Traditional Chinese medicine probiotic composition for preventing and treating colorectal cancer and application thereof

Abstract

The invention belongs to the field of traditional Chinese medicines, and particularly relates to a traditional Chinese medicine probiotic composition for preventing and treating colorectal cancer and application thereof. The traditional Chinese medicine probiotic composition is prepared from a traditional Chinese medicine composition and probiotics, the traditional Chinese medicine composition is prepared from the following raw materials in parts by mass: 15g of Chinese pulsatilla root, 12g of cortex fraxini, 12g of cortex phellodendri and 6g of rhizoma coptidis. The feed is characterized in that the probiotics are Bifidobacterium pseudolongum (B.p), and the preservation number of the probiotics is ATCC (American Type Culture Collection) 25526. The invention finds that the combined application of the Chinese pulsatilla root decoction compound and the bifidobacterium pseudolongum shows obvious synergistic and excellent effects in the aspect of inhibiting the colorectal cancer, which is superior to the single application of the traditional Chinese medicine compound or the probiotics, and the invention provides a novel, safe and effective traditional Chinese medicine and traditional Chinese medicine-probiotic combined intervention scheme for preventing and treating the colorectal cancer. Good application prospects are realized.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine, and in particular relates to a traditional Chinese medicine probiotic composition for the prevention and treatment of colorectal cancer and its application. Background Technology

[0002] Colorectal cancer (CRC) remains a significant global health challenge, being one of the most common malignancies and the second leading cause of cancer-related mortality. The prognosis of CRC patients is highly correlated with clinical stage; the 5-year survival rates for early-stage and mid-stage CRC patients are 91% and 73%, respectively, while the 5-year survival rate for patients with advanced disease is only 14%. Therefore, shifting the intervention focus to earlier stages of CRC is of great importance for its prevention and control.

[0003] The development and progression of colorectal cancer (CRC) are closely related to multiple factors, including genetic factors, dietary structure, inflammatory responses, and gut microbiota imbalance. Among these, chronic inflammation-driven "inflammatory-cancer transformation" is a key pathological progression in CRC. Chronic inflammation damages the intestinal epithelial barrier, accelerating the transformation of intestinal epithelial cells into tumor cells. Simultaneously, immune cells, including macrophages, are recruited to the intestine, where they release pro-inflammatory cytokines. This signaling triggers a vicious cycle of exacerbated inflammation, leading to epithelial barrier dysfunction and disruption of intestinal homeostasis. Studies and statistical analyses show that long-term intestinal inflammation increases the risk of colorectal cancer by 6-8 times. In particular, chronic intestinal inflammation accompanied by intestinal gland dysplasia accelerates the progression from normal epithelium to adenoma to adenocarcinoma. Furthermore, inflammation also accelerates the carcinogenesis process associated with APC gene mutations. Therefore, inhibiting inflammation-driven colorectal cancer transformation is an important strategy for reducing the incidence of CRC. Although studies have shown that nonsteroidal anti-inflammatory drugs (NSAIDs) and cyclooxygenase-2 (COX-2) inhibitors can reduce the incidence of CRC by suppressing inflammation, their use is limited by certain adverse reactions. Therefore, finding safer and more effective strategies for the prevention and treatment of CRC has significant clinical implications.

[0004] The rich and long-standing clinical history of Traditional Chinese Medicine (TCM) provides valuable resources for identifying effective treatments for a range of diseases. Baitouweng Decoction (BTW) is a commonly used traditional Chinese medicine compound, composed of four herbs—Pulsatilla chinensis, Fraxinus rhynchophylla, Phellodendron chinense, and Coptis chinensis—in a 5:4:4:2 ratio. BTW possesses various pharmacological effects, including anti-inflammatory, antioxidant, and autophagy-enhancing properties. Due to its significant clinical efficacy in treating inflammatory bowel diseases, it has attracted increasing attention. Furthermore, animal studies have shown that BTW can improve DSS-induced colitis by regulating immune cells and gut microbiota, demonstrating therapeutic potential for chronic rheumatoid arthritis (CRC). However, the exact role of BTW in the prevention and treatment of CRC remains to be elucidated.

