Use of mulberry concentrate in the preparation of a medicament for preventing and / or treating ulcerative colitis-associated colorectal cancer

Abstract

The application belongs to the field of medicine and health, and relates to application of mulberry concentrated solution in preparation of a medicine for preventing and / or treating ulcerative colitis related colorectal cancer. The mulberry concentrated solution is prepared by mixing mulberry branches, mulberry leaves and mulberry roots, and then extracting, filtering and concentrating. The mulberry concentrated solution is rich in active ingredients such as flavonoids and polyphenols, can improve the body weight loss and colon shortening of model mice in an ulcerative colitis related colorectal cancer (UC-CAC) animal model induced by azoxymethane / dextran sulfate sodium, reduce the tumorigenic rate, tumor number and tumor area, reduce the pathological damage of colon tissue, and down-regulate the expression of Ki67, up-regulate the expression of barrier related proteins ZO-1 and MUC2, indicating that the mulberry concentrated solution has the effects of inhibiting abnormal proliferation, protecting the intestinal mucosal barrier and delaying the inflammation-cancer evolution. The mulberry concentrated solution does not show a stable and significant treatment advantage in a DSS induced ulcerative colitis model, but shows a more explicit prevention and treatment effect in the UC-CAC model, indicating that the advantage indication of the mulberry concentrated solution is more suitable for positioning in the prevention and treatment of UC-CAC.

Description

Technical Field

[0001] This invention belongs to the field of medicine and health, and relates to the application of natural product active preparations in digestive system diseases, specifically to the application of a mulberry concentrate in the preparation of drugs for the prevention and / or treatment of ulcerative colitis-related colorectal cancer. Background Technology

[0002] Ulcerative colitis (UC) is a chronic, relapsing inflammatory bowel disease. Long-term, recurrent ulcerative colitis can lead to persistent damage to the intestinal mucosal barrier, imbalance of the inflammatory microenvironment, and abnormal proliferation and accumulation, thereby inducing UC-associated colorectal cancer (UC-CAC). UC-CAC is one of the serious complications of UC and a significant factor affecting the long-term prognosis of patients.

[0003] Currently, clinical treatment of ulcerative colitis (UC) mainly relies on positivity agents such as 5-aminosalicylic acids, glucocorticoids, immunosuppressants, and biologics. While these drugs can control active inflammation to some extent, they primarily focus on "inflammation control," and their effects on mucosal barrier reconstruction, inhibition of abnormal proliferation, and blocking the progression of UC to UC-CAC under long-term inflammatory conditions remain limited. Furthermore, long-term use also presents challenges such as adverse reactions, decreased adherence, and difficulties in maintaining interventions. Therefore, developing safe, natural, and long-term suitable novel formulations that can intervene in the inflammatory-cancer transformation process is of great significance.

[0004] Mulberry is a plant used for both food and medicine, rich in flavonoids, polysaccharides, alkaloids, vitamins, and various trace elements, possessing high development value. In existing technologies, some mulberry-derived preparations have been explored for applications in ulcerative colitis or tumor-related fields. For example, a patent (CN114601865A) reports that mulberry extract can be used to prepare products for the treatment and / or prevention of ulcerative colitis. Its raw materials are mainly mulberry branches, mulberry bark, and / or mulberry leaves, prepared using resin separation and purification processes. The technical effects are mainly focused on improving inflammation-related indicators such as body weight, disease activity index, colon length, and pathological damage in UC models. However, the experimental evaluation of this existing technology mainly focuses on inflammation-related indicators in the UC model induced by sodium dextran sulfate (DSS), without further disclosing its improvement effects on tumorigenesis rate, tumor number, tumor area, abnormal proliferation, and intestinal mucosal barrier-related indicators in the UC-CAC model induced by azomethane / sodium dextran sulfate (AOM / DSS). Therefore, it cannot be directly deduced that it has the technical effect of blocking the evolution of UC to UC-CAC.

[0005] Another patent (CN104800204A) reports that mulberry-derived monomer component morin has anti-malignant tumor uses. Although the claims list solid tumors in a general way and include colorectal cancer, the experimental focus of its specification and examples is mainly on tumor types such as glioma. Whether it is effective against colorectal cancer, especially ulcerative colitis-related colorectal cancer, is still unknown. According to the mechanism of action revealed in the prior art, it is not applicable to colorectal cancer, especially ulcerative colitis-related colorectal cancer.

