Application of radix puerariae exosome-shaped vesicles in preparation of medicine for preventing and / or treating ulcerative colitis and related colorectal cancer thereof

Abstract

The invention belongs to the field of medicine and health, and particularly relates to application of fresh traditional Chinese medicine radix puerariae exosome-shaped vesicles P-ELNs in medicine or health food for preventing and treating irritable bowel syndrome, the P-ELNs serving as an active ingredient can improve expression of colonic intestinal mucus protein MUC2 and tight junction protein ZO-1 and improve mucous membrane barrier integrity; the inhibition of the expression level of proinflammatory factors (such as TNF-alpha, IL-6, IL-1beta and NF-kappa B) indicates that the action mechanism may involve the synergistic effect of inflammation inhibition and barrier repair. Toxicological experiments show that the P-ELNs have no toxicity after long-term intragastric administration, have no adverse effects on heart, liver, spleen, lung and kidney functions, and have good oral safety and tissue tolerance. The indications comprise anti-inflammatory treatment of active-stage UC patients; maintenance and barrier repair of patients in the UC remission period; the UC disease course is more than or equal to 8 years, and a preventive medicine is applied to high-risk people with cancerization family history and the like; the invention relates to auxiliary treatment of UC-CAC early cancerization patients.

Description

Technical Field

[0001] This invention belongs to the field of medicine and health, and relates to a drug or health product for the preparation of prevention, treatment and / or relief of digestive tract diseases, and particularly to the application of fresh kudzu root exosome-like vesicles in the prevention and treatment of irritable bowel syndrome in drugs or health foods. Background Technology

[0002] Ulcerative colitis (UC) is a chronic, relapsing inflammatory bowel disease that commonly affects the rectum and colon, typically presenting with mucus and bloody stools, abdominal pain, and weight loss. Long-term chronic inflammation damages the intestinal epithelial barrier, leading to persistent immune activation and cell damage, ultimately inducing colorectal cancer, known as ulcerative colitis-associated colorectal cancer (UC-CAC). UC-CAC is one of the leading causes of death in UC patients, accounting for approximately 10%–15% of all UC deaths. Statistics show that in UC patients with a disease duration exceeding 20 years, especially those with involvement of the entire colon, the cumulative incidence of colorectal cancer can reach as high as 18%. Once cancer develops, clinical intervention usually requires combined surgery and radiotherapy / chemotherapy, but the efficacy is limited and significantly impacts the patient's quality of life. Therefore, timely control of the inflammatory response in UC and effective prevention of its progression to cancer are crucial for delaying the progression of UC-CAC and improving patient prognosis.

[0003] Currently, the treatment of ulcerative colitis (UC) mainly relies on 5-aminosalicylic acids (5-ASA), glucocorticoids, immunosuppressants (such as azathioprine), and anti-TNF-α biologics. While these drugs play a role in controlling inflammation, long-term use often leads to immunosuppression, drug resistance, and other adverse reactions, and they remain ineffective in preventing UC from progressing to cancer. Many drugs are currently available for treating UC, but they have the following limitations:

[0004] 1. Existing drugs are unable to achieve the "three-in-one" therapeutic effect of relieving inflammation, repairing the barrier, and preventing cancer.

[0005] 2. Long-term use of hormones or immunosuppressants can easily lead to serious side effects;

[0006] 3. There is a lack of drugs that can be delivered orally, target the colon locally, and have multiple synergistic effects;

[0007] 4. Although anti-TNF-α and other biological agents are effective, they are expensive, have poor compliance, and cannot completely block the progression of UC to CAC.

[0008] 5. There is a lack of safe, natural, and effective drugs suitable for early prevention and long-term intervention.

[0009] 6. Currently, there are no naturally derived plant ELNs preparations that have been translated and applied in the prevention and intervention of UC-CAC.

[0010] In particular, the lack of natural and safe drugs that can both alleviate inflammation and protect the intestinal barrier, as well as intervene in the process of inflammation-cancer transformation at multiple targets, remains a major bottleneck in the prevention and treatment of UC / UC-CAC.

