Euphorbiaceae baliospermum plant baliospermum cordifolium stem ethyl acetate extract as well as preparation method and application thereof

Abstract

The invention relates to an ethyl acetate extract of stems of Euphorbiaceae Baliospermum as well as a preparation method and application of the ethyl acetate extract in the technical field of biological medicine, the ethyl acetate extract is prepared by the following steps: fully soaking Baliospermum cornutum with 95% ethanol, then heating and refluxing and extracting with ethanol, concentrating an extracting solution until no alcohol smell exists, and then suspending with water to obtain the ethyl acetate extract of the stems of the Euphorbiaceae Baliospermum cornutum. Adding petroleum ether, ethyl acetate and n-butyl alcohol which are equal to the suspension in volume for extraction respectively, collecting supernatant liquid, and concentrating the obtained ethyl acetate extract phase under the condition of reduced pressure or normal pressure to remove the solvent, so as to obtain the ethyl acetate extract. The viburnum cordifolium stem ethyl acetate extract disclosed by the invention can be used for preparing a medicine for treating colitis or colon cancer. The provided ethyl acetate extract of the fordia cordata stems can inhibit colon cancer and improve the enterotoxicity of chemotherapeutic drugs. The medicine source is natural, the preparation process is mature, and the extraction process is convenient and easy to operate.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to a plant of the genus *Euphorbia* (family Euphorbiaceae). Baliospermum yui Preparation method and application of ethyl acetate extract of stem. This extract can be used to prepare drugs for the prevention and treatment of intestinal mucosal damage and colon cancer induced by the chemotherapy drug irinotecan (CPT-11), and has a comprehensive effect of repairing the intestinal mucosal barrier, regulating intestinal immune homeostasis and synergistically enhancing anti-tumor activity. Background Technology

[0002] Irinotecan (CPT-11) is a widely used first-line chemotherapy drug for the treatment of advanced colorectal cancer, playing a key role in the FOLFIRI combination regimen. However, in clinical application, irinotecan often causes serious adverse reactions, especially dose-limiting toxicity—irinotecan-induced colitis (IIC). This adverse reaction manifests as diarrhea, intestinal mucosal damage, and significant inflammatory response, severely impacting patients' quality of life and chemotherapy adherence, becoming one of the main factors limiting its clinical application. Currently, research on the prevention and treatment of IIC induced by the chemotherapy drug irinotecan (CPT-11) mainly focuses on intestinal microecological regulators, anti-inflammatory drugs, and traditional Chinese medicine monomers. Existing studies have shown that natural products such as probiotics, resveratrol, flavonoids, and triterpenoid saponins can alleviate chemotherapy-related intestinal mucosal damage to some extent. However, most studies only focus on improving enteritis symptoms, lacking a comprehensive approach that considers both anti-tumor efficacy and protection against chemotherapy toxicity.

[0003] Plants of the Euphorbiaceae family are a rich resource of medicinal plants with diverse pharmacological activities, first recorded in the *Shennong Bencao Jing* (Shennong's Classic of Materia Medica). The chemical composition of these plants is complex and diverse, mainly including terpenes, phenanthrenes, flavonoids, and polyphenols. Their active components exhibit certain regularities and similarities across different genera and species, providing important scientific evidence for systematic research and medicinal development. Numerous studies have shown that natural compounds in Euphorbiaceae plants possess significant anti-inflammatory, antibacterial, antitumor, and antiviral pharmacological effects, thus holding significant application value in the modernization of traditional Chinese medicine research and new drug development.

[0004] With the deepening of research on plants in the Euphorbiaceae family, the genus *Euphorbia* (*Euphorbia*) has become increasingly important. Baliospermum Due to its unique pharmacological activity and potential application value, *Symplocos rubrum* (also known as spotted shrub) is gradually gaining attention. Baliospermum solanifolium*Symplocos rubrum* is a representative species of this genus and is explicitly recorded as an important medicinal plant in the *Flora of China*. The entire plant of *Symplocos rubrum* can be used medicinally, possessing traditional effects such as purgation, diuresis, swelling reduction, heat clearing and detoxification, and pain relief. Clinically, it is used to treat asthma, bronchitis, edema, jaundice, leprosy, leukemia, and various skin diseases. Modern pharmacological studies have confirmed that *Symplocos rubrum* extract possesses various biological activities, including anti-inflammatory, antibacterial, antioxidant, antitumor, and hepatoprotective effects, laying an important foundation for its application in the development of herbal medicines.

