Ulva pertusa homogeneous polysaccharide as well as preparation method and application thereof

Abstract

The invention discloses ulva pertusa polysaccharide UPP and ulva pertusa homogeneous polysaccharide UPP-5 as well as a preparation method and application thereof. The single-component polysaccharide UPP-5 is obtained by adopting DEAE-52 anion exchange chromatography in combination with ultrafiltration purification. The structure of the UPP-5 is systematically characterized by high performance gel permeation chromatography, infrared spectroscopy, methylation analysis, nuclear magnetic resonance and other methods, and the result shows that the UPP-5 is acidic heteropolysaccharide with a novel structure. Based on an AOM / DSS induced colorectal cancer mouse model, the effect of the mouse model is evaluated from the aspects of disease activity index, colon length, tumor number, histological observation, gene and protein expression and the like. Results show that the UPP can significantly improve inflammation and pathological states and reduce the incidence of tumors, and has the potential of preventing and treating colorectal cancer. The invention provides a new thought and theoretical basis for structural research and medical function development of algal polysaccharides.

Description

Technical Field

[0001] This invention relates to the field of polysaccharide technology, specifically to a homogeneous polysaccharide, UPP-5, isolated from *Ulva perforatum*, whose structure was determined by infrared spectroscopy, methylation, and NMR, and identified as a novel polysaccharide. Furthermore, by constructing an AOM / DSS mouse model, it was demonstrated that this *Ulva perforatum* polysaccharide can prevent the development of colorectal cancer, providing a new direction for the prevention of colorectal cancer using *Ulva perforatum* polysaccharides. Background Technology

[0002] Ulvapertusa Kjellm is a common green algae in the genus Ulvapertus, also known as sea lettuce, sea lettuce moss, etc., and is an economically abundant algae in my country (Sun Yuyu, 2009). Due to its high nutritional value, it has been approved for human consumption by the French authorities (Lahaye et al., 1993). Ulvapertusa Kjellm polysaccharide is a sulfated heteropolysaccharide (Li, 2018) with the following skeletal structure: →4)-β-D-Xylp-(1→4)-α-L-Rhap3S(1→3,4)-β-L-Arap(1→3)-α-L-Rhap(1→2,4)-α-L-Rhap(1→4)-α-D-Galp(1→4), and two main branches. Physicochemical analysis shows that Ulvapertusa Kjellm polysaccharide is a semi-crystalline polymer and does not possess a triple helix structure (Gao et al., 2020). Qi et al. (2005a, b) studied the antioxidant activity of Ulva perforata polysaccharide derivatives with different sulfate contents, finding that derivatives with high sulfate contents exhibited significant antioxidant activity in vitro. Lin Long (2013) demonstrated that Ulva perforata polysaccharide had a good hypoglycemic effect on alloxan-induced diabetic mice and investigated its main mechanism. Yu et al. (2003) studied the lipid-lowering activity of sulfated polysaccharides obtained from Ulva perforata, finding that low molecular weight polysaccharides (151.6–28.2 kDa) were more effective in lowering blood lipids than high molecular weight polysaccharides. Sun Xifeng et al. (2014) explored the moisturizing activity of Ulva perforata polysaccharides with different molecular weights, showing that undegraded Ulva perforata polysaccharides had stronger moisturizing ability, indicating that the moisturizing activity of Ulva perforata polysaccharides is closely related to molecular weight. Numerous studies (Yoon et al., 2015; Manzoor et al., 2016; Ali et al., 2017) have shown that substances with strong biological activity can be isolated from Ulva perforatum.

[0003] However, the extraction yield, structural characteristics, and biological functions of polysaccharides are significantly influenced by extraction process conditions. Existing studies have shown that factors such as extraction temperature, time, pH, solid-liquid ratio, solvent type, and extraction steps, as well as different extraction methods (e.g., hot water extraction, acid-base extraction, enzymatic hydrolysis, ultrasound-assisted extraction, and combined extraction processes), all significantly affect the yield, composition, and physicochemical properties of polysaccharides. Generally, increasing the extraction temperature and extending the extraction time can promote the release of cell wall polysaccharides, but excessive heating can easily cause glycosidic bond breakage or deacetylation, thereby reducing the average molecular weight and weakening the activity.

[0004] Different extraction methods play a crucial role in regulating the structural diversity of polysaccharides. Acid extraction can significantly improve extraction efficiency, but excessively acidic conditions can easily lead to the degradation of polysaccharide chains. Alkaline extraction helps release acidic polysaccharides bound to the cell wall, but may cause damage to branched structures. Enzymatic hydrolysis, due to its mild reaction and high specificity, can better maintain the integrity of the main glycan structure, thus obtaining components with a more uniform molecular weight distribution (Line et al., 2019). In addition, ultrasound-assisted extraction (UAE) disrupts algal cell walls through acoustic cavitation, accelerating the mass transfer rate, but excessive acoustic energy may cause partial main chain breakage. In recent years, combined extraction technologies (such as "enzymatic hydrolysis-ultrasound method" and "microwave-hot water method") have become an important development direction for optimizing polysaccharide extraction, balancing high yield and structural fidelity.

[0005] Differences in extraction processes and conditions not only affect the chemical structural parameters of polysaccharides (such as molecular weight, degree of branching, and sulfate content), but also significantly alter their biological activities. Zhang et al. (2021) found in their study of lentinan that the molecular weights of polysaccharides obtained under different subcritical water extraction conditions varied significantly (28-120 kDa), and the branching ratio was positively correlated with antioxidant activity. Further studies by Yoone et al. (2015) and Li et al. (2024) indicated that sulfated polysaccharides with medium molecular weight (approximately 50–150 kDa) and moderate branching exhibited the most significant immunomodulatory and antitumor activities, with the presence of sulfate and carboxyl groups playing a crucial role in enhancing their physiological activity.

