Application of safflower extract in preparation of anti-esophageal cancer drugs

Abstract

The application discloses application of a safflower extract in preparation of an anti-esophageal cancer drug and belongs to the technical field of medicines.The application proves that the safflower extract can dose-dependently inhibit esophageal cancer transplanted tumor growth through construction of a subcutaneous tumor-bearing mouse model of esophageal cancer, and the tumor inhibition rate of a high-dose group reaches 59.57%; the safflower extract can also significantly reduce the serum pro-inflammatory factor TNF-alpha level of the tumor-bearing mouse and the IL-1beta and IL-6 levels of tumor tissue, improve the local inflammatory microenvironment of the tumor, effectively reverse the liver and kidney function damage caused by tumor bearing, and antagonize the liver damage caused by cisplatin; and the safflower extract can also play an anti-tumor role by regulating the intestinal flora structure and restoring the intestinal microecological homeostasis.The application provides a new drug source for the treatment of esophageal cancer and also provides an experimental basis for the safflower as a chemotherapy adjuvant drug and for reducing chemotherapy-related organ toxicity.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to new uses of Rhizoma Serratum extract, and particularly to its application in the preparation of anti-esophageal cancer drugs. Background Technology

[0002] Esophageal cancer is one of the most common malignant tumors of the digestive tract worldwide, ranking among the top cancer incidence rates globally and having a high mortality rate. Esophageal squamous cell carcinoma (ESCC) is the dominant histological type, especially prevalent in developing countries, accounting for approximately 80%–90% of all esophageal cancer cases. As a highly aggressive upper gastrointestinal malignancy, ESCC is closely related to the abnormal proliferation of esophageal epithelial cells. Due to the lack of rapid and sensitive diagnostic tools, most patients are diagnosed at an intermediate or advanced stage, resulting in extremely poor prognosis and a persistently high mortality rate. Clinical treatment still faces bottlenecks such as limited efficacy and significant toxic side effects, necessitating the exploration of safe and effective natural drug intervention strategies.

[0003] Tumor development is often accompanied by gut microbiota dysbiosis, and imbalance of gut microbiota homeostasis is a key driver of tumor progression. There is increasing interest in improving the effectiveness of cancer treatment by regulating gut microbiota. The gut is the largest immune organ in the human body, rich in immune cells, and plays a crucial role in systemic immune responses and tumor immune regulation. Therefore, regulating gut microbiota composition holds promise as a novel strategy to enhance the efficacy of cancer treatment.

[0004] *Inula salsoloides* (Turcz.) Ostenf., a plant belonging to the genus *Inula* in the family Asteraceae, is primarily used in traditional Chinese medicine for its anti-tumor and anti-inflammatory effects. Current research indicates that *Inula salsoloides* extract can significantly inhibit the proliferation of esophageal cancer cells (ESCC) and possesses anti-esophageal cancer potential. Screening and isolating natural active ingredients with anti-tumor activity from traditional Chinese medicine and natural medicinal plants has become a new research direction and potential breakthrough in the field of cancer prevention and treatment, and is also a core trend in the current development of innovative anti-tumor drugs. However, while the in vitro anti-tumor effect of *Inula salsoloides* has been preliminarily confirmed, its in vivo tumor-suppressing effect and organ-protective safety remain unclear. Furthermore, whether this drug can improve tumor-borne intestinal flora imbalance and whether it exerts its in vivo anti-tumor effect by regulating the intestinal microecology, the related molecular and microbial mechanisms, have not been systematically elucidated.

[0005] Therefore, developing new medicinal uses for *Vaccaria segetalis* has significant clinical value and broad application prospects. Summary of the Invention

[0006] In view of this, the purpose of this invention is to provide the application of *Rhizoma Scutellariae* extract in the preparation of drugs for treating esophageal cancer. This invention constructs a mouse model of subcutaneous esophageal cancer and conducts experiments from multiple dimensions, including overall animal signs, tumor growth kinetics, tumor histopathological morphology, systemic and local inflammatory factors, liver and kidney function biochemical indicators, and gut microbiota, to systematically evaluate the in vivo anti-esophageal cancer efficacy and safety of *Rhizoma Scutellariae* extract, and elucidates its mechanism of anti-tumor effect through regulation of gut microbiota.

[0007] To achieve the above objectives, the present invention provides the following technical solution: This invention provides the application of *Rubus sarmentosus* extract in the preparation of a medicament for treating esophageal cancer.

[0008] Furthermore, the extract of *Rubus sarmentosus* is one of a petroleum ether extract, an ethyl acetate extract, or a water extract.

[0009] Furthermore, the esophageal cancer is esophageal squamous cell carcinoma.

[0010] Furthermore, the drug comprises an effective amount of Senna rhododendron extract and pharmaceutically acceptable excipients.

[0011] Furthermore, the drug is an oral preparation.

[0012] The present invention also provides the use of Senna rhododendron extract in the preparation of a medicament for alleviating liver and kidney damage caused by chemotherapy drugs.

[0013] Furthermore, the chemotherapy drug is cisplatin.

[0014] The present invention also provides a pharmaceutical composition for treating esophageal cancer, comprising an effective amount of raspberry extract and pharmaceutically acceptable excipients.

[0015] Furthermore, the pharmaceutical composition is an oral formulation.

[0016] The present invention also provides the application of the ethyl acetate extract of Rhodiola rosea in the preparation of a drug for improving intestinal flora imbalance, characterized in that the intestinal flora imbalance is associated with esophageal cancer.

[0017] It contains at least the following beneficial technical effects: This invention is the first to demonstrate the in vivo anti-esophageal cancer activity of ethyl acetate extract of Rhizoma Senna. Experiments using a mouse model of subcutaneous esophageal cancer tumors showed that ethyl acetate extract of Rhizoma Senna can dose-dependently inhibit the growth of esophageal cancer xenografts, with a tumor inhibition rate of up to 59.57% in the high-dose group, demonstrating clear in vivo anti-tumor activity.

[0018] This invention confirms that the ethyl acetate extract of *Rhizoma Scutellariae* has good drug safety. At an effective tumor-suppressing dose, *Rhizoma Scutellariae* can effectively improve the overall condition of tumor-bearing mice and restore body weight; it can significantly downregulate abnormally elevated ALT, AST, and UREA levels in tumor-bearing mice, effectively alleviate tumor-induced liver and kidney damage, and antagonize cisplatin-induced liver damage.

[0019] This invention confirms that the ethyl acetate extract of *Rhizoma Scutellariae* exerts anti-tumor effects by inhibiting systemic inflammation and excessive local inflammation in tumors. ELISA results show that *Rhizoma Scutellariae* can dose-dependently downregulate serum levels of the pro-inflammatory factor TNF-α and tumor tissue levels of IL-1β and IL-6 in tumor-bearing mice, thereby improving the local inflammatory microenvironment of tumors.

