography column is a DEAE-agarose gel FF column, the flow rate of the ion exchange column chromatography is 50 mL / h, and the gradient elution program is: 0–30 min water, 20–40 min 0.1 mol / L NaCl, 20–30 min 0.3 mol / L NaCl; the packing material of the gel column is Superdex-200.

[0013] In another aspect, the present invention provides the application of the above-mentioned homogeneous polysaccharide PCPs from Polygonum cuspidatum in the preparation of drugs for treating ulcerative colitis.

[0014] Compared with the prior art, the beneficial effects of the present invention are as follows: The homogeneous polysaccharide PCPs of Polygonum cuspidatum in this invention are novel homogeneous polysaccharide compounds discovered for the first time. They have fixed components and a high yield, and have good effects in preventing and alleviating colitis. This invention provides a basis for the quality control and standardized production of homogeneous polysaccharide of Polygonum cuspidatum. Attached Figure Description

[0015] Figure 1 Schematic diagram of the preparation of homogeneous polysaccharide PCPS from Polygonum cuspidatum and its application in the treatment of IBD.

[0016] Figure 2 The hydrogen spectrum of the homogeneous polysaccharide PCPS of Polygonum cuspidatum.

[0017] Figure 3 This is the carbon spectrum of PCPS, a homogeneous polysaccharide from Polygonum cuspidatum.

[0018] Figure 4 The HH-COSY profile of homogeneous polysaccharide PCPS from Polygonum cuspidatum.

[0019] Figure 5 The NOESY spectrum of homogeneous polysaccharide PCPS from Polygonum cuspidatum.

[0020] Figure 6 The HSQC spectrum of homogeneous polysaccharide PCPS from Polygonum cuspidatum.

[0021] Figure 7 The HMBC map of homogeneous polysaccharide PCPS from Polygonum cuspidatum.

[0022] Figure 8The role of Polygonum cuspidatum polysaccharide PCPs in the treatment of DSS-related IBD is shown in the following figures: (A) Mouse body weight on day 13; (B) Disease Activity Index (DAI) score; (C, D) Colon length; (E) Typical histopathological sections of five colon tissues. Arrows indicate areas of crypt structure loss, goblet cell reduction, and exacerbated inflammation. (FK) Serum biochemical analysis, including myeloperoxidase (MPO), malondialdehyde (MDA), total superoxide dismutase (T-SOD), catalase (CAT), interleukin-1β (IL-1β), and interleukin-6 (IL-6). Detailed Implementation

[0023] The following embodiments are used to illustrate the present invention, but are not intended to limit the scope of protection of the present invention. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art. Unless otherwise specified, the test methods in the following embodiments are conventional methods.

[0024] Example 1: Extraction of homogeneous polysaccharides (PCPs) from Polygonum cuspidatum (1) Extraction of total polysaccharides from Polygonum cuspidatum Dried Polygonum cuspidatum (from Ji'an, Jiangxi Province) slices were collected. 5 kg of Polygonum cuspidatum was chopped, added to 10 times its volume of distilled water, and decocted at 100℃ for 1 hour. After cooling, the mixture was filtered, and the filtrate was collected to obtain a Polygonum cuspidatum polysaccharide extract. The polysaccharide extract was concentrated using a rotary thin-film evaporator to obtain a fluid extract. The fluid extract was dispersed and dissolved in 1 L of distilled water, and 90% ethanol was added. The mixture was allowed to stand at 4℃ for 24 hours, centrifuged, and the precipitate was obtained. The ethanol was evaporated to obtain crude polysaccharide precipitated with alcohol.

[0025] Add one volume of distilled water to the ethanol-precipitated crude polysaccharide, and extract using the Sevag method by adding 1 / 5 of the solution volume of a chloroform-n-butanol mixture (volume ratio 4:1). Shake on a shaker for 20 min, transfer to a separatory funnel, let stand, collect the supernatant, and centrifuge for 1 min to remove residual protein precipitate. Repeat 3-5 times to obtain a crude Polygonum cuspidatum polysaccharide solution after protein removal. Concentrate and dry under reduced pressure to obtain 0.82 kg of total Polygonum cuspidatum polysaccharide.

