Nanchuan ancient tree tea polysaccharide NATP-60a for preventing ulcerative colitis and application thereof
By extracting polysaccharide NATP-60a from ancient tea trees in Nanchuan, the safety and efficacy issues of ulcerative colitis have been resolved, achieving anti-inflammatory, immune-modulating, and gut microbiota remodeling effects, and significantly alleviating colitis symptoms.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- CHONGQING RES INST OF HARBIN UNIV OF TECH
- Filing Date
- 2026-01-20
- Publication Date
- 2026-04-28
AI Technical Summary
Existing treatments for ulcerative colitis have safety and efficacy issues. Exogenous interventions such as probiotics or fecal microbiota transplantation may cause adverse reactions, and there is a lack of safe and effective strategies for regulating gut microbiota.
The polysaccharide NATP-60a was isolated and purified from ancient tea trees in Nanchuan. It is used to prevent ulcerative colitis through anti-inflammatory, immunomodulatory and gut microbiota remodeling processes. The specific steps include hot water extraction, deproteinization, dialysis, ethanol precipitation and ion exchange chromatography purification.
The polysaccharide NATP-60a from ancient tea trees in Nanchuan significantly reduces the level of pro-inflammatory cytokines, increases the abundance of beneficial bacteria, restores the production of short-chain fatty acids, corrects metabolic disorders, and alleviates intestinal inflammation, providing a new treatment option for ulcerative colitis.
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Abstract
Description
Technical Field
[0001] This invention relates to the fields of biomedicine and functional food technology, specifically to a polysaccharide NATP-60a isolated and purified from ancient tea trees in Nanchuan, and its application in drugs or health foods for the prevention and treatment of ulcerative colitis. Background Technology
[0002] Ulcerative colitis is a major subtype of inflammatory bowel disease (IBD), a chronic and complex inflammatory bowel disease. It has a long clinical course, is prone to relapse, severely impairs patients' quality of life, and can lead to various long-term complications. Studies have shown that gut microbiota dysbiosis is a crucial mechanism driving the occurrence and development of colitis. Based on this, probiotics or fecal microbiota transplantation (FMT) are commonly used clinically to reshape the gut microbiota and alleviate symptoms. However, these exogenous interventions may cause adverse reactions such as bloating, abdominal pain, and even infection. Therefore, developing novel therapeutic strategies that can safely and effectively modulate the host's own gut microbiota has become an urgent research need.
[0003] Tea is one of the world's three major non-alcoholic beverages, second only to water in consumption. Tea polysaccharides are the main active components of tea, possessing various biological activities, including anti-tumor, immunomodulatory, antioxidant, anti-diabetic, anti-aging, and hepatoprotective effects. Recent studies have found that tea polysaccharides can significantly reduce the incidence of colitis-related colorectal cancer in mice induced by azomethane and sodium dextran sulfate. Furthermore, Fu brick tea polysaccharides have been shown to effectively alleviate the symptoms of experimental colitis; rhubarb tea polysaccharides can improve physical barrier function by maintaining intestinal mucosal integrity and enhancing tight junction protein expression, and strengthen the chemical barrier by upregulating glucagon-like peptide-1 levels and increasing the number of intestinal goblet cells. They can also inhibit the transformation of immune cells and cytokines into pro-inflammatory phenotypes, thereby enhancing the intestinal immune barrier.
[0004] Mounting evidence suggests that tea polysaccharides can serve as a carbon source for gut microbiota, effectively increasing microbial diversity, regulating microbial structure, and promoting the production of short-chain fatty acids, thereby improving gut health. For example, green brick tea polysaccharides exert anti-colitis effects by increasing the content of short-chain fatty acids such as acetic acid, propionic acid, and butyric acid, and promoting changes in the abundance of Bacteroidetes and Firmicutes. Crude Tieguanyin oolong tea polysaccharides can reverse the proliferation of pathogenic Helicobacter pylori while increasing the relative abundance of beneficial bacteria such as Akkermansia, Trichophyton, and Heterobacter. Crude Fu brick tea polysaccharides can increase the expression of tight junction proteins and promote the growth of probiotics such as Bacteroidetes, Parapsilosis, and Collins, thereby enhancing intestinal barrier function and alleviating colitis. Therefore, compared with polysaccharides from other sources, tea polysaccharides have significant advantages in terms of low cost and easy availability, making them a highly promising dietary supplement or therapeutic agent for alleviating colitis.