[0020] This invention reveals for the first time the mechanism by which the ethyl acetate extract of Rhizoma Senna exerts its antitumor effect by regulating the gut microbiota. High-throughput 16S rDNA sequencing analysis showed that Rhizoma Senna can restore the gut microbiota structure in tumor-bearing mice, increase the abundance of beneficial bacteria, improve gut microecological homeostasis, and exert its antitumor effect by restoring metabolic functions such as secondary bile acid biosynthesis.

[0021] This invention provides complete in vivo experimental evidence for the development of *Symplocos salsa* as an adjuvant therapy for esophageal cancer and a new natural low-toxicity anti-tumor drug, and also lays the foundation for the study of the mechanism of anti-tumor immunity mediated by intestinal flora in traditional Chinese medicine. Attached Figure Description

[0022] Figure 1 The graph shows the changes in body weight of mice in each group.

[0023] Figure 2 The tumor growth curves are shown for each group of tumor-bearing mice.

[0024] Figure 3 This is a comparison chart of tumor weight in different groups of tumor-bearing mice.

[0025] Figure 4 Photos of tumors in each group of tumor-bearing mice.

[0026] Figure 5 The pathological morphology of tumor tissues from each group of tumor-bearing mice after H&E staining (×200).

[0027] Figure 6 A comparison chart of tumor tissue pathological scores for each group of tumor-bearing mice.

[0028] Figure 7 A comparison chart of serum biochemical levels (ALP, ALT, AST, CREA, UREA) in mice from different groups.

[0029] Figure 8A graph comparing the levels of inflammatory factors (TNF-α, IL-10, IL-1β, IL-6) in the serum and tumor tissues of mice in each group.

[0030] Figure 9 Venn diagram showing the distribution of ASVs in the gut microbiota of mice in each group.

[0031] Figure 10 The cumulative species curves of the gut microbiota in each group of mice are shown.

[0032] Figure 11 Principal coordinate analysis (PCoA) of gut microbiota in each group of mice and the difference in Beta diversity between groups are presented.

[0033] Figure 12 A diagram showing the species composition analysis of the gut microbiota at the phylum level for each group of mice.

[0034] Figure 13 A diagram showing the family-level species composition of the gut microbiota in each group of mice.

[0035] Figure 14 A diagram showing the species composition of the gut microbiota at the genus level in each group of mice.

[0036] Figure 15 A bar chart showing the distribution of LDA values ​​in the gut microbiota of mice in each group using LEfSe analysis.

[0037] Figure 16 Heatmap showing the distribution of KEGG metabolic function in the gut microbiota of mice in each group.

[0038] Figure 17 A bar chart showing the relative abundance of KEGG functional entries in the gut microbiota of mice in each group. Detailed Implementation

[0039] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0040] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0041] Unless otherwise specified, all raw materials or instruments used in the following embodiments of the present invention are commercially available.

[0042] Example 1 1. Experimental Materials 1.1 Experimental Animals A total of 48 male SPF-grade C57BL / 6 mice, weighing 18~22g, were purchased from Beijing Huafukang Bioscience Co., Ltd., with the animal production license number: SCXK (Jing) 2024-0003. All mice were housed in the SPF-grade Laboratory Animal Center of Xinjiang Uygur Autonomous Region Institute of Materia Medica. The ambient temperature was controlled at 22±2℃, the relative humidity was 50%±10%, with a 12h light / 12h dark cycle. The feed and drinking water were sterilized, and the mice had free access to food and water. The formal experiment was carried out after 7 days of adaptive acclimation.

[0043] The laboratory animal use license number is: SYXK (Xin) 2024-0002. This experiment was reviewed and approved by the Laboratory Animal Ethics Committee of Xinjiang Uygur Autonomous Region Institute of Materia Medica (Ethics Approval No.: XJIMM-20250703).

[0044] 1.2 Cell Strain The mouse AKR esophageal cancer cell line was purchased from Mirror & Point (Shanghai) Cell Technology Co., Ltd.

[0045] 1.3 Drugs The Inula salsoloides (Turcz.) Ostenf. medicinal material used in this experiment was identified by Researcher He Jiang from the New Drug Research Center of Xinjiang Uygur Autonomous Region Institute of Materia Medica as the whole herb of Inula salsoloides, a plant belonging to the genus Inula in the Asteraceae family Inula salsoloides (Turcz.) Ostenf. The specimen of Inula salsoloides is preserved in Xinjiang Uygur Autonomous Region Institute of Materia Medica.

[0046] 1.4 Reagents Phosphate-buffered saline (PBS, catalog number PB180327), Wuhan Pronosei Life Sciences Co., Ltd.; DMEM high glucose medium (catalog number PYG0073), Wuhan Boster Biological Engineering Co., Ltd.; Fetal bovine serum (FBS, catalog number SH30406.05), HyClone New Zealand; 0.25% trypsin (catalog number FG301-01), Beijing TransGen Biotech Co., Ltd.; Sodium carboxymethyl cellulose (CMC-Na, catalog number 30036328), Sinopharm Chemical Reagent Co., Ltd.; 4% PFA fixative (general tissue fixative, catalog number 25423982), Beijing Lanjieke Technology Co., Ltd.; Hematoxylin staining solution (catalog number BA4097) and eosin staining solution (catalog number BA4098) were purchased from Zhuhai Beso Biotechnology Co., Ltd.; Tumor necrosis factor-α (TNF-α), interleukin-10 (IL-10), and interleukin-1β (IL-1β) were purchased from Zhuhai Beso Biotechnology Co., Ltd. IL-1β and interleukin-6 (IL-6) kits (Wuhan E-EL-M3063, E-MSEL-M0031, E-EL-M0037, E-EL-M0044, Wuhan E-EL-M Biotechnology Co., Ltd.); alkaline phosphatase (ALP) assay kit (AMP buffer method), alanine aminotransferase (ALT) assay kit (IFCC method), aspartate aminotransferase (AST) assay kit (IFCC method), creatinine (CREA) assay kit (sarcosine oxidase method), and urea (UREA) assay kit (UV-glutamate dehydrogenase method), batch numbers 140325002, 140125008, 140225007, 141124006, 141323017, all purchased from Shenzhen Mindray Bio-Medical Electronics Co., Ltd.; ethanol, petroleum ether, ethyl acetate, and Tween-80 were all analytical grade; distilled water was used for the experiments.