[0005] With spotted seed wood ( Baliospermum solanifolium ) of the same genus, Heartleaf Spotted Seed Tree ( Baliospermum yui The high morphological and systematic similarity among plants suggests potential commonalities in chemical composition and pharmacological activity. However, current research on *Euphorbia pulcherrima* remains limited, and its chemical composition, pharmacodynamic material basis, and mechanism of action have not been systematically elucidated. Existing literature indicates that terpenes, flavonoids, and polyphenols in Euphorbiaceae plants play important roles in anti-inflammation, antioxidant activity, and immunomodulation. Terpenes are considered the key material basis for their anti-inflammatory activity, while flavonoids and polyphenols are closely related to immune homeostasis regulation. Currently, there is no information on *Euphorbia pulcherrima* (a type of Euphorbiaceae plant). Baliospermum This study reports on the systematic research and application of extracts from the genus *Hemiberlesia lataniae* in an irinotecan-induced colitis and tumor comorbidity model. Therefore, exploring the mechanism of action and application value of ethyl acetate extract of *Hemiberlesia lataniae* in the prevention and treatment of gastrointestinal tumors and chemotherapy-related intestinal injury has significant innovative and potential clinical translational value.

[0006] Irinotecan-induced colitis (IIC) has a complex pathogenesis involving multiple factors such as immune dysregulation, oxidative stress, and impaired intestinal barrier function. Currently, there is a lack of safe, effective, and long-term clinical interventions, thus necessitating the search for natural drugs with good anti-inflammatory and intestinal protective effects. Previous studies have shown that extracts from *Heliotropium indicum* plants possess significant anti-inflammatory and antitumor activities, providing a theoretical basis for the application of these plants in the protection against chemotherapeutic drug toxicity and in synergistic therapy. However, specific applications of *Heliotropium indicum* (heartleaf *Heliotropium indicum*) remain to be explored. Baliospermum yui The systematic pharmacological studies of irinotecan are still lacking, and its potential value in the prevention and treatment of irinotecan-induced colitis and in the synergistic treatment of colorectal cancer has not yet been verified. It is urgent to conduct systematic evaluation through experimental studies. Summary of the Invention

[0007] To address the toxic side effects such as dose-limiting colitis caused by existing chemotherapy drugs for colon cancer, irinotecan (CPT-11), this invention aims to overcome the shortcomings of the prior art and provide a drug derived from natural sources. Specifically, this invention provides an ethyl acetate extract of the stem of *Euphorbia pulcherrima*, a plant in the Euphorbiaceae family, its preparation method, and its uses, which can be used to prepare drugs for treating colitis or colon cancer.

[0008] The technical solution provided by this invention is: a method for preparing an ethyl acetate extract from the stem of *Euphorbia pulcherrima*, a plant in the Euphorbiaceae family, comprising the following steps: S1: Extract from the ethyl acetate fraction of the stem of the heart-leafed variegated seed tree: Take the crushed stems of the heartleaf variegated seed tree, add 8-10 times its weight of 95% ethanol, soak at room temperature for 30-45 minutes, then reflux extract at 70-80℃ 2-5 times, 45-75 minutes each time; combine the extracts, filter coarsely through a filter cloth, and concentrate under reduced pressure by rotary evaporation at 40-50℃ until no alcohol odor remains, to obtain a concentrated extract; disperse the obtained concentrated extract in distilled water, and perform gradient extraction with petroleum ether and ethyl acetate in sequence, collect the ethyl acetate extract, concentrate under reduced pressure, and dry in an oven at 60-70℃ to obtain the ethyl acetate extract of the heartleaf variegated seed tree stem; S2: Extract of ethyl acetate fraction from the stem of *Heartleaf variegated* seed: The ethyl acetate extract of the stem of *Symplocos cuspidata* was dispersed in distilled water and then subjected to gradient extraction with equal volumes of petroleum ether, ethyl acetate, and n-butanol, three times for each solvent, each time for 25-35 min. The ethyl acetate extracts were combined and concentrated under reduced pressure at 40-50℃ using a rotary evaporator to a thick paste, and then dried in an oven at 60-70℃ to constant weight to finally obtain the ethyl acetate extract of the stem of *Symplocos cuspidata*.

[0009] Furthermore, in step S1, the sample is refluxed at 75°C three times, each time for 60 minutes.