[0005] In recent years, numerous studies have demonstrated the crucial role of the gut microbiota and its metabolites in maintaining intestinal homeostasis, and their dysregulation is closely related to the pathogenesis of colitis and colorectal cancer. On the one hand, regulating the gut microbiota is a key mechanism by which traditional Chinese medicine (TCM) exerts its therapeutic effects. On the other hand, the gut microbiota can metabolize TCM compounds, improving their bioavailability and overall efficacy. However, current probiotic intervention programs for colorectal cancer (CRC) are still in the exploratory stage, and specific effective strains and their combined application with drugs remain unclear. Therefore, developing a novel CRC prevention and treatment program based on BTW (biomicrobial-mediated transformation) formulas combined with gut microbiota regulation has significant scientific and practical value. Summary of the Invention

[0006] The purpose of this invention is to provide a traditional Chinese medicine probiotic composition for the prevention and treatment of colorectal cancer and its application, so as to overcome the problems of limited efficacy, obvious side effects and lack of combined intervention programs of traditional Chinese medicine and probiotics in the existing colorectal cancer prevention and treatment methods.

[0007] This invention provides a traditional Chinese medicine probiotic composition, which is composed of a traditional Chinese medicine composition and probiotics; The traditional Chinese medicine composition is made from the following raw materials in parts by weight: 15g of Pulsatilla chinensis, 12g of Fraxinus chinensis, 12g of Phellodendron chinense, and 6g of Coptis chinensis; The probiotic is Bifidobacterium pseudolongum (Bifidobacterium pseudolongum) Bifidobacterium pseudolongum (Bp), with accession number ATCC 25526.

[0008] This invention provides the application of a traditional Chinese medicine probiotic composition in the preparation of drugs for the prevention and treatment of colorectal cancer; Furthermore, the traditional Chinese medicine composition is administered simultaneously or sequentially with the Bifidobacterium pseudolongum.

[0009] Furthermore, the dosage ratio of the traditional Chinese medicine composition to Bifidobacterium pseudolongum is 30-90g / 10g. 9 -10 11 CFU.

[0010] Furthermore, the dosage of the *Bifidobacterium pseudolongum* is 10... 9 -10 11 CFU / 200ul.

[0011] Furthermore, the *Bifidobacterium pseudolongum* is a live bacterium, a freeze-dried bacterium, or a metabolite thereof.

[0012] This invention provides a preparation made from a traditional Chinese medicine probiotic composition, wherein the preparation is in the form of pills, capsules, ointment, or granules.

[0013] This invention provides an application of a formulation in the preparation of drugs for the prevention and treatment of colorectal cancer.

[0014] Compared with the prior art, the present invention has the following beneficial effects: 1. For the first time in basic experiments, it was confirmed that Pulsatilla chinensis decoction has a clear anti-colorectal cancer effect, without obvious systemic toxicity, and can regulate intestinal flora, inhibit inflammatory response and enhance intestinal barrier function; 2. This study is the first to reveal that Pulsatilla chinensis decoction can inhibit colorectal cancer by regulating gut microbiota; 3. For the first time, it was discovered and verified that Pulsatilla chinensis decoction can significantly increase the abundance of Bifidobacterium pseudolongum, and that Bifidobacterium pseudolongum has a significant inhibitory effect on colorectal cancer; 4. This study first proposed and confirmed the superior efficacy of the combined use of Pulsatilla chinensis decoction and Bifidobacterium pseudolongum in the treatment of colorectal cancer, thus improving the efficacy of single treatment regimens. 5. Depending on actual clinical needs, Pulsatilla Decoction and Bifidobacterium pseudolongum can be used alone or in combination; this combined and highly effective regimen has high safety, few toxic side effects, and good prospects for clinical translation and industrialization. Attached Figure Description