[0006] However, preparations derived from different mulberry sources differ significantly in raw material composition, preparation process, and active ingredient profile, and their pharmacodynamic characteristics and advantageous indications may also differ. The mulberry concentrate described in this application is not a single active ingredient, nor is it a mulberry extract obtained through resin purification. Rather, it is a compound concentrated preparation obtained by mixing mulberry branches, leaves, and roots, followed by extraction, filtration, and high-temperature concentration, namely, mulberry concentrate (SC).

[0007] Previous comparative studies have found that mulberry concentrate (mainly derived from mulberry branches, leaves, and roots), a non-single-active-ingredient preparation, only showed a certain improvement trend in the DSS-induced UC model, without demonstrating a stable and significant therapeutic advantage. Based on the results of the UC model alone, those skilled in the art would find it difficult to expect that it could further reduce tumorigenesis rate and tumor burden, and improve abnormal proliferation and barrier damage in the AOM / DSS-induced UC-CAC model. Furthermore, there are no existing reports on the use of mulberry concentrate for the prevention and / or treatment of UC-CAC.

[0008] Therefore, it is necessary to conduct relevant research on this mulberry concentrate and propose new uses for its protection. Summary of the Invention

[0009] The purpose of this invention is to provide the application of mulberry concentrate in the preparation of drugs for the prevention and / or treatment of ulcerative colitis-associated colorectal cancer, in order to solve the problem of the lack of safe, natural, and suitable candidate formulations for long-term intervention in UC-CAC in the prior art.

[0010] To achieve the above objectives, the present invention provides the following technical solution: the application of mulberry concentrate in the preparation of drugs for the prevention and / or treatment of ulcerative colitis-related colorectal cancer. The mulberry concentrate is prepared by water extraction and concentration of mulberry branches, leaves and roots mixed in a specific ratio. The application achieves intestinal mucosal barrier protection and inhibition of abnormal proliferation by downregulating Ki67 expression in colonic tissue and upregulating ZO-1 and MUC2 expression, rather than simply inhibiting inflammation.

[0011] This invention further clarifies the chemical composition and in vitro activity of the mulberry concentrate. Systematic analysis of the mulberry concentrate using broad-target metabolomics technology identified 2,286 primary metabolites, mainly including terpenoids, amino acids and their derivatives, flavonoids, phenolic acids, and alkaloids.

[0012] This invention is based on the following discovery: Mulberry concentrate is rich in active ingredients such as flavonoids and polyphenols. In the UC-CAC mouse model induced by azomethane / dextran sulfate (AOM / DSS), compared with the model group, treatment with mulberry concentrate can improve the loss of body weight in the model animals, alleviate colon shortening, significantly reduce the tumor formation rate (from 88.89% to 40%), the number of tumors and the area of ​​tumors, and alleviate histopathological damage.

[0013] Furthermore, immunohistochemical results showed that mulberry concentrate could reduce the expression of the tumor proliferation marker Ki67 and increase the expression levels of intestinal barrier-related proteins ZO-1 and MUC2. These results suggest that mulberry concentrate may exert its preventive and / or therapeutic effects on UC-CAC by inhibiting abnormal proliferation, improving intestinal mucosal barrier homeostasis, and regulating the expression of related protective factors. Combined with its rich content of flavonoids, polyphenols, terpenes, and other active ingredients and its strong antioxidant activity, this suggests that mulberry concentrate may participate in the regulation of the inflammatory-cancer transformation process by alleviating oxidative stress.

[0014] Furthermore, the applicant evaluated the mulberry extract in a DSS-induced UC model, showing a certain trend of improvement in some inflammatory or barrier indicators, but overall it did not exhibit a stable and significant therapeutic advantage. In contrast, its tumor-suppressing and barrier-protective effects in the UC-CAC model were more clearly defined. Based on this, the present invention focuses its application protection on UC-CAC, rather than UC alone.

[0015] The mulberry concentrate described in this application is not a single active ingredient, nor is it a mulberry extract obtained through resin purification. Instead, it is a complex concentrated preparation obtained by extracting, filtering, and concentrating a mixture of mulberry branches, leaves, and roots at high temperatures. Broad-target metabolomics and activity assays further demonstrate that this mulberry concentrate is rich in various active ingredients such as terpenes, flavonoids, phenolic acids, and alkaloids, and possesses significant in vitro antioxidant activity. This supports its use as a natural complex active preparation for intervention in inflammation and cancer transformation at the material level.