[0006] This study obtained a novel and homogeneous sulfated polysaccharide from Ulva perforatum by optimizing the extraction and purification process. Although previous studies have reported that Ulva perforatum polysaccharide possesses various biological activities such as hypoglycemic, hypolipidemic, immunomodulatory, and antioxidant effects, its mechanism of action in the prevention of colorectal cancer remains poorly understood and requires further in-depth investigation. Summary of the Invention

[0007] To address the aforementioned technical problems, this invention provides a homogeneous polysaccharide from Ulva perforatum, wherein the highest peak weight-average molecular weight (Mw) of the homogeneous polysaccharide UPP-5 is 5.7 × 10⁻⁶. 4±0.3×10 4 Da.

[0008] Furthermore, the molecular weight is 5.7 × 10⁻⁶. 4 ±0.2×10 4 Da.

[0009] Furthermore, the molecular weight is 57491 Da.

[0010] The use of "about" or "approximately" for molecular weight indicates a value within ±5%, but explicitly includes the exact number. For example, "about 5.7 × 10⁻⁶". 4 This refers to 5.5 × 10 4 Up to 6.0×10 4 However, it also explicitly includes 5.7 × 10 4 Da.

[0011] Furthermore, the homogeneous polysaccharide UPP-5 of *Ulva perforatum* is mainly composed of rhamnose, galactose, xylose, glucuronic acid and mannuronic acid, with molar percentages of 55.65%, 0.78%, 9.83%, 29.35% and 4.39%, respectively.

[0012] The homogeneous polysaccharide UPP-5 from Ulva perforatum provided by this invention has a structure represented by the following general formula:

[0013]

[0014] Where n = 34.

[0015] This invention also provides a method for preparing homogeneous polysaccharides from Ulva perforatum, comprising the following steps:

[0016] (1) Hot water extraction: Ulva pore powder was extracted with hot water at a temperature of 80-90℃ to obtain an extract;

[0017] (2) The extract was filtered, concentrated and centrifuged to remove insoluble matter, and the supernatant was obtained.

[0018] (3) Pass the supernatant through a macroporous resin to remove protein and pigment, and obtain the treated sample;

[0019] (4) Dialyze the processed sample, freeze-dry the contents of the dialysis bag to obtain crude polysaccharide UPP;

[0020] (5) Dissolve the crude polysaccharide UPP in water, separate it using a DEAE-52 anion exchange chromatography column, elute with water and 0.1M to 1.2M sodium chloride solution, detect the sugar peaks and collect the sugar-containing components, freeze dry to obtain different components of Ulva polysaccharide.

[0021] (6) Dissolve different components of Ulva polysaccharide in water, purify by ultrafiltration, freeze dry to obtain Ulva homogeneous polysaccharide UPP-5.

[0022] Furthermore, the method includes any one or more of the following items [1] to

[16] :

[0023] [1] Dry Ulva perforata and pulverize it to obtain Ulva perforata powder;

[0024] [2] In step (1), the liquid-to-solid ratio of Ulva perforatum powder to hot water extraction is 1:18-22 g / mL;

[0025] [3] The water used in step (1) is distilled water or deionized water;

[0026] [4] The extraction time in step (1) is 1.5-2.5 hours;

[0027] [5] The extraction operation in step (1) is repeated once or multiple times, and the extract is obtained by combining the results;

[0028] [6] In step (2), the filtration is carried out by sieve or gauze filtration, with a mesh size of 100-600 mesh, to obtain filtrate;

[0029] [7] The filtrate concentrated in step (2) is evaporated under reduced pressure at 60-70℃ to 1 / 6-1 / 3 of its original volume;

[0030] [8] In step (2), the centrifugation conditions are 8000rpm-12000rpm and the centrifugation time is 10-15min to obtain the supernatant;

[0031] [9] The macroporous resin in step (3) is NKA-9;

[0032]

[10] In step (3), the supernatant is diluted to 2-3 times the original volume to obtain the first loading solution. The ratio of the wet volume (BV) of the first loading solution to the macroporous resin is 1:2-5.

[0033]

[11] The molecular weight cutoff of the dialysis bag in step (4) is 7000-10000 Da;

[0034]

[12] The water used for dialysis in step (4) is tap water or grade 1-3 water, and the dialysis time is 48-96 hours;

[0035]

[13] In step (5), the crude polysaccharide UPP is dissolved in water and then filtered through a microporous membrane;

[0036]

[14] In step (5), crude polysaccharide UPP is dissolved in water to a concentration of 50–100 mg / mL to obtain a second loading solution. The volume ratio (BV) of the second loading solution to the DEAE-52 anion exchange chromatography column bed is 1:2-5.

[0037]

[15] The concentrations of the sodium chloride solution in step (5) are 0.1M, 0.3M, 0.6M, 0.9M, and 1.2M, respectively;

[0038]

[16] In step (6), the eluent with a sodium chloride solution concentration of 0.9M was dialyzed, freeze-dried, reconstituted, and purified by ultrafiltration.

[0039] Furthermore, the pore size of the microporous filter membrane in

[13] is 0.22 μm, or:

[0040] In the

[16] ultrafiltration purification, ultrafiltration tubes with molecular weight cutoffs of 100 kDa and 10 kDa were used in sequence.

