Anti-tumor composition of irinotecan and licorice extract or licorice total yellow and metabonomics detection method thereof

Abstract

The invention discloses an anti-tumor composition of irinotecan and liquorice extract or liquorice total flavonoids and a metabonomics detection method of the anti-tumor composition of irinotecan and the liquorice extract or the liquorice total flavonoids. The traditional Chinese medicine composition has an adjusting effect on various enteritis indexes such as body weight, disease activity index score, colon length and reduction of inflammatory factors caused by irinotecan, and has an obvious effect of reducing intestinal toxic and side effects of irinotecan. The invention develops a rapid and efficient UPLC-TQ-MS / MS (ultra performance liquid chromatography-tandem mass spectrometry / mass spectrometry) method capable of synchronously detecting. According to the method, the tissue distribution condition of irinotecan and main metabolites thereof in a mouse body can be detected and quantified with high sensitivity, and the influence of liquorice total flavonoids and liquorice total extracts on the metabolic process and distribution characteristics of irinotecan and metabolites thereof is evaluated; not only can the possible effect of the liquorice in the process of reducing irinotecan be revealed, but also a theoretical basis can be provided for optimization of a medicine compatibility scheme.

Description

Technical Field

[0001] The present invention relates to an anti-tumor drug, in particular to an anti-tumor composition of irinotecan and liquorice extract or liquorice root rhizome and a metabolomics detection method thereof. Background Art

[0002] Due to the widespread clinical use of irinotecan and its toxic side effects, patients receiving irinotecan treatment may experience severe delayed diarrhea, a side effect that prevents patients from receiving higher doses of irinotecan and impairs the effectiveness of chemotherapy. With the in-depth study of irinotecan in recent years, neutropenia and delayed diarrhea are the most common side effects, regardless of the dosing regimen, and they vary greatly between individuals. Irinotecan also has significant side effects, including severe gastrointestinal reactions such as diarrhea, nausea, and vomiting, with diarrhea in particular often considered one of its most challenging side effects. In addition, bone marrow suppression, hair loss, and weight loss are also common.

[0003] To reduce these side effects, medications are often used in combination to alleviate gastrointestinal reactions. Delayed diarrhea is a more serious adverse reaction to irinotecan, and drugs commonly used to treat chemotherapy-induced diarrhea (primarily loperamide and octreotide) are not sufficient to effectively prevent all severe diarrhea.

[0004] Licorice contains a rich array of active ingredients, primarily triterpenoids, flavonoids, and polysaccharides. Modern pharmacological research has revealed its multifaceted effects, including anti-inflammatory, analgesic, anticancer, antiviral, antioxidant, and hepatoprotective activities. Licorice's active ingredients may intervene in the treatment of inflammatory bowel disease-associated colon cancer by regulating cancer cell apoptosis, proliferation, migration, and angiogenesis, affecting cancer cell glycolysis, reducing metastasis and invasion, and modulating autophagy. Furthermore, licorice can alleviate the side effects of chemotherapy drugs used to treat colorectal cancer. Summary of the Invention

[0005] Purpose of the invention: The purpose of the present invention is to use total flavonoids of licorice or total extract of licorice in combination with irinotecan as an anti-tumor drug. Experiments have found that licorice combined with irinotecan can reduce the intestinal toxic side effects of irinotecan.

[0006] In addition, the present invention selected irinotecan and its metabolites SN-38 and SN-38G as research subjects, and developed a rapid, efficient, and simultaneously detectable UPLC-TQ-MS / MS method using camptothecin as an internal standard. This method can highly sensitively detect and quantify the tissue distribution of irinotecan and its major metabolites in mice. By comparing distribution changes between different groups, it evaluates the effects of total flavonoids and total extracts of glycyrrhiza on the metabolic processes and distribution characteristics of irinotecan and its metabolites. This method not only helps to reveal the possible role of glycyrrhiza in the detoxification process, but also provides a theoretical basis for optimizing drug combination regimens and provides more scientific support for clinical treatment.

[0007] Technical solution: In order to achieve the above objectives, the technical solution adopted by the present invention is: an anti-tumor composition, characterized in that it includes irinotecan and licorice extract or licorice total flavonoids.

[0008] As a preferred embodiment, in the above-mentioned anti-tumor composition, the weight ratio of irinotecan to licorice extract is 1:50-200; the mass ratio of irinotecan to total flavonoids of licorice is 1:2.5-1:10.

[0009] Licorice total flavonoids can be prepared or purchased commercially (purity ≥80%, Shanghai Ronghe Pharmaceutical Technology Development Co., Ltd., catalog number: RH1006-3g).

[0010] As a preferred embodiment, the above-mentioned anti-tumor composition, wherein the total extract of licorice is prepared by the following method: washing and drying raw licorice, first extracting it with 50-85% ethanol three times, each time for 0.5-2 hours, then extracting the residue with water three times, each time for 0.5-2 hours, combining the ethanol extract and the water extract, concentrating under reduced pressure, and setting aside; As a more preferred embodiment, the above-mentioned anti-tumor composition, the total extract of licorice is prepared by the following method: washing and drying raw licorice, first extracting it three times with 85% ethanol at a material-liquid ratio of 1:8, 1:6, and 1:6, each time for 2 hours, then taking the residue and extracting it three times with water at a material-liquid ratio of 1:8, 1:6, and 1:6, each time for 2 hours, combining the ethanol extract and the water extract, concentrating under reduced pressure, and setting aside; the above-mentioned extraction methods include reflux extraction, decoction, maceration, percolation and ultrasonic extraction.