[0016] The applicant's preliminary comparative study found that the mulberry concentrate did not show a stable and significant therapeutic advantage in the DSS-induced UC model, indicating that it is not a drug for controlling active inflammation. On the contrary, it showed a more obvious protective effect in the azomethane / dextran sulfate (AOM / DSS)-induced UC-CAC model, suggesting that its superior indication is not UC treatment, but rather the prevention and treatment of UC-related cancers.

[0017] The systematic experimental evidence provided in this application demonstrates that the mulberry concentrate can inhibit the progression of inflammation and cancer by reducing tumor burden (tumor formation rate from 88.89% to 40%), improving pathological damage, and regulating Ki67 and barrier-related indicators (ZO-1, MUC2). Compared to the limitations of existing positive control drugs that mainly focus on controlling inflammation, this invention provides a natural preparation more suitable for long-term intervention in the process of inflammation and cancer transformation, providing a basis for subsequent mechanism research and product development. Attached Figure Description

[0018] Figure 1 Schematic diagram of the quantity and relative content of primary metabolites in mulberry concentrate broad-target metabolomics;

[0019] Figure 2 Schematic diagram of the DPPH free radical scavenging effect of mulberry concentrate;

[0020] Figure 3 A schematic diagram of the results in the model of preventing DSS-induced UC using mulberry concentrate;

[0021] Figure 4 A schematic diagram of the results in the DSS-induced UC model using mulberry concentrate for prevention and treatment;

[0022] Figure 5 Schematic diagram of mouse weight changes during the treatment of UC-CAC with mulberry concentrate;

[0023] Figure 6 Schematic diagram showing the statistical results of changes in colon length, tumor formation rate, tumor area, and tumor number in mice during the treatment of UC-CAC with mulberry concentrate;

[0024] Figure 7 A schematic diagram of the pathological changes in the colonic HE of mice during the treatment of UC-CAC with mulberry concentrate;

[0025] Figure 8 A schematic diagram of the immunohistochemical results of Ki67 and barrier-related proteins (ZO-1, MUC2) in the mouse colon during the prevention and treatment of UC-CAC with mulberry concentrate. Detailed Implementation

[0026] Preparation of mulberry concentrate:

[0027] The mulberry concentrate in this example can be prepared according to the "Mulberry Concentrate and its Preparation Process" disclosed in patent CN108125236A. Specifically, by mass, 90 parts of mulberry leaves, 12 parts of mulberry branches, and 5 parts of mulberry roots are taken as raw materials, cleaned and set aside. First, add an appropriate amount of purified water to the extraction pot and raise the water temperature to 85-90℃. Then, mix the above raw materials and put them into the extraction pot for hot water extraction to obtain the extract. After the obtained extract is slowly cooled to 50-70℃, it is placed in a filter tank for filtration to obtain the filtrate. The filtrate is concentrated at 60-70℃ for 3-5 hours and then cooled to room temperature. The protein concentration is quantified by BCA (diquinoline carboxylic acid method) to reach 56.89 ± 2.68 mg / mL, which is the mulberry concentrate.

[0028] Figure 1 This diagram illustrates the number and relative content of primary metabolites in the mulberry concentrate from this example, based on broad-target metabolomics. A total of 2,286 primary metabolites were identified, belonging to 13 categories. Among them, terpenoids (396, 17.32%), amino acids and their derivatives (334, 14.61%), flavonoids (275, 12.03%), phenolic acids (255, 11.15%), and alkaloids (253, 11.07%) were the main metabolites, accounting for 66.18% of the total and constituting the main chemical composition characteristics of the mulberry concentrate.

[0029] Quantitative analysis of the main active substances in the mulberry concentrate was performed. Table 1 shows that the total flavonoid content was 2.65 ± 0.06 g / 100 mL, the total polyphenol content was 3.90 ± 0.11 g / 100 mL, and the proportion of total flavonoids to total polyphenols was 67.97 ± 1.31%. The results indicate that flavonoids are the main component of polyphenols in the mulberry concentrate, accounting for nearly 70%, suggesting that they may be key functional active ingredients.