[0041] The *Ulvapertusa* powder described in this invention is derived from the genus *Ulvapertusa* of the family Ulvaceae in the phylum Ulvophyceae. It can be collected from naturally growing or artificially cultivated *Ulvapertusa* thallus in shallow coastal rocky reef areas of China. The *Ulvapertusa* powder is obtained by washing, removing impurities, drying at a low temperature below 60°C, and then pulverizing fresh *Ulvapertusa*. It is a light green or yellowish-green fine powder with a characteristic seaweed odor and no obvious impurities. The *Ulvapertusa* powder is commercially available and can be used directly; for example, it can be purchased from Xufeng Aquatic Products Co., Ltd.

[0042] The structural composition and biological activity of polysaccharides from Ulva perforatum are highly sensitive to extraction conditions and separation and purification processes. Different researchers often employ separation and purification steps and specific parameters (including but not limited to protein removal methods, extraction temperature, salt concentration gradient, and elution methods) which lead to fundamental differences in the composition and structure of the obtained polysaccharides, and their biological effects cannot be reliably predicted by current technologies.

[0043] For example, in the paper "Preparation and Bioactivity Study of Polysaccharide from Ulva prostrata," by Han Yu, published in the China Excellent Master's Thesis Full-text Database, under the category of Medical and Health Science and Technology, a relatively high extraction temperature was used, and the Sevag method (multiple extractions with chloroform / n-butanol) was employed for protein removal to obtain crude polysaccharide UPP from Ulva prostrata. This crude polysaccharide was separated by DEAE-Sepharose Fast Flow ion exchange chromatography, yielding three polysaccharide fractions, which eluted under elution conditions of 0, 1.0, and 1.1 mol / L NaCl, and were named UPP-1, UPP-2, and UPP-3, respectively. The chromatograms provided in the paper (…) Figure 9 As shown in the figure, a clear elution peak appears when eluted with pure water (0 mol / L NaCl), indicating that the crude polysaccharide UPP contains neutral or weakly acidic polysaccharide components that can be eluted under low ionic strength.

[0044] However, the extraction and purification strategies employed in this invention are significantly different. In the extraction stage, this invention uses hot water extraction at a lower temperature to reduce the degradation or transformation of heat-sensitive sugar residues and sulfate groups in the polysaccharide structure; in the impurity removal stage, a macroporous resin method for protein removal and decolorization is used instead of the Sevag method, achieving a gentler and more controllable protein removal process. The obtained crude polysaccharide UPP is then separated by DEAE-52 anion exchange cellulose column chromatography, using a gradient elution with 0.1, 0.3, 0.6, 0.9, and 1.2 mol / L NaCl solutions. The results show that, from the chromatogram (… Figure 1 As can be seen, almost no peaks appeared in the pure water (0 mol / L NaCl) elution stage, while obvious peaks appeared in the 0.9 mol / L and 1.2 mol / L elution stages. This indicates that the polysaccharides obtained in this invention are mainly composed of acidic sugar residues with a strong negative charge, and there are no neutral polysaccharide components.

[0045] In summary, this invention is not a simple repetition or equivalent replacement of existing extraction and separation steps, but rather, through the synergistic optimization of process parameters and purification pathways, it yields novel Ulva perforata polysaccharide components with significant differences in both structure and activity, exhibiting outstanding unpredictability.

[0046] In another aspect, the present invention provides a homogeneous polysaccharide of Ulva perforatum, which is prepared by the above-described method.

[0047] Another aspect of the present invention provides a polysaccharide preparation of Ulva perforata, comprising the above-mentioned homogeneous polysaccharide of Ulva perforata, and optionally pharmaceutically acceptable excipients.

[0048] Furthermore, the Ulva perforata polysaccharide preparation further contains other agents and / or drugs with preventive and / or therapeutic effects on colorectal cancer.

[0049] Studies have shown that seaweed polysaccharides possess significant antitumor activity, exerting their effects through direct or indirect pathways, and are of great importance to human health. Currently, research on the anti-colorectal cancer activity of Ulva perforatum polysaccharides mainly focuses on cellular levels, gut microbiota regulation, and tumor-bearing mouse models. However, systematic studies on its therapeutic effects on colorectal cancer in animal experiments have not been reported. Based on this, this invention further utilizes Ulva perforatum polysaccharide preparations to explore whether they have a preventive effect against colorectal cancer.

[0050] Furthermore, the *Ulva perforatum* preparation is an oral preparation, an injectable preparation, or a lyophilized powder injection.

[0051] Another aspect of the present invention provides the use of the aforementioned Ulva perforatum pharmaceutical preparation in the preparation of pharmaceuticals, foods and / or health products for the prevention and / or treatment of colorectal cancer.

[0052] Furthermore, the colorectal cancer is AOM / DSS-induced mouse colorectal cancer.

[0053] Furthermore, the prevention and / or treatment include improving weight loss, stool characteristics, reducing bloody stool score, lengthening colon length, and reducing the incidence and number of colon tumors in the Disease Activity Index (DAI).

[0054] Another aspect of the present invention provides a method for preventing and / or treating colorectal cancer in a subject, comprising administering to the subject an effective amount of the above-mentioned Ulva perforata homogeneous polysaccharide or the above-mentioned Ulva perforata pharmaceutical preparation.

[0055] The present invention has the following advantages over the prior art:

[0056] (1) This invention extracts and purifies a novel homogeneous polysaccharide UPP-5 with a single component from Ulva prostrata, and systematically characterizes its structural features using various chemical and physical analysis methods. The molecular weight of this polysaccharide is 5.7 × 10⁻⁶. 4 Da is mainly composed of rhamnose, galactose, xylose, glucuronic acid, and mannuronic acid, with molar percentages of 55.65%, 0.78%, 9.83%, 29.35%, and 4.39%, respectively. Its connectivity, as characterized by its structure, is as follows: Figure 8 As shown.