[0047] 1.5 Instruments HERA cell-150i CO2 cell incubator, Thermo Fisher Scientific, USA; AB135-S electronic analytical balance, Mettler Toledo Instruments Ltd.; TGL-16K benchtop high-speed refrigerated centrifuge, Hunan Xiangyi Laboratory Instrument Development Co., Ltd.; CX21 optical microscope, OLYMPUS, Japan; IN014-2010 pathological sectioner, LEICA GmbH, Germany; BMJ-A embedding machine, Changzhou Zhongwei Electronic Instruments Co., Ltd.; BS-480 fully automated biochemical analyzer, Mindray.

[0048] 2. Methods 2.1 Drug Preparation An appropriate amount of whole herb powder of *Inula japonica* was extracted with 95% ethanol and 50% ethanol as extraction solvents at a material-to-liquid ratio of 1:20, and then extracted three times for 2 hours each time. The extracts were combined, and the solvent was recovered under reduced pressure to obtain 2000 mL of the herbal solution. Subsequently, a gradient extraction method was used to extract the above-prepared herbal solution sequentially with 2000 mL each of equal volumes of petroleum ether and ethyl acetate, three times for 2 hours each time. The corresponding extracts were combined, and the solvent was recovered under reduced pressure to obtain the petroleum ether extract and the ethyl acetate extract. The ethyl acetate extract (yield 12.74%) was ground, sieved, and an appropriate amount was accurately weighed and placed in a centrifuge tube. Anhydrous ethanol was used as the solvent, and the solution was vortexed and continuously stirred to ensure complete dissolution. To obtain a uniform and stable drug solution, Tween 80 and 0.5% sodium carboxymethyl cellulose (CMC-Na) were added as suspending and thickening agents, and the solution was brought to the required concentration, with the final volume fractions of anhydrous ethanol and Tween 80 both controlled below 10%. The mass of drug powder weighed for different concentration groups varied, but the total volume of solvent used remained consistent.

[0049] 2.2 Cell Culture Logarithmic growth phase mouse AKR esophageal cancer cells were taken, washed 3 times with PBS (3 ml each time), and 500 μL of trypsin was added and digested in an incubator for 2 min. Digestion was terminated by adding DMEM high glucose complete medium containing 10% fetal bovine serum and 1% penicillin-streptomycin. The cells were then pipetted and mixed to prepare a single-cell suspension for counting and use.

[0050] 2.3 Animal grouping, model establishment, and drug administration Forty-eight SPF-grade experimental C57BL / 6 mice with balanced body weight were selected. After acclimatization, eight mice were randomly selected as blank control group, and the remaining 40 mice were used to establish a subcutaneous xenograft model of esophageal cancer.

[0051] Model establishment: Moisten the skin of the mouse's right hind limb with an alcohol swab to fully expose the pre-injection site. After strict disinfection of the operator's hands and the mouse injection site, a cell suspension was drawn and injected subcutaneously into the mice. The concentration of the cell suspension for axillary inoculation was 0.5 × 10⁻⁶. 7 Cells / mL. During injection, ensure a small needle insertion angle and press the injection site briefly after needle withdrawal to prevent cell suspension from overflowing from the injection site and affecting the modeling effect.

[0052] The dosages of *Symplocos salsa* in the SXF-L, SXF-M, and SXF-H groups were 0.2, 0.4, and 0.8 g / kg, respectively (determined based on preliminary experiments), administered via gavage (ig). The positive control group received cisplatin 5 mg / kg (determined based on preliminary experiments) via injection (iv). Administration was once daily, starting the day after tumor inoculation. The blank control group and model group mice received an equal volume of physiological saline via gavage once daily. All groups underwent intervention for 12 consecutive days. Body weight and tumor volume were measured daily, and mortality was observed.

[0053] 2.4 Measurement of body weight and behavioral observation of mice in each group During the experiment, the body weight of the mice was observed and recorded daily. The survival status of the mice in each group was observed using indicators such as body weight, mental state, food and water intake, stool, activity level, and fur.

[0054] 2.5 Measurement of tumor volume in each group of mice Tumor growth was observed approximately 6 days after inoculation, with a palpable tumor mass about the size of a soybean at the inoculation site in the mouse's armpit. The modeling success rate was 100%. Starting from day 6 of drug administration, the long axis (a) and short axis (b) of the subcutaneous xenograft were measured daily using a vernier caliper, and the tumor volume (V) was calculated using the formula V(mm²). 3 )=(a×b 2 ) / 2, and finally plot the tumor growth curve.

[0055] 2.6 Sample Collection 2.6.1 Fecal Sample Collection Fecal samples were collected from mice one day before sacrifice, and the entire process was performed aseptically in a laminar flow hood in the animal facility. Before collection, the laminar flow hood was pre-sterilized for 30 minutes, ventilated, and then disinfected with 75% ethanol. 1.5 mL sterile cryovials were pre-numbered according to experimental groups. Abdominal massage was used to induce defecation in mice, and 2–4 fecal pellets were collected from each mouse and placed into the corresponding cryovial. These pellets were then rapidly frozen in liquid nitrogen and subsequently transferred to an ultra-low temperature freezer at -80°C for storage.

[0056] 2.6.2 Tissue Sample Collection Mice were fasted for 12 hours prior to sample collection, but had free access to water. Blood was drawn from the carotid artery and placed in a 1.5 mL EP tube to collect serum. After blood collection, the mice were euthanized by cervical dislocation, the tumor was quickly dissected, and the tissue was cleaned with physiological saline. After blotting with filter paper, each tumor was weighed and the weight recorded. The tumor tissue was divided into two parts: one part was fixed with 4% paraformaldehyde fixative for H&E staining; the other part was frozen in liquid nitrogen for later use.

[0057] 2.7 H&E staining of tumor tissue: The prepared paraffin sections were first dewaxed and then subjected to a gradient rehydration process: the sections were immersed in xylene I and xylene II for 10 min each, followed by two stages of treatment with anhydrous ethanol for 5 min each, and then successively treated with 95%, 90%, 80%, and 70% ethanol solutions for 5 min each. Finally, they were rinsed with distilled water for later use. For nuclear staining, Harris hematoxylin was used, with the staining time controlled at 3–8 min. After rinsing with tap water, the sections underwent brief differentiation with 1% hydrochloric acid-alcohol solution, rinsed with running water, and then blued again with 0.6% ammonia solution, followed by thorough rinsing with running water. Subsequently, eosin staining was performed on the cytoplasm, with the sections immersed in eosin working solution for 1–3 min. After staining, a gradient dehydration and clearing process was carried out: the sections were dehydrated for 5 min each with two stages of 95% ethanol and two stages of anhydrous ethanol, and then cleared with xylene I and xylene II for 5 min each. After the sections were slightly dried, they were mounted with neutral resin, and the tissue morphology was observed under an optical microscope. Field images were acquired and subsequent statistical analysis was performed.