[0030] Table 1. Flavonoid and Polyphenol Content and Proportion in Mulberry Concentrate (SC)

[0031]

[0032] In other embodiments of the present invention, the mulberry concentrate can be appropriately adjusted within the range of the disclosed process. For example, it is preferred to use 90 parts by weight of mulberry leaves, 12 parts by weight of mulberry branches, and 5 parts by weight of mulberry roots as raw materials; in the extraction step, the extraction temperature can be controlled at 85-90°C, the extraction time can be controlled at 2-4 hours, and the slow cooling time of the extract can be controlled at 8-13 hours; in the high-temperature concentration step, the concentration time can be controlled at 3.5-4.5 hours, and the finished product is obtained after cooling to room temperature. The amount of purified water used is sufficient to fully submerge the raw materials and complete the hot water extraction. The concentration endpoint can also be controlled in conjunction with the concentration time, the properties of the finished product, and quality indicators such as total flavonoids / total polyphenols; in the subsequent broad-target metabolomics, activity detection, and pharmacodynamic experiments of this application, the mulberry concentrate prepared according to the above process and confirmed by quality testing is used as the test substance.

[0033] Experiment 1: DPPH Free Radical Scavenging Effect of Mulberry Concentrate

[0034] The in vitro antioxidant capacity of the prepared mulberry concentrate was evaluated using the DPPH free radical scavenging method, with vitamin C as a positive control. Figure 2 This diagram illustrates the DPPH free radical scavenging effect of mulberry concentrate in this example. Part A compares the DPPH free radical scavenging results of vitamin C and 1 / 500 mulberry concentrate (SC), while Part B compares the DPPH free radical scavenging capacity of vitamin C and 1 / 500 mulberry concentrate (SC). The results show that at a 1 / 500 dilution, the DPPH free radical scavenging rate of mulberry concentrate is approximately 84%, with a scavenging capacity of approximately 29.8 μg Trolox / mL, which is close to the level of vitamin C (10 mg / mL). Considering the important role of oxidative stress in the progression of ulcerative colitis to colorectal cancer, the aforementioned material basis and antioxidant properties may be important material bases for the protective effect of this mulberry concentrate in the UC-CAC model.

[0035] Experiment 2: Evaluation of mulberry concentrate in DSS-induced UC model:

[0036] A mouse UC model was established using DSS induction to evaluate the preventive or therapeutic effects of the mulberry concentrate in Example 1.

[0037] Figure 3 A schematic diagram of the results in the UC model for preventing DSS-induced cytotoxicity with mulberry concentrate, wherein:

[0038] Part A shows the weight change curve of mice under the preventive drug administration mode. After DSS treatment, the weight of mice showed a decreasing trend, indicating that the UC model was successfully established. The weight loss of the mulberry concentrate intervention group (DSS + SC, the dose of mulberry concentrate (SC) was 10 mL / kg per gavage, the quantitative concentration of BCA protein in mulberry concentrate was 56.89±2.68 mg / mL, the total flavonoid content was 2.65 ± 0.06 g / 100 mL, and the total polyphenol content was 3.90 ± 0.11 g / 100 mL) was slightly alleviated compared with the DSS + PBS model group, but the overall improvement was limited. Compared with the positive drug 5-aminosalicylic acid (5-ASA) or other controls, the advantage was not obvious, suggesting that SC has a weak protective effect against DSS-induced weight loss in UC.

[0039] Part B shows the changes in the gross morphology and length of the colon in mice under the prophylactic drug administration mode. DSS treatment led to shortening of the colon in mice, which is consistent with the characteristics of an acute colitis model. After SC intervention, the length of the colon showed a certain recovery trend compared with the model group, but the degree of improvement was not very prominent, suggesting that it has a certain alleviating effect on DSS-induced colon shortening, but the effect is limited.

[0040] Part C shows the results of HE staining of the colon under the prophylactic drug administration mode. In the DSS model group, the colonic mucosal structure was destroyed, crypt damage and inflammatory cell infiltration were observed. After SC intervention, the local tissue structure and inflammatory damage were improved compared with the model group, but significant mucosal damage was still observed, suggesting that SC has a certain alleviating trend on UC pathological damage, but the overall protective effect is not strong.