[0057] (2) This invention is the first to verify that Ulva perforatum polysaccharide has the efficacy of preventing and treating colorectal cancer. Animal experiments show that the extract can reduce the Disease Activity Index (DAI) score, reduce the number of tumors, prolong the colon length, improve the colon tissue structure, and has a regulatory effect on immune organs such as the spleen and thymus. It can downregulate the expression levels of inflammatory factors Tnf-α, Il-1β, Inos, and Cox-2 in a colorectal cancer model, indicating that it has a significant anti-inflammatory effect, can alleviate chronic intestinal inflammation, and thus inhibit tumor formation and development. It can upregulate the mRNA and protein expression levels of tight junction-related proteins (OCCLUDIN, ZO-1, E-CADHERIN), effectively repair the intestinal barrier structure, improve epithelial integrity, and enhance the defense function of colon tissue at the molecular level. Attached Figure Description

[0058] Figure 1 The DEAE-52 elution curve of Ulva perforatum homogeneous polysaccharide UPP-5 in Example 1 is shown.

[0059] Figure 2 This is a graph showing the molecular weight of UPP-5.

[0060] Figure 3 This is a UPP-5 ultraviolet scan image.

[0061] Figure 4This is a chromatogram of the monosaccharide composition of UPP-5, where a is the standard and b is the chromatogram of the detected ions of UPP-5.

[0062] Figure 5 This is the infrared spectrum of UPP-5.

[0063] Figure 6 This is a UPP-5 GCMS chromatogram.

[0064] Figure 7 This is the NMR spectrum of UPP-5 (a) 1 H spectrum; b) 13 C spectrum; c) COSY spectrum; d) HSQC spectrum.

[0065] Figure 8 It has the structure of UPP-5.

[0066] Figure 9 This is another comparative method for separating polysaccharides from Ulva prostrata.

[0067] Figure 10 This refers to the process of establishing a mouse AOM / DSS model.

[0068] Figure 11 The AOM / DSS model phenotypic diagrams are as follows: a) Disease Activity Index (DAI) score; b) Tumor number; c) Spleen index; d) Kidney index; e) Thymus index. * P < 0.05 ** P < 0.01, *** P < 0.001.

[0069] Figure 12 The diagram shows: a) Macroscopic view of colon length; b) Statistical comparison of colon length. *** P < 0.001.

[0070] Figure 13 This is an H&E staining image of colon tissue.

[0071] Figure 14 The relative expression of inflammatory factors is: a) Tnf-α, b) Il-1β, c) Inos, d) Cox-2. * P < 0.05 ** P < 0.01, *** P < 0.001, **** P < 0.0001.

[0072] Figure 15 The relative expression levels of intestinal barrier-related factors are: a) Zo-1; b) Occludin; c) Cdh1. * P < 0.05 ** P < 0.01, ***P < 0.001.

[0073] Figure 16 This represents the expression levels of different intestinal barrier-related proteins obtained from Western blot analysis. Detailed Implementation

[0074] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0075] Example 1: Preparation of homogeneous polysaccharide UPP-5 from Ulva perforatum:

[0076] 1. Hot water extraction: Take 150g of naturally dried and crushed Ulva prostrata, mix it with grade III water at a ratio of 1:20 (g / mL) and stir evenly. Then, extract at 90℃ for 2 hours, stirring once every half hour. Repeat the extraction twice.

[0077] 2. Filtration and concentration: Filter the above extract through a 500-mesh sieve. The filtrate is then evaporated under reduced pressure at 60-70℃ to 1 / 6 of its original volume, and centrifuged at 8000rpm for 10-15min. The supernatant is collected.

[0078] 3. Protein and pigment removal: Dilute the supernatant to 2-3 times its original volume and pass it through macroporous resin to remove protein and pigment, and obtain a protein and pigment-removed sample.

[0079] 4. Dialysis freeze-drying: The above sample was placed in an 8000 da dialysis bag and dialyzed with tap water for 24 hours, then with tertiary water for 24 hours, and then with primary water for 24 hours, changing the dialysate every 4 hours. After removal, the sample was placed in a vacuum freeze dryer for freeze-drying to obtain the sample, which was named UPP.

[0080] 5. Preparation and Purification: A 2.6cm × 50cm glass chromatography column was used. Using a glass rod as a guide, the DEAE-52 packing material, which had been ultrasonically degassed, was slowly added to the column in one go, avoiding the generation of air bubbles. After standing until stable, ultrapure water was added and washed until no ethanol odor remained. 500mg of UPP was weighed and dissolved in 5-10mL of ultrapure water. After complete dissolution, the solution was filtered through a 0.22μm aqueous microporous membrane. The filtered sample was then added to the DEAE-52 chromatography column. Elution was performed sequentially with ultrapure water, 0.1M, 0.3M, 0.6M, 0.9M, and 1.2M NaCl solutions, with a flow rate controlled at 1.0mL / min. 10mL was collected from each tube, and the collected solution was analyzed using the phenol-sulfuric acid method. The absorbance was measured at 490nm, and an elution curve was plotted. Figure 1As shown, based on the elution curve, the collected solutions from the corresponding tubes were combined and lyophilized by dialyzing. The lyophilized sample eluted with 0.9M NaCl was subjected to GPC analysis to estimate the purity and molecular weight of the lyophilized sample. 100kda and 10kda ultrafiltration tubes were used for further purification of the lyophilized sample. The portion of the sample remaining on the 10kda ultrafiltration membrane was collected, lyophilized, and named UPP-5.