[0058] 2.8 Serum Biochemical Indicators Detection Take the collected serum as described above, and apply it at 3000 rpm. -1 Centrifuge for 15 minutes, collect the supernatant serum, and use a fully automated biochemical analyzer to detect ALP, ALT, AST, CREA, and UREA levels. Store the remaining serum at -80 °C for ELISA testing.

[0059] 2.9 ELISA method for detecting TNF-α, IL-10, IL-6, and IL-1β levels Enzyme-linked immunosorbent assay (ELISA) was used, strictly following the kit instructions, to detect the levels of TNF-α, IL-10, IL-6, and IL-1β in each group of samples. To simultaneously reflect the systemic inflammatory state and the inflammatory level of the tumor microenvironment, this invention uses two sample sources for detection: TNF-α and IL-10 are detected using serum samples to assess the level of inflammatory factors in systemic circulation, while IL-6 and IL-1β are detected using tumor tissue homogenate supernatant. This approach not only compensates for the insufficient amount of serum samples but also more directly reflects the inflammatory response characteristics of the tumor microenvironment.

[0060] Tumor tissue homogenate preparation: The tissue was rinsed with pre-cooled PBS, weighed, and placed in a clean centrifuge tube. The tissue was then minced, and the corresponding volume of PBS and a certain amount of steel balls were added. The homogenate was then homogenized in a high-speed homogenizer. Finally, the homogenate was centrifuged at 2-8℃ and 8000 r / min for 5-10 min, and the supernatant was collected for analysis.

[0061] 2.10 Intestinal flora detection Fecal samples from section "2.6.1" were sent to Shanghai Baipu Biotechnology Co., Ltd. for high-throughput sequencing analysis of the 16S rDNA gene of gut microbiota. Total genomic DNA was extracted from the fecal samples using a specialized kit, and its purity and integrity were verified by agarose gel electrophoresis and nucleic acid quantification. Using the total DNA as a template, universal primers (with barcode tags) were designed for PCR amplification targeting the V3-V4 variable region of the 16S rDNA gene. The upstream primer was 338F (ACTCCTACGGGAGGCAGCA), and the downstream primer was 806R (GGACTACHVGGGTWTCTAAT), with an amplification product length of 480 bp. The amplification product was purified by magnetic bead method, and a library was constructed. After passing the library test, paired-end sequencing was performed using the Illumina platform (read length 2×250 bp). The raw data was de-noising and cleaned to obtain CleanReads, which were then compared with the SILVA database for species annotation, obtaining species composition information at the phylum, class, order, family, and genus levels. Subsequently, through species composition, alpha diversity, beta diversity, LEfSe analysis and differential microbial screening, the richness, evenness, intergroup differences and metabolic function prediction of the gut microbiota in the samples were clarified, and a comprehensive detection and analysis of the gut microbiota was completed.

[0062] 2.11 Statistical Methods GraphPad Prism 9 software was used for statistical analysis of the data. Quantitative data that conformed to normality and homogeneity of variance were expressed as mean ± standard deviation. Mean squared (±s) indicates the statistical significance of data. One-way ANOVA was used for comparisons among multiple groups. The least significant difference t-test (LSD-t test) was used for pairwise comparisons between groups. The Kruskal-Wallis test was used for non-normally distributed continuous data. The significance level was set at α=0.05.

[0063] 3. Results 3.1 Effects of *Capsella bursa-pastoris* on the growth of subcutaneous xenografts in tumor-bearing mice Seven days after mice were inoculated with AKR cells under their armpits, soybean-shaped tumor nodules were palpable in all animals, with a tumor formation rate of 100%, indicating that the subcutaneous xenograft model had been successfully established.

[0064] like Figure 1As shown, mice in the Control group were in good condition and active, with smooth fur. In the Model group, as the tumor volume gradually increased, the mice became lethargic, less active, had rough fur, and experienced hair loss. Mice in the treatment groups were in better condition than those in the Model group. Compared with the Control group, the Model group showed a decrease in body weight, starting from day 7 (P<0.01). Compared with the Model group, the DDP group showed a decrease in body weight, with a difference starting from day 8 (P<0.05), the SXF-M group showed an increase in body weight, with a difference starting from day 10 (P<0.01), and the SXF-H group showed an increase in body weight, with a difference starting from day 9 (P<0.05).

[0065] Tumor growth curve (see) Figure 2 The results showed that the tumor volume in the Model group increased rapidly, while the growth of each treatment group was inhibited in a dose-dependent manner. Compared with the Model group, the tumor growth of the DDP group was significantly slowed from day 8 of administration, the SXF-H group from day 9, and the SXF-M group from day 12 (P<0.05); the tumor inhibition effect at the end of the experiment was best in the SXF-H group and the DDP group (P<0.01).

[0066] Data on tumor weight and tumor inhibition rate in each group ( Figure 3 , Figure 4 Table 1 shows that the average tumor weight of the SXF-L, SXF-M, SXF-H and DDP groups was significantly lower than that of the model group (P<0.01), and the corresponding tumor inhibition rates were 34.57%, 46.28%, 59.57% and 72.34%, respectively.

[0067] In summary, *Rhizoma Scutellariae* can inhibit the growth of esophageal cancer tumors in tumor-bearing mice in a dose-dependent manner, demonstrating clear in vivo antitumor activity.

[0068] Table 1. Tumor weight and tumor inhibition rate in mice (Mean±SD, n=6)

[0069] Note: Compared to the model group, P <0.05, P <0.01 3.2 Morphological changes in tumor tissue of tumor-bearing mice The effects of *Rhizoma Senna* on tumor cell morphology were investigated using H&E staining. Results (see...) Figure 2The results showed that in the Model group, tumor cells were densely packed, with deeply stained nuclei, active mitotic figures, significant microvascular proliferation, and sparse necrotic areas. With increasing doses of *Rubus idaeus*, the extent of tumor necrosis gradually increased, tumor cell density decreased, and inflammatory cell infiltration was observed in the tumor stroma. The degree of pathological morphological improvement in the SXF-H group was similar to that in the positive control DDP group. 3.3 Effects of *Rubus idaeus* on liver and kidney function indicators in mice. Serum alkaline phosphatase (ALP), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) are important biochemical indicators for assessing liver function, while creatinine (CREA) and blood urea nitrogen (UREA) are important biochemical indicators for assessing kidney function. To evaluate the effects of *Vaccaria segetalis* on liver and kidney function in mice, this invention detected the levels of ALP, ALT, AST, CREA, and UREA in mouse serum. The results are as follows: Figure 7 As shown in Table 2, compared with the Control group, the Model group showed significantly increased levels of ALT, AST, and UREA (P<0.01), and ALP and CREA levels showed an increasing trend, but the differences were not statistically significant (P>0.05), indicating that tumors in the right axilla can cause secondary liver and kidney function damage in mice, and the tumor-bearing model of this invention was successfully constructed. Compared with the Model group, the DDP group showed a further significant increase in AST level (P<0.01), while ALP, ALT, CREA, and UREA levels increased slightly, but there were no statistically significant differences (P>0.05). The SXF-H group significantly downregulated ALT (P<0.05), AST (P<0.05), and UREA (P<0.01) levels, while the SXF-L and SXF-M groups had no significant effect on any of the indicators (P>0.05). None of the drug treatments significantly regulated ALP and CREA levels (P>0.05).