[0011] The metabolomics detection method of the anti-tumor composition of the present invention comprises the following steps: (1) Preparation of mixed reference solution and internal standard solution Accurately weigh irinotecan, 7-ethyl-10-hydroxycamptothecin, SN-38 glucuronide, and camptothecin standards and dissolve them in methanol to prepare reference stock solutions of certain concentrations. Take each reference substance stock solution and dilute it with methanol to prepare a series of concentrations of mixed reference substance solutions; Camptothecin was added with methanol to prepare an internal standard solution; (2) Preparation of standard curve Mixed reference solutions containing internal standard solutions at different concentrations were injected into UPLC-TQ-MS / MS for analysis, with the concentration of the mixed reference solution as the abscissa and the ratio of the reference peak area to the internal standard peak area as the ordinate to draw the standard curve equation; (3) Experimental animal modeling The experimental mice were adaptively fed for one week, with standard food and drinking water provided daily. After one week of adaptive feeding, the model group mice received a single intraperitoneal injection of azomethane (AOM). One week after that, the model group mice drank DSS (dextran sulfate sodium) solution for 7 days and normal drinking water for 14 days, and this cycle lasted for three consecutive days. (4) Sample processing: After modeling, mice were divided into groups and given medication. After blood was collected from the mice's orbits, they were killed by cervical dislocation and dissected. The liver, kidney, spleen, testicle, jejunum, ileum, and colon tissues of the mice were completely removed and placed in tissue fixative and stored at room temperature. Plasma samples: Plasma samples were placed in blank centrifuge tubes. Proteins were precipitated with a formic acid-methanol solution containing a certain amount of camptothecin, vortexed, and all samples were allowed to stand for a while before centrifugation. The supernatant was transferred to a clean centrifuge tube and concentrated by vacuum centrifugation to dryness. Tissue samples: Accurately weigh liver, kidney, spleen, testis, jejunum, ileum, and colon tissues. Add a certain amount of formic acid-methanol solution containing camptothecin to the above samples to precipitate protein. Vortex and let all samples stand for a while. Centrifuge and transfer the supernatant to a clean centrifuge tube. Concentrate and evaporate to dryness in a vacuum centrifuge. (5) Metabolite analysis Plasma and tissue samples were reconstituted with cold methanol, vortexed, and centrifuged. The supernatant was placed in an intubation tube for UPLC-TQ-MS / MS analysis.

[0012] As a preferred embodiment, in the above-mentioned metabolomics detection method of irinotecan, the preparation method of the mixed reference solution and internal standard solution in step (1) is: Accurately weigh irinotecan, 7-ethyl-10-hydroxycamptothecin, SN-38 glucuronide, and camptothecin standards and dissolve them in methanol to prepare a reference stock solution with a concentration of 1 mg / mL; A series of mixed reference solutions were prepared by diluting each reference stock solution with methanol, with irinotecan concentrations of 10000, 5000, 2500, 1250, 625, 312.5, 78.13, 19.53, 9.77, and 2.44 ng / mL, respectively; The concentrations of 7-ethyl-10-hydroxycamptothecin were: 2000, 1000, 500, 250, 125, 62.5, 31.25, 15.56, 7.81, and 1.95 ng / mL; The concentrations of SN-38 glucuronide were: 10000, 5000, 2500, 1250, 625, 312.5, 78.13, 9.77, 4.88, and 2.44 ng / mL; Camptothecin was dissolved in methanol to prepare an internal standard solution; the internal standard solution was added to the mixed reference solution of each concentration so that the concentration of camptothecin in each mixed reference solution was 125 ng / mL; As a more preferred solution, in the above-mentioned metabolomics detection method of irinotecan, the experimental animal modeling and grouping method in step (3) is: The mice were adaptively fed for one week and given standard food and drinking water every day. After one week of adaptive feeding, the mice in the model group received a single intraperitoneal injection of 10 mg / kg azomethane. One week later, the mice in the model group drank 2.0%~2.5% DSS (dextran sulfate sodium) solution for 7 days and normal drinking water for 14 days, which lasted for 3 consecutive cycles. Except for the control group, after the modeling, the mice were divided into the model group, irinotecan group, low-dose total flavonoids of glycyrrhiza combined with irinotecan group, medium-dose total flavonoids of glycyrrhiza combined with irinotecan group, high-dose total flavonoids of glycyrrhiza combined with irinotecan group, and total extract of glycyrrhiza combined with irinotecan group.

[0013] As a preferred embodiment, in the above-mentioned metabolomics detection method of irinotecan, the sample processing method in step (4) is: After orbital blood collection, mice were killed by cervical dislocation and dissected. The liver, kidney, spleen, testis, jejunum, ileum, and colon tissues of the mice were completely removed and placed in tissue fixative and stored at room temperature. Plasma samples: 50 μL of plasma sample was placed in a blank centrifuge tube. Protein was precipitated with 350 μL of 0.1% formic acid in methanol (containing 125 ng / mL camptothecin) and vortexed for 3 min. All samples were allowed to stand for a while and then centrifuged at 13,000 rpm / min at 4°C for 10 min. The supernatant was transferred to a clean centrifuge tube and concentrated by vacuum centrifugation at 40°C to dryness. Tissue samples: Accurately weigh 50 mg of liver, kidney, spleen, testis, jejunum, ileum, and colon tissues, respectively. Add 350 μL of 0.1% formic acid in methanol (containing 250 ng / mL camptothecin) to each sample to precipitate protein. Vortex and let all samples rest for a while before centrifuging at 13,000 rpm / min for 10-15 min. Transfer the supernatant to a clean centrifuge tube and evaporate to dryness by vacuum centrifugation at 40°C. As a preferred embodiment, in the above-mentioned metabolomics detection method of irinotecan, the chromatographic conditions for the preparation of the standard curve in step (2) and the UPLC-TQ-MS / MS analysis of the metabolites in step (5) are as follows: Chromatographic separation was performed on an ACQUITY UPLC HSS T3 column with dimensions of 2.1 mm × 100 mm and 1.8 μm. The mobile phase consisted of 0.1% formic acid in water (B) and acetonitrile (A) for gradient elution. The injection volume was 1.0 μL, the flow rate was 0.3 mL / min, the column temperature was 35 °C, the autosampler temperature was 5 °C, and the analysis time was 11 min. The gradient elution program was as follows: Time / min A% B% 0.0-1.0 10 90 1.0-4.0 10-45 90-55 4.0-5.6 45-80 55-20 5.6-7.0 80-90 20-10 7.0-8.0 90 10 8.0-10.0 90-10 10-90 10.0-11.0 10 90 The mass spectrometry conditions were as follows: electrospray ionization positive ion source (ESI + ) and multiple reaction monitoring (MRM) scan mode for quantification, with a capillary voltage of 3.0 kV, a desolvation temperature of 350 °C, and a conical gas flow rate of 150 L·h -1 , desolvation gas flow rate 1000 L·h -1 , the collision gas is argon.