[0011] With in-depth research into the pathogenesis of UC-CAC, increasing attention is being focused on how to slow its progression through early, multi-dimensional interventions, particularly in areas such as inflammation regulation, barrier repair, and microenvironment stabilization. Natural products are demonstrating broad application potential in these areas. Kudzu root (Pueraria lobata), a legume, is widely used in traditional Chinese medicine for its heat-clearing, detoxifying, yin-nourishing, and fluid-generating effects. Current technologies have revealed that puerarin (an isoflavone), the main active ingredient in kudzu root, has good antioxidant, gut microbiota-regulating, and enteritis-relieving effects. On the other hand, plant-derived exosome-like nanovesicles (ELNs) are a newly emerging natural delivery system with advantages such as small particle size, good biocompatibility, oral administration, and richness in natural active ingredients. In existing technologies, it has been disclosed that fresh pueraria lobate root-derived exosome-like vesicles (P-ELNs) are extracted using physical methods. However, thin-layer chromatography (TLC) has revealed that P-ELNs contain very little puerarin compared to the P-ELNs supernatant (P-ELNsSNF) (see Representative Paper 1 for details). From the perspective of existing technologies, P-ELNs cannot play a role in the preparation of drugs for the prevention and / or treatment of ulcerative colitis and related colorectal cancer. Summary of the Invention

[0012] This invention aims to overcome the biases in the prior art and innovatively proposes the application of kudzu root exosome-like vesicles in the preparation of drugs for the prevention and / or treatment of ulcerative colitis and related colorectal cancer.

[0013] The inventors discovered that P-ELNs exhibited significant therapeutic effects in UC and UC-CAC animal models, effectively alleviating intestinal inflammation, repairing the epithelial barrier, and significantly reducing the occurrence of carcinogenesis in long-term intervention. This suggests that kudzu exosome-like vesicles have broad medicinal prospects in the prevention and / or treatment of ulcerative colitis and its related carcinogenesis.

[0014] The inventors systematically verified the biological effects of P-ELNs in UC and UC-CAC mouse models.

[0015] In a DSS-induced acute colitis model, P-ELNs gavage significantly improved weight loss, reduced bloody stools, lowered DAI scores, and prolonged colon length. HE staining showed reduced inflammatory cell infiltration and intact glandular structure.

[0016] In the AOM / DSS-induced UC-CAC model, after intermittent administration, the number of tumors in the P-ELNs-treated group mice was significantly reduced, the average tumor diameter was reduced, the expression of Ki-67 tumor proliferation marker decreased, the tissue structure remained intact, and focal dysplasia was reduced.

[0017] Meanwhile, P-ELNs increased the expression of colonic mucin MUC2 and tight junction protein ZO-1, improving mucosal barrier integrity; and inhibited the expression levels of pro-inflammatory factors (such as TNF-α, IL-6, IL-1β, and NF-κB), suggesting that their mechanism of action may involve a synergistic effect of inflammation suppression and barrier repair.

[0018] Toxicological experiments showed that P-ELNs exhibited no toxicity with long-term oral administration, had no adverse effects on the function of the heart, liver, spleen, lungs, and kidneys, and possessed good oral safety and tissue tolerability.

[0019] The indications for P-ELNs include:

[0020] 1. Anti-inflammatory treatment for patients with active UC;

[0021] 2. Maintenance and barrier repair in UC patients in remission;

[0022] 3. Prophylactic medication for high-risk groups such as those with UC disease duration ≥ 8 years or a family history of cancer;

[0023] 4. Adjuvant therapy for patients with early-stage UC-CAC cancer.

[0024] Furthermore, the P-ELNs are derived from the juice extract of kudzu root, obtained through a series of physical extraction and purification processes, and possess a stable particle size range and structural characteristics. Since this patent does not involve characterization, the physicochemical properties are not specifically described; however, it is a well-established fact that the P-ELNs do not contain any toxic chemical additives.

[0025] Furthermore, the P-ELNs can be used as active ingredients, formulated alone into capsules, microcapsules, emulsions, oral solutions, and other dosage forms, or used in combination with other drugs to enhance efficacy. These drugs are suitable for patients with ulcerative colitis in both active and remission phases, and can also be used for long-term preventative medication in individuals at high risk of cancer. Attached Figure Description

[0026] Figure 1Schematic diagram showing the changes in body weight in UC mice treated with P-ELNs;

[0027] Figure 2 Schematic diagram showing changes in colorectal length in UC mice treated with P-ELNs;

[0028] Figure 3 Schematic diagram of pathological changes in the colorectal HE of UC mice treated with P-ELNs;

[0029] Figure 4 Schematic diagram showing changes in colorectal inflammatory factors in UC mice treated with P-ELNs;

[0030] Figure 5 Schematic diagram of mouse weight changes during P-ELNs prevention of UC;

[0031] Figure 6 Schematic diagram of changes in colorectal length in mice during P-ELNs prevention of UC;

[0032] Figure 7 Schematic diagram of P-ELNs preventing pathological changes of HE in the colorectal region of UC mice.