[0010] Furthermore, in step S1, the rotary evaporation temperature is 45°C, and the drying temperature in the oven is 65°C.

[0011] Furthermore, in step S2, the rotary evaporation temperature is 45°C, and the drying temperature in the oven is 65°C.

[0012] The use of an ethyl acetate extract of the stem of *Euphorbia pulcherrima*, a plant in the Euphorbiaceae family, prepared according to the above method, is for the preparation of drugs for treating colitis or colon cancer. The colitis is induced by the chemotherapy drug irinotecan.

[0013] Compared with existing technologies, the ethyl acetate extract of the stem of the heartleaf variegated seed provided by this invention exhibits multiple beneficial effects in the fields of natural medicine and adjuvant therapy for anti-tumor drugs, specifically reflected in the following aspects: 1. It is derived from natural sources and has a mature preparation process. The heartleaf variegated sapwood used in this invention belongs to the Euphorbiaceae family and the Euphorbiaceae genus. This family of plants is abundant, and the extraction process is convenient, easy to operate, and highly controllable, which helps to ensure the stability and batch consistency of the extract.

[0014] 2. Highly targeted, addressing clinical pain points This invention directly addresses the thorny problem of dose-limiting colitis (IIC) caused by the clinical chemotherapy drug irinotecan (CPT-11). Severe diarrhea and intestinal mucosal damage caused by irinotecan often force the interruption or reduction of chemotherapy, severely impacting treatment efficacy and patients' quality of life. Currently, there is a lack of ideal drugs that can effectively protect against intestinal mucosal damage without affecting its anti-tumor efficacy. The extract of this invention aims to prepare a related drug that can prevent and treat irinotecan-induced intestinal mucosal damage and has a therapeutic effect on colon cancer, providing a potential new adjuvant therapy option for clinical practice and helping patients better complete chemotherapy cycles. Attached Figure Description

[0015] Figure 1 Flowchart for the preparation of ethyl acetate extract from the stem of the heartleaf variegated seed.

[0016] Figure 2 An experimental flowchart illustrating the use of ethyl acetate extract from the stems of *Heliotropium indicum* for the treatment of chemotherapy-induced intestinal mucosal damage.

[0017] Figure 3 A schematic diagram of the colon length in mice treated with ethyl acetate extract of the stem of the heartleaf variegated seed to treat intestinal mucosal damage induced by chemotherapy drugs.

[0018] Figure 4 Histological images of intestinal mucosal pathological damage induced by chemotherapy drugs, obtained from the ethyl acetate extract of the stem of the heartleaf variegated seed.

[0019] Figure 5 Flowchart of an experimental study on the use of ethyl acetate extract from the stems of the heartleaf variegated seed for the treatment of subcutaneous colon cancer.

[0020] Figure 6 This is a schematic diagram showing the tumor volume and size in mice treated with ethyl acetate extract of the stem of the heartleaf variegated seed for subcutaneous colon cancer.

[0021] Figure 7 Experimental flowchart for the treatment of in situ colon cancer with ethyl acetate extract of the stem of the heartleaf variegated seed.

[0022] Figure 8 This image shows the appearance of the colon, cecum, and tumor as a result of the use of ethyl acetate extract from the stem of the heartleaf variegated seed for the treatment of in situ colon cancer.

[0023] Figure 9 The image shows the results of colonic leakage index detection in mice with orthotopic colon cancer treated with ethyl acetate extract of the stem of the heartleaf variegated seed.

[0024] Figure 10 This is a schematic diagram of the in vivo imaging results of the colon treated with ethyl acetate extract of the stem of the heartleaf variegated seed for in situ colon cancer.

[0025] Figure 11The positive mode UPLC-Q-TOF-MS spectrum of the ethyl acetate extract of the stem of the heartleaf variegated seed is shown.