[0014] The mass spectrometry conditions for each compound are: Compound tR (min) Precursor ion (m / z) Product ion (m / z) Cone voltage (V) Collision energy (eV) Ion mode Irinotecan 2.90 587.52 124.03 50.0 32.0 <![CDATA[ES + ]]> 7-Ethyl-10-hydroxycamptothecin 3.36 393.30 249.11 10.0 46.0 <![CDATA[ES + ]]> SN-38 glucuronide 2.65 569.55 393.23 10.0 26.0 <![CDATA[ES + ]]> Camptothecin 3.54 349.19 219.02 8.0 48.0 <![CDATA[ES + ]]> Beneficial effects: The metabolomics detection method for irinotecan provided by the present invention has the following advantages compared with the prior art: Irinotecan's dose-limiting adverse events, such as myelosuppression and gastrointestinal toxicity, particularly unpredictable severe diarrhea, have greatly limited its clinical use. According to the National Cancer Institute's Common Toxicity Criteria, 87% of patients receiving irinotecan-based chemotherapy experience delayed diarrhea, and approximately 40% experience severe diarrhea. Therefore, irinotecan-induced diarrhea is the most common obstacle in cancer treatment, often leading to treatment delays, dose reductions, or treatment interruptions. On the other hand, severe diarrhea may lead to severe sepsis, dehydration, electrolyte imbalance, or hemodynamic disturbances, and may even be life-threatening.

[0015] The present invention adopts total flavonoids of glycyrrhiza or licorice extract prepared by a specific extraction method to be combined with irinotecan for investigation through experimental screening. The experimental results show that the total flavonoids of glycyrrhiza or licorice extract prepared by a specific extraction method have a regulating effect on various enteritis indicators such as body weight, DAI score, colon length, and reduction of inflammatory factors caused by irinotecan, and have a significant effect on reducing the intestinal toxic side effects of irinotecan.

[0016] Furthermore, the present invention selected irinotecan and its metabolites SN-38 and SN-38G as research subjects and developed a rapid, efficient, and simultaneously detectable UPLC-TQ-MS / MS method using CPT as the internal standard. This method can highly sensitively detect and quantify the tissue distribution of irinotecan and its major metabolites in mice. By comparing distribution changes between different groups, it evaluates the effects of total flavonoids and total extracts of glycyrrhiza on the metabolic processes and distribution characteristics of irinotecan and its metabolites. This method not only helps to reveal the possible role of glycyrrhiza in reducing irinotecan levels, but also provides a theoretical basis for optimizing drug combination regimens and further scientific support for clinical treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 To establish the AOM / DSS colorectal cancer model mice, the changes in body weight and disease activity index ( ± SD , Control = 6, Model = 42) Note: Compared with the control group, ### p <0.001, ## p <0.01.

[0018] Figure 2 Effects of irinotecan on body weight and disease activity index in AOM / DSS colorectal cancer mice ( ± SD , n = 6) Note: Compared with the control group, ### p <0.001.

[0019] Figure 3 is the length of the mouse colon and rectum ( ± SD , n = 6) Note: Compared with the control group, ## p <0.01; compared with the model group, *** p <0.001, ** p <0.01, * p <0.05.

[0020] Figure 4 This is the H&E staining image of mouse intestinal tissue.

[0021] Figure 5 is the expression level of biochemical index proteins in mouse colon tissue ( ± SD , n = 6), Note: Compared with the control group, ### p <0.001, ## p <0.01; compared with the model group, *** p <0.001, ** p <0.01, * p <0.05.

[0022] Figure 6 is the β-glucuronidase activity of mouse colon contents ( ± SD , n =6) Note: Compared with the model group, ### p <0.001, ## p <0.01, # p <0.05; compared with CPT-11, **** p <0.0001, *** p <0.001, ** p <0.01, * p <0.05.

[0023] Figure 7 These are the MRM chromatograms of CPT-11, SN-38, SN-38G, and CPT.

[0024] Figure 8 is the content of irinotecan and its metabolites in plasma and testis ( ± SEM , n = 6), compared with the CPT-11 group, *** p <0.001, ** p <0.01, * p <0.05.

[0025] Figure 9is the content of irinotecan and its metabolites in the kidney and spleen ( ± SEM , n = 6) compared with the CPT-11 group, *** p <0.001, ** p <0.01, * p <0.05.

[0026] Figure 10 is the content of irinotecan and its metabolites in the liver and jejunum ( ± SEM , n = 6) Compared with the CPT-11 group, *** p <0.001, ** p <0.01, * p <0.05.

[0027] Figure 11 is the content of irinotecan and its metabolites in the ileum and colon ( ± SEM , n = 6) Compared with the CPT-11 group, *** p <0.001, ** p <0.01, * p <0.05. DETAILED DESCRIPTION

[0028] The present invention is further illustrated below with reference to specific examples. It should be understood that these examples are only used to illustrate the present invention and are not used to limit the scope of the present invention. After reading the present invention, modifications of various equivalent forms of the present invention made by those skilled in the art all fall within the scope defined by the claims attached to this application.

[0029] Example 1 Experiment on the toxicity reduction of irinotecan by combining total flavonoids of licorice and licorice extract 1 Experimental Materials 1.1 Experimental Animals Forty-eight 8-week-old SPF C57BL / 6 male mice were purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd. (certificate number SCXK (Beijing) 2021-0006). The animal experiments were approved by the Ethics Committee of Shaanxi University of Chinese Medicine, and animal husbandry and experimental procedures complied with the regulations for experimental management.