[0005] Nanchuan Ancient Tree Tea is a unique tea tree variety originating from Nanchuan District, Chongqing, China, which enjoyed a high reputation as a tribute tea in ancient times. Its unique growing environment and genetic characteristics endow it with significant research value. However, current research on Nanchuan Ancient Tree Tea mainly focuses on sensory quality and aroma components, while research on its functional bioactive components remains lacking. Summary of the Invention
[0006] Based on the aforementioned challenges in treating ulcerative colitis and the research potential of tea polysaccharides, this invention aims to provide a novel polysaccharide derived from ancient tea trees in Nanchuan, possessing a well-defined structure and highly effective anti-colitis activity. To this end, this invention provides Nanchuan ancient tea polysaccharide NATP-60a and its application in the prevention of ulcerative colitis. This invention is the first to discover and confirm that the Nanchuan ancient tea polysaccharide NATP-60a has significant preventive and therapeutic effects on ulcerative colitis. Its effects were verified using a DSS-induced colitis mouse model, and its effectiveness was elucidated from multiple perspectives, including anti-inflammatory effects, immune regulation, gut microbiota remodeling, and correction of metabolic disorders, providing a new solution for the treatment of ulcerative colitis.
[0007] In a first aspect, the present invention provides the use of the Nanchuan ancient tree tea polysaccharide NATP-60a in a medicament for the prevention of ulcerative colitis.
[0008] In some embodiments of the present invention, the relative molecular mass of the Nanchuan ancient tree tea polysaccharide NATP-60a is 8.4 kDa.
[0009] In some embodiments of the present invention, the Nanchuan ancient tree tea polysaccharide NATP-60a is composed of rhamnose, arabinose, galactose, glucose, xylose and mannose.
[0010] In some embodiments of the present invention, the total sugar content of the Nanchuan ancient tree tea polysaccharide NATP-60a is 93.38%.
[0011] In some embodiments of the present invention, the Nanchuan ancient tree tea polysaccharide NATP-60a contains the following sugar residue types: T-α-Araf-(1→、→6)-β-Galp-(1→、→3,6)-β-Galp-(1→、→2,4)-α-Rhap-(1→、→4)-α-D-Glcp-(1→、→4)-β-D-Xylp-(1→、→6)-α-D-Manp-(1→、→5)-α-L-Araf-(1→ and→3)-α-D-Manp-(1→、→3)-α-D-Manp-(1→、→6)-α-D-Manp-(1→、→5)-α-L-Araf-(1→ and→3)-α-D-Manp-(1→、→6 ...
[0012] In some embodiments of the present invention, the sugar residue sequence of the Nanchuan ancient tree tea polysaccharide NATP-60a is as follows: T-β-Rhap-1→3-α-D-Manp-1→6-α-D-Manp-1→3,6-β-Galp-1→5-α-L-Araf-1→4-β-D-Xylp-1→6-β-Galp-(1→
[0013] In some embodiments of the present invention, the extraction method of the polysaccharide NATP-60a from ancient tea trees in Nanchuan includes the following steps:
[0014] (1) Extract crude polysaccharides from ancient tea trees in Nanchuan using hot water;
[0015] (2) The crude extract was subjected to deproteinization, decolorization and dialysis treatment;
[0016] (3) Use ethanol for fractional precipitation, and collect the precipitates at 30%, 60% and 80% ethanol concentrations in sequence;
[0017] (4) The fraction obtained by precipitation with 60% ethanol was purified by passing it through a DEAE-52 anion exchange column and eluted with 0.1 mol / L NaCl solution. The target fraction was then collected.
[0018] (5) The above components were further purified by passing them through a Sephacryl S-300HR dextran gel column, eluted with 0.1 mol / L NaCl solution, and the main peak components were collected. After concentration, dialysis and lyophilization, NATP-60a was obtained.
[0019] In some embodiments of the present invention, the deproteinization process includes deproteinization using the Sevag method in combination with a protease.
[0020] In some embodiments of the present invention, the dialysis uses a dialysis bag with a molecular weight cutoff of 3.5 kDa.
[0021] In some embodiments of the present invention, the ion exchange chromatography is DEAE-52 anion exchange chromatography.
[0022] In some embodiments of the present invention, the purification process uses NaCl solution as the eluent, with an elution concentration gradient of 0, 0.05, 0.1, 0.3, and 0.5 mol / L, and the target component NATP-60a is collected at the 0.1 mol / L NaCl elution peak.
[0023] In some embodiments of the present invention, the extraction method further includes using gel permeation chromatography to further verify its purity and molecular weight.
[0024] A second aspect of the present invention provides the application of the aforementioned Nanchuan ancient tree tea polysaccharide NATP-60a in the prevention of ulcerative colitis.
