Preparation method and application of portulaca oleracea polysaccharide

Abstract

The invention discloses a preparation method of portulaca oleracea polysaccharide. The preparation method comprises the steps of degreasing, water extraction, deproteinization, alcohol precipitation, drying, crushing and the like. The preparation method of the portulaca oleracea polysaccharide has the remarkable advantages of simple and convenient process, low cost, safety, high efficiency and the like. Pharmacological experiments prove that the portulaca oleracea polysaccharide prepared by the preparation method disclosed by the invention has obvious effects of resisting ulcerative colitis and secondary osteoporosis, and can be used for preparing healthy products, including medicines, health foods and foods, for resisting ulcerative colitis and secondary osteoporosis.

Description

Technical Field

[0001] The invention relates to a preparation method of purslane polysaccharide and application of the purslane polysaccharide in health products for resisting osteoporosis secondary to ulcerative colitis. Background Art

[0002] Ulcerative colitis (UC) is a type of chronic inflammatory bowel disease. The etiology and pathogenesis are still unclear. Persistent chronic inflammation of the intestinal mucosa caused by intestinal immune system disorders plays an important role in ulcerative colitis. [1] Patients with inflammatory bowel disease are often found to have extraintestinal changes, most of which affect the musculoskeletal system. Among them, secondary osteoporosis is the most common manifestation of bone and joint damage [2]. Currently, the main clinical treatment for secondary osteoporosis of ulcerative colitis is the combination of anti-bone resorption drugs such as bisphosphonates. [3] However, due to the hidden nature of secondary osteoporosis caused by UC, it is difficult to clinically identify the interventional point for anti-resorptive drugs, which ultimately leads to unexpected fractures. Therefore, there is an urgent need to develop a product that can simultaneously treat intestinal inflammation and bone loss in UC patients.

[0003] Portulaca oleracea is a common medicinal and edible plant in my country. It has a wide range of pharmacological effects, including anti-inflammatory, antibacterial, gastrointestinal protection, and regulation of intestinal flora homeostasis [4]. In clinical practice, Portulaca oleracea is believed to have the effects of clearing heat and detoxifying, cooling blood and stopping bleeding, and stopping diarrhea. Traditional Chinese medicine formulas with Portulaca oleracea as the main ingredient have a good effect on the treatment of UC. [5] The Compendium of Materia Medica states that "for pain in the muscles and bones, regardless of rheumatism, syphilitic rashes, or women's menstrual diseases, this medicine should be used first to relieve the pain." Portulaca oleracea water extract can inhibit LPS-induced bone loss in vivo and inhibit osteoclast development and bone resorption capacity in vitro by inhibiting the Akt / GSK3β-c-Fos-NFATc1 signaling pathway. [6] The anti-osteoporosis effect of Portulaca oleracea in treating ulcerative colitis makes it very promising in treating osteoporosis secondary to UC.

[0004] The chemical composition of purslane is complex, including flavonoids, alkaloids, organic acids, polysaccharides, polysaccharides, cerebrosides and various nutrients. Due to the unclear efficacy of the ingredients, patients often need to take purslane in excess of the pharmacopoeial dosage, which limits its application in treating osteoporosis secondary to ulcerative colitis. Isolating and purifying the main efficacy ingredients from purslane, retaining the efficacy while achieving dosage reduction is an effective way to solve this problem. In recent years, the inventors have focused on solving this problem. After repeated experimental studies and process optimization, they have clarified that purslane polysaccharides (PO polysaccharides, POP) are the main active ingredients of purslane in treating osteoporosis secondary to ulcerative colitis, and have constructed a method for extracting and preparing them to confirm their efficacy. The present invention can lay the foundation for the research and development of new products for treating osteoporosis secondary to ulcerative colitis and the high value-added industrial development of purslane.

[0005] [1] SU Xiaolu, DONG Chi, ZHANG Jing, MAO Jie, GUO Jiwu, WANG Xiang, XIAOLU S, CHID, et al. Clinical pathological characteristics and differential diagnosis of ulcerative colitis[J]. Biomedical Translation, 2022, 3(2): 66-74.