[0030] 1.2 Drugs and reagents Raw licorice slices were purchased from the School Hospital of Shaanxi University of Chinese Medicine; azoxymethane (Sigma-Aldrich (Shanghai) Trading Co., Ltd., catalog number: A5486-25MG); dextran sulfate sodium (Yisheng Biotechnology (Shanghai) Co., Ltd., catalog number: 60316ES60); 0.9% sodium chloride injection (Xi'an Jingxi Shuanghe Pharmaceutical Co., Ltd., batch number: 2406B); 4% tissue fixative (Xi'an Hete Biotechnology Co., Ltd., catalog number: HT115); ciprofloxacin hydrochloride monohydrate (Shanghai Aladdin Biochemical Technology Co., Ltd., catalog number: I2126156); hematoxylin stain (Wuhan Sewell Biotechnology Co., Ltd., catalog number: G1 004); Hematoxylin staining solution set (Biosharp, catalog number: BL700B); Irinotecan hydrochloride injection (Qilu Pharmaceutical (Hainan) Co., Ltd., catalog number: EB7E4001); Mouse tumor necrosis factor α (TNF-α) ELISA research kit (Jiangsu ELISA Industrial Co., Ltd., catalog number: MM-0132M2); Mouse interleukin 6 (IL-6) ELISA research kit (Jiangsu ELISA Industrial Co., Ltd., catalog number: MM-0163M2); Mouse interleukin 1β (IL-1β) ELISA research kit (Jiangsu ELISA Industrial Co., Ltd., catalog number: MM-0040M2); Mouse CEA ELISA KIT (Shanghai ELISA Biotechnology Co., Ltd., catalog number: YJ003265); β-glucuronidase (β-GD) activity (Shanghai ELISA Biotechnology Co., Ltd., catalog number: mlsh0219).

[0031] 1.3 Instruments and Equipment The instrument and equipment information is as follows:

[0032] 2 Experimental methods 2.1 Preparation of main experimental reagents AOM solution: Prepare AOM solution with normal saline to a concentration of 10 mg mL -1 AOM stock solution was stored at -20°C for future use.

[0033] DSS Solution: Accurately weigh 25.0 g of DSS powder into a clean beaker. Add an appropriate amount of ultrapure water and stir on a magnetic stirrer to dissolve. Once the DSS is completely dissolved, add ultrapure water to a volume of 1000 mL to obtain a 2.5% DSS solution. Refrigerate the resulting DSS solution at 4°C until ready to use.

[0034] Irinotecan injection: Irinotecan injection (CPT-11): Irinotecan hydrochloride injection is diluted in infusion solution (0.9% sodium chloride solution) before intraperitoneal injection and stored in a 4°C refrigerator away from light until ready for use.

[0035] Ciprofloxacin hydrochloride monohydrate: Accurately weigh a certain amount of ciprofloxacin hydrochloride monohydrate and prepare a 3 mg / mL ciprofloxacin solution with normal saline.

[0036] Licorice total extract: Wash and dry the raw licorice root, then extract it with 85% ethanol at a material-liquid ratio of 1:8, 1:6, and 1:6 (g / mL) three times under reflux for 2 h each time. Then, take the medicinal residue and decoct it with water at a material-liquid ratio of 1:8, 1:6, and 1:6 (g / mL) three times for 2 h each time. Combine the ethanol extract and the water extract, concentrate under reduced pressure at 60°C to a crude drug concentration of 0.5 g / mL, and set aside. Glycyrrhiza uralensis total flavonoids: Glycyrrhiza uralensis total flavonoids (purchased from Shanghai Ronghe Pharmaceutical Technology Development Co., Ltd., product number: RH1006-3g) were suspended in 0.5% CMC-Na solution to prepare a drug solution, which was stored in a refrigerator at 4°C for future use.

[0037] 2.2 Experimental animal model The animal experiment process adopted a scheme of modeling first and then grouping and drug administration. The modeling scheme of the AOM / DSS mouse colorectal cancer model was as follows: the mice were adaptively fed for one week and given standard food and drinking water every day; after one week of adaptive feeding, the mice in the model group received a single intraperitoneal injection of 10 mg / kg azomethane. One week later, the mice in the model group drank 2.0%~2.5% DSS (dextran sulfate sodium) solution for 7 days and normal drinking water for 14 days, and continued for 3 consecutive cycles. After the AOM / DSS mouse colorectal cancer model was established, 42 C57BL / 6 mice induced with AOM / DSS were randomly divided into six groups according to body weight, including the model group (Model), irinotecan group (CPT-11), positive drug group (CPT-11+CF), low-dose total flavonoids from Glycyrrhizae combined with irinotecan group (CPT-11+LTFGU), medium-dose total flavonoids from Glycyrrhizae combined with irinotecan group (CPT-11+MTFGU), high-dose total flavonoids from Glycyrrhizae combined with irinotecan group (CPT-11+HTFGU), and total extract of Glycyrrhizae combined with irinotecan group (CPT-11+TEGU). The body weight of the mice was recorded before administration, and the mice were observed for diarrhea. From the day of administration, mice in each group were given 0.2 mL of licorice extract. 20 g 1 d 1The dose of CPT-11+low-dose total flavonoids from Glycyrrhizae LTFGU group (CPT-11: 40 mg kg -1 , LTFGU: 100 mg kg -1 ); CPT-11 + medium-dose total flavonoids from Glycyrrhizae MTFGU group (CPT-11: 40 mg kg -1 , MTFGU: 200 mg kg -1 ); CPT-11 + high-dose total flavonoids from Glycyrrhiza uralensis HTFGU administration group dose (CPT-11: 40 mg kg -1 HTFGU: 400 mg kg -1 ); CPT-11 + licorice extract TEGU administration group dose (CPT-11: 40 mg kg -1 , TEGU: 4.5 g kg -1 ); CPT-11+ciprofloxacin CF administration group dose (CPT-11: 40 mg kg -1 , CF: 40 mg kg -1 During the administration period, mice in the control group and model group were intraperitoneally injected with an equal volume of normal saline.

[0038] 2.3 Experimental Animal Collection After the dosing period, mice were sacrificed by cervical dislocation after orbital blood collection. The small intestine and colon (including the ileocecal valve) were completely removed. The colon surface morphology was observed and photographed. The intestinal tissue length of each group of mice was recorded, and the ileocecal valve was discarded. The cecal and colon contents were collected and placed in sterile cryovials. Residual intestinal contents were rinsed with pre-chilled saline. Approximately 1 cm of ileum and colon were placed in 4% tissue fixative and stored at room temperature. Organ tissues from the jejunum, ileum, colon, liver, spleen, kidney, and testicles were also collected. All biological samples were stored at -80°C until further use.

[0039] 2.4 Disease Activity Index The disease activity index (DAI) combines weight changes and diarrhea in mice. Weight and diarrhea were recorded regularly throughout the experiment. The DAI score was calculated using the weight and diarrhea scoring methods described in the literature. DAI = (weight loss score + stool consistency score + fecal occult blood score) / 3. The DAI scoring system is as follows:

[0040] 2.5 H&E staining of pathological sections The intestinal tissue was stained with H&E according to the literature, and the images of the sections were collected using an optical microscope.