[0026] (2) Isolation and purification of total polysaccharides from Polygonum cuspidatum The ion exchange chromatography column used was a DEAE-agarose gel FF column, connected to a fraction collector and a peristaltic pump. Total polysaccharides from Polygonum cuspidatum were eluted sequentially with distilled water, 0.1 mol / L NaCl, and 0.3 mol / L NaCl at a flow rate of 50 mL / h for elution times of 25 min, 30 min, and 25 min, respectively. The eluent was collected in 10 mL tubes and labeled. The absorbance was measured at 490 nm using the phenol-sulfuric acid method, and a scatter plot was plotted. 30–50 tubes of eluent (eluent with 0.1 mol / L NaCl) were collected, concentrated, dialyzed through a 3500 Da dialysis bag, and freeze-dried to obtain the eluted fraction PCPs-B, weighing 182 g.

[0027] Take 100 mg of PCPs-B fraction, dissolve it in 3 mL of distilled water, centrifuge (12000 rpm) for 10 min, and further separate and purify the supernatant by passing it through a dextran gel Superdex-200 column. Combine the solutions and concentrate them by rotary evaporation and freeze-dry to obtain the fraction named PCPs with a mass of 35 mg.

[0028] Example 2: Structural identification of homogeneous polysaccharide PCPs from Polygonum cuspidatum (1) The molecular weight of polysaccharides was determined by HPGPC. The optimized conditions were: Shimadzu LC-10A high performance liquid chromatograph, RI-20A differential detector, BRT105-103-101 tandem gel chromatography column (8×300 mm); mobile phase: 0.2 mol / L NaCl solution, flow rate: 0.8 mL / min, column temperature: 40 ℃; injection volume: 25 μL. Regression curves were established using dextran standards with different molecular weights, and the molecular weight was calculated. Dextran standards (molecular weights: 1152, 5000, 11600, 23800, 48600, 80900, 148000, 273000) were prepared into solutions with a concentration of 5 mg / mL with the mobile phase before use. A standard curve was plotted with the molecular weight (lg) as the abscissa and the retention time (RT) as the ordinate. The equation of the lgMp-RT correction curve was: Y =-0.207 X +11.06 (R²=0.999); the equation of the lgMw-RT correction curve is: Y =-0.204 X +10.99 ( R 2 =0.999); the equation of the lgMn-RT correction curve is: Y =-0.205 X +11.01 R 2 =0.999); PCP S Prepared using the same method, PCP was calculated from the regression equation of the standard curve.S The molecular weight of PCP was obtained. S Its weight-average molecular weight is approximately 93.245 kDa.

[0029] (2) Analysis of PCP by ion chromatography S Monosaccharide composition: a. A mixture of monosaccharide standards including fucose, rhamnose, arabinose, galactose, glucose, xylose, mannose, fructose, ribose, galacturonic acid (GalA), glucuronic acid (GlcA), galactosyl hydrochloride, glucosamine hydrochloride, N-acetyl-D-glucosamine, guluronic acid (GulA), and mannuronic acid (ManA) were used as controls.

[0030] b. Accurately weigh 5 mg of sample into an ampoule, add 2 mL of 3 mol / L trifluoroacetic acid (TFA), and hydrolyze at 120 °C for 3 h. Accurately pipette the acid-hydrolyzed solution into a tube and dry under nitrogen. Add 5 mL of water and vortex to mix. Pipe 200 µL of the solution into 800 µL of deionized water and centrifuge at 12000 rpm for 5 min. Collect the supernatant for analysis using an ion chromatograph (ICS5000). Chromatographic conditions: Dionex... TM Carbopac TM PA20 column (3×150 mm), column temperature 30 ℃, injection volume 25 µL, flow rate: 0.3 mL / min, mobile phase: A: H2O; B: 15 mol / L NaOH; C: 15 mol / L NaOH & 100 mol / L NaOAC, detector: electrochemical detector.

[0031] The results showed that the homogeneous polysaccharide PCPs of Polygonum cuspidatum were mainly composed of fucose, rhamnose, arabinose, galactose and glucose in a molar ratio of 1.17:3.94:1.58:6.72:86.59.

[0032] (3) Gas chromatography-mass spectrometry (GC-MS) analysis of samples after methylation, hydrolysis, and acetylation, as well as infrared spectroscopy and 1H NMR spectroscopy. Figure 2 ), 13C NMR spectrum ( Figure 32D (¹H-¹HCOSY, HSQC, HMBC, NOESY) NMR spectra were used to assign glycosidic bond signals to the polysaccharide. The analysis results are shown in Table 1-2. The main glycosidic bond structure of the polysaccharide was determined to be as follows: the main chain is connected by glycosidic bonds of →6)-α-d-glcp-(1→) and a small number of →3,6)-α-d-galp-(1→), while the side chains are mainly connected by α-D-Glcp-(1→) at the O-6 position of SR→3,6)-α-d-galp-(1→).