[0081] Example 2: Determination of molecular weight and purity of UPP-5 in Example 1 by HPGPC

[0082] Determination of relative molecular weight: The molecular weight and purity of the polysaccharide component UPP-5 were determined by high-performance gel permeation chromatography (HPGPC). UPP-5 was dissolved in a mobile phase of 0.05M NaCl solution to a final concentration of 5 mg / mL. After vortexing, the solution was centrifuged at 12,000 rpm for 10 min. The supernatant was collected and filtered through a 0.22 μm aqueous microporous membrane. The sample was then transferred to a 1.8 mL vial. The chromatographic conditions were as follows: mobile phase: 0.05M NaCl solution; column: BRT105-103-101 tandem gel column (8 × 300 mm); flow rate: 0.7 mL / min; column temperature: 40℃; injection volume: 25 μL; detector: RID-20A differential detector; analysis time: 70 min. A regression equation was established using the logarithm of the relative molecular mass of the dextran standard against the elution volume to obtain a standard curve. The relative molecular weight of UPP-5 was calculated based on the standard curve. The weight-average molecular weight of UPP-5 was determined to be 57491 Da by HPGPC. The data are as follows: Figure 2 As shown in Table 1.

[0083] Table 1. Molecular weight of UPP-5

[0084] Sample Name RT(min) lgMp lgMw lgMn Mp Mw Mn Peak area ratio % UPP-5 34.880· 4.8· 4.8· 4.7· 60167· 57491· 56182· 100

[0085] Example 3: Basic component analysis of UPP-5 from Example 1

[0086] Determination of basic components: The total sugar content of component polysaccharide UPP-5 was determined using the phenol-sulfuric acid method with rhamnose as a standard; the protein content of component polysaccharide UPP-5 was determined using the Coomassie brilliant blue method with bovine serum albumin as a standard; the uronic acid content of component polysaccharide UPP-5 was determined using the m-hydroxybiphenyl method with glucuronic acid as a standard; and the sulfate content of component polysaccharide UPP-5 was determined using the barium chloride-gelatin method with potassium sulfate as a standard. UPP-5 was prepared into a 500 μg / mL solution with ultrapure water, with ultrapure water serving as a blank control. The UV-Vis spectrophotometer was used to scan at wavelengths of 190-400 nm. The UV scanning results of seaweed polysaccharide UPP-5 are as follows: Figure 3As shown in the figure, the total sugar content of UPP-5 was 67.13% ± 0.35%, the glucuronic acid content was 17.0% ± 0.8%, and the sulfate content was 23.3% ± 0.1%. No protein was detected by the Coomassie Brilliant Blue method, and no absorption peaks were observed at 280 nm and 260 nm, indicating that the seaweed polysaccharide UPP-5 does not contain protein or nucleic acid and has high purity.

[0087] Example 4: Structural characterization of the seaweed polysaccharide UPP-5 obtained in Example 1 of the present invention.

[0088] 1. Determination of Monosaccharide Composition: The monosaccharide composition of UPP-5 was determined using high-performance anion exchange chromatography-amperometric detection (HPAEC-PAD). Take a clean chromatographic vial, weigh 5 mg of the polysaccharide sample, add 1 ml of 2 MtFA acid solution, and heat at 121°C for 2 hours. Purge with nitrogen and dry. Wash with 99.99% methanol, then dry again; repeat the methanol washing 2-3 times. Dissolve in sterile water and transfer to a chromatographic vial for analysis. Accurately weigh the required standards for this project (fucose, rhamnose, arabinose, galactose, glucose, xylose, mannose, fructose, ribose, galacturonic acid, glucuronic acid, mannulic acid, and guluronic acid), add water to prepare a 10 mg / mL standard stock solution. Then, take an appropriate amount of the stock solution and mix them to prepare a standard mixture with a maximum index concentration of 60 μg / mL, 50 μg / mL, or 40 μg / mL. Prepare the series of standards required for the instrument according to the following concentration gradient (where the concentration of fucose, rhamnose, arabinose, galactose, glucose, and xylose is 40 μg / mL, the concentration of mannose is 50 μg / mL, and the concentration of fructose, ribose, galacturonic acid, glucuronic acid, mannulic acid, and guluronic acid is 60 μg / mL).

[0089] Ion chromatography analysis results as follows Figure 4 As shown in Table 2, a comparison with the monosaccharide standard revealed that UPP-5 in this invention is composed of rhamnose (55.65%), galactose (0.78%), xylose (9.83%), glucuronic acid (29.35%), and mannuronic acid (4.39%).

[0090] Table 2 Monosaccharide composition of UPP-5

[0091]

[0092] 2. Infrared spectroscopy analysis: Weigh 1 mg of fully dried UPP-5 sample, mix and grind it evenly with 100 mg of dried potassium bromide under a heating lamp, place it in a tablet press and press it into a transparent tablet, then place the tablet on a VERTEX 70 infrared spectrometer and observe it at 4000-500 cm⁻¹. -1 Infrared data is collected within the interval.

[0093] In Fourier transform infrared spectroscopy, characteristic vibrations of sulfate ester groups and uronic acid groups (such as...) were observed. Figure 5 The absorption peak at 1619.11 cm⁻¹ originates from the C=O vibration of uronic acid. 1260.54 cm⁻¹ -1 The absorption peaks at 848.43 cm⁻¹ and 790.34 cm⁻¹ are observed. -1 The two shoulder peaks at the location indicate the presence of sulfate ester substituents: the former is 1260.54 cm⁻¹. -1 The absorption at this point is attributed to the COS stretching vibration. 1055.16 cm -1 The band at that location corresponds to the stretching vibration of uronic acid C=O and the vibration of the COC bridge of the glycosidic bond. Additionally, at approximately 3451.90 cm⁻¹... -1 The main absorption peak at 2938.56 cm⁻¹ is attributed to the stretching vibration of the hydroxyl group. -1 The absorption peak at that point originates from the CH stretching vibration of the CH group.