[0041] 2.6 Biochemical index determination Colonic tissue was rinsed with pre-chilled saline and dried with filter paper. Colonic tissue was precisely weighed and placed in a homogenizer tube. An appropriate amount of saline was added and the homogenate was ground into a 10% colonic tissue homogenate using a tissue homogenizer. The homogenate was centrifuged at 4°C and 12,000 rpm for 5 minutes, and the supernatant was collected. ELISA kits were used according to the kit's protocol to measure the levels of TNF-α, IL-6, IL-1β, and CEA in the colonic tissue homogenates of each group of mice.

[0042] 2.7 Intestinal β-glucuronidase assay Accurately weigh the colonic contents into a sterile, enzyme-free homogenizer tube. Add an appropriate amount of homogenate and grind to a 10% colonic homogenate. Centrifuge at 4°C, 12,000 rpm / min for 5 min, and collect the supernatant. β-glucuronidase activity in the colonic contents of each group of mice was assayed according to the kit's instructions.

[0043] 2.6 Statistical Analysis The experimental data are all based on ± SEM Statistical analysis was performed using GraphPad Prism 8.3.0. t test, p <0.05 was statistically significant.

[0044] 3 Experimental Results 3.1 Observation of general condition, weight changes, and disease activity index of mice during modeling like Figure 1 As shown, after the first dose of DSS solution, the model group mice experienced weight loss, diarrhea, and occult blood in their stools, and the disease activity index increased. After the second dose of DSS solution, the mice experienced loose stools and bloody stools, which worsened compared to the first stage. After the third dose of DSS solution, the mice lost significant weight, and symptoms such as loose stools and bloody stools were significantly aggravated. During the modeling process, these symptoms were alleviated to a certain extent after the model group mice resumed normal drinking water. During the modeling process, the control group mice had shiny fur and were more responsive. Their feces were grain-like, with no visible blood in their stools. Their weight steadily increased with prolonged feeding.

[0045] 3.2 Observation of general condition, weight changes, and disease activity index of mice during grouping and drug administration pass Figure 2 As shown, both the total flavonoids and total extracts of licorice improved irinotecan-induced weight loss in mice, with overall effects comparable to those in the active drug group. The DAI score, a composite score based on the rate of weight change, severity of diarrhea, and blood in the stool, was calculated. Irinotecan significantly impacted the DAI score in mice, with scores increasing significantly with increasing days of irinotecan administration, indicating that irinotecan-induced diarrhea and blood in the stool were responsible. The DAI scores in the total flavonoids and total extract groups were comparable to those in the active drug group and were lower than those in the CPT-11 group.

[0046] 3.3 Colorectal length The appearance and length of intestinal tissue are important indicators for evaluating intestinal inflammation caused by irinotecan. In this experiment, the length of the colon of mice was measured. Figure 3 It can be seen that irinotecan administration can significantly shorten the length of the mouse colon, while the licorice total flavonoids and licorice total extract groups have a significant improvement effect. The effects of licorice total flavonoids and licorice total extract are slightly better than the positive drug ciprofloxacin.

[0047] 3.4 Mouse pathological H&E staining like Figure 4 As shown, in order to further verify the compatibility and detoxification effect of total flavonoids and total extracts of licorice on irinotecan, H&E staining was performed on the intestinal segments fixed with 4% tissue fixative. Figure 4 As shown, the control group mice had intact intestinal wall structure, orderly arranged villi, and intact mucosa. Mice in the model group exhibited pathological changes including necrosis of the mucosal lamina propria, inflammatory cell infiltration, dilated intestinal glands, and a decrease in goblet cells. Tumor tissue broke through the muscularis mucosa and invaded the submucosa, leading to cancerous transformation. The irinotecan group showed swollen colonic mucosa, severe destruction of crypt structure, shortened or lost villi, and extensive infiltration of inflammatory cells, particularly lymphocytes. Compared to the irinotecan group, the glycyrrhizic total flavonoids and glycyrrhizic total extract groups showed relatively intact intestinal structure, with relatively orderly but slightly diffused perivillary structures.

[0048] 3.5 Biochemical indicators according to Figure 5 As shown in the results, compared with the CPT-11 group, the protein expression levels of pro-inflammatory factors TNF-α, IL-6, and IL-1β were significantly reduced after administration of licorice total flavonoids and licorice total extracts. The ELISA experimental results once again verified the regulatory effects of licorice total flavonoids and licorice total extracts on irinotecan-induced intestinal inflammatory response.

[0049] 3.6 Intestinal β-glucuronidase activity assay like Figure 6 As shown in the figure, the β-glucuronidase activity in the colon contents of mice was measured. As expected, the flavonoids and total extracts of Glycyrrhizae Radix inhibited the β-glucuronidase activity in the colon contents to varying degrees compared with the CPT-11 group.

[0050] Based on the existing results of the present invention, total flavonoids of licorice and licorice extract have a regulatory effect on various enteritis indicators such as body weight, DAI score, colon length, and reducing inflammatory factors caused by CPT-11. Licorice combined with irinotecan can significantly inhibit the activity of β-glucuronidase, achieving the purpose of reducing the intestinal toxic side effects of irinotecan.

[0051] Example 2 In vivo metabolic chemistry study of the detoxification effect of licorice combined with irinotecan based on UPLC-TQ-MS / MS 1 Experimental Materials 1.1 Experimental Animals Forty-eight 8-week-old SPF C57BL / 6 male mice were purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd. (certificate number SCXK (Beijing) 2021-0006). The animal experiments were approved by the Ethics Committee of Shaanxi University of Chinese Medicine, and animal husbandry and experimental procedures complied with the regulations for experimental management.