[0006] [2]BJARNASON I,MACPHERSON A,MACKINTOSH C,et al.Reduced bone density inpatients with inflammatory bowel disease[J / OL].Gut,1997,40(2):228-233.DOI:10.1136 / gut.40.2.228.

[0007] [3]BERNSTEIN CN,TARGOWNIK LE,LESLIE W D.What is the role for bisphosphonates in IBD? [J / OL].Gut,2014,63(9):1369-1370.DOI:10.1136 / gutjnl-2013-306141.

[0008] [4] LI K,

[0009] [5] Zhang Yun'e, Liu Yao. Treatment of 38 cases of ulcerative colitis with modified Baitouweng decoction combined with sulfasalazine enteric-coated tablets[J]. Shaanxi Journal of Traditional Chinese Medicine, 2014, 35(1):5-6.

[0010] [6] KIM JY, OH HM, KWAK SC, et al. Purslane suppresses osteoclastdifferentiation and bone resorbing activity via inhibition of Akt / GSK3β-c-Fos-NFATc1 signaling in vitro and prevents lipopolysaccharide-induced boneloss in vivo[J / OL]. Biological&Pharmaceutical Bulletin,2015,38(1):66-74.DOI:10.1248 / bpb.b14-00567. Summary of the Invention

[0011] In view of the above-mentioned deficiencies in the prior art, according to the embodiments and test examples of the present invention, it is intended to provide a method for preparing purslane polysaccharide with simple process, low cost, safety and high efficiency, and to propose the application of purslane polysaccharide in the preparation of health products for treating osteoporosis secondary to ulcerative colitis.

[0012] According to an embodiment, the present invention provides a method for preparing purslane polysaccharide, comprising the following steps:

[0013] (A)Degreasing

[0014] Weigh 100 g of dry purslane powder, add 200 mL of petroleum ether, soak at room temperature for 12 h to defat, repeat 3 times, wash with distilled water several times, and then filter under reduced pressure to recover the purslane powder.

[0015] (B) Water extraction

[0016] The defatted purslane powder was added to 1 L of distilled water and extracted at room temperature using 100% ultrasonic power for 1 hour, repeated three times. The two filtrates were combined and concentrated to 200 mL.

[0017] (C) Deproteinization

[0018] The above concentrate was added with 10 g of trichloroacetic acid, placed on a decolorizing shaker and shaken for 10 minutes, and then centrifuged at 4000 rpm for 10 minutes. The protein precipitate in the lower layer was discarded and the supernatant was retained.

[0019] (D) Alcohol precipitation

[0020] The supernatant was neutralized with NaOH solution, and then 4 volumes of anhydrous ethanol were added and incubated at 4°C overnight. The precipitate was collected after centrifugation at 4000 rpm for 10 min at 4°C.

[0021] (E) Drying

[0022] The precipitate was dried and crushed, washed several times with 95% ethanol, anhydrous ethanol, and acetone, and dried in a drying oven at 60°C to a constant mass to obtain crude purslane polysaccharide. The crude purslane polysaccharide was stored at -20°C for later use.

[0023] Animal experiments have found that the crude purslane polysaccharide prepared by the method of the present invention can effectively improve disease-related indicators of mice with ulcerative colitis, including disease activity index (DAI), intestinal weight index, liver index, spleen index, colon HE staining, histological score, serum bone metabolism markers, inflammatory factor levels and femoral bone microstructure. The crude purslane polysaccharide prepared by the method of the present invention has significant anti-ulcerative colitis and secondary osteoporosis effects, and has a smaller dosage than crude drugs and better efficacy. Therefore, the purslane polysaccharide prepared by the method of the present invention can be used for the development of health products for anti-ulcerative colitis secondary osteoporosis, including medicines, health foods, and foods, and has good application prospects in related fields. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This figure shows that purslane polysaccharide attenuates ulcerative colitis activity in mice. Figure A shows the changes in DAI scores over time over 8 weeks; B shows the DAI scores at the end of the 8-week modeling period; C shows representative images of the colons of mice in each group; D shows the colon length at the end of the 8-week modeling period; and E shows the intestinal weight index (intestinal weight index = colon weight / colon length) at the end of the 8-week modeling period. Data are presented as mean ± standard deviation (error bars) from biological replicates. Sample size for each group was 8. # P<0.05, ## P<0.01, ### P<0.001, #### P < 0.0001 compared with the control group.* P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001 compared with the model group.