[0026] Figure 12 The negative mode UPLC-Q-TOF-MS spectrum of the ethyl acetate extract of the stem of the heartleaf variegated seed. Detailed Implementation Example 1

[0027] A method for preparing an ethyl acetate extract from the stem of *Haloxylon ammodendron*, a plant in the Euphorbiaceae family, comprises the following steps: S1: Extraction method of ethyl acetate extract from the stem of *Symplocos cuspidatum* (heart-leaved variegated seed): Take 20 kg of pulverized plant material, add 10 kg of 95% ethanol, soak at room temperature for 30 min, and then reflux extract three times at 75℃ for 60 min each time. Combine the extracts, coarsely filter through a filter cloth, and concentrate under reduced pressure at 45℃ using a rotary evaporator until no alcohol odor remains, obtaining a concentrated extract. Disperse the concentrated extract in distilled water, and perform gradient extraction with petroleum ether and ethyl acetate sequentially. Collect the ethyl acetate extract, concentrate under reduced pressure, and dry in an oven at 65℃ to obtain the ethyl acetate extract of the heart-leafed variegated seed stem. (See flowchart) Figure 1 ) S2: Extraction method of ethyl acetate extract from the stem of *Symplocos cuspidatum* (heart-leaved variegated seed): The ethyl acetate extract of the stem of *Symplocos cuspidata* was dispersed in distilled water and then subjected to a gradient extraction with equal volumes of petroleum ether, ethyl acetate, and n-butanol, three times for each solvent, for 30 min each time. The combined ethyl acetate extracts were concentrated to a thick paste under reduced pressure at 45°C using a rotary evaporator, and then dried to constant weight in a 65°C oven to obtain 83.4 g of the ethyl acetate extract of *Symplocos cuspidata* stem.

[0028] Detection method for ethyl acetate extract of heart-leafed variegated seed stem: The ethyl acetate extract of the stem of the heartleaf variegated seed was analyzed using ultra-high performance liquid chromatography-ion mobility-quadrupole time-of-flight mass spectrometry to obtain the UPLC-Q-TOF-MS spectrum of the ethyl acetate extract.

[0029] Mass spectrometry conditions were as follows: ESI ion source, positive ion mode detection; nebulizer N pressure 50 psi; dry gas N; flow rate 15 L / min; mass scan range 50-1200; automated 3-stage mass spectrometry.

[0030] The chromatographic conditions were as follows: column: ACQUITY UPLC® BEH C18 (100 mm × 2.1 mm id, 1.7 µm); binary gradient elution, phase A was 0.1% formic acid aqueous solution; phase B was acetonitrile, flow rate was 0.3 mL / min, column temperature was 40 ℃, injection volume was 2 μL, and the gradient elution program was as follows:

[0031] The results of the ethyl acetate extract of the heart-leafed variegated seed stem are shown in the figure. Figure 11 , Figure 12 As shown.

[0032] The ethyl acetate extract of the heartleaf variegated seed stem is used to prepare drugs for the treatment of colitis or colon cancer, especially for colitis induced by the chemotherapy drug irinotecan. Example 2

[0033] Evaluation of the in vivo anti-inflammatory activity of the ethyl acetate extract of the heart-leafed variegated seed stem: Drugs and reagents: Irinotecan injection (CPT-11) was purchased from Hainan Jinrui Pharmaceutical Co., Ltd. The test drug was the ethyl acetate extract of the stem of the heartleaf variegated seed tree (EtOAc), which was dissolved in physiological saline before use to prepare a solution of 100 mg / mL.

[0034] Laboratory animals: Twenty-four SPF-grade female C57BL / 6 mice (6–8 weeks old, weighing 18–22 g) were purchased from the Comparative Medicine Center of Yangzhou University. The mice were housed in a standard environment with a temperature of 22±2℃ and a 12 h / 12 ​​h light / dark cycle, with free access to food and water, and underwent acclimatization for at least 7 days before the experiment. All animal experimental protocols were approved by the Institutional Animal Care and Use Committee (IACUC), approval number YXYLL-2024-86. At the end of the experiment, the mice were anesthetized with isoflurane and then euthanized by carbon dioxide asphyxiation.

[0035] Experimental methods: Animal grouping: Mice were randomly divided into 4 groups (n=6 in each group): ① Blank control group (not subjected to CPT-11 modeling); ② Model group (subjected to CPT-11 modeling and administered physiological saline by gavage); ③ EtOAc group (not subjected to CPT-11 modeling, administered EtOAc 100 mg / kg by gavage only); ④ Treatment group (subjected to CPT-11 modeling and administered EtOAc 100 mg / kg by gavage).