[0041] Part D shows the immunohistochemical results of ZO-1 and MUC2 under the prophylactic administration mode. After DSS treatment, the expression of intestinal mucosal barrier-related proteins ZO-1 and MUC2 decreased, indicating that the barrier function was impaired. After SC intervention, the expression of ZO-1 and MUC2 showed a certain recovery trend, indicating that SC may have a certain protective effect on the intestinal mucosal barrier. However, based on statistics and staining intensity, the improvement was not very stable.

[0042] Figure 4 A schematic diagram of the results in the model for the prevention and treatment of DSS-induced UC using mulberry concentrate, wherein:

[0043] Part A shows the weight change curves of mice under the prevention and treatment model. After DSS induction, the weight of mice decreased significantly. After intervention with different doses of SC, the weight loss trend was alleviated in some groups. Four concentrations were set: SC stock solution, 0.75 SC (SC stock solution diluted to 0.75 times with sterile water), 0.5 SC (SC stock solution diluted to 0.5 times with sterile water), and 0.25 SC (SC stock solution diluted to 0.25 times with sterile water). The gavage dose was 10 mL / kg each time. However, no very clear and stable dose-dependent relationship was observed between different doses, and the overall weight recovery effect was limited, suggesting that SC does not have a prominent therapeutic effect on the improvement of ulcerative colitis (UC) damage.

[0044] Part B presents the gross morphology and colon length statistics of the mouse colon under the prevention and treatment model. The colon in the DSS model group was significantly shortened, while the colon length in the SC intervention group showed a certain improvement trend compared to the model group. However, the differences between different doses of SC were not stable, and the overall recovery was limited. These results suggest that SC has a certain alleviating effect on DSS-induced colon shortening, but it is difficult to use as primary evidence for a potent treatment of UC.

[0045] Part C presents the colonic HE staining results under the prevention and treatment model. The DSS model group exhibited typical colitis pathological changes, including mucosal structural disorder, crypt damage, and inflammatory cell infiltration. After SC intervention, some pathological damage was alleviated, but the degree of improvement was not entirely consistent across different dose groups, and overall pathological recovery was insufficient, suggesting that SC has a relatively limited therapeutic effect on inflammatory lesions in UC.

[0046] The results above indicate that mulberry concentrate showed a certain trend of improvement in some indicators, but overall did not demonstrate a stable and significant therapeutic advantage. These results suggest that the indications for the mulberry concentrate described in this application are not limited to UC, but are more suitable for further research on the prevention and treatment of UC-related carcinogenesis.

[0047] Experiment 3 Evaluation of the preventive and therapeutic effects of mulberry concentrate in the AOM / DSS-induced UC-CAC model

[0048] A mouse UC-CAC model was established using AOM / DSS induction. The mice were divided into a normal control group, a model group, and a mulberry concentrate intervention group. The mulberry concentrate prepared in Example 1 was used, and the dose administered by gavage was 10 mL / kg each time.

[0049] Figure 5 To investigate the changes in mouse body weight during the treatment of UC-CAC with mulberry concentrate, after AOM / DSS modeling, the body weight of mice showed a phased decrease and fluctuation; the decrease in body weight in the mulberry concentrate intervention group was less than that in the model group, suggesting that it can improve the overall condition during the UC-CAC modeling process.

[0050] Figure 6This diagram illustrates the changes in colon length, tumorigenesis rate, tumor area, and tumor number in mice during the treatment of UC-CAC with mulberry concentrate. AOM / DSS treatment led to colon shortening and the formation of significant intestinal tumors in mice. After intervention with mulberry concentrate, colon length recovered somewhat, and the tumor incidence (tumorigenesis rate decreased from 88.89% to 40%), tumor area, and tumor number all showed a decreasing trend, suggesting that it has an inhibitory effect on the occurrence and development of UC-CAC.

[0051] Figure 7 A schematic diagram of the pathological changes in the colonic HE of mice during the treatment of UC-CAC with mulberry concentrate; the colonic mucosa structure of mice in the model group was disordered, the glands were arranged abnormally, and there were obvious inflammation and tumor-like pathological changes; after intervention with mulberry concentrate, the pathological damage of colonic tissue was reduced and the mucosal structure was relatively improved.