[0025] Figure 2 The results show that purslane polysaccharide reduces DSS-induced colon histological damage. In the figures: AE show representative images of colon HE staining (200X); F shows the colon histological score based on HE staining.

[0026] Figure 3 The results showed that purslane polysaccharide reduced the systemic inflammation level in UC mice. In the figure: A shows the liver index at the end of 8 weeks of modeling (liver index = liver weight / mouse body weight × 100%); B shows a representative image of the liver; C shows the spleen index at the end of 8 weeks of modeling (spleen index = spleen weight / mouse body weight × 1000‰); D shows a representative image of the spleen; E shows the TNF-α content in colon tissue homogenate; F shows the TNF-α content in serum; G shows the TNF-α content in bone tissue homogenate.

[0027] Figure 4 It shows that purslane polysaccharide prevents DSS-induced bone loss and elevation of serum bone metabolism markers. In the figure: AF shows quantitative analysis of mouse bone microarchitecture parameters; G shows representative reconstructed images of mouse femur micro-CT; HJ shows quantitative analysis of serum bone metabolism markers. DETAILED DESCRIPTION

[0028] The present invention will be further described below in conjunction with the accompanying drawings and specific examples. These embodiments should be understood to be merely illustrative of the present invention and not intended to limit the scope of protection of the present invention. After reading the contents described herein, those skilled in the art may make various changes or modifications to the present invention, and these equivalent variations and modifications also fall within the scope defined by the claims of the present invention.

[0029] Example 1 - Preparation of Portulaca Oleracea Polysaccharide

[0030] Take 100g of purslane powder and process it according to the following steps: (1) Degreasing: Add 200mL of petroleum ether and soak for 12h at room temperature for degreasing, repeat 3 times, wash with distilled water several times, and then filter under reduced pressure to recover the defatted purslane powder. (2) Water extraction: Add 100g of defatted purslane powder to 10L of distilled water and extract at 100% ultrasonic power at room temperature for 1h, repeat 3 times. After filtering under reduced pressure again, combine the 3 filtrates and concentrate the filtrate to 200mL. (3) Deproteinization: Add 10g of trichloroacetic acid to the above concentrate, place it on a decolorizing shaker and shake for 10min, then centrifuge at 4000rpm for 10min, discard the lower protein precipitate, and keep the supernatant. (4) Alcohol precipitation: Neutralize the above supernatant with NaOH solution and add 4 times the volume of anhydrous ethanol to precipitate polysaccharides at 4℃ overnight. On the second day, after discarding the supernatant, the lower suspension was centrifuged at 4°C, 4000 rpm for 10 min, and the precipitate was collected. (5) Drying and pulverization: The precipitate was washed several times with 95% ethanol, anhydrous ethanol, and acetone, and dried in a drying oven at 60°C to a constant mass. 7.01 g of crude purslane polysaccharide was obtained. The polysaccharide content was determined to be 57.81% using the phenol-sulfuric acid method.

[0031] Example 2 - Preparation of Portulaca Oleracea Polysaccharide

[0032] The material-liquid ratio in the water extraction step was 1:30, the water extraction time was 0.5 h, and the extraction was performed once. The remaining steps were the same as in Example 1, and 5.34 g of dry powder of purslane polysaccharide was obtained. The polysaccharide content was 53.25% as determined by the phenol-sulfuric acid method.

[0033] Example 3 - Preparation of Portulaca Oleracea Polysaccharide

[0034] The material-liquid ratio in the water extraction step was 1:30, the water extraction time was 1 h, and the extraction was repeated twice. The remaining steps were the same as in Example 1, and 5.77 g of dry powder of purslane polysaccharide was obtained. The polysaccharide content was 51.33% as determined by the phenol-sulfuric acid method.

[0035] Example 4 - Preparation of Portulaca Oleracea Polysaccharide

[0036] The material-liquid ratio in the water extraction step was 1:30, the water extraction time was 1.5 h, and extraction was performed three times. The remaining steps were the same as in Example 1, and 5.71 g of dry purslane polysaccharide powder was obtained. The polysaccharide content was 55.89% as determined by the phenol-sulfuric acid method.