[0094] 3. Methylation Analysis: Weigh 2-3 mg of the polysaccharide sample and place it in a glass reaction flask. Add 1 mL of anhydrous DMSO, then quickly add methylation reagent A, seal the flask, and dissolve the polysaccharide under sonication. Add methylation reagent B. React in a magnetically stirred water bath at 30°C for 60 min. Finally, add 2 mL of ultrapure water to the mixture to terminate the methylation reaction. Dialyze the mixture using a 1000 Da dialysis bag for 24 hours, then freeze-dry. Accurately weigh 2 mg of the dialyzed sample and 200 mg of potassium bromide, compress them into tablets, and use potassium bromide powder tablets as a blank control. Scan the tablets using a Fourier transform infrared spectrometer (FT-IR650, Tianjin Gangdong Technology Development Co., Ltd.) to confirm complete methylation before proceeding with subsequent experiments.

[0095] Take the methylated polysaccharide, add 1 ml of 2M trifluoroacetic acid (TFA) for hydrolysis for 90 min, and evaporate to dryness using a rotary evaporator. Add 2 ml of double-distilled water to the residues, reduce with 60 mg of sodium borohydride for 8 hours, neutralize with glacial acetic acid, rotary evaporate, dry in a 101°C oven, then add 1 ml of acetic anhydride for acetylation, react at 100°C for 1 h, and cool. Finally, add 10 ml of pure water to terminate the reaction.

[0096] The acetylated product was dissolved in 3 mL of CH2Cl2 and transferred to a separatory funnel. A small amount of distilled water was added, and the mixture was shaken thoroughly. The supernatant was removed, and this process was repeated four times. The CH2Cl2 layer was dried with an appropriate amount of anhydrous sodium sulfate, concentrated to 1 mL, and placed in a liquid chromatography vial. Analysis of the acetylated product sample was performed using a Thermo Scientific 1300-7000 gas chromatograph-mass spectrometer.

[0097] GC-MS conditions: HP-INNOWAX column 30m*0.32mm*0.25um; temperature program conditions: initial temperature 140℃, increased to 230℃ at 1℃ / min; injection port temperature 250℃, detector temperature 250℃, carrier gas helium, flow rate 1mL / min.

[0098] Experimental results: The results of the methylated monosaccharide form of UPP-5 are as follows Figure 6 As shown in Table 3, methylation treatment and GC-MS analysis of sugar residues and their proportions revealed that the guava oligosaccharide UPP-5 may have nine monosaccharide residue linkages: 2,3,4-Me3-Rhap, 2,3,4,6-Me4-Glcp, 2,3-Me2-Xylp, 3-Me1-Rhap, 2-Me1-Rhap, 2,3,6-Me3-Manp, 2,3,6-Me3-Glcp, 2,6-Me2-Glcp, and 2,4-Me2-Galp. These nine monosaccharide residue linkages are consistent with the monosaccharide composition. The results regarding the analysis of UPP-5 methylated sugar alcohol acetyl esters (PMAA) are shown in Table 3.

[0099] Table 3. Analysis of UPP-5 methylated sugar alcohol acetyl ester (PMAA) results

[0100]

[0101] 4. Nuclear Magnetic Resonance Analysis: The structure of UPP-5 in Example 1 was deduced using a superconducting nuclear magnetic resonance spectrometer. 30 mg of UPP-5 sample was weighed, dissolved in 0.55 ml of D2O, placed in a water bath to fully dissolve, centrifuged, and the supernatant was placed in an NMR tube for one-dimensional NMR analysis on a Bruker 600 MHz NMR spectrometer. 1 H NMR and 13 Determination of C NMR and two-dimensional (COSY, HSQC and HMBC) spectra.

[0102] Experimental results: The NMR results of UPP-5 are as follows Figure 7 As shown, combining the results of methylation, from Figure 7 Chemical migration signals for terminal hydrogens and carbons belonging to nine monosaccharide residues were found in residues a and 7b, and their configurations were determined based on these assigned chemical migration signals. Furthermore, from... Figure 7 In c) and 7d), the chemical migration signals of the remaining hydrogen and carbon in 9 sugar residues were identified (Table 4). Finally, combined with Figure 7 e), thus obtaining the structure of UPP-5 ( Figure 8 )

[0103] Table 4. Chemical shift assignments of UPP-5 monosaccharide residues.

[0104]

[0105] Example 5: The preventive effect of UPP in an AOM / DSS-induced mouse model of colorectal cancer:

[0106] The UPP of Example 1 of this invention was experimentally verified to have a preventive effect against colorectal cancer. The experimental methods and results are as follows:

[0107] Experimental methods:

[0108] (1) Grouping and Modeling: Six-week-old male C57BL / 6J mice were used for the experiment. After one week of acclimatization, the mice were randomly divided into four groups (CON, MOD, ULM, and UHM groups). The CON and MOD groups were administered sterile saline via gavage for one week prior to the experiment, while the ULM and UHM groups were administered 200 mg / kg and 400 mg / kg UPP via gavage for one week, respectively. Subsequently, the CON group was injected intraperitoneally with sterile saline on days 8 and 13, while the other three groups were injected directly into the peritoneum with 10 mg / kg AOM (azomethane). Except for the CON group, the other three groups had 2 wt% DSS (sodium dextran sulfate) added to their drinking water for one week starting from day 8, and continued to drink normal water for two weeks. This constituted one cycle, which was continued for three cycles. During this period, the mice were administered the above-mentioned gavage method daily, and the modeling was completed on day 91 (e.g., Figure 10 Daily observations and records were kept of the mice's weight changes, stool characteristics, and other general conditions. The weight and stool characteristics of each group of mice were scored according to the following criteria, and the sum of the scores for each indicator was used as the Disease Activity Index (DAI). Specific scoring criteria are shown in Table 5. Mice were fasted for 8 hours before the end of the experiment. After euthanasia via ocular and cervical dislocation, the colon was dissected and its length measured. After washing with PBS, the colon length and tumor formation were observed, and the location, size, and number of tumors were recorded.