[0070] In summary, *Vigna angularis* significantly alleviated abnormally elevated ALT, AST, and UREA levels in tumor-bearing mice, with a clear dose-dependent effect. Only the SXF-H group showed a significant protective effect, while the SXF-L and SXF-M groups showed insufficient efficacy, which is a common pattern in the in vivo effects of natural plant extracts. The results indicate that *Vigna angularis* can effectively improve tumor-induced liver and kidney function damage and antagonize cisplatin-induced liver injury, demonstrating potential value as an adjuvant therapy for tumors and in reducing chemotherapy-related organ toxicity.

[0071] Table 2 Liver and kidney function indices of mice in each group (Mean ± SD, n=6)

[0072] Note: Compared with the control group, P <0.05, P <0.01; compared with the model group, # P <0.05, ## P <0.01 3.4 ELISA detection of TNF-α, IL-10, IL-6, and IL-1β levels Serum inflammatory factor levels: Compared with the control group, the serum pro-inflammatory factor TNF-α level in the model group mice was significantly increased (P < 0.01), suggesting that tumor-bearing status can induce a significant systemic inflammatory response in the body. Compared with the model group, the serum TNF-α level in the treatment group was significantly decreased (P < 0.05 or P < 0.01), and the TNF-α level decreased significantly in a dose-dependent manner with increasing dosage. Figure 8 A). Serum anti-inflammatory factor IL-10 levels showed that the IL-10 level in the Model group was slightly higher than that in the Control group, but the difference was not statistically significant; compared with the Model group, there were no significant changes in IL-10 levels in any of the treatment groups (P>0.05), indicating that *Symplocos edulis* had no significant regulatory effect on serum IL-10. Figure 8 B).

[0073] Tumor tissue inflammatory factor levels: Compared with the Model group, the levels of IL-1β and IL-6 in the treatment group decreased in a dose-dependent manner. Figure 8 (C, D). Among them, the IL-1β levels in the SXF-M group, DDP group, and SXF-H group were significantly reduced (P < 0.05 or P < 0.01); in terms of IL-6 levels, the SXF-H group and DDP group were significantly reduced (P < 0.05 or P < 0.01), indicating that *Symplocos rubra* can effectively inhibit the overexpression of pro-inflammatory factors in the tumor microenvironment and improve the local inflammatory state of the tumor.

[0074] Table 3 Effects of *Capsella bursa-pastoris* on serum TNF-α and IL-10 levels in mice and on IL-1β and IL-6 levels in mouse tumor tissues (Mean ± SD, n=6)

[0075] Note: Compared with the control group, P <0.05, P <0.01; compared with the model group, # P <0.05, ## P <0.01 3.5 16S rDNA Investigation of the Effects of Radix Rubra salsa Intervention on Gut Microbiota in Tumor-Bearing Mice 3.5.1 ASV / OUT Cluster Analysis Venn diagram analysis was used to illustrate the distribution and overlap of amplicon sequence variants (ASVs) among the Control, Model, and SXF-H groups. A total of 686 shared ASVs were found across the three groups. The Control group contained 2539 unique ASVs, while the Model and SXF-H groups contained 2611 and 3554 unique ASVs, respectively. Pairwise comparisons showed 243 overlapping ASVs between the Control and Model groups, 313 overlapping ASVs between the Control and SXF-H groups, and 322 overlapping ASVs between the Model and SXF-H groups.

[0076] 3.5.2 Alpha Diversity Analysis 3.5.2.1 Species Accumulation Curve like Figure 10 As shown, the sparse curve gradually stabilizes with increasing sequencing depth, indicating that the current sequencing depth is sufficient to cover most of the microbial diversity in each sample.

[0077] 3.5.2.2 Alpha Diversity Index Alpha diversity analysis showed no statistically significant difference in Shannon index among the groups. Compared to the Control group, the Model group showed a significantly lower Simpson index (P<0.01), indicating a decrease in community evenness under modeling conditions. Notably, neither the Shannon nor Simpson index of the SXF-H group differed significantly from the Control group. Regarding richness estimation, the Chao1 and ACE indices of the Model and SXF-H groups were slightly higher than the control group, but the differences between groups were not statistically significant (P>0.05). See Table 4.

[0078] Table 4 Comparison of gut microbiota abundance and diversity in mice of different groups (Mean±SD, n=5)

[0079] Note: Compared with the blank group, represent P <0.01 3.5.3 Beta Diversity Analysis β-diversity analysis based on principal coordinate analysis (PCoA) revealed significant segregation of the gut microbiota among the three groups. PC1 and PC2 explained 43.42% and 17.89% of the total variance, respectively. The Model and Control groups formed distinct clusters, indicating that model induction significantly altered the gut microbiota structure. The SXF-H group was clearly separated from the Model group and more closely resembled the Control group, suggesting that the intervention reshaped the gut microbiota composition. See [link to relevant documentation] Figure 11 .

[0080] 3.5.4 Species Composition Analysis At the phylum level, the fecal microbial communities in all groups were dominated by Bacteroidetes and Firmicutes. In the Control group, Bacteroidetes had the highest abundance, followed by Firmicutes and Verrucomicrobia. In the Model group, Bacteroidetes abundance decreased, Firmicutes abundance increased accordingly, while Verrucomicrobia abundance decreased significantly. In the SXF-H group, Bacteroidetes were significantly enriched and became the absolutely dominant phylum, while Firmicutes abundance decreased compared to the Model group. See [link to relevant documentation]. Figure 12 .

[0081] At the family level, Xanthomonadaceae was one of the major families in all groups. Its relative abundance decreased slightly in the Model group compared to the Control group, but increased in the SXF-H group. Conversely, Vibrionaceae was significantly enriched in the Model group, while Verrucomicrobiaceae was significantly reduced. See Figure 13 .

[0082] At the genus level, the Control group is characterized by *Isobacterium* (…). Allobaculum ) and Akkermansia ( Akkermansia ) is the main genus, while Lactobacillus ( Lactobacillus ) and Bacteroides ( Bacteroides The abundance of *Lactobacillus* was relatively low. In the Model group, *Lactobacillus* was significantly enriched, while the abundance of *Isobacterium* and *Ackermania* decreased significantly. In contrast, the abundance of *Lactobacillus* decreased in the SXF-H group, while the abundance of *Bacteroides* and *Prevotella* was higher. Prevotella The abundance of *Akermansia* and *Akermansia* increased, indicating that the microbial composition had partially recovered to levels close to the Control group. Overall, these results suggest that modeling induced disruption of the gut microbial composition, while drug administration partially restored the microbial structure to the Control group state. See [link to relevant documentation] Figure 14 .