[0052] 1.2 Drugs and reagents Raw licorice slices were purchased from the School Hospital of Shaanxi University of Chinese Medicine; total flavonoids from licorice (purity ≥80%, Shanghai Ronghe Pharmaceutical Technology Development Co., Ltd., catalog number: RH1006-3g); azoxymethane (Sigma-Aldrich (Shanghai) Trading Co., Ltd., catalog number: A5486-25MG); sodium dextran sulfate (Yisheng Biotechnology (Shanghai) Co., Ltd., catalog number: 60316ES60); 0.9% sodium chloride injection (Xi'an Jingxi Shuanghe Pharmaceutical Co., Ltd., batch number: 2406B); 4% tissue fixative (Xi'an Hete Biotechnology Co., Ltd., catalog number: HT115); ciprofloxacin hydrochloride monohydrate (Shanghai Aladdin Biochemical Technology Co., Ltd., catalog number: I2126156); hematoxylin staining =Staining solution (Wuhan Sewell Biotechnology Co., Ltd., catalog number: G1004); Hematoxylin staining solution set (Biosharp, catalog number: BL700B); Irinotecan hydrochloride injection (Qilu Pharmaceutical (Hainan) Co., Ltd., catalog number: EB7E4001); Mouse tumor necrosis factor α (TNF-α) ELISA research kit (Jiangsu ELISA Industrial Co., Ltd., catalog number: MM-0132M2); Mouse interleukin 6 (IL-6) ELISA research kit (Jiangsu ELISA Industrial Co., Ltd., catalog number: MM-0163M2); Mouse interleukin 1β (IL-1β) ELISA research kit (Jiangsu ELISA Industrial Co., Ltd., catalog number: MM-0040M2); Mouse CEA ELISA KIT (Shanghai ELISA Biotechnology Co., Ltd., catalog number: YJ003265); β-glucuronidase (β-GD) activity (Shanghai ELISA Biotechnology Co., Ltd., catalog number: mlsh0219).

[0053] 1.3 Instruments and Equipment The instrument and equipment information is as follows:

[0054] 2 Experimental methods 2.1 Modeling grouping and sample processing The animal experiment process adopted a scheme of modeling first and then grouping and drug administration. The modeling scheme of the AOM / DSS mouse colorectal cancer model was as follows: the mice were adaptively fed for one week and given standard food and drinking water every day; after one week of adaptive feeding, the mice in the model group received a single intraperitoneal injection of 10 mg / kg azomethane. One week later, the mice in the model group drank 2.0%~2.5% DSS (dextran sulfate sodium) solution for 7 days and normal drinking water for 14 days, and continued for 3 consecutive cycles. After the AOM / DSS mouse colorectal cancer model was established, except for the control group mice, the remaining C57BL / 6 mice that had been modeled with AOM / DSS were randomly divided into 6 groups according to body weight, namely model group (Model), irinotecan group (CPT-11), positive drug group (CPT-11+CF), low-dose total flavonoids of glycyrrhiza combined with irinotecan group (CPT-11+LTFGU), medium-dose total flavonoids of glycyrrhiza combined with irinotecan group (CPT-11+MTFGU), high-dose total flavonoids of glycyrrhiza combined with irinotecan group (CPT-11+HTFGU), and total extract of glycyrrhiza combined with irinotecan group (CPT-11+TEGU). The body weight of the mice was recorded before administration, and the mice were observed for diarrhea. From the day of administration, the mice in each group were given 0.2 mL of 20 g 1 d 1 The dose of CPT-11+LTFGU group (CPT-11: 40 mg kg -1 , LTFGU: 100 mg kg -1 ); CPT-11+MTFGU administration group dose (CPT-11: 40 mg kg -1 , MTFGU: 200 mg kg -1 ); CPT-11+HTFGU administration group dose (CPT-11: 40 mg kg -1 HTFGU: 400 mg kg -1 ); CPT-11+TEGU administration group dose (CPT-11: 40 mg kg -1 , TEGU: 4.5 g kg -1 ); CPT-11+CF administration group dose (CPT-11: 40 mg kg -1 , CF: 40 mg kg-1 During the administration period, mice in the control and model groups were intraperitoneally injected with equal volumes of normal saline. After the administration, the mice in each group were killed.

[0055] Plasma sample: 50 μL of plasma sample was placed in a blank centrifuge tube. Protein was precipitated with 350 μL of 0.1% formic acid methanol solution (containing 125 ng / mL CPT) and vortexed for 3 min. All samples were allowed to stand for a while and then centrifuged at 13,000 rpm / min at 4°C for 10 min. The supernatant was transferred to a clean centrifuge tube and concentrated by vacuum centrifugation at 40°C to dryness.

[0056] Tissue samples: Accurately weigh 50 mg of liver, kidney, spleen, testis, jejunum, ileum, and colon tissues, respectively. Add 350 μL of 0.1% formic acid in methanol (containing 250 ng / mL camptothecin) to each sample to precipitate protein. Vortex and let all samples rest for a while before centrifuging at 13,000 rpm / min for 10-15 min. Transfer the supernatant to a clean centrifuge tube and evaporate to dryness by vacuum centrifugation at 40°C. Before analysis, all samples were reconstituted with 100 μL of 50% cold methanol, vortexed for 3 minutes, and centrifuged at 13,000 rpm / min at 4°C for 10 minutes. The supernatant was then transferred to an insert for UPLC-TQ-MS / MS analysis. Data were analyzed using MassLynx software.

[0057] 2.2 Preparation of reference solution and internal standard solution Accurately weigh CPT-11, SN-38, SN-38G, and CPT standards, dissolve them in methanol to prepare a stock solution with a concentration of 1 mg / mL, and store in a refrigerator at 4°C until use. A series of mixed standard solutions were prepared by dilution with methanol, including 10 concentration points: CPT-11 concentrations of 10,000, 5,000, 2,500, 1,250, 625, 312.5, 78.13, 19.53, 9.77, and 2.44 ng / mL; SN-38 concentrations of 2,000, 1,000, 500, 250, 125, 62.5, 31.25, 15.56, 7.81, and 1.95 ng / mL; and SN-38G concentrations of 10,000, 5,000, 2,500, 1,250, 625, 312.5, 78.13, 9.77, 4.88, and 2.44 ng / mL. CPT solution was diluted to 125 ng / mL as an internal standard. All standard solutions were stored at 4°C until ready for analysis.

[0058] 2.3 Chromatographic conditions Chromatographic separation was performed on an ACQUITY UPLC HSS T3 column (2.1 mm × 100 mm, 1.8 μm); the mobile phase consisted of 0.1% formic acid in water [v / v] (B)-acetonitrile (A) for gradient elution. The gradient elution program is shown in Table 1. The injection volume was 1.0 μL, the flow rate was 0.3 mL / min, the column temperature was 35°C, the autosampler temperature was 5°C, and the analysis time was 11 min.