[0015] Figure 1 The diagram shows the microbial structure of the colorectal inflammation-cancer transformation and its abnormal regulation in the BTW-induced AOM / DSS-induced mouse model in Example 1; (A) Schematic diagram of the experimental protocol (CON - normal control group, MOD - AOM / DSS model control group, BTW - Pulsatilla chinensis decoction treatment group); (B) Representative images of the mouse intestines in different groups; (C) Statistical graph of the number and size of intestinal tumors; (D) Representative images of mouse intestines stained with HE; (E) Representative images of mouse intestines stained with Ki-67; (F) Differences in PCNA protein expression in different groups; (G) PCA analysis of microbial community at OTU level; (H) Differential analysis of microbial community at OTU level. Figure 2 The diagram shows the effect of BTW treatment on the inhibition of colitis-related tumorigenesis in mice in Example 1; (A) Schematic diagram of the experimental protocol (NS - physiological saline, ABX - antibiotic depletion; N-FMT: mice transplanted with normal flora, B-FMT: mice transplanted with Pulsatilla chinensis decoction intervention bacteria, M-FMT: mice transplanted with inflammatory cancer disorder bacteria, MB-FMT: mice transplanted with Pulsatilla chinensis decoction therapeutic effect bacteria); (B) Curves of weight change of mice in each group during the experiment; (C) Typical colonoscopy images of mice in different groups; (D) Representative intestinal images of the four groups of mice and statistical graph of length differences; (E) Statistical analysis of the difference in tumor number between groups; (FH) Representative images of mouse intestines stained with HE and Ki-67 and pathological scores, statistical analysis of differences in Ki-67 staining; Figure 3 Bifidobacterium pseudolongum in Example 2 ( Bifidobacterium pseudolongumBp (ATCC25526) is a key dominant bacterial species in BTW regulation. (A) LDA score plot of bacterial communities with rich taxonomic features identified by LEfSe analysis between the M-FMT and MB-FMT groups (feature selection criteria were log-LDA score > 3); (B) Heatmap of the microbial species with the greatest differences between the two groups; (C, D) Differential analysis of Bp abundance after BTW treatment and fecal transplantation; (E) Correlation analysis of Bp abundance and tumor number in microbial community transplantation recipient mice; (F) Correlation analysis of Bp abundance and tumor number in AOM / DSS mice; Figure 4 The diagram shows the inhibitory effect of Bifidobacterium pseudolongum on colitis-associated colorectal cancer in Example 2; (A) Schematic diagram of mouse experimental design (NS - physiological saline; Ctrl - normal control group; Model - model control group; Bp (a) Bifidobacterium pseudolongum treatment group); (b) Mouse weight change curve; (c) Representative images of the colon in each group; (d) Differences in intestinal length between groups; (e) Quantification of the total number of intestinal tumors and tumors with a diameter greater than 3 mm; (f) Representative H&E and Ki-67 immunohistochemical (IHC) staining of colon sections; (g) ELISA determination of the levels of inflammatory factors IL-1β, IL-6 and TNF-α in colonic serum; (h) Protein expression of pro-inflammatory factors in colonic tissue; (i) Representative mucus barrier-related histochemical staining of colon sections: PAS, Alcian Blue and MUC2; (j) Immunofluorescence staining of tight junction proteins ZO-1 and Occludin in colonic tissue; Figure 5 The diagram shows the combined application of Pulsatilla chinensis decoction and Bifidobacterium pseudolongum in Example 3; (A) Schematic diagram of mouse experimental design (NS - physiological saline; Con - normal control group; M - model control group; Bp - Bifidobacterium pseudolongum treatment group; BTW- Pulsatilla chinensis decoction treatment group; B.p+ (B) Mouse weight change curve; (C) Representative images of the colon in each group and differences in intestinal length between groups; (D) Quantification of the total number of intestinal tumors and tumors with a diameter greater than 3 mm; (E) Representative H&E and Ki-67 immunohistochemical (IHC) staining of colon sections; (F) Comparison of pathological changes between groups; (G) ELISA measurement of the levels of inflammatory factors IL-1β, IL-6 and TNF-α in colonic serum; (H) Representative histochemical staining of mucus barrier-related components in colon sections: PAS, Alcian Blue and MUC2; (I) Difference analysis of Bp abundance in feces; Figure 6 In Example 3, the combination of Pulsatilla chinensis decoction and Bifidobacterium pseudolongum, alone or in combination, showed good safety. Detailed Implementation