[0109] Table 5 DAI Scoring Table

[0110]

[0111] (2) H&E staining: After dissecting the mouse colon, it was washed with PBS, fixed with 4% paraformaldehyde and embedded in paraffin. Serial sections were prepared and stained with hematoxylin and eosin (HE). Finally, the sections were mounted and the pathological morphological changes of the colon tissue were observed under an optical microscope. The images were taken and analyzed under the microscope.

[0112] (3) Real-time quantitative PCR was used to detect the mRNA levels of related factors in colon tissue: 25 mg of frozen colon tissue was taken, washed with pre-cooled PBS, and placed in a 1.5 ml enzyme-free centrifuge tube. 1 ml of Trizol was added, and the tissue was homogenized in a cryogenic homogenizer. After standing for 10 min, 200 μL of chloroform was added, and the mixture was inverted and mixed. After standing for 5 min, the mixture was centrifuged at 12000 × g at 4 °C for 15 min. 400 μL of the upper aqueous phase was carefully aspirated. 500 μL of isopropanol was added, and the mixture was stood at room temperature for 10 min. After centrifuging at 12000 × g at 4 °C for 10 min, RNA precipitate was observed at the bottom of the centrifuge tube. The isopropanol in the centrifuge tube was discarded, and 1 mL of 75% ethanol was added. The mixture was centrifuged at 7500 × g at 4 °C for 5 min twice. The 75% ethanol in the tube was removed with a pipette, and the tube was dried. 50 μL of enzyme-free water was added to the dried RNA, mixed, and stored at -80 °C. The extracted RNA was analyzed for quality and concentration; the A260 / A280 ratio of all extracted RNAs was between 1.8 and 2.2. 1000 ng of the extracted RNA was taken and reverse transcribed according to the reverse transcription instructions to obtain cDNA. Furthermore, the levels of Tnf-α, Il-1β, Inos, Cox-2, Zo-1, Occludin, and Ddh1 were measured.

[0113] (4) Western blot analysis of colon tissue protein expression: 25 mg of frozen colon tissue was taken, washed with pre-cooled PBS, and placed in a 1.5 ml enzyme-free centrifuge tube. 200 μL of a mixture of RIPA, protease inhibitor, phosphorylase inhibitor, and EDTA was added in a specific ratio to extract proteins. Protein concentration was determined using the BCA method. After adjusting the protein concentration, the protein was denatured in a 100℃ water bath for 5 min. Protein samples were added to the wells for gel electrophoresis, transferred to a membrane, and blocked. After adding the appropriate primary and secondary antibodies, ECL chemiluminescence buffer was used for development, and ImageJ software was used to analyze the protein bands. The ratio of the gray value of the target protein to that of the internal control (GAPDH) protein in each group reflected the relative expression level of the target protein, and the differences between groups were compared.

[0114] Experimental results

[0115] (1) The modeling of mice is as follows: Figure 11 As shown. The DAI score can reflect the severity of disease in mice to some extent. Figure 11As shown in Figure a, the DAI score in the CON group remained around 1 with slight fluctuations as body weight changed. In the MOD group, the DAI score gradually increased after AOM injection, rapidly increased after DSS administration of drinking water, and then slowly decreased due to intestinal adaptation, eventually stabilizing but still remaining above normal levels. The DAI scores in the ULM and UHM groups also showed an increasing trend after AOM injection. Subsequent DSS and drug treatments also resulted in an increasing DAI score, but the increase was smaller compared to the MOD group. Figure 11 b. The ULM and UHM groups significantly reduced the number of tumors in mice to some extent. The effects on different organs also varied, such as... Figure 11 As shown in Figure 1, the MOD group caused significant enlargement of the spleen and thymus, while the ULM and UHM groups showed a trend towards recovery to normal levels after intervention (not statistically significant). Furthermore, it was found that modeling had no significant effect on the kidneys. Figure 12 The colon length in the CON group was (6.9±0.72) cm, in the MOD group it was (3.85±0.27) cm, in the ULM group it was (6.0±0.08) cm, and in the UHM group it was (4.82±0.66) cm. Compared with the CON group, the colon length in the MOD group was significantly shorter (P<0.001). Intervention in the ULM and UHM groups could inhibit the shortening of colon length in mice to some extent, but the effect was not significant.

[0116] (2) H&E staining of colon tissue sections as follows Figure 13 As shown, in the CON group, the colonic villi were intact, the cells were uniform in size and complete, the epithelial glands were neatly arranged, and there were more goblet cells. Compared with the CON group, the colonic epithelial villi structure in the MOD group was destroyed, the structure was tumor-like, the glandular structure had unclear edges, the cell size was different, and the number of goblet cells was reduced. Compared with the MOD group, the ULM and UHM groups reversed the colonic damage, and the cell structure was relatively compact.

[0117] (3) RT-qPCR experiments showed that in the AOM / DSS mouse model, MOD significantly upregulated inflammatory factors Tnf-α, Il-1β, Inos and Cox-2 (e.g., Figure 14 UPP significantly downregulated ULM and UHM in mice, indicating that UPP can alleviate DSS-induced inflammation in mice. RT-qPCR was used to detect intestinal barrier-related factors in mice, and the results are as follows: Figure 15 The MOD group significantly downregulated Zo-1, Occludin, and Cdh1, disrupting the intestinal barrier. Furthermore, the ULM and UHM groups significantly upregulated these substances, although the upregulation in the UHM group was not significant, it showed an upward trend. These results indicate that UPP can restore the intestinal barrier in AOM / DSS model mice.