[0033] Figure 8 Schematic diagram of immunohistochemical changes in the colorectal region of mice during P-ELNs prevention of UC;

[0034] Figure 9 Schematic diagram of changes in inflammatory factors in mouse colorectal cells during P-ELNs prevention of UC;

[0035] Figure 10 Schematic diagram of weight changes during P-ELNs treatment of UC-CAC;

[0036] Figure 11 Schematic diagram of mouse colorectal length and tumor growth at the end of modeling during P-ELNs treatment of UC-CAC;

[0037] Figure 12 Schematic diagram of pathological changes in the colorectal HE of mice during P-ELNs treatment of UC-CAC;

[0038] Figure 13 Immunohistochemical staining of mouse colorectal region during P-ELNs treatment of UC-CAC;

[0039] Figure 14 A schematic diagram of the pathological changes in the colorectal HE of wild-type normal mice during the safety evaluation of oral P-ELNs. Detailed Implementation

[0040] In this example, differential centrifugation was used to extract and separate natural kudzu root exosome-like vesicles (P-ELNs). The vesicles were nanoscale, and the diameter of the separated P-ELNs ranged from 150.7 to 82.8 nm. The zeta potential values ​​were all negative, and the surface potential values ​​of the vesicles were mainly concentrated at -31 mV, all exhibiting typical exosome-vesicle structures.

[0041] Proteins, RNA, and lipids were obtained from pueraria lobata ELNs through purification methods. A series of protein bands were observed upon separation on an SDS polypropylene gel and development with Coomassie staining. The naked RNA purified from pueraria lobata ELNs was mostly small-sized and sensitive to RNase degradation. Thin-layer chromatography (TLC) analysis showed that P-ELNs contained different lipid types. Furthermore, the corresponding position of puerarin (flavonoid) standard was found to be extremely low in P-ELNs, but abundant in P-ELN supernatant (P-ELNs SNF). For more similar P-ELN preparation, characterization, and principal component analysis, please refer to the literature: Zhang W, Song Q, Bi X, et al. Preparation of Pueraria lobata Root-Derived Exosome-Like Nanovesicles and Evaluation of Their Effects on Mitigating Alcoholic Intoxication and Promoting Alcohol Metabolism in Mice. Int J Nanomedicine. 2024; 19:4907-4921. Published 2024 May 27. doi:10.2147 / IJN.S462602.

[0042] [Experimental Example 1] Experimental Method for Treating Inflammatory Phenotypic Effects of P-ELNs in UC Mice: C57BL / 6 mice were given free access to 2.5% DSS to establish a colitis model. At the start of the experiment, the DSS solution (2.5% sodium dextran sulfate, w / v) was changed every other day for 6 consecutive days. From day 6 to day 13, mice were administered P-ELNs via gavage before 10:00 AM daily. The gavage volume was 10 ml / kg bw P-ELNs. The three groups were: a stock solution with a concentration of 6.0 ± 1.5 mg / ml (this concentration was used to quantify the protein content in the obtained P-ELNs stock solution), a 5-fold dilution of the stock solution, and a 10-fold dilution of the stock solution. The gavage volume for the P-ELNs SNF group was 10 ml / (kg bw), and the P-ELNs SNF stock solution concentration was 28 ± 1.5 mg / ml. The initial concentration in this experiment was a 4-fold dilution of the stock solution. The control group (WT) was given an equal volume of PBS for 7 consecutive days. The changes in body weight, colon length, inflammatory markers, and pathological scores under HE staining were assessed in mice. Results: Mice in the P-ELNs group showed reduced body weight loss, increased colon length, alleviated colonic inflammation, and enhanced structural integrity.

[0043] (A) Changes in body weight in UC mice treated with P-ELNs

[0044] Changes in the weight gain rate of laboratory animals, such as Figure 1 As shown, compared with the DSS group, the DSS+P-ELNs group (undiluted solution) and the DSS+0.2P-ELNs group (undiluted solution diluted 5 times) showed significant differences (p<0.05). The weight gain rate of the DSS+0.2P-ELNs group was better than that of the DSS+P-ELNs group, while there were no significant differences in the DSS+0.1P-ELNs group (undiluted solution diluted 10 times) and the P-ELNsSNF group containing a large amount of puerarin.

[0045] (B) P-ELNs treatment of colorectal length and HE pathological changes in UC mice

[0046] Changes in intestinal length in UC mice, as follows Figure 2 As shown, there were no significant changes in the overall length of the intestine, but the changes in the colon were as follows: the DSS+0.2P-ELNs group was longer than the DSS group (p<0.05). From Figure 3 Analysis of HE staining results of the colon showed that the pathological changes in the DSS+0.2P-ELNs group were better than those in other experimental groups, and the mucosal structure was more intact.