[0036] The experiment employed a pre-drug regimen, with both the EtOAc group and the treatment group receiving EtOAc via gavage for three consecutive days. On day 4, CPT-11 was administered to establish the colitis model, and the same dose of EtOAc (100 mg / kg) was given concurrently during the modeling process. The colitis model was established via intraperitoneal injection of CPT-11 at a dose of 75 mg / kg for five consecutive days (see flowchart). Figure 2 ) Detection indicators and methods: Mice body weight was recorded daily during the experiment. Diarrhea severity was scored according to established criteria (Cooper et al., 1993; Lin et al., 2019): 0 points indicated normal stool, 1 point indicated mildly moist and soft stool, and 2 points indicated moderately moist and unformed stool. The Disease Activity Index (DAI) was calculated daily.

[0037] Table 1 Disease Activity Index (DAI) Table 1 Disease Activity Index (DAI)

[0038] Mice were sacrificed after the experiment, and cecal and colonic tissues were collected to measure intestinal length and assess the degree of colonic inflammation. Analysis of the in vivo anti-colitis evaluation results of the ethyl acetate extract of *Heliotropium indicum* stem: Table 1 shows that in the CPT-11-induced colitis model, the DAI score of mice in the model group gradually increased with the modeling time, indicating poorer fecal formation and varying degrees of rectal bleeding. Compared with the model group, the DAI score of the CPT-11 combined with EtOAc treatment group was significantly reduced, fecal moisture and diarrhea severity were significantly improved, the incidence of rectal bleeding was reduced, and overall inflammatory symptoms were effectively alleviated. The results indicate that the ethyl acetate extract of *Heliotropium indicum* stem can reduce CPT-11-induced intestinal mucosal damage and improve the disease activity index.

[0039] Compared with the control group, the colon length of mice in the chemotherapy group was significantly shortened, while mice in the EtOAc-only group showed no significant change. The colon length of mice in the combined chemotherapy and traditional Chinese medicine group was significantly longer than that of mice in the chemotherapy-only group (see...). Figure 3 ).

[0040] In HE-stained tissue sections, the model group mice exhibited significant intestinal mucosal pathological damage, mainly manifested as villus structure destruction, significant shortening or even breakage, disordered crypt arrangement, partial epithelial cell shedding, and high edema in the submucosa, accompanied by a large number of neutrophils and lymphocytes infiltrating, showing typical characteristics of chemotherapy-induced mucositis. In contrast, the intestinal tissue structure of mice treated with EtOAc combined with CPT-11 was significantly improved, with relatively intact villus length and morphology, clearer crypt structure, significantly reduced epithelial cell shedding, reduced submucosa edema, significantly reduced inflammatory cell infiltration, and a more intact overall mucosal barrier structure, indicating that EtOAc extract can effectively alleviate chemotherapy-induced intestinal mucosal pathological damage. Meanwhile, no abnormal pathological changes were observed in the EtOAc-treated group, indicating that the extract has good tissue safety (see...). Figure 4 ). Example 3

[0041] In vivo colon cancer activity evaluation of ethyl acetate extract of *Heliotropium indicum* stem: Drugs and reagents: The test drug was the ethyl acetate extract of the stem of the heartleaf variegated seed tree (EtOAc), which was dissolved in physiological saline before use to prepare a solution of 100 mg / mL.

[0042] Laboratory animals: Twelve SPF-grade female C57BL / 6 mice (6–8 weeks old, weighing 18–22 g) were purchased from the Comparative Medicine Center of Yangzhou University. The mice were housed in a standard environment with a temperature of 22±2℃ and a 12 h / 12 ​​h light / dark cycle, with free access to food and water, and underwent acclimatization for at least 7 days before the experiment. All animal experimental protocols were approved by the Institutional Animal Care and Use Committee (IACUC), approval number YXYLL-2024-86. At the end of the experiment, the mice were anesthetized with isoflurane and then euthanized by carbon dioxide asphyxiation.

[0043] Experimental methods: Animal grouping: Mice were randomly divided into 4 groups (n=6 per group): ① Model group (administered saline by gavage after modeling); ② Treatment group (administered EtOAc 100 mg / kg by gavage after modeling). Drug administration was continuous for 21 days throughout the experiment. The colon cancer model was established by subcutaneous inoculation of MC38 colon cancer cells, with a cell number of 1×10⁻⁶. 6 Each mouse was administered a dose of the extract solution (100 mg / kg) via gavage. The model group was administered physiological saline by gavage, while the EtOAc group was administered the extract solution (100 mg / kg) via gavage (see [link to relevant documentation]). Figure 5 ).