[0033] Table 1. Analysis of methylated sugar alcohol acetyl ester (PMAA) results for PCPs Table 2. Hydrogen and carbon signal attribution of PCPs The present invention also performed a series of structural characterizations, including: methylation assays to analyze its sugar residues and NMR spectra analysis to determine the glycosidic bond linkage mode. Figure 4 The HH-COSY spectrum of homogeneous polysaccharide PCPs from Polygonum cuspidatum. Figure 5 The NOESY spectrum of homogeneous polysaccharide PCPs from Polygonum cuspidatum. Figure 6 The HSQC spectrum of homogeneous polysaccharide PCPs from Polygonum cuspidatum. Figure 7 The HMBC diagram of homogeneous polysaccharide PCPs from Polygonum cuspidatum is shown. The structural formulas of the PCPs structural units were obtained using various analytical methods and are shown below: .

[0034] Example 3: Therapeutic effect of Polygonum cuspidatum homogenized polysaccharide on IBD model mice 3.1 Animal Model Fifty 6-week-old male C57BL / 6 mice (Nanjing University Model Animal Research Center, Production License No.: SCXK(SU) 2018-0008) were housed under a 12 h:12 h light-dark cycle at a temperature of 24±1 ℃ and a humidity of 50-70%, and were provided with food and drinking water to establish a DSS-induced inflammatory bowel disease (IBD) model for intervention and detection experiments.

[0035] 3.2 Reagents Sodium dextran sulfate (Yisheng Biotechnology Co., Ltd.), total superoxide dismutase (Merck Life Sciences Co., Ltd.), catalase (Shanghai Aladdin Biochemical Technology Co., Ltd.), malondialdehyde (Shanghai Cent Biotechnology Co., Ltd.) detection kit, 4% paraformaldehyde (Jiangsu Kaiji Biotechnology Co., Ltd.), hematoxylin-eosin (HE) staining kit (for staining colon tissue pathological sections), enzyme-linked immunosorbent assay (ELISA) detection kit (Jiangsu ELISA); 3.3 Consumables and Instruments 3.3.1 Analytical balance (Restaurant Scientific Instruments Co., Ltd.), Thermo clean bench, microplate reader, freeze dryer, optical microscope.

[0036] 3.3.2 Pipettes, pipettes (5 ml and 10 ml respectively), 6-well plates, 96-well plates, and Transwell 24-well plates.

[0037] 3.4 Experimental Methods 3.4.1 Therapeutic effect of PCPs on DSS-induced IBD model mice 3.4.1.1 Construction of IBD mouse model A mouse IBD model was constructed using the DSS induction method. It is hypothesized that by allowing mice to freely drink water containing a specific concentration of DSS for a certain period of time (usually 7-10 days), the damaging effect of DSS on the intestinal mucosa can be used to simulate the pathological features of human IBD, such as intestinal inflammation and barrier disruption.

[0038] 3.4.1.2 Core Indicator Detection and Sample Collection After model construction and the intervention period (usually 7-14 days after intervention, depending on the pathological progression of IBD), mice were treated and samples were collected for indicator detection: 3.4.1.2.1 General Condition and Disease Activity Index (DAI) Score The general condition of mice in each group was observed and recorded daily, including changes in body weight (calculation of weight loss rate), fecal characteristics (e.g., formed / loose / bloody stool), and activity level. The mice in each group were scored according to the standard DAI scoring system (weight loss, fecal characteristics, and bloody stool were assigned values ​​separately, with a total score of 0-12 points) to quantify the severity of intestinal inflammation.

[0039] 3.4.1.2.2 Colon tissue-related tests After euthanizing the mice, the intact colon was quickly dissected, its contents removed, and the colon length measured with a ruler (IBD model mice exhibit colonic shortening due to intestinal inflammation; PCP intervention may alleviate this shortening trend). Histopathological analysis: Tissue from the mid-colon was taken, fixed in 4% paraformaldehyde, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (HE). The colonic mucosal structure was observed under a light microscope (e.g., mucosal integrity, degree of epithelial cell damage, and inflammatory cell infiltration), and pathological scoring was performed according to pathological scoring criteria (e.g., degree of mucosal damage, extent of inflammatory infiltration, etc.).