[0047] (C) Changes in colorectal inflammatory factors in UC mice treated with P-ELNs

[0048] Changes in the mRNA levels of pro-inflammatory cytokines TNFα / IL-6 / IL-1β and anti-inflammatory cytokines IL-10 in colon cells of UC mice are as follows: Figure 4 As shown, compared with the DSS group, the pro-inflammatory factors TNFα / IL-6 / IL-1β in the DSS+0.2P-ELNs group were significantly decreased (p<0.05). The other two P-ELNs groups showed varying degrees of decrease in pro-inflammatory factors, with weaker effects than the DSS+0.2P-ELNs group. Compared with the DSS group, the anti-inflammatory factor IL-10 in the DSS+0.2P-ELNs group was significantly increased (p<0.05). However, the anti-inflammatory effect of P-ELNsSNF containing a large amount of puerarin was poor.

[0049] In summary, P-ELNs were found to have a therapeutic effect on DSS-induced ulcerative colitis (UC), and the effect was better with 0.2 P-ELNs (P-ELNs stock solution diluted 5 times). Therefore, subsequent trials all used 0.2 P-ELNs (P-ELNs stock solution diluted 5 times) for enrollment, and were uniformly referred to as P-ELNs.

[0050] [Experimental Example 2] Experimental Method for P-ELNs Preventing Inflammatory Phenotypic Effects in UC Mice: C57BL / 6 mice were given free access to 2.5% DSS to establish a colitis model. Starting on the fourth day of the experiment, the DSS solution (2.5%, w / v) was changed every other day for 7 consecutive days to establish DSS-induced UC. From the beginning to the end of the experiment, mice were administered 10 ml / (kg.bw) of P-ELNs by gavage before 10:00 AM every day. The control group was given an equal volume of PBS for 11 consecutive days. The changes in mouse body weight, colon length, inflammatory markers, intestinal barrier markers, and pathological scores under HE staining were evaluated. Results: The DSS+P-ELNs group showed slower weight loss, longer colon length, reduced colonic inflammation, increased intestinal barrier markers, and enhanced colonic structural integrity.

[0051] (A) Changes in mouse body weight during P-ELNs prevention of UC

[0052] Changes in the weight gain rate of laboratory animals Figure 5 As shown, compared with DSS, the DSS+P-ELNs group had a slight increase in body weight, but it was not significant.

[0053] (B) Changes in colon and rectum length in mice during P-ELNs prevention of UC

[0054] like Figure 6 As shown, regarding changes in colorectal length, compared to DSS, the colorectal length in the DSS+P-ELNs group was slightly longer but not significantly so.

[0055] (C) Changes in HE staining of the colorectal region of mice during P-ELNs prevention of UC

[0056] According to such Figure 7The analysis of colon HE staining results showed that, compared with the DSS group, the DSS+P-ELNs group had significantly improved inflammation and mucosal layer damage.

[0057] (D) Immunohistochemical changes in the colorectal region of mice during P-ELNs prevention of UC

[0058] According to such Figure 8 The colonic IHC results analysis showed that, compared with the DSS group, the expression of the intestinal barrier tight junction protein ZO-1 in the DSS+P-ELNs group was significantly upregulated in the colonic tissue, and the expression of the intestinal barrier mucin MUC2 showed an upward trend but was not significant.

[0059] (E) Changes in inflammatory factors in mouse colorectal cells during UC prevention with P-ELNs

[0060] Changes in the pro-inflammatory factors TNFα / IL-6 / IL-1β in colon cells of UC mice are as follows: Figure 9 As shown, compared with the DSS group, the pro-inflammatory factors TNFα / IL-6 / IL-1β in the DSS+P-ELNs group were significantly decreased (p<0.05), indicating that kudzu ELNs have anti-inflammatory effects.

[0061] In summary, P-ELNs have been shown to have a role in preventing DSS-induced ulcerative colitis (UC).

[0062] [Experimental Example 3] Establishment of a P-ELNs-Inhibited UC-CAC Colon Cancer Model: C57BL / 6 mice were intraperitoneally injected with azomethane (AOM, 10 mg / kg), combined with intermittent periodic DSS drinking water to induce a UC-CAC model. Mice were pre-administered with P-ELNs (10 ml / (kg.bw)) by gavage for 7 days. Starting on day 8, a single intraperitoneal injection of 10 mg / kg azomethane (AOM) was administered, followed by one week of standard diet feeding. DSS solution (2.5%, w / v) was given for one week, and P-ELNs were administered by gavage before 10:00 AM daily during this period, followed by two weeks of normal drinking water. The above steps were repeated for a total of 3 cycles, successfully establishing a drug-treated UC-CAC mouse model. After the third cycle, mice were given normal drinking water for two weeks, followed by two weeks of P-ELNs treatment, for a total experimental period of 98 days. Changes in mouse body weight, tumor number and volume, colon length, inflammatory markers, intestinal barrier markers, and pathological scores under HE staining were then compared. Results: P-ELNs mice showed slower weight loss, longer colon length, reduced colonic tissue inflammation, significantly reduced tumor number, decreased mucosal pathological score, reduced Ki-67 positive cells, and enhanced colonic structural integrity.