[0044] Detection indicators and methods: During the experiment, changes in mouse body weight and tumor volume were monitored and recorded daily to dynamically assess the antitumor effect of the drug. After the experiment, the mice were sacrificed, tumor tissue was removed, and tumor size was measured to further evaluate the treatment effect.

[0045] Analysis of in vivo anti-colon cancer results of ethyl acetate extract of *Heliotropium indicum* stem: The results showed that the subcutaneous tumor volume in mice treated with EtOAc was significantly smaller than that in the control group, indicating that it has clear in vivo antitumor activity (see...). Figure 6 ). Example 4

[0046] Simultaneous investigation and verification of the anti-tumor and anti-inflammatory effects of ethyl acetate extract of *Heliotropium indicum* stem in in situ colon cancer: Drugs and reagents: Irinotecan injection (CPT-11) was purchased from Hainan Jinrui Pharmaceutical Co., Ltd.

[0047] The test drug was the ethyl acetate extract of the stem of the heartleaf variegated seed tree (EtOAc), which was dissolved in physiological saline before use to prepare a solution of 100 mg / mL.

[0048] Laboratory animals: Twenty-four SPF-grade female C57BL / 6 mice (6–8 weeks old, weighing 18–22 g) were purchased from the Comparative Medicine Center of Yangzhou University. The mice were housed in a standard environment with a temperature of 22±2℃ and a 12 h / 12 ​​h light / dark cycle, with free access to food and water, and underwent acclimatization for at least 7 days before the experiment. All animal experimental protocols were approved by the Institutional Animal Care and Use Committee (IACUC), approval number YXYLL-2024-86. At the end of the experiment, the mice were anesthetized with isoflurane and then euthanized by carbon dioxide asphyxiation.

[0049] Experimental methods: Animal grouping: C57BL / 6 mouse colon cancer cells (5×10⁻⁶) were grouped together. 6(Number of mice / mouse) were inoculated into the cecum of mice. Once the tumor was detectable by an in vivo imaging system, the mice were randomly divided into four groups (n = 6 per group): ① Model group (inoculated with MC38 colon cancer cells and administered saline via gavage); ② Chemotherapy group (inoculated with MC38 colon cancer cells to establish a tumor model and administered CPT-11 intraperitoneally); ③ EtOAc group (inoculated with MC38 cells to establish a tumor model and administered EtOAc 100 mg / kg via gavage); ④ Combined treatment group (inoculated with MC38 colon cancer cells to establish a tumor model and administered CPT-11 combined with EtOAc 100 mg / kg). During the experiment, mice in the EtOAc treatment group and the combined treatment group were administered EtOAc (100 mg / kg) daily via gavage from day 1 to day 21; mice in the chemotherapy model group and the combined treatment group were administered CPT-11 (75 mg / kg) intraperitoneally twice weekly throughout the experiment. (See flowchart) Figure 7 ) Detection indicators and methods: Mice body weight was recorded daily during the experiment, and the long diameter (L) and short diameter (W) of the tumor were measured using calipers. Diarrhea severity was scored according to established criteria (Cooper et al., 1993; Lin et al., 2019): 0 points indicated normal stool, 1 point indicated mildly moist and soft stool, and 2 points indicated moderately moist and unformed stool. Stool consistency and rectal bleeding were monitored every three days, and the Disease Activity Index (DAI) was calculated.

[0050] Table 2 Disease Activity Index (DAI) Table 2 Disease Activity Index (DAI)

[0051] Analysis of in vivo anti-colon cancer efficacy of ethyl acetate extract from the stem of *Heliotropium indicum*: Further in vivo schematic diagrams of the colon, cecum, and tumor appearance in a mouse model of orthotopic colon cancer showed that, compared with the model group, mice treated with CPT-11 alone and mice treated with CPT-11 in combination with EtOAc exhibited significantly reduced tumor volume. The combination therapy group showed particularly strong tumor suppression, demonstrating a potential synergistic anti-tumor effect (see [link to relevant documentation]). Figure 8 ).

[0052] The FITC-dextran intestinal leakage assay showed that CPT-11 treatment led to significant intestinal barrier damage and increased intestinal leakage in mice; however, in the combined treatment group, EtOAc effectively reduced FITC fluorescence intensity and significantly improved chemotherapy-induced intestinal mucosal barrier damage, indicating that it has a clear intestinal protective effect (see...). Figure 9 ).