[0083] 3.5.5 LEfSe Analysis The linear discriminant analysis (LEfSe) method was used (LDA score > 2.0). P The differentially enriched microbial groups among the Control, Model, and SXF-H groups (<0.05) were identified. The results showed that each group had different microbial characteristics.

[0084] In the Control group, the discriminative taxa primarily belong to the phylum Verruciformis, including the genus *Akermansia* and the family Verruciformisaceae. Additionally, members of the family *Erysipelotrichaceae*, particularly *Iserobacteria*, and groups belonging to the phylum *Deferribacterota* were also identified. The families *Enterobacteriaceae* and *Helicobacter* were also included. Helicobacter ) and Spirulina genus ( Mucispirillum Features such as ) were also identified as discriminative features in this group.

[0085] The Model group is characterized by enrichment of multiple taxa within the Firmicutes phylum, particularly those belonging to the class Bacilli and order Lactobacillales. Among these, *Lactobacillus* was identified as a key discriminant genus. Furthermore, other Firmicutes-related taxa, including members of the families Lachnospiraceae, Streptococcaceae, and Clostridiumles, as well as the candidate phylum TM7, were also significantly enriched.

[0086] In contrast, the SXF-H group showed a significant enrichment of taxa associated with the phylum Bacteroidetes, including the orders Bacteroidales and families Bacteroidaceae, with the genus Bacteroides identified as a key biomarker. Furthermore, the family Bifidobacteriaceae and its representative genus Bifidobacterium (…) were also enriched. Bifidobacterium ), and members of the phylum Fusobacteriota, including the family Fusobacteriaceae and the genus Cetacea ( Cetobacterium Enrichment also occurs. See Figure 15 .

[0087] 3.5.6 KEGG Function Prediction Based on the KEGG database, the PICRUSt2 assay was used to predict the metabolic functional potential of the gut microbiota. Differential pathways were screened using ANOVA, and post-hoc comparisons were performed using the Tukey-Kramer test, with Bonferroni analysis also conducted. P Value correction, P<0.05 indicates a significant difference. The functional pathways in the Control group, Model group, and SXF-H group showed significant clustering differences.

[0088] The Control group is primarily enriched in pathways related to core metabolic processes, including amino acid biosynthesis (such as phenylalanine, tyrosine, and tryptophan biosynthesis; glycine, serine, and threonine metabolism), energy metabolism (oxidative phosphorylation and the tricarboxylic acid cycle), and cofactor biosynthesis (folate and biotin metabolism). Pathways related to cellular homeostasis are also prominent, such as the NOD-like receptor signaling pathway, peroxisome function, endoplasmic reticulum protein processing, and lipopolysaccharide biosynthesis.

[0089] In contrast, the Model group showed significant enrichment in pathways related to carbohydrate metabolism (starch and sucrose metabolism, glycolysis / gluconeogenesis, propionic acid metabolism), nucleotide metabolism (purine and pyrimidine metabolism), lipid metabolism (fatty acid biosynthesis), and amino acid metabolism (D-alanine, β-alanine, and tyrosine metabolism). Furthermore, pathways related to microbial adaptation and environmental response were upregulated, including the phosphotransferase system (PTS), protein secretion, and pathways associated with microbial infection and degradation of exogenous substances.

[0090] The functional characteristics of the SXF-H group were intermediate between the Control and Model groups: some pathways recovered to the Control group pattern, while others remained close to the Model group. These results indicate that the intervention remodeled the metabolic function of the gut microbiota to some extent, contributing to the restoration of functional homeostasis. See [link to relevant documentation]. Figure 16 .

[0091] Compared to the Model group, the Control group was primarily enriched in pathways related to anesamcinolone biosynthesis, secondary bile acid biosynthesis, and vancomycin-like antibiotic biosynthesis. Notably, these pathways showed a recovery trend in the SXF-H group, indicating that the functional potential of the microorganisms was partially restored after intervention.

[0092] Conversely, pathways related to D-amino acid metabolism (such as D-alanine metabolism, D-glutamine, and D-glutamate metabolism) were relatively more abundant in the Model group. These pathways are closely related to bacterial cell wall synthesis and stress adaptation, indicating that the microbial community is shifting towards a stress-responsive model under tumor conditions.

[0093] Furthermore, several core metabolic pathways remained relatively stable across groups, including alanine, aspartic acid, and glutamate metabolism; branched-chain amino acid (valine, leucine, and isoleucine) biosynthesis; peptidoglycan biosynthesis; folate-mediated one-carbon metabolism; and the degradation of other carbohydrates. This indicates that despite changes in composition, the core metabolic functions of the gut microbiota were largely maintained.

[0094] Overall, these findings suggest that tumor induction is associated with alterations in microbial metabolic function, particularly in pathways related to stress response and amino acid metabolism, and that drugs may partially restore specific metabolic functions, including those involved in bile acid metabolism. See Figure 17 .

[0095] 4. Discussion Esophageal cancer is characterized by its strong malignant proliferative capacity and poor prognosis. Current clinical treatments suffer from significant toxic side effects and drug resistance. Therefore, the discovery of safe and effective natural anti-tumor drugs is of great research value. This invention utilizes transplantable tumors to establish tumor models, which offer advantages such as rapid tumor formation, easy control of transplanted cell numbers, and minimal individual variability. A C57BL / 6 mouse esophageal cancer xenograft model was successfully constructed with a 100% tumor formation rate. The intervention effect of *Vigna angularis* on the growth of xenografts in tumor-bearing mice was observed, clarifying its in vivo anti-esophageal cancer efficacy and providing experimental evidence for its subsequent development and application.