[0052] The results showed that, compared with the model group, the mulberry concentrate intervention group experienced improved weight loss and restored colon length shortening. Further statistical analysis revealed that the mulberry concentrate treatment group significantly reduced tumor formation rate, tumor area, and tumor number. HE staining results showed that the model group exhibited significant colonic tissue structure disorder, with inflammatory infiltration and abnormal lesions; while the mulberry concentrate treatment group showed less tissue damage than the model group.

[0053] Experiment 4 Effects of mulberry concentrate on the expression of Ki67 and barrier-related proteins (ZO-1, MUC2)

[0054] Immunohistochemistry was used to detect the expression of relevant indicators in colon tissue. Figure 8 This image shows the immunohistochemical results of Ki67 and barrier-related proteins ZO-1 and MUC2 in the mouse colon during the treatment of UC-CAC with mulberry concentrate. The image also shows the immunohistochemical results of Ki67 and barrier-related proteins ZO-1 and MUC2 in the mouse colon during the treatment of UC-CAC with mulberry concentrate (undiluted solution, 10 mL / kg per gavage). Increased Ki67 expression in the AOM / DSS model group indicates enhanced abnormal cell proliferation; simultaneously, decreased ZO-1 and MUC2 expression suggests impaired intestinal mucosal barrier. After intervention with mulberry concentrate, Ki67 expression decreased, while ZO-1 and MUC2 expression recovered somewhat, suggesting that it may exert its role in preventing and treating UC-CAC by inhibiting abnormal proliferation and improving the intestinal mucosal barrier.

[0055] The results showed that, compared with the model group, Ki67 expression decreased and ZO-1 expression increased after treatment with mulberry concentrate, while MUC2 also showed a recovery trend. These results suggest that mulberry concentrate can exert a protective effect by reducing abnormal proliferation and improving the expression of intestinal mucosal barrier-related molecules.

[0056] In summary, mulberry concentrate has promising applications in the prevention and / or treatment of ulcerative colitis-associated colorectal cancer and can be used as an active ingredient in related drug development. Typical dosage forms may include, but are not limited to, oral liquids, granules, capsules, tablets, or lyophilized powders.

Claims

1. The use of mulberry concentrate in the preparation of drugs for the prevention and / or treatment of ulcerative colitis-related colorectal cancer, wherein the mulberry concentrate is obtained by mixing mulberry branches, mulberry leaves and mulberry roots in a specific ratio and then extracting and concentrating them, and the mulberry concentrate is used as a medicinal component to reduce the tumor burden in ulcerative colitis-related colorectal cancer models and improve intestinal mucosal barrier damage, rather than simply inhibiting inflammation.

2. The application according to claim 1, characterized in that, The mulberry concentrate is prepared by mixing 8-12 parts by weight of mulberry branches, 3-5 parts by weight of mulberry roots, and 80-90 parts by weight of mulberry leaves, followed by extraction, filtration, and concentration processes.

3. The application according to claim 1 or 2, characterized in that, The reduction in tumor burden is manifested as a decrease in tumorigenesis rate, a reduction in the number of tumors, and / or a reduction in tumor area.

4. The application according to claim 1 or 2, characterized in that, The improvement in intestinal mucosal barrier damage is manifested by increased expression of ZO-1 and / or MUC2 in colon tissue.

5. The application according to claim 1 or 2, characterized in that, The mulberry concentrate can reduce Ki67 expression in colon tissue, thereby inhibiting abnormal proliferation.

6. The application according to claim 1 or 2, characterized in that, The mulberry concentrate contains one or more of the following: terpenoids, amino acids and their derivatives, flavonoids, phenolic acids, and alkaloids.

7. The application according to claim 1 or 2, characterized in that, The total flavonoid content in the mulberry concentrate is 2.0–3.0 g / 100 mL.

8. The application according to claim 1 or 2, characterized in that, The mulberry concentrate has in vitro antioxidant activity; the mulberry concentrate still has DPPH free radical scavenging ability after dilution.

9. A pharmaceutical composition for the prevention and / or treatment of ulcerative colitis-associated colorectal cancer, characterized in that, The pharmaceutical composition comprises mulberry concentrate as the active ingredient, and pharmaceutically acceptable excipients.

10. The pharmaceutical composition according to claim 9, characterized in that, The dosage form of the pharmaceutical composition is oral liquid, granules, capsules, tablets or lyophilized powder.

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