[0053] Imaging results in mice showed that, compared with the model group, the tumor volume of mice in the CPT-11 treatment group and the CPT-11 combined with EtOAc treatment group was significantly reduced. (See...) Figure 10 ) The combined results of the above in vivo and in vivo experiments demonstrate that EtOAc not only directly inhibits the growth of colon cancer cells but also significantly improves intestinal mucosal damage, reduces intestinal leakage, and enhances anti-tumor efficacy in the context of chemotherapy. These findings indicate that EtOAc exhibits excellent efficacy in the prevention and treatment of colitis and colon cancer, and possesses significant clinical application potential. This invention systematically reveals for the first time the dual protective and anti-tumor effects of EtOAc in treating chemotherapy-related intestinal mucosal damage and colon cancer, providing a novel natural drug candidate for these diseases.

[0054] The above embodiments provide a detailed description of the present invention and its specific implementation methods. The accompanying drawings are only one preferred embodiment and are not intended to limit the invention. Without departing from the design intent and core principles of the present invention, any obvious modifications, substitutions, or improvements made by those skilled in the art based on the technical teachings of the present invention, such as in step S1, adding 8-10 times the weight of the heartleaf variegated seed stem to ethanol, soaking at room temperature for 30-45 minutes, and then reflux extraction at 70-80°C 2-5 times, each time for 45-75 minutes; the rotary evaporation temperature is 40-50°C, and the oven drying temperature is 60-70°C; in step S2, each solvent is extracted 3 times, each time for 25-35 minutes; the rotary evaporation temperature is 40-50°C, and the oven drying temperature is 60-70°C. All adjustments to the above ranges should be considered to fall within the protection scope of the present invention.

Claims

1. A method for preparing an ethyl acetate extract of the stem of Euphorbia maculata L. of the family Euphorbiaceae, characterized by, Comprising the following steps: S1: extracting the ethyl acetate fraction of the stem of Maytenus hookeri: The pulverized stem of Maytenus hookeri is added with 8-10 times the weight of 95% ethanol, and after being soaked at room temperature for 30-45 min, it is extracted by refluxing at 70-80℃ for 2-5 times, each time for 45-75 min. The extract is combined, and after being coarsely filtered through filter cloth, it is concentrated by rotary evaporation under reduced pressure at 40-50℃ until no alcohol taste is left. The concentrated extract is dispersed in distilled water, and gradient extraction is performed using petroleum ether and ethyl acetate in sequence. The ethyl acetate extract is collected, concentrated under reduced pressure, and dried in an oven at 60-70℃ to obtain the ethyl acetate fraction of the stem of Maytenus hookeri; S2: extracting the ethyl acetate fraction of the stem of Maytenus hookeri: The ethyl acetate fraction of the stem of Maytenus hookeri is dispersed in distilled water, and gradient extraction is performed using equal volumes of petroleum ether, ethyl acetate and n-butanol in sequence, each solvent being extracted for 3 times, each time for 25-35 min. The ethyl acetate extract is combined, concentrated by rotary evaporation under reduced pressure at 40-50℃ until it becomes a thick paste, and then transferred to an oven at 60-70℃ for drying until the weight is constant, to obtain the ethyl acetate extract of the stem of Maytenus hookeri.

2. A method for preparing the ethyl acetate extract of the stem of Euphorbia maculata L. according to claim 1, characterized by, In step S1, the extraction is performed by refluxing at 75℃ for 3 times, each time for 60 min.

3. A method for preparing the ethyl acetate extract of the stem of Euphorbia maculata L. according to claim 1, characterized by, In step S1, the rotary evaporation temperature is 45℃, and the drying temperature in the oven is 65℃.

4. The method for preparing an ethyl acetate extract of the stem of *Euphorbia pulcherrima*, a plant in the Euphorbiaceae family, according to claim 1, is characterized in that... In step S2, the rotary evaporation temperature is 45℃, and the drying temperature in the oven is 65℃.

5. An acetic ether extract of the stem of Euphorbia maculata of the family Euphorbiaceae, characterized by, Obtained by the method according to any one of claims 1-4.

6. Use of the ethyl acetate extract of the stem of Euphorbia maculata L. according to claim 5, characterized in that: A medicament for treating colitis or colon cancer.

7. Use of the ethyl acetate extract of the stem of Euphorbia maculata L. of the family Euphorbiaceae according to claim 6, characterized by: The colitis is induced by the chemotherapeutic drug irinotecan. The colitis is induced by the chemotherapeutic drug irinotecan.

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