[0025] In some embodiments of the present invention, the NATP-60a is applicable to mammals, including humans, mice, and rats.
[0026] In some embodiments of the present invention, NATP-60a can reduce the levels of pro-inflammatory cytokines (including TNF-α, IL-6, IL-1β) and inhibit the production of nitric oxide (NO).
[0027] In some embodiments of the present invention, the NATP-60a can improve gut microbiota diversity, increase the abundance of beneficial bacteria (such as Muribauculaceae, Lepagella, Ligilactobacillus, Lachnospiraceae), and restore the level of short-chain fatty acids (such as acetic acid, propionic acid, and butyric acid).
[0028] This invention, using enzyme-linked immunosorbent assay (ELISA), 16S rRNA gene sequencing, gas chromatography-mass spectrometry (GC-MS), and metabolomics, confirms that NATP-60a, a polysaccharide from ancient tea trees in Nanchuan, can significantly inhibit the excessive production of pro-inflammatory cytokines and nitric oxide, and alleviate DSS-induced decrease in gut microbiota diversity and metabolic disorders. This indicates that NATP-60a can alleviate intestinal inflammation through multiple synergistic pathways, possessing the potential to act as an immunomodulator, gut microbiota regulator, and metabolic regulator.
[0029] The beneficial effects of this invention are:
[0030] This invention marks the first time that a novel polysaccharide, NATP-60a, has been isolated and purified from ancient tea trees in Nanchuan, and its significant effects in the prevention and treatment of ulcerative colitis have been systematically demonstrated. Its mechanism of action involves multiple pathways, including anti-inflammation, immune regulation, remodeling of the intestinal microecology, and correction of metabolic disorders, providing new candidate substances and theoretical basis for the development of therapeutic drugs or health foods for ulcerative colitis. Attached Figure Description
[0031] Figure 1 This diagram illustrates the effects of different crude NATP polysaccharides on the proliferation activity of RAW264.7 cells and their inhibitory effects on the secretion levels of inflammatory factors NO, TNF-α, IL-6, and IL-1β under LPS stimulation.
[0032] Figure 2 DEAE-52 purification elution curve, molecular weight determination chromatogram of NATP-60a, and ion chromatogram for monosaccharide composition analysis of crude polysaccharides from ancient tea trees in Nanchuan.
[0033] Figure 3 The images show the one-dimensional proton (¹H NMR) and carbon (¹³C NMR) spectra of NATP-60a.
[0034] Figure 4 The two-dimensional nuclear magnetic resonance spectra of NATP-60a include the HSQC spectrum, COSY spectrum, HMBC spectrum, and their inferred repeating unit structure.
[0035] Figure 5 The morphological characterization of NATP-60a includes scanning electron microscope images, atomic force microscope images, and X-ray diffraction patterns.
[0036] Figure 6 The figure shows the effects of NATP-60a on body weight changes, survival rate, disease activity index score, colon length, and colon tissue morphology in DSS-induced colitis mice.
[0037] Figure 7 The figure shows the effect of NATP-60a on the levels of inflammatory factors NO, TNF-α, IL-6 and IL-1β in the colon tissue of mice with DSS-induced colitis.
[0038] Figure 8 The figure shows the effect of NATP-60a on the content of short-chain fatty acids (including propionic acid, valeric acid, acetic acid, butyric acid, isobutyric acid, isovaleric acid, and hexanoic acid) in the feces of mice with DSS-induced colitis.
[0039] Figure 9 The figure shows the effects of NATP-60a on the α-diversity index, β-diversity, and phylum-level community composition of the gut microbiota in DSS-induced colitis mice.
[0040] Figure 10 The figure shows the effects of NATP-60a on key differentially expressed species at the phylum to genus level and their abundance changes in the gut microbiota of mice with DSS-induced colitis, as demonstrated by LEfSe analysis.
[0041] Figure 11 This figure shows the effect of NATP-60a on the metabolite profile of intestinal contents in mice with DSS-induced colitis, based on principal component analysis, and the statistical results of the differential metabolite counts between groups.
[0042] Figure 12 This is a schematic diagram illustrating the effect of NATP-60a on the levels of key differential metabolites in the intestinal contents of mice with DSS-induced colitis.
[0043] Figure 13This is a heatmap showing the correlation between key differentially expressed species and key differentially expressed metabolites in the gut microbiota of colitis mice under NATP-60a intervention.