[0037] Example 5 - Preparation of Portulaca Oleracea Polysaccharide

[0038] The material-liquid ratio in the water extraction step was 1:50, the water extraction time was 1 h, and extraction was performed 3 times. The remaining steps were the same as in Example 1, and 5.91 g of dry powder of purslane polysaccharide was obtained. The polysaccharide content was 52.11% as determined by the phenol-sulfuric acid method.

[0039] Example 6 - Preparation of Portulaca Oleracea Polysaccharide

[0040] The material-liquid ratio in the water extraction step was 1:50, the water extraction time was 0.5 h, and the extraction was repeated twice. The remaining steps were the same as in Example 1, and 6.33 g of dry purslane polysaccharide powder was obtained. The polysaccharide content was 56.91% as determined by the phenol-sulfuric acid method.

[0041] Example 7 - Preparation of Portulaca Oleracea Polysaccharide

[0042] The material-liquid ratio in the water extraction step was 1:50, the water extraction time was 1.5 h, and the extraction was performed once. The remaining steps were the same as in Example 1, and 6.55 g of dry powder of purslane polysaccharide was obtained. The polysaccharide content was 55.47% as determined by the phenol-sulfuric acid method.

[0043] Example 8 - Preparation of Portulaca Oleracea Polysaccharide

[0044] The material-liquid ratio in the water extraction step was 1:100, the water extraction time was 1 h, and the extraction was performed once. The remaining steps were the same as in Example 1, and 6.22 g of dry powder of purslane polysaccharide was obtained. The polysaccharide content was 53.65% as determined by the phenol-sulfuric acid method.

[0045] Example 9 - Preparation of Portulaca Oleracea Polysaccharide

[0046] The material-liquid ratio in the water extraction step was 1:10, the water extraction time was 1.5 h, and the extraction was repeated twice. The remaining steps were the same as in Example 1, and 6.94 g of dry purslane polysaccharide powder was obtained. The polysaccharide content was 50.53% as determined by the phenol-sulfuric acid method.

[0047] Experimental Example - Confirmation of the efficacy of Portulaca oleracea polysaccharide in treating ulcerative colitis and secondary osteoporosis

[0048] In order to confirm the efficacy of the purslane polysaccharide of the present invention, animal experiments were conducted. The specific experimental contents are as follows:

[0049] 1. Purpose of the experiment

[0050] The protective effect of Portulaca oleracea polysaccharide on ulcerative colitis (UC) and secondary bone loss was observed using dextran sodium sulfate (DSS).

[0051] 2. Materials

[0052] 2.1 Instruments: Micro-CT (Micview V2.1.2 software, GE, USA), ultrasonic cleaner (DL-1000B, Shanghai Zhixin), electric constant temperature blast drying oven (DHG-9240A, Shanghai Yiheng), electronic balance (1 / 100,000) (BT25SSartorius, Germany), microplate reader (ELx800 BIO-TEX, USA), dehydrator (JJ-12J, Wuhan Junjie), embedding machine (JB-P5, Wuhan Junjie), pathology slicer (RM2016, Shanghai Leica), optical microscope (ECLIPSE E100, Nikon, Japan), imaging system (DS-U3, Nikon, Japan), circulating water vacuum pump (SHB-III, Changsha Mingjie), rotary evaporator (RE-52AA, Shanghai Yarong).

[0053] 2.2 Drugs and reagents Mesalazine enteric-coated tablets (Mesalazine, MS) (Fok Pharmaceuticals, Germany, batch number: H20171358, specification: 0.5 g / tablet), dextran sulfate sodium (DSS) (MW: 36000-50000, Shanghai Yisheng), urine and fecal occult blood test kit (benzidine method) (Shanghai Yisheng), osteocalcin, CTX-1 and TRACP-5b enzyme-linked immunosorbent assay kits (Shanghai ELISA), TNF-α enzyme-linked immunosorbent assay kit (Wuhan Elaruite), paraformaldehyde (Shanghai Boguang), anhydrous ethanol (Shanghai Sinopharm), xylene (Shanghai Sinopharm), HE staining solution kit (Shanghai Zhuocai Biological).