[0063] (A) Weight changes during P-ELNs treatment of UC-CAC

[0064] like Figure 10 As shown, after 3 months of modeling, the experimental results showed that, compared with the AOM / DSS group, the AOM / DSS+P-ELNs group significantly increased the weight gain rate (p<0.05).

[0065] (B) Colorectal length and tumor growth in mice during P-ELNs treatment of UC-CAC and after model establishment

[0066] like Figure 11 As shown, regarding changes in colonic length and tumor growth, compared with the AOM / DSS group, the AOM / DSS+P-ELNs group showed a significant increase in colonic length (p<0.05), and also a significant reduction in tumor burden, i.e., tumor number and tumor area (p<0.05). In particular, the tumor formation rate in mice decreased from 88.89% in the AOM / DSS group to 20% after P-ELNs (AOM / DSS+P-ELNs group) intervention.

[0067] (C) HE staining of mouse colon and rectum during P-ELNs treatment of UC-CAC

[0068] In histopathology, such as Figure 12 As shown, the colon tissue of mice in the model group (AOM / DSS group) showed obvious inflammatory cell infiltration, crypt structure destruction, glandular dysplasia, and tumor nodules, which were significantly alleviated after administration of P-ELNs (AOM / DSS+P-ELNs group).

[0069] (D) Immunohistochemical staining of mouse colorectal intestine during P-ELNs treatment of UC-CAC

[0070] like Figure 13 The IHC results shown indicate that, compared with the model group, the expression of tumor marker ki67 was significantly decreased in the P-ELNs administration group (p<0.05), while the expression of KLB and intestinal barrier indicators (ZO-1, MUC2) was significantly increased (p<0.05).

[0071] In summary, P-ELNs play a role in the prevention and treatment of UC-CAC by protecting the intestinal barrier, reducing inflammatory responses, and lowering the rate of cancer transformation.

[0072] [Experimental Example 4] Long-term oral safety evaluation of P-ELNs: C57BL / 6 mice were administered 10 ml / (kg.bw) of P-ELNs by gavage for 3 weeks. Body weight and behavioral status were recorded, and major organs (heart, liver, spleen, lungs, and kidneys) were observed by HE staining. Results are as follows: Figure 14As shown: No obvious toxicity was observed in the P-ELNs group, and no pathological damage was observed in organs and tissues.

[0073] In this invention, the P-ELNs can be used as active ingredients in the preparation of drugs / health products for the prevention and / or treatment of ulcerative colitis and related colorectal cancer. Specifically, P-ELNs can be formulated alone, but not limited to, capsules, microcapsules, emulsions, oral liquids, etc., or can be used in combination with other drug components to enhance efficacy.

[0074] The above descriptions are merely embodiments of the present invention, and common knowledge such as specific technical solutions and / or characteristics are not described in detail here. It should be noted that those skilled in the art can make various modifications and improvements without departing from the technical solutions of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications and improvements will not affect the effectiveness of the implementation of the present invention or the practicality of the patent. The scope of protection claimed by the present invention should be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims.

Claims

1. Application of kudzu root exosome-like vesicles in the preparation of drugs / health products for the prevention and / or treatment of ulcerative colitis and related colorectal cancer.

2. The application according to claim 1, characterized in that, The kudzu exosome-like vesicles are derived from the juice extract of kudzu root and are obtained through a series of physical extraction and purification processes.

3. The application according to claim 1, characterized in that, The diameter of the kudzu exosome-like vesicles is 150.7±82.8 nm.

4. A drug / health product for the prevention and / or treatment of ulcerative colitis and related colorectal cancer, characterized in that, It contains kudzu root exosomes as its active ingredient.

5. The drug / health product according to claim 4, characterized in that, The drug also contains other pharmaceutical ingredients used to enhance the prevention and / or treatment of ulcerative colitis and its associated colorectal cancer.

6. The drug / health product according to claim 4 or 5, characterized in that, The dosage form of the drug / health product is capsule, microcapsule, emulsion or oral liquid.

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