[0044] Figure 14 Diagram illustrating the mechanism of action of NATP-60a in preventing ulcerative colitis. Detailed Implementation
[0045] The present invention will be further described in detail below through specific embodiments. Unless otherwise specified, the raw materials, reagents, or apparatus used in the embodiments can be obtained from conventional commercial sources or by existing technical methods. Unless otherwise specified, the experimental or testing methods are conventional methods in the art.
[0046] In the following embodiments, the ancient tree tea from Nanchuan was purchased from Nanchuan District, Chongqing, China.
[0047] Extraction and purification of NATP-60a, a polysaccharide from ancient tea trees in Nanchuan:
[0048] The extraction of natural NATP from ancient tea trees in Nanchuan was performed according to the method of Shan et al. (2022). NATP was ground and treated with 90% ethanol at 80°C for 2 hours to remove pigments and small molecules. Distilled water was added to the ethanol-treated material, and extraction was performed in a 90°C water bath for 2 hours, repeated three times. The extracts were combined, filtered, and centrifuged. The clarified liquid phase was concentrated, then deproteinized and decolorized, and dialyzed using a dialysis bag with a molecular weight cutoff of 3.5 kDa. The dialyzed solution was concentrated and then subjected to sequential ethanol precipitation using 30%, 60%, and 80% ethanol concentrations. The precipitates at each concentration were separated by centrifugation and freeze-dried to obtain different crude NATP fractions.
[0049] Results: Three crude polysaccharide fractions, NATP-30, NATP-60, and NATP-80, were successfully obtained through sequential ethanol precipitation. The anti-inflammatory activities of these three fractions were preliminarily screened, and NATP-60 showed the best anti-inflammatory activity. Figure 1 ).
[0050] The fraction obtained by precipitation with 60% ethanol was used to further purify NATP-60 using a DEAE-52 cellulose column.
[0051] Results: The target fraction was collected by DEAE-52 column chromatography with gradient elution using NaCl solution, at the elution peak of 0.1 mol / L NaCl. Figure 2 A), named NATP-60a.
[0052] The structure of the NATP-60a obtained above was characterized as follows:
[0053] Its relative molecular mass was determined by high performance gel permeation chromatography.
[0054] Results: The results show that the relative molecular mass of NATP-60a is 8.4 kDa ( Figure 2 B).
[0055] The monosaccharide composition of 1-phenyl-3-methyl-5-pyrazolone was determined by pre-column derivatization-high performance liquid chromatography.
[0056] Results: The results showed that NATP-60a is composed of rhamnose, arabinose, galactose, glucose, xylose, and mannose. Figure 2 C, D).
[0057] The total sugar content was determined using the phenol-sulfuric acid method.
[0058] Results: The results showed that the total sugar content of NATP-60a was 93.38%.
[0059] Its fine structure was determined by methylation analysis and nuclear magnetic resonance spectroscopy (including 1H NMR, 13C NMR, HSQC, COSY, and HMBC).
[0060] Results: Methylation analysis revealed ten glycosidic bond linkages in NATP-60a. Nuclear magnetic resonance analysis (NMR analysis) Figure 3 , Figure 4 These bond types were further confirmed, and their repeating unit structure was derived. Figure 4 D).
[0061] The morphological features of NATP-60a were characterized by SEM, AFM, and XRD.
[0062] Results: The results showed that NATP-60a exhibited a plate-like morphology, characterized by a porous and loosely structured structure. NATP-60a consisted of irregularly shaped aggregates of particles, with a height of approximately 4.1 nm. NATP-60a had an amorphous structure and contained numerous disordered regions. Figure 5 ).
[0063] Therapeutic effects of NATP-60a on a mouse model of colitis:
[0064] Preparation of NATP-60a solution: Take 100 mg of the purified NATP-60a and add it to PBS buffer to prepare a working solution of the required concentration. After sterilization by filtration through a 0.22 μm microporous membrane, store at 4 °C for later use.
[0065] Preparation and administration of a mouse model of colitis: Male Kunming mice (5 weeks old) were randomly divided into four cohorts (n=8 / group): normal control (CK), colitis model (MC), NATP-60a intervention (HTP60-a), and positive control (YC). All procedures strictly followed national laboratory animal care and usage guidelines. After a one-week acclimatization period, the MC, NATP-60a, and YC groups were given free access to 4% sodium dextran sulfate (DSS) in drinking water to induce colitis, while the CK group drank normal water. Administration was performed by gavage daily for 14 consecutive days: the NATP-60a group received 400 mg / kg·bw NATP-60a, the YC group received 100 mg / kg·bw 5-aminosalicylic acid, while the CK and MC groups received physiological saline. After model establishment, the drinking water was changed to normal, and administration continued for 2 days. Mice were then sacrificed, and colonic tissue, spleen, and colonic contents were collected for later use.