[0118] (4) Figure 16 As shown, at the translational level, the MOD group severely disrupted the protein levels of tight junction proteins such as OCCLUDIN, E-CADHERIN, and ZO-1. Compared with MOD, the ULM and UHM groups significantly upregulated the levels of these tight junction proteins, but in no concentration-dependent manner. This indicates that ULM and UHM can restore intestinal barrier damage caused by AOM / DSS.

[0119] The above are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above content. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. Homogeneous polysaccharide from Ulva perforatum, characterized in that: The molecular weight of the homogeneous polysaccharide UPP-5 from Ulva perforatum is 5.5 × 10⁻⁶. 4 It has a molecular weight of ~6.0×10⁴ Da and is mainly composed of rhamnose, galactose, xylose, glucuronic acid and mannuronic acid, with molar percentages of 55.65%, 0.78%, 9.83%, 29.35% and 4.39%, respectively.

2. The homogeneous polysaccharide of Ulva perforatum according to claim 1, characterized in that: The molecular weight of the polysaccharide from *Ulva perforatum* is 57491 Da.

3. The homogeneous polysaccharide of Ulva perforatum according to claim 1, characterized in that: The homogeneous polysaccharide from *Ulva perforatum* is UPP-5, and has a structure represented by the following general formula: Where n = 34.

4. A method for preparing the homogeneous polysaccharide of Ulva perforatum according to any one of claims 1, 2 or 3, characterized in that: Includes the following steps: (1) Hot water extraction: Ulva pore powder was extracted with hot water at a temperature of 80-90℃ to obtain an extract; (2) The extract was filtered, concentrated and centrifuged to remove insoluble matter, and the supernatant was obtained. (3) Pass the supernatant through a macroporous resin to remove protein and pigment, and obtain the treated sample; (4) Dialyze the processed sample, freeze-dry the contents of the dialysis bag to obtain crude polysaccharide UPP; (5) Dissolve the crude polysaccharide UPP in water, separate it using a DEAE-52 anion exchange column, elute with water and 0.1M to 1.2M sodium chloride solution, detect the sugar peak and collect the sugar-containing fractions, freeze dry to obtain different fractions of Ulva polysaccharide. (6) Dissolve different fractions of Ulva polysaccharide in water, purify by ultrafiltration, freeze dry to obtain Ulva homogeneous polysaccharide UPP-5.

5. The method according to claim 4, characterized in that: The method includes any one or more of the following items [1] to [16]: [1] Dry Ulva perforata and pulverize it to obtain Ulva perforata powder; [2] In step (1), the liquid-to-solid ratio of Ulva perforatum powder to hot water extraction is 1:18-22 g / mL; [3] The water used in step (1) is distilled water or deionized water; [4] The extraction time in step (1) is 1.5-2.5 hours; [5] The extraction operation in step (1) is repeated once or multiple times, and the extracts are combined to obtain the aqueous extract; [6] In step (2), the filtration is carried out by sieve or gauze filtration, with a mesh size of 100-600 mesh, to obtain filtrate; [7] The filtrate concentrated in step (2) is evaporated under reduced pressure at 60-70℃ to 1 / 6-1 / 3 of its original volume; [8] In step (2), the centrifugation conditions are 8000rpm-12000rpm and the centrifugation time is 10-15min to obtain the supernatant; [9] The macroporous resin in step (3) is NKA-9; [10] In step (3), the supernatant is diluted to 2-3 times the original volume to obtain the first loading solution. The ratio of the wet volume (BV) of the first loading solution to the macroporous resin is 1:2-5. [11] The molecular weight cutoff of the dialysis bag in step (4) is 7000-10000 Da; [12] In step (4), the water used for dialysis is tap water or grade 1-3 water, and the dialysis time is 48-96 hours; [13] In step (5), the crude polysaccharide UPP is dissolved in water and then filtered through a microporous membrane; [14] In step (5), crude polysaccharide UPP is dissolved in water to a concentration of 50-100 mg / mL to obtain a second loading solution. The volume ratio (BV) of the second loading solution to the DEAE-52 anion exchange chromatography column bed is 1:2-5. [15] The concentrations of the sodium chloride solution in step (5) are 0.1M, 0.3M, 0.6M, 0.9M, and 1.2M, respectively; [16] In step (6), the elution buffer with a sodium chloride solution concentration of 0.9M was freeze-dried, reconstituted, and purified by ultrafiltration.

6. The method according to claim 5, characterized in that: The microporous filter membrane in [13] has a pore size of 0.22 μm, or: In the [16] ultrafiltration purification, ultrafiltration tubes with molecular weight cutoffs of 100 kDa and 10 kDa were used in sequence.

7. A homogeneous polysaccharide of Ulva perforatum, prepared by the method described in any one of claims 4-6.

8. A pharmaceutical preparation of Ulva perforata, comprising the homogeneous polysaccharide of Ulva perforata as described in any one of claims 1 to 3 or the homogeneous polysaccharide of Ulva perforata as described in claim 7, and optionally pharmaceutically acceptable excipients.

9. The use of the homogeneous polysaccharide of Ulva perforatum according to any one of claims 1 to 3, or the homogeneous polysaccharide of Ulva perforatum according to claim 7, or the pharmaceutical preparation of Ulva perforatum according to claim 8, in the preparation of pharmaceuticals, foods, and / or health products for the prevention and / or treatment of colorectal cancer.

10. The use according to claim 9, characterized in that: The colorectal cancer in question is AOM / DSS-induced mouse colorectal cancer.

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