[0054] 2.3 Experimental animals: 40 6-week-old SPF male C57BL / 6J mice, weighing 20-25 g, were purchased from Hangzhou Ziyuan Experimental Animal Technology Co., Ltd. (certificate number: 20230731Abzz0105000308, experimental animal production license number: SCXK (Zhejiang) 2019-0004).

[0055] 3. Test sample

[0056] Purslane polysaccharide: prepared according to the method of Example 1.

[0057] 4. Solution Preparation

[0058] Mesalazine gavage solution (Mesalazine, MS): Take 100 mg of mesalazine enteric-coated tablet powder, add distilled water to 10 mL, and prepare a 10 mg / mL mesalazine solution.

[0059] Low-dose and high-dose purslane polysaccharide groups: weigh 0.2 g and 0.4 g purslane polysaccharide powder, add distilled water to 10 mL, respectively, to prepare 20 mg / mL and 40 mg / mL purslane polysaccharide solutions.

[0060] 5. Animal Grouping and Dosage Method

[0061] Animals were housed in separate cages at the Experimental Animal Center of the Naval Medical University at an ambient temperature of 22 ± 0.5°C, with a 12-h light-dark cycle and free access to food and water. All studies were conducted in accordance with the principles of experimental animal ethics and care of the Naval Medical University.

[0062] Mice were weighed the day before dosing and randomly divided into five groups (Table 1), each consisting of eight mice: a control group (CTRL), a DSS model group (DSS), a mesalamine group (MS), and low- and high-dose purslane polysaccharide groups (POPL and POPH). Modeling lasted for eight weeks. Except for the control group, mice in the model, positive drug, and different POP dose groups received drinking water supplemented with 3% DSS for the first two weeks and 1.5% DSS for weeks 6-8. Starting from the second week, mice in the CTRL and DSS groups received normal saline, mice in the MS group received 100 mg / kg MS solution, and mice in the POPL and POPH groups received 200 mg / kg and 400 mg / kg POP, respectively, by oral gavage.

[0063] Table 1. Experimental animal groups and drug administration methods

[0064]

[0065] 6. Experimental Methods

[0066] 6.1 Assessment of Inflammation Severity in Mice: Drinking water was changed every two days, and changes in mouse weight, stool characteristics, and fecal occult blood were observed and recorded to calculate the Disease Activity Index (DAI). The severity of inflammation in mice was assessed using a total score of 0-4 for mild inflammation, 4-8 for moderate inflammation, and 8-12 for severe inflammation. The scoring criteria are shown in Table 2. Fecal occult blood was determined using a urine and fecal occult blood test kit (benzidine method): a certain amount of freshly collected mouse feces was evenly applied to a white porcelain plate, and 1-2 drops of reagent 1 and reagent 2 were added in sequence. An immediate dark blue color indicates a strong positive, an immediate bluish-green color indicates a positive, a blue color within 30-60 seconds indicates a weak positive, and no color development within 3 minutes indicates a negative.

[0067] Table 2. DAI scoring details

[0068]

[0069] 6.2 Preparation of tissue samples: 8 weeks after modeling, the mice were anesthetized with 3% chloral hydrate, and the eyeballs were removed to draw blood before being killed. After separating the liver and spleen of the mice, the blood, water and other dirt on the surface were gently absorbed with sterile paper towels or absorbent paper, and the weight and length of the colon were weighed and recorded. The colon of the mice was taken out and placed in normal saline for flushing, and the residual feces were removed, and then the weight and length of the colon were measured. A small part of the colon about 1 cm above the anus was selected and placed in 4% paraformaldehyde for fixation. The remaining part of the colon was quickly frozen with liquid nitrogen and stored at -80°C for use. After separating the soft tissue on the tibia and fibula, the bone tissue was placed in 4% paraformaldehyde and stored at room temperature.

[0070] 6.3 HE staining of colon samples: After dewaxing and washing, paraffin sections were stained with hematoxylin and eosin, dehydrated, and mounted for microscopic examination, image acquisition, and analysis.