[0059] Table 1 Mobile phase gradient elution program Time (min) Flow Rate (mL / min) A% B% 0.0-1.0 0.3 10 90 1.0-4.0 0.3 10-45 90-55 4.0-5.6 0.3 45-80 55-20 5.6-7.0 0.3 80-90 20-10 7.0-8.0 0.3 90 10 8.0-10.0 0.3 90-10 10-90 10.0-11.0 0.3 10 90 2.4 Mass spectrometry conditions The mass spectrometry conditions were as follows: electrospray ionization positive ion source (ESI + ) and multiple reaction monitoring (MRM) scan mode for quantification, with a capillary voltage of 3.0 kV, a desolvation temperature of 350 °C, and a conical gas flow rate of 150 L·h -1 , desolvation gas flow rate 1000 L·h -1 , the collision gas was argon.

[0060] 2.5 Statistical analysis The experimental data are based on ± SEM Statistical analysis was performed using GraphPad Prism 8.3.0. t test, p <0.05 was statistically significant.

[0061] 3 Experimental Results 3.1 The optimized mass spectrometry conditions are shown in Table 2.

[0062] Table 2 Compound tR (min) Precursor ion (m / z) Product ion (m / z) Cone voltage (V) Collision energy (eV) Ion mode Irinotecan 2.90 587.52 124.03 50.0 32.0 <![CDATA[ES + ]]> 7-Ethyl-10-hydroxycamptothecin 3.36 393.30 249.11 10.0 46.0 <![CDATA[ES + ]]> SN-38 glucuronide 2.65 569.55 393.23 10.0 26.0 <![CDATA[ES + ]]> Camptothecin 3.54 349.19 219.02 8.0 48.0 <![CDATA[ES + ]]> 3.2 Linear Relationship The regression equation, linear coefficient, and linear range of irinotecan and its metabolites SN-38 and SN-38G in plasma are shown in Table 3. The regression equation of irinotecan and its metabolites SN-38 and SN-38G has a good linear coefficient ( r 2 The lowest concentration of the analyte that can be detected by this analytical method is defined as the lower limit of quantification. Therefore, the lower limits of quantification for CPT-11 and SN-38 are 2.44 ng / mL, and the lower limit of quantification for SN-38G is 1.95 ng / mL.

[0063] Table 3 Linear range, regression equation and linear coefficient of irinotecan and its metabolites Compound Regression equation Linear range (ng / mL) <![CDATA[ r 2 ]]> CPT-11 =0.0195+0.0666 2.44-10000 0.9939 SN-38 =0.0029-0.0099 2.44-10000 0.9995 SN-38G =0.0258+0.0010 1.95-2000 0.9997 3.3 Tissue distribution of irinotecan in mice The concentrations of irinotecan and its metabolites SN-38 and SN-38G in mouse plasma, testis, kidney, spleen, liver, jejunum, ileum, and colon were determined. Figure 7 shown.

[0064] Glycyrrhiza uralensis total flavonoids and total extracts may have a toxicity-reducing effect by affecting the tissue distribution of irinotecan. To test this hypothesis, a UPLC-TQ-MS / MS analytical method was developed to detect irinotecan and its metabolites. Furthermore, the distribution characteristics of irinotecan and its metabolites in various tissues were systematically compared.

[0065] according to Figures 8-11 It can be seen that when glycyrrhizic acid total flavonoids and glycyrrhizic acid total extracts were combined with irinotecan, the concentration of SN-38 decreased to varying degrees. Combined with previous research results, glycyrrhizic acid total flavonoids and glycyrrhizic acid total extracts can effectively reduce β-glucuronidase activity and alleviate irinotecan-induced diarrhea in mice. It is speculated that glycyrrhizic acid total flavonoids and glycyrrhizic acid total extracts can reduce SN-38 concentration by inhibiting β-glucuronidase activity, thereby alleviating the intestinal toxicity caused by irinotecan.

[0066] This study investigated irinotecan and its metabolites SN-38 and SN-38G, using CPT as an internal standard. A rapid, efficient, and simultaneously detectable UPLC-TQ-MS / MS method was developed. UPLC-TQ-MS / MS tissue distribution analysis of irinotecan revealed that both total flavonoids and total extracts from glycyrrhizae (Glycyrrhizae Radix) inhibited the hydrolysis of SN-38G in intestinal tissue to varying degrees, reducing SN-38 exposure within the intestine. It is hypothesized that total flavonoids and total extracts from Glycyrrhizae Radix (Glycyrrhizae Radix) may protect against irinotecan-induced enterotoxicity by inhibiting β-glucuronidase activity in the intestine.

Claims

1. An anti-tumor composition, characterized in that: It includes irinotecan and licorice extract or total flavonoids from licorice.

2. The anti-tumor composition according to claim 1, characterized in that The weight ratio of irinotecan to licorice extract is 1:50-200; The mass ratio of irinotecan to total flavonoids in licorice is 1:2.5~1:

10.

3. The anti-tumor composition according to claim 1, characterized in that The licorice total extract is prepared by the following method: washing and drying raw licorice, first extracting it with 85% ethanol at a material-liquid ratio of 1:8, 1:6, and 1:6 for three times, each time for 2 hours; then taking the medicinal residue and extracting it with water at a material-liquid ratio of 1:8, 1:6, and 1:6 for three times, each time for 2 hours; combining the ethanol extract and the water extract, concentrating under reduced pressure, and setting aside.

4. The anti-tumor composition according to claim 1, characterized in that Application of licorice extract or total flavonoids in reducing the intestinal toxicity of irinotecan.