[0096] In the previous stage of this application, multi-stage separation and purification were carried out on the total ethanol extract of *Rhizoma Scutellariae*, resulting in the isolation of 18 monomeric compounds. Among them, two sesquiterpenes, *Eupatorium fortunei* lactone and ovomycin, were isolated from this plant for the first time. In vitro MTS proliferation inhibition experiments showed that the above two monomers, as well as the petroleum ether and ethyl acetate fractions of *Rhizoma Scutellariae*, exhibited significant inhibitory activity against the proliferation of A549 human lung cancer, BEL-7402 human liver cancer, and HT-29 human colon cancer cells. The core active pharmaceutical ingredients of *Rhizoma Scutellariae* that exert its antitumor effect are mainly moderately polar sesquiterpenes and flavonoids, with these active components primarily concentrated in the ethyl acetate extract. This application further conducted in vitro activity screening using human esophageal cancer cells (Eca109), comparing the inhibitory effects of the petroleum ether, ethyl acetate, and water fractions on the proliferation of esophageal cancer cells. The results showed that the ethyl acetate fraction exhibited the best antitumor activity. Based on the previous material basis and in vitro cell activity screening results, this invention selected the ethyl acetate fraction of *Rhizoma Scutellariae* as the test drug for in vivo experiments. The LD50 of the ethyl acetate fraction was determined accordingly. 50 Acute toxicity data were used to determine the recommended dosages: low dose 0.2 g / kg, medium dose 0.4 g / kg, and high dose 0.8 g / kg; the axillary tumor cell inoculation concentration was 0.5 × 10⁻⁶. 7 The concentration of cells / mL is the appropriate concentration selected from previous preliminary experiments.

[0097] Body weight and general condition in mice are important macroscopic indicators for evaluating drug toxicity and tolerance. In this experiment, after 12 days of continuous drug administration, mice in the model group exhibited abnormalities such as lethargy, reduced activity, and stunted growth due to tumor burden, with significantly suppressed physiological metabolism. Cisplatin further reduced body weight, posing a risk of systemic damage. In contrast, medium and high doses of *Rhizoma Scutellariae* effectively improved the overall condition of the mice and restored their body weight. This indicates that *Rhizoma Scutellariae* has good safety at effective tumor-suppressing doses, effectively alleviating tumor-mediated systemic consumption and maintaining physiological homeostasis in tumor-bearing mice.

[0098] Tumor growth curves, tumor weight, and tumor inhibition rate can dynamically and quantitatively evaluate the in vivo antitumor activity of drugs. The results of this experiment showed that the tumor volume in the model group exhibited a sustained and rapid proliferation, while each dose of *Rhizoma Scutellariae* (a type of herb) dose-dependently inhibited tumor proliferation, with higher doses showing earlier onset of tumor inhibition and significantly greater final tumor inhibition effect. Statistical analysis indicated that *Rhizoma Scutellariae* significantly inhibited the growth of esophageal cancer xenografts (P<0.01), clarifying its good in vivo anti-esophageal cancer proliferation pharmacological activity and demonstrating potential for further research and application.

[0099] HE staining of tumor tissue can directly reflect tumor proliferative activity, microangiogenesis, and the degree of tissue damage. The results of this invention show that the tumor cells in the model group were densely packed, exhibiting vigorous mitosis and significant angiogenesis, displaying typical malignant proliferative characteristics. Intervention with *Rhizoma Scutellariae* significantly improved tumor pathological morphology, effectively reduced tumor cell mitotic activity, inhibited intratumoral angiogenesis, remodeled the tumor stromal immune microenvironment, increased local inflammatory immune infiltration, and inhibited malignant tumor growth. Furthermore, this pathological improvement effect gradually increased with increasing dose, with the high-dose group showing improvement similar to the cisplatin-positive group. From a histological perspective, *Rhizoma Scutellariae* can exert its anti-esophageal cancer effect by inhibiting tumor proliferation, reducing angiogenesis, and regulating the local inflammatory microenvironment, with high-dose intervention achieving a better tumor-suppressive effect.

[0100] The abnormal inflammatory microenvironment of tumors is closely related to tumor proliferation and invasion. Classical pathways such as PI3K-Akt, NF-κB, and JAK-STAT can participate in the regulation of tumor inflammation and immune disorders by modulating the transcription of inflammatory factors such as TNF-α, IL-6, and IL-1β. The ELISA results of this invention showed that serum pro-inflammatory factor TNF-α was significantly upregulated in tumor-bearing mouse models, confirming that tumor burden can induce a systemic inflammatory response, consistent with the characteristics of tumor-mediated systemic inflammatory disorders. *Rhizoma Scutellariae* intervention dose-dependently downregulated serum TNF-α levels in mice, effectively alleviating the systemic inflammatory state in tumor-bearing mice, while having no significant regulatory effect on serum anti-inflammatory factor IL-10, indicating that its improvement of peripheral inflammation is mainly achieved by inhibiting pro-inflammatory pathways, rather than upregulating the expression of anti-inflammatory factors. In the local tumor microenvironment, *Rhizoma Scutellariae* significantly reduced the levels of pro-inflammatory factors IL-1β and IL-6 in tumor tissue, and the inhibitory effect was dose-dependent, with particularly significant improvement in the medium and high dose groups, similar to the effect of positive drug intervention. In summary, *Rhizoma Scutellariae* can simultaneously inhibit systemic inflammation and excessive local inflammation in the tumor, thereby reshaping the tumor inflammatory microenvironment and inhibiting malignant tumor progression. This is one of the important mechanisms by which it exerts its antitumor effect in vivo.

[0101] Liver and kidney function biochemical indicators are core criteria for evaluating drug safety in vivo and the degree of organ damage. The results of this invention show that tumor-bearing mice can cause abnormal increases in ALT, AST, and UREA, confirming that tumor burden can induce secondary liver and kidney function damage, further validating the model's effectiveness. The positive control drug cisplatin further increased AST levels in mice, suggesting its hepatotoxicity. However, intervention with *Rhizoma Scutellariae* (a type of rhizome) dose-dependently improved abnormal liver and kidney function in tumor-bearing mice. Only high-dose *Rhizoma Scutellariae* significantly downregulated ALT, AST, and UREA levels, effectively alleviating tumor-induced liver and kidney function damage and antagonizing cisplatin-induced liver damage; low and medium doses showed no significant effect. These results indicate that *Rhizoma Scutellariae* has good organ protection and drug safety, can effectively improve liver and kidney damage induced by tumors and chemotherapy drugs, and has potential applications in adjuvant tumor therapy and alleviating chemotherapy toxicity.

[0102] The results showed that tumor progression significantly disrupts gut microbiota homeostasis, and intervention with *Rhizoma Scutellariae* can restore gut microbiota structure and metabolic function to some extent. Alpha diversity analysis showed a significant decrease in the Simpson index in the model group, indicating reduced evenness of the microbial community under tumor conditions; while the Chao1 and ACE indices showed no significant changes, suggesting that tumor burden mainly affects microbial structure and dominant bacterial distribution, rather than overall species richness, which is consistent with some previous findings. Beta diversity analysis further confirmed significant clustering differences in the microbial community among the groups, indicating that tumor induction significantly remodels the gut microbiota structure. Notably, after drug intervention, the microbial structure partially shifted towards the control group, suggesting that *Rhizoma Scutellariae* has a certain gut microbiota restoration effect.