[0071] 6.4 Scoring of histological changes in colon samples: Observe the colon HE-stained sections to evaluate tissue morphological changes and score according to Table 3, with a total score of 16.

[0072] Table 3. Histological scoring details

[0073]

[0074] 6.5 Enzyme-linked immunosorbent assay (ELISA): Mouse blood was allowed to rest at room temperature for 2 hours, then centrifuged at 3000 rpm for 10 minutes at 4°C. Serum was collected and stored at -80°C. Serum levels of osteocalcin, CTX-1, TRACP-5b, and TNF-α were determined according to the procedures specified in the corresponding kit instructions.

[0075] 6.6 Bone Microarchitectural Parameters: The distal femur was scanned using high-resolution micro-CT, with the 100th layer above the growth plate as the ROI to assess bone mass and bone microarchitecture. Measured parameters included bone mineral density (BMD), relative bone volume (BV / TV), bone surface area / bone volume (BS / BV), trabecular number (Tb.N), trabecular thickness (Tb.Th), and trabecular separation (Tb.Sp).

[0076] 6.7 Statistical Methods: Graphpad Pism 9.5 software was used to analyze and plot experimental data. Quantitative data were expressed as mean ± standard deviation (X ± S). One-way analysis of variance was used to compare multiple groups of quantitative data that conformed to a normal distribution and homogeneity of variance. The Kruskal-Wallis test was used to compare multiple groups of quantitative data or discrete variables that did not conform to a normal distribution or homogeneity of variance, with the FDR correction method for multiple comparisons. P < 0.05 and / or FDR < 0.05 were considered significant.

[0077] 7. Experimental Results

[0078] 7.1 Effect of Portulaca oleracea polysaccharide on intestinal inflammatory activity in DSS-induced UC mice.

[0079] The DAI score is an indicator for evaluating the activity of UC, and the higher the value, the more severe the intestinal inflammation. In the first week of using DSS to induce UC in mice, except for the CTRL group whose DAI score remained basically unchanged, the DAI scores of the four groups of mice, DSS, MS, POPL, and POPH, all increased significantly, indicating that the mice were in the acute UC period. After drug gavage intervention starting in the second week, the DAI score of the DSS group continued to rise significantly, and was higher than 7 points at some time. The DAI scores of the other three drug-treated groups decreased slightly, and remained in the range of 5-7 points. From the beginning of the third week to the end of the eighth week, the DSS content in the drinking water was adjusted to 1.5%. The DAI scores of the mice in the DSS group decreased slightly between 24 and 30 days, and remained between 6 and 7 points. After 30 days, they gradually increased to more than 7 points, indicating that the ulcerative colitis of the mice in the DSS group gradually worsened after becoming chronic. The DAI scores of the mice in the MS, POPL, and POPH groups showed an overall downward trend between 3 and 8 weeks, among which the MS group decreased the most significantly ( Figure 1 A). At the end of the 8th week of modeling, the DAI score of the DSS group was significantly higher than that of the CTRL group, while the DAI scores of the MS and POP groups at different doses were lower than those of the DSS group ( Figure 1 B).

[0080] Long-term chronic inflammation of the intestinal mucosa promotes interstitial fibrosis, which leads to shortened colon length, increased weight, and thickening of the intestinal wall. Therefore, the severity of UC can be judged by colon length and weight. Compared with the CTRL group, the colon length of mice in the DSS group was significantly shortened ( Figure 1 C, Figure 1 D), the intestinal weight index increased significantly ( Figure 1 E). Compared with the DSS group, the colon length of mice in the MS, POPL, and POPH groups increased significantly ( Figure 1 C, Figure 1 D), the intestinal weight index decreased significantly ( Figure 1E) The changes in DAI score and intestinal weight index indicated that POP and MS effectively improved the disease activity and severity of DSS-induced ulcerative colitis in mice.

[0081] 7.2 Effect of Portulaca oleracea polysaccharide on the pathological morphology of colon tissue in DSS-induced UC mice.