[0016] Example 1 1. Preparation for BTW BTW granules were purchased from China Xinlv Pharmaceutical Co., Ltd. The daily dose for a normal adult (60 kg) is 15 g of Pulsatilla chinensis, 12 g of Phellodendron chinense, 12 g of Fraxinus chinensis, and 6 g of Coptis chinensis, totaling 45 g / dose. Based on the equivalent dose ratio of body surface area conversion between humans and mice, the dose for mice is 45 g / 60 kg × 10 = 7.5 g / kg. BTW was dissolved in 0.9% NaCl solution (NS) to prepare a stock solution.

[0017] 2. Animal models Male C57BL / 6J mice (6 weeks old) were purchased from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd. All experimental procedures involving animal research were approved by the Animal Ethics Committee of Shanghai University of Traditional Chinese Medicine (Approval No.: PZSHTCM23070500006). Mice were housed under specific pathogen-free conditions at 25±2 ℃ and 45%-50% humidity, with a 12-hour light / dark cycle and free access to food and water. Experiments began after a 3-day acclimatization period.

[0018] Methods for establishing mouse models of colorectal cancer (e.g.) Figure 1 A) The model mice first received a single intraperitoneal injection of AOM (12.5 mg / kg). One week later, they began four cycles of DSS intervention. Each cycle began with drinking water containing 2.5% DSS for 5 days, followed by 16 days of normal water (21 days per cycle).

[0019] The intervention protocol for BTW was as follows: starting from the first day of the experimental protocol DSS, mice were orally administered BTW (7.5 g / kg) or an equal volume of NS daily.

[0020] 3. Inhibitory effect of BTW on CRC model mice A mouse model of CRC induced by AOM / DSS was used, and BTW was administered by gavage. The results showed that, compared with the model group, BTW significantly improved the shortening of intestinal length in mice. Figure 1 B), the number and volume of colorectal tumors were significantly reduced in the BTW treatment group ( Figure 1 C), intestinal pathological damage was significantly improved ( Figure 1 D), the malignant proliferation of tumor cells was significantly inhibited ( Figure 1 E, F). Meanwhile, BTW treatment improved gut microbiota dysbiosis in the model group mice (E, F). Figure 1 These results indicate that BTW inhibited colorectal inflammation-cancer transformation in an AOM / DSS-induced mouse model and modulated aberrant gut microbiota structure.

[0021] 4. Fecal microbiota transplantation experiment after BTW treatment The fecal microbiota transplantation (FMT) experimental method was as follows: donor feces were collected from four different groups: normal mice (N), AOM / DSS-induced mice (MOD), BTW-treated normal mice (B), and BTW-treated AOM / DSS mice (BTW). Figure 2 A). Subsequently, the samples were homogenized in sterile saline and then centrifuged to prepare fecal suspensions. Starting from day one of the DSS protocol, recipient mice were administered the resulting supernatant (200 µl per dose) three times a week until three DSS treatment cycles were completed. Results showed that colorectal cancer progression in recipient mice was significantly inhibited. BTW-regulated microbiota transplantation significantly improved weight loss in mice (…). Figure 2 B) significantly inhibited intestinal mucosal lesions ( Figure 2 C), alleviated the shortening of intestinal length ( Figure 2 D), the number and size of tumors were significantly reduced ( Figure 2 E), intestinal pathological damage was significantly improved ( Figure 2 F, G), the malignant proliferation of tumor cells was significantly inhibited (F, G), Figure 2 (F, H). The above results indicate that BTW treatment of the gut microbiota in mice can inhibit the occurrence of colitis-related tumors.