5. The metabolomics detection method for the anti-tumor composition according to any one of claims 1 to 4, characterized in that: The following steps are included: (1) Preparation of mixed reference solution and internal standard solution Accurately weigh irinotecan, 7-ethyl-10-hydroxycamptothecin, SN-38 glucuronide, and camptothecin standards and dissolve them in methanol to prepare reference stock solutions of certain concentrations. Take each reference substance stock solution and dilute it with methanol to prepare a series of concentrations of mixed reference substance solutions; Camptothecin was added with methanol to prepare an internal standard solution; (2) Preparation of standard curve Mixed reference solutions containing internal standard solutions at different concentrations were injected into UPLC-TQ-MS / MS for analysis. The standard curve equation was drawn with the concentration of the mixed reference solution as the abscissa and the ratio of the peak area of the reference solution to the peak area of the internal standard solution as the ordinate. (3) Experimental animal modeling The experimental mice were adaptively fed for one week, with standard food and drinking water provided daily. After one week of adaptive feeding, the model group mice received a single intraperitoneal injection of azomethane (AOM). One week after that, the model group mice drank dextran sulfate sodium DSS solution for 7 days and normal drinking water for 14 days, for three consecutive cycles. (4) Sample processing: After modeling, mice were divided into groups and given medication. After blood was collected from the mice's orbits, they were killed by cervical dislocation and dissected. The liver, kidney, spleen, testicle, jejunum, ileum, and colon tissues of the mice were completely removed and placed in tissue fixative and stored at room temperature. Plasma samples: Plasma samples were placed in blank centrifuge tubes. Proteins were precipitated with a formic acid-methanol solution containing a certain amount of camptothecin, vortexed, and all samples were allowed to stand for a while before centrifugation. The supernatant was transferred to a clean centrifuge tube and concentrated by vacuum centrifugation to dryness. Tissue samples: Accurately weigh liver, kidney, spleen, testis, jejunum, ileum, and colon tissues. Add a certain amount of formic acid-methanol solution containing camptothecin to the above samples to precipitate protein. Vortex and let all samples stand for a while. Centrifuge and transfer the supernatant to a clean centrifuge tube. Concentrate and evaporate to dryness in a vacuum centrifuge. (5) Metabolite analysis Plasma and tissue samples were reconstituted with cold methanol, vortexed, and centrifuged. The supernatant was placed in an intubation tube for UPLC-TQ-MS / MS analysis.

6. The metabolomics detection method for irinotecan according to claim 5, characterized in that: The preparation method of the mixed reference solution and internal standard solution in step (1) is as follows: Accurately weigh irinotecan, 7-ethyl-10-hydroxycamptothecin, SN-38 glucuronide, and camptothecin standards and dissolve them in methanol to prepare a reference stock solution with a concentration of 1 mg / mL; A series of mixed reference solutions were prepared by diluting each reference stock solution with methanol, with irinotecan concentrations of 10000, 5000, 2500, 1250, 625, 312.5, 78.13, 19.53, 9.77, and 2.44 ng / mL, respectively; The concentrations of 7-ethyl-10-hydroxycamptothecin were: 2000, 1000, 500, 250, 125, 62.5, 31.25, 15.56, 7.81, and 1.95 ng / mL; The concentrations of SN-38 glucuronide were: 10000, 5000, 2500, 1250, 625, 312.5, 78.13, 9.77, 4.88, and 2.44 ng / mL; Camptothecin was dissolved in methanol to prepare an internal standard solution; The internal standard solution was added to the mixed reference solution of each concentration so that the concentration of camptothecin in each mixed reference solution was 125 ng / mL.

7. The metabolomics detection method for irinotecan according to claim 1, characterized in that: The experimental animal modeling and grouping methods in step (3) are as follows: The mice were adaptively fed for one week and given standard food and drinking water every day. After one week of adaptive feeding, the mice in the model group received a single intraperitoneal injection of 10 mg / kg azomethane. One week later, the mice in the model group drank 2.0%-2.5% dextran sulfate sodium DSS solution for 7 days and normal drinking water for 14 days, which lasted for 3 consecutive cycles. After the modeling was completed, the mice were divided into the model group, irinotecan group, low-dose total flavonoids of glycyrrhiza combined with irinotecan group, medium-dose total flavonoids of glycyrrhiza combined with irinotecan group, high-dose total flavonoids of glycyrrhiza combined with irinotecan group, and total extract of glycyrrhiza combined with irinotecan group.

8. The metabolomics detection method for irinotecan according to claim 7, characterized in that: The sample processing method in step (4) is: After orbital blood collection, mice were killed by cervical dislocation and dissected. The liver, kidney, spleen, testis, jejunum, ileum, and colon tissues of the mice were completely removed and placed in tissue fixative and stored at room temperature. Plasma samples: 50 μL of plasma sample was placed in a blank centrifuge tube. Protein was precipitated with 350 μL of 0.1% formic acid in methanol (containing 125 ng / mL camptothecin) and vortexed for 3 min. All samples were allowed to stand for a while and then centrifuged at 13,000 rpm / min at 4°C for 10 min. The supernatant was transferred to a clean centrifuge tube and concentrated by vacuum centrifugation at 40°C to dryness. Tissue samples: Accurately weigh 50 mg of liver, kidney, spleen, testis, jejunum, ileum, and colon tissues respectively. Add 350 μL of 0.1% formic acid in methanol (containing 250 ng / mL camptothecin) to each sample to precipitate protein. Vortex and let all samples stand for a while before centrifuging at 13,000 rpm / min for 10-15 min. Transfer the supernatant to a clean centrifuge tube and evaporate to dryness by vacuum centrifugation at 40°C.

9. The metabolomics detection method for irinotecan according to claim 1, characterized in that: The chromatographic conditions for the preparation of the standard curve in step (2) and the UPLC-TQ-MS / MS analysis in step (5) of the metabolite analysis were as follows: Chromatographic separation was performed on an ACQUITY UPLC HSS T3 column with dimensions of 2.1 mm × 100 mm and 1.8 μm. The mobile phase consisted of 0.1% formic acid in water (B) and acetonitrile (A) for gradient elution. The injection volume was 1.0 μL, the flow rate was 0.3 mL / min, the column temperature was 35 °C, the autosampler temperature was 5 °C, and the analysis time was 11 min. The gradient elution program was as follows: The mass spectrometry conditions were as follows: electrospray ionization positive ion source (ESI + ) and multiple reaction monitoring (MRM) scan mode for quantification, with a capillary voltage of 3.0 kV, a desolvation temperature of 350 °C, and a conical gas flow rate of 150 L·h -1 , desolvation gas flow rate 1000 L·h -1 , the collision gas was argon.

10. The metabolomics detection method for irinotecan according to claim 1, characterized in that: The mass spectrometry conditions for each compound in step (5) are: 。

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