[0082] The pathological manifestations of continuous diffuse chronic active inflammation in the colonic mucosa of UC mice can be evaluated by observing the pathological changes. After HE staining, the cell structure of the colon tissue in the CTRL group was intact and clear, without ulcers, and the intestinal glands were neatly arranged with a large number of goblet cells in between. There was no invasion of inflammatory cells and no lymph nodes ( Figure 2 A). In the DSS group, the colonic mucosal layer showed incomplete detachment of cell tissue, with multiple focal superficial ulcers and large-scale loss of crypts. A large number of inflammatory factors in the mucosal layer invaded the serosal layer, and the mucosal layer was significantly thickened. There were scattered lymph nodes ( Figure 2 B). In the MS group, the colonic mucosal layer had occasional detachment of cell tissue, no superficial ulcers, neatly arranged crypts, a small amount of inflammatory cell infiltration around the crypts, no obvious thickening of the mucosal layer, and occasional scattered lymph nodes ( Figure 2 C). In the POPL and POPH groups, the epithelial cells of the mucosa recovered, the glands were neatly arranged, and a large number of goblet cells appeared. There was no significant difference compared with the positive drug group ( Figure 2 D, Figure 2 E). Compared with the CTRL group, the colon histological score of the DSS group was significantly increased. Compared with the DSS group, the colon histological score of the MS, POPL, and POPH groups was significantly decreased ( Figure 2 F) Colon HE staining and histological score changes indicated that POP and MS effectively resisted DSS-induced colon histological changes.

[0083] 7.3 Effect of Portulaca oleracea polysaccharide on systemic inflammation levels in DSS-induced UC mice.

[0084] The pathophysiological process of UC includes uncontrolled chronic inflammation. Liver index and spleen index are indicators for evaluating systemic inflammation in the body. Compared with the CTRL group, the liver index and spleen index in the DSS group increased significantly ( Figure 3 A, Figure 3 C), liver and spleen slightly increased in size ( Figure 3 B, Figure 3 D). Compared with the DSS group, the liver index and spleen index of the MS, POPL, and POPH groups were significantly decreased ( Figure 3 A, Figure 3 C), liver and spleen were slightly reduced in size ( Figure 3 B, Figure 3 D). Compared with the control group, the TNF-α levels in the serum, colon tissue homogenate, and bone tissue homogenate of the mice in the DSS group were significantly increased ( Figure 3 EG). Compared with the DSS group, the TNF-α levels in the serum, colon tissue homogenate, and bone tissue homogenate of the mice in the MS, POPL, and POPH groups were significantly decreased ( Figure 3 E- Figure 3 G). In summary, DSS may induce the local release of a large amount of TNF-α in the colon, which enters the blood through the damaged colonic mucosa, induces systemic inflammation, and then reaches the bone marrow microenvironment through the blood circulation, while POP and MS reduce the level of systemic inflammation in mice.

[0085] 7.4 Effects of purslane polysaccharide on bone microstructure and serum bone metabolism markers in DSS-induced UC mice.

[0086] Bone microstructure parameters measured by Micro-CT are key indicators for measuring changes in mouse bone mass. Compared with the CTRL group, the BMD and BV / TV ( Figure 4 A, Figure 4 B), and the trabecular morphological parameters Tb.N and Tb.Th were significantly reduced ( Figure 4 D, Figure 4 E), BS / BV and Tb.Sp values ​​were significantly increased ( Figure 4 C, Figure 4 F). After administration, compared with the DSS model group, the BMD and BV / TV ( Figure 4 A, Figure 4 B), and the trabecular morphological parameters Tb.N and Tb.Th were significantly increased ( Figure 4 D, Figure 4 E), BS / BV, Tb.Sp values ​​were significantly reduced ( Figure 4 C, Figure 4 F). Compared with the MS model group, the bone microstructure parameters of the mice in the high-dose POP group were more similar to those of the CTRL group, but the difference with the MS group was not statistically significant. Compared with the CTRL group, the femurs of the mice in the DSS model group showed obvious bone cavities in the selected areas, and the number and area of ​​trabeculae were significantly reduced. After drug treatment, MS and low- and high-dose POP could significantly improve the bone microstructure of the mouse trabeculae, indicating that POP can prevent DSS-induced bone loss ( Figure 4 G). Osteocalcin, CTX-1 and TRACP-5b are common serum bone metabolism markers that can indirectly reflect the bone metabolism in vivo. Compared with the CTRL group, the serum levels of Osteocalcin, CTX-1 and TRACP-5b in the DSS group were significantly increased ( Figure 4 H- Figure 4 J). Compared with the DSS group, the serum levels of osteocalcin, CTX-1, and TRACP-5b in the MS and POPH groups were significantly decreased ( Figure 4H- Figure 4 J). Compared with the DSS group, the serum levels of Osteocalcin and CTX-1 in the POPL group were significantly lower ( Figure 4 H, Figure 4 I), TRACP-5b levels decreased, but not statistically significant ( Figure 4 J).