[0066] Examination of the general condition and colonic tissue of mice: During the experiment, the weight, stool characteristics, and hematochezia of mice were recorded daily, and the disease activity index was calculated. After sacrifice, the length of the colon was measured, and a portion of the colonic tissue was fixed in 4% paraformaldehyde, embedded in paraffin, sectioned, and stained with H&E to observe pathological changes.
[0067] Results: Compared with the model group (MC), NATP-60a intervention significantly alleviated the weight loss induced by DSS in mice. Figure 6 A), improve survival rate ( Figure 6 B), reduce the disease activity index score ( Figure 6 C), and inhibits DSS-induced colonic shortening ( Figure 6 D). H&E staining of colon tissue sections showed that NATP-60a significantly reduced pathological damage such as inflammatory cell infiltration, epithelial erosion, and goblet cell reduction. Figure 6 E).
[0068] Detection of inflammatory factor levels: Another portion of colon tissue was homogenized, and the levels of tumor necrosis factor-α, interleukin-6, and interleukin-1β were detected using a commercial ELISA kit. The level of nitric oxide was detected using the Griess method.
[0069] Results: NATP-60a significantly reduced the levels of nitric oxide (NO), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-1β (IL-1β) in colon tissue. Figure 7 ).
[0070] Detection of gut microbiota: Collected colon contents were used for 16S rRNA gene sequencing analysis of gut microbiota composition.
[0071] Results: Gut microbiota analysis showed that NATP-60a alleviated the DSS-induced decrease in the Chao1, Shannon, and Simpson indices. Figure 9 AC), modulated microbial β diversity ( Figure 9 D), reversed the change in the Bacteroidota / Bacillota ratio caused by DSS ( Figure 9 E), and significantly increased the abundance of beneficial bacteria such as Muribauculaceae, Lepagella, Ligilactobacillus, and Lachnospiraceae. Figure 10 ).
[0072] Detection of short-chain fatty acids: The collected colon contents were used to detect the content of short-chain fatty acids using gas chromatography-mass spectrometry.
[0073] Results: Short-chain fatty acid assays showed that NATP-60a intervention effectively increased the contents of propionic acid, valeric acid, acetic acid, butyric acid, isobutyric acid, isovaleric acid, and hexanoic acid. Figure 8 ).
[0074] Metabolomics detection: The collected colon contents were used for non-targeted metabolomics analysis using liquid chromatography-mass spectrometry.
[0075] Results: Metabolomics analysis confirmed that NATP-60a can regulate DSS-induced metabolic disorders, mainly involving key pathways such as amino acid metabolism, tryptophan metabolism, and tyrosine metabolism. Figure 11 , Figure 12 Correlation analysis showed a significant association between key differentially expressed species and key differentially expressed metabolites in the gut microbiota. Figure 13 ).
[0076] Comprehensive analysis of all omics: combined analysis of gut microbiota, inflammatory factor levels, short-chain fatty acids, and metabolomics.
[0077] Results: NATP-60a remodeled the metabolic profile of colitis-infected mice by reducing the levels of inflammatory factors, increasing short-chain fatty acids, and balancing the gut microbiota, thereby alleviating colitis. Figure 14 ).
[0078] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. 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. A polysaccharide NATP-60a from ancient tea trees in Nanchuan, used for the prevention of ulcerative colitis, characterized in that... The sugar residue sequence is T-β-Rhap-1→3-α-D-Manp-1→6-α-D-Manp-1→3,6-β-Galp-1→5-α-L-Araf-1→4-β-D-Xylp-1→6-β-Galp-(1→).
2. The NATP-60a polysaccharide from ancient tea trees in Nanchuan, as described in claim 1, is characterized in that... The relative molecular mass of NATP-60a is 8.4 kDa.
3. The NATP-60a polysaccharide from ancient tea trees in Nanchuan, as described in claim 1, is characterized in that... The NATP-60a is composed of rhamnose, arabinose, galactose, glucose, xylose, and mannose.
4. The application of NATP-60a, a polysaccharide from ancient tea trees in Nanchuan, in the prevention of ulcerative colitis.
5. The application according to claim 4, characterized in that, The NATP-60a can enhance gut microbiota α diversity, increase the abundance of at least one of Muribauculaceae, Lepagella, Ligilactobacillus and Lachnospiraceae, and / or restore the level of at least one short-chain fatty acid of acetic acid, propionic acid and butyric acid.