[0022] Example 2 1. Source and culture of Bifidobacterium pseudolongum Bifidobacterium pseudolongum ( Bifidobacterium pseudolongum Bp (ATCC 25526) was obtained from BioBio Biotechnology Co., Ltd. and cultured: Bp was cultured in BS medium (HB0394-1, hopebio, Qingdao, China) supplemented with 0.1% (v / v) Tween 80 and incubated at 37 °C in an anaerobic workstation (YY-XXL, MAWORDE, Qiqihaer, China) filled with mixed nitrogen (10% H2, 10% CO2, 80% N2).

[0023] 2. Identify Bifidobacterium pseudolongum ( Bifidobacterium pseudolongum , Bp)(ATCC 25526) is the key dominant strain for BTW regulation.

[0024] To further identify the key microbiota for the beneficial effects of BTW on CAC, differential analysis was performed on the post-transplant gut microbiota from step 4 of Example 1. Comparative analysis using linear discriminant analysis of effect size (LEfSe) revealed that *Bifidobacterium* (from Actinobacteria to Bifidobacteriaceae) was identified as a key taxa potentially associated with BTW's improvement of CAC. Figure 3A). Further analysis using the T-test and other methods to determine the differences in bacteria between groups showed that *Bifidobacterium pseudolongum* (… Bifidobacterium pseudolongum ,Bp)(ATCC 25526) is one of the most diverse strains ( Figure 3 Bp (B) may be a key dominant bacterial species in BTW regulation. Abundance analysis of Bp in feces indicates that BTW can increase Bp abundance in vivo, and this abundance dominance can be transferred to recipient mice. Figure 3 C, D). Correlation analysis between fecal Bp abundance and tumor number revealed a negative correlation between Bp abundance and tumor number. Figure 3 E, F). The above results indicate that Bp is a key dominant bacterial species for BTW regulation and may have the potential to inhibit CRC.

[0025] 3. Inhibitory effect of Bp on colorectal cancer The Bp intervention method for the model mice was as follows: starting from the first day of the DSS experimental protocol, AOM / DSS model mice were given Bp (10g) by gavage every three days. 9 CFU / 200ul), while other mice received the same amount of NS.

[0026] Intestinal tissue and other major organs (heart, liver, spleen, lung, kidney) were fixed, embedded in paraffin, and sectioned to 4 µm. After dewaxing in xylene and rehydration with a series of graded ethanol solutions (95%, 80%, and 70%), sections were stained with hematoxylin for 10 minutes and eosin for 3 minutes at room temperature. After a brief rinse in distilled water, sections were dehydrated in 95% absolute ethanol, cleaned in xylene, and finally mounted on Per-mount medium.

[0027] Intestinal tissue samples were embedded in paraffin and sectioned to a thickness of 4 µm. For all staining procedures, sections were first dewaxed in xylene, then rehydrated via a series of graded ethanol solutions, and finally rinsed in distilled water. Antigen retrieval was performed using citrate buffer. After endogenous peroxidase and BSA blockade, sections were incubated overnight with primary antibody at 4°C, followed by incubation with biotinylated secondary antibody and DAB development. Hematoxylin was used for counterstaining.

[0028] Protein extracts from cells and frozen colon tissue were prepared using RIPA buffer containing protease and phosphatase inhibitors. Protein concentrations were determined using a BCA kit. Equal volumes of protein were separated by SDS-PAGE and transferred to a PVDF membrane. After blocking, the membrane was incubated overnight at 4 °C with primary antibody.