[0087] The results of this study showed that a certain dose of purslane polysaccharide can effectively increase colon length, improve colon histological changes, and reduce the DAI score and intestinal weight index in DSS-induced UC mice, fully demonstrating the anti-UC effect of purslane polysaccharide. Purslane polysaccharide can also effectively prevent DSS-induced bone microarchitecture changes and bone loss, and reduce the levels of bone metabolism markers in serum, suggesting that purslane polysaccharide may exert its anti-osteoporosis effect by inhibiting the high bone turnover state.

[0088] In summary, DSS-induced UC mice showed significant increases in DAI scores and systemic inflammation levels, obvious colonic tissue pathological damage, increased serum bone metabolism markers osteocalcin, CTX-1, and TRACP-5b levels, significant bone loss, and significant decreases in BMD, BV / TV, Tb.N, and Tb.Th, while significantly increasing BS / BV and Tb.Sp. Compared with the DSS model group, oral administration of MS and different doses of POP effectively reversed the changes in these indicators. This suggests that the purslane polysaccharide prepared by the present method can significantly inhibit the severity of colitis in UC mice, reduce systemic inflammation levels, and inhibit high levels of bone metabolism, thereby exerting anti-ulcerative colitis and secondary osteoporosis effects. This suggests that the purslane polysaccharide has the potential to further develop products for the treatment of ulcerative colitis and secondary osteoporosis.

Claims

1. A method for preparing purslane polysaccharide, characterized in that: The steps include: (A)Degreasing Take purslane powder and add 200 mL of petroleum ether, soak at room temperature for 12 hours to defatted, wash with distilled water several times, and then filter under reduced pressure to recover defatted purslane powder; (B) Water extraction Defatted purslane powder was added to 1 L of distilled water and extracted with 100% ultrasonic power. The filtrates were combined and concentrated to 200 mL after filtration under reduced pressure again. (C) Deproteinization The above concentrate was added with 10 g of trichloroacetic acid, placed on a decolorizing shaker and shaken for 10 min, then centrifuged at 4000 rpm for 10 min, the lower protein precipitate was discarded, and the supernatant was retained; (D) Alcohol precipitation The supernatant was neutralized with NaOH solution and then 4 times the volume of anhydrous ethanol was added and incubated at 4°C overnight; the precipitate was collected after centrifugation at 4000 rpm for 10 min at 4°C; (E) Drying and crushing The product was washed several times with 95% ethanol, anhydrous ethanol and acetone, and dried in a drying oven at 60° C. to a constant mass to obtain crude purslane polysaccharide.

2. The preparation method according to claim 1, wherein In the step (A), petroleum ether was added repeatedly three times.

3. The preparation method according to claim 1, wherein In the step (B), the water extraction temperature is room temperature and the extraction is repeated 1-3 times.

4. The preparation method according to claim 1, wherein In the step (B), the material-liquid ratio is 1:10-1:

100.

5. The preparation method according to claim 1, wherein In the step (B), the water extraction time is 0.5-1.5h.

6. The preparation method according to claim 1, wherein In the step (D), the polysaccharide is precipitated by standing at 4° C. for 8-12 hours.

7. Portulaca oleracea polysaccharide prepared by the preparation method according to any one of claims 1 to 6.

8. Use of the purslane polysaccharide according to claim 7 in the preparation of a medicament for treating ulcerative colitis and secondary osteoporosis.

9. The purslane polysaccharide according to claim 7 is used as a health food or food for treating ulcerative colitis and secondary osteoporosis.

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