[0103] At the phylum level, Bacteroidota and Firmicutes were the dominant microbiota in all groups. In the model group, Bacteroidota abundance decreased while Firmicutes relatively increased, indicating that microbial metabolic homeostasis was disrupted under tumor conditions. Bacteroidota is typically associated with the degradation of complex polysaccharides and the generation of short-chain fatty acids (SCFAs), while Firmicutes have a strong energy extraction capacity; therefore, the imbalance between the two may reflect tumor-related metabolic reprogramming and altered host nutrient utilization. After intervention with *Rhizoma Scutellariae*, Bacteroidota abundance recovered somewhat, which may have improved gut microbial metabolic homeostasis to some extent.

[0104] In addition, Verrucomicrobiota, especially in the model group Akkermansia Significantly reduced. Akkermansia Closely related to mucin degradation, mucus layer renewal, and intestinal barrier maintenance, a decrease in its abundance may indicate impaired intestinal epithelial integrity and increased inflammatory sensitivity in tumor states. Conversely, after drug intervention... Akkermansia Partial recovery suggests improved intestinal mucosal homeostasis. Furthermore, in the model group... Allobaculum The reduction may also mean a decrease in the production of SCFAs, especially butyrate, which plays an important role in maintaining epithelial integrity and regulating inflammatory responses.

[0105] It is worth noting that in the model group Lactobacillus Significant enrichment. Although Lactobacillus While often considered beneficial bacteria, their abnormal proliferation under tumor conditions is more likely an adaptive response to altered gut environment conditions than a purely protective one. Tumor-related gut environment changes, such as increased fermentable substrates, altered gut pH, and inflammatory stress, may all promote the growth of acid-tolerant and fermentative bacteria, such as... LactobacillusThe amplification of [something]. Furthermore, excessive lactate accumulation may also participate in the formation of an immunosuppressive microenvironment and metabolic imbalance. Therefore, in the model group... Lactobacillus An increase in these levels is more likely to reflect a gut microbiota imbalance and stress adaptation.

[0106] LEfSe analysis further revealed the specific microbial biomarkers in each group. The model group was mainly enriched in Firmicutes-related taxa, especially... Lactobacillus The control group, on the other hand, was enriched with... Akkermansia and Allobaculum Mucosal-associated flora, including those found in the mucosa. After drug intervention, Bacteroides , Prevotella and Akkermansia Beneficial bacteria showed a certain degree of recovery. Bacteroides and Prevotella were involved in polysaccharide fermentation and SCFAs production, indicating that Rheum palmatum may promote intestinal metabolic function and epithelial homeostasis by improving microbial fermentation capacity.

[0107] PICRUSt2 functional prediction analysis showed that the model group was enriched with pathways related to carbohydrate metabolism, including glycolysis / gluconeogenesis, starch and sucrose metabolism, and the phosphotransferase system (PTS), suggesting enhanced microbial fermentation metabolism and accelerated carbon source utilization under tumor conditions. This result is consistent with the model group. Lactobacillus The amplification was consistent with that of the model group. In addition, pathways such as D-alanine metabolism and D-glutamine / D-glutamate metabolism were also enriched in the model group, suggesting enhanced bacterial stress adaptation and cell wall remodeling under tumor-associated microbiota imbalance.

[0108] Conversely, pathways related to oxidative phosphorylation, the tricarboxylic acid cycle, secondary bile acid biosynthesis, and amino acid biosynthesis were relatively enriched in both the control and drug groups. Secondary bile acids, as important microbial metabolites, participate in host lipid metabolism, immune regulation, and the maintenance of intestinal homeostasis. The partial recovery of these pathways after *Scutellaria baicalensis* intervention suggests that it may help improve microbial metabolic balance and intestinal physiological function. Furthermore, the recovery of the flora associated with SCFA generation further supports the view that drug intervention helps improve epithelial barrier function and suppress inflammatory responses.

[0109] However, caution is still needed when interpreting the predicted microbial functions. The KEGG functional analysis in this invention was inferred from 16S rRNA gene sequencing data using a predictive algorithm, rather than being directly determined through metagenomics or metabolomics. Therefore, these results reflect potential functional capabilities rather than actual metabolic activity. Furthermore, although this invention observed a significant association between changes in the gut microbiota and tumor status, the causal relationship between specific microbiota and antitumor effects remains unclear. Future research should combine metagenomics, metabolomics, and host immunology analyses to further elucidate the specific mechanisms by which *Rhizoma Scutellariae* regulates the gut microbiota and exerts its antitumor effects.

[0110] In vivo experiments of this invention have confirmed that tumor burden can significantly induce intestinal flora structure disorder, dominant flora imbalance and abnormal microecological function in tumor-bearing mice, while causing liver and kidney function damage and local inflammatory microenvironment disorder in the tumor. Senna rhubarb can safely and effectively inhibit the proliferation of subcutaneous esophageal cancer xenografts, reduce the expression of pro-inflammatory factors in the body and tumor tissues in a dose-dependent manner, improve the malignant pathological phenotype of tumors, regulate the intestinal flora community structure, restore intestinal microecological homeostasis, and thus exert a significant in vivo anti-tumor effect.

[0111] This invention preliminarily confirms that *Rhizoma Scutellariae* has good in vivo anti-esophageal cancer effects and drug safety. It can effectively inhibit the growth of transplanted tumors by improving the inflammatory microenvironment of tumors, reversing the intestinal flora disorder and organ damage caused by tumor bearing, and provides an experimental basis for further in-depth research on its anti-esophageal cancer effects and microecological regulation mechanisms.

[0112] The above description is only a preferred embodiment of the present invention. It should be noted that those skilled in the art can make several improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Application of Rhododendron simsii extract in the preparation of anti-esophageal cancer drugs.

2. The application according to claim 1, characterized in that, The extract of *Solanum tuberosum* is one of petroleum ether extract, ethyl acetate extract, or water extract.

3. The application according to claim 1, characterized in that, The esophageal cancer mentioned is esophageal squamous cell carcinoma.

4. The application according to any one of claims 3, characterized in that, The drug contains an effective amount of Senna rhododendron extract and pharmaceutically acceptable excipients.

5. The application according to claim 4, characterized in that, The drug is an oral preparation.

6. Application of *Rubus idaeus* extract in the preparation of drugs for alleviating liver and kidney damage caused by chemotherapy drugs.

7. The application according to claim 6, characterized in that, The chemotherapy drug is cisplatin.

8. A pharmaceutical composition for treating esophageal cancer, characterized in that, It contains an effective amount of raspberry extract and pharmaceutically acceptable excipients.

9. The pharmaceutical composition according to claim 8, characterized in that, The pharmaceutical composition is an oral preparation.

10. Application of Ethyl Acetate Extract of Rhizoma Serratum in the Preparation of Drugs for Improving Intestinal Flora Disorders.

Images