[0029] After washing away nonspecific binding, the membrane was incubated with horseradish peroxidase-conjugated secondary antibody at room temperature for 1 hour. Protein bands were visualized using an enhanced chemiluminescence (ECL) detection kit and imaged using a Tanon 5200 system. Band density was quantified using ImageJ software.

[0030] The results showed that Bp could alleviate weight loss in mice. Figure 4 B), reducing intestinal shortening ( Figure 4 C, D), inhibiting the occurrence and progression of tumors ( Figure 4 E), reduces intestinal pathological damage and inhibits the malignant proliferation of tumor cells ( Figure 4 F). Furthermore, Bp significantly alleviated systemic and intestinal inflammation, manifested by reduced levels of pro-inflammatory factors IL-1β, IL-6, and TNF-α (F). Figure 4 G, H), and protect the intestinal mucus barrier and mechanical barrier ( Figure 4 I and J) improved intestinal barrier function. These results suggest that Bp may be a potential treatment strategy for CRC.

[0031] Example 3 Validation of the superiority of BTW and BP combined application This experiment was divided into five groups: a negative control group, a model control group, a Bp monotherapy group, a BTW monotherapy group, and a BTW and Bp combination therapy group. The specific modeling methods and administration routes were the same as in Examples 1 and 2. However, when BTW and Bp were used in combination, the dosage and frequency of both remained unchanged at a ratio of 150 mg / 10 ml. 9 CFU ( Figure 5 A).

[0032] The results showed that the combined treatment group reduced the body weight of mice ( Figure 5 B) Shortened intestinal length ( Figure 5 C), inhibiting the occurrence and progression of tumors ( Figure 5 D), reduces intestinal pathological changes and malignant proliferation ( Figure 5 E, F), reduce inflammation ( Figure 5 G) and protection of the intestinal barrier ( Figure 5 Both H and I showed superior efficacy compared to the single-drug groups, and the abundance of Bp in feces was also higher in the combined group. These results indicate that the tumor suppression effect of the combined treatment group was significantly better than that of the single-drug group, demonstrating a clear synergistic and superior effect.

[0033] To test the safety of BTW and Bp alone or in combination, liver, heart, spleen, lung, and kidney samples from mice in each group were collected for pathological staining (same as step 3 in Example 2). The results are as follows: Figure 6As shown, both BTW and Bp, whether used alone or in combination, exhibit good biocompatibility.

[0034] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A traditional Chinese medicine probiotic composition, characterized in that, Composed of traditional Chinese medicine and probiotics; The traditional Chinese medicine composition is made from the following raw materials in parts by weight: 15g of Pulsatilla chinensis, 12g of Fraxinus chinensis, 12g of Phellodendron chinense, and 6g of Coptis chinensis; The probiotic is Bifidobacterium pseudolongum (Bifidobacterium pseudolongum) Bifidobacterium pseudolongum (Bp), with accession number ATCC25526.

2. The traditional Chinese medicine probiotic composition according to claim 1, characterized in that, The dosage ratio of the traditional Chinese medicine composition to Bifidobacterium pseudolongum is 30-90g / 10g. 9 -10 11 CFU.

3. The traditional Chinese medicine probiotic composition according to claim 2, characterized in that, The dosage of the *Bifidobacterium pseudolongum* was 10. 9 -10 11 CFU / 200ul.

4. The use of a traditional Chinese medicine probiotic composition as described in any one of claims 1-3 in the preparation of a drug for the prevention and treatment of colorectal cancer, characterized in that, The traditional Chinese medicine composition is administered simultaneously or sequentially with the Bifidobacterium pseudolongum.

5. The application according to claim 4, characterized in that, The *Bifidobacterium pseudolongum* is a live bacterium, a freeze-dried bacterium, or its metabolites.

6. A preparation made from the traditional Chinese medicine probiotic composition according to any one of claims 1-3, characterized in that, The preparations are pills, capsules, ointments, or granules.

7. The use of the formulation as described in claim 6 in the preparation of a drug for the prevention and treatment of colorectal cancer.

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