[0040] 3.4.1.2.3 Detection of Oxidative Stress and Inflammatory Factors A portion of colonic tissue was collected, added with physiological saline or lysis buffer, homogenized, and centrifuged to obtain a colonic tissue homogenate. Oxidative stress markers were detected: total superoxide dismutase (T-SOD) activity, catalase (CAT) activity, and malondialdehyde (MDA) content in the homogenate were measured using colorimetric or enzyme-linked immunosorbent assay (ELISA) to assess the effect of PCPs on intestinal oxidative stress in IBD mice. Inflammatory factor detection: the levels of inflammatory factors in the colonic tissue homogenate or serum were detected using ELISA, including pro-inflammatory factors such as tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-1β (IL-1β), as well as anti-inflammatory factors such as interleukin-10 (IL-10), to analyze the anti-inflammatory effect of PCPs.

[0041] 3.4.1.2.4 Detection of intestinal barrier function-related proteins Immunohistochemistry or Western blotting was used to detect the expression levels of tight junction proteins (such as Occludin and ZO-1) in colon tissue. The effect of PCPs on the repair of intestinal barrier integrity in IBD mice was evaluated by protein expression levels (such as the gray value of Western blotting and the intensity of positive staining by immunohistochemistry).

[0042] 3.5 Experimental Results In a therapeutic experiment on PCPs in DSS-induced IBD model mice, it was found that Polygonum cuspidatum polysaccharides (PCPs) could alleviate DAI symptoms, prolong colon length, and repair colon tissue damage in mice. Figure 8 AE); reduce intestinal oxidative stress (increase T-SOD and CAT activity, reduce MDA), regulate the balance of inflammatory factors (downregulate pro-inflammatory factors such as TNF-α, upregulate IL-10) Figure 8 FK).

[0043] The embodiments described above are merely preferred embodiments of the present invention and are only used to explain the present invention. They are not intended to limit the scope of the present invention. For those skilled in the art, other implementation methods can be easily made by substitution or modification based on the technical content disclosed in this specification. Therefore, all changes and improvements made on the principle of the present invention should be included within the scope of the patent application of the present invention.

Claims

1. A homogeneous polysaccharide PCPs from Polygonum cuspidatum, characterized in that, It is mainly composed of fucose, rhamnose, arabinose, galactose, and glucose in a molar ratio of 1.17:3.94:1.58:6.72:86.59, with a weight-average molecular weight of 93.245 kDa. Its structural unit has the following structural formula: 。 2. The method for preparing homogeneous polysaccharide PCPs from Polygonum cuspidatum according to claim 1, characterized in that, Includes the following steps: 1) Boil the chopped Japanese knotweed in water, cool, filter, and obtain Japanese knotweed polysaccharide extract; 2) The extract of Polygonum cuspidatum polysaccharide was subjected to alcohol precipitation to obtain crude polysaccharide precipitated with alcohol; 3) The crude polysaccharide precipitated with alcohol was deproteinized using the Sevag method to obtain total polysaccharide of Polygonum cuspidatum; 4) The total polysaccharides of Polygonum cuspidatum were separated and purified by ion exchange chromatography and gel column chromatography, then concentrated and freeze-dried to obtain homogeneous polysaccharides PCPs of Polygonum cuspidatum.

3. The method for preparing homogeneous polysaccharide PCPs from Polygonum cuspidatum according to claim 2, characterized in that, The boiling time mentioned in step 1) is 1~2 hours.

4. The method for preparing homogeneous polysaccharide PCPs from Polygonum cuspidatum according to claim 2, characterized in that, The alcohol precipitation in step 2) uses ethanol with a concentration of 80-95%, the precipitation temperature is 4-25 ℃, and the time is 2-12 h.

5. The method for preparing homogeneous polysaccharide PCPs from Polygonum cuspidatum according to claim 2, characterized in that, In step 3), when removing protein using the Sevag method, a mixture of chloroform and n-butanol is used for extraction; the volume ratio of chloroform to n-butanol is 3~5:

1.

6. The method for preparing homogeneous polysaccharide PCPs from Polygonum cuspidatum according to claim 2, characterized in that, The ion exchange chromatography column mentioned in step 4) is a DEAE-agarose gel FF column, the flow rate of the ion exchange column chromatography is 50 mL / h, and the gradient elution program is: 0-30 min water, 20-40 min 0.1 mol / L NaCl, 20-30 min 0.3 mol / L NaCl; the packing material of the gel column is Superdex-200.

7. The use of the homogeneous polysaccharide PCPs of Polygonum cuspidatum according to claim 1 in the preparation of a drug for treating ulcerative colitis.

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