Application of Wantongkang in preparation of antitumor drugs

Abstract

The invention discloses an application of Wantongkang in preparation of an anti-tumor drug. Experiments show that Wantongyankang significantly reduces the weight and size of solid tumors of pancreatic cancer, shows a stronger growth inhibition effect on pancreatic cancer cells, induces programmed death of pancreatic cancer cells and has an anti-tumor effect, so that Wantongyankang can be used for resisting tumors.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine, specifically involving the application of Wantong Yankang in the preparation of anti-tumor drugs. Background Technology

[0002] Pancreatic cancer is a malignant tumor that primarily occurs in the pancreatic ductal epithelium (such as pancreatic ductal adenocarcinoma) or acinar cells, located deep in the abdominal cavity (retroperitoneum). It often manifests as malignant transformation of glandular epithelial cells, forming dense tumors with significant fibrosis, which readily invade nerves and blood vessels. Pancreatic cancer is a type of solid tumor, with early symptoms often being insidious, and metastasis is frequently present by the time it is discovered. First-line clinical treatment often involves 5-FU + oxaliplatin + irinotecan or gemcitabine + albumin-bound paclitaxel. Both pancreatic cancer and breast cancer are solid tumors, but the former is deep within the abdominal cavity and rapidly invades driven by KRAS, while the latter is easily detected on the body surface and relies on hormones or pathway targets. Ascites is actually a terminal stage manifestation of cancer, essentially abdominal effusion caused by metastatic cancer; treatment requires a combination of local drainage and systemic antitumor therapy. Leukemia is a hematologic malignancy characterized by arrested differentiation of hematopoietic cells, requiring epigenetic or metabolic targeted drugs to restore differentiation capacity. Although all four types of diseases are malignant, their origins, mechanisms, and drug choices are different—solid tumors require the inhibition of proliferation pathways (such as KRAS, CDK4 / 6), ascites requires the blocking of exudative factors (such as VEGF), and leukemia requires the reversal of differentiation disorders (such as IDH2, menin). Summary of the Invention

[0003] The purpose of this invention is to provide the application of Wantong Yankang in the preparation of anti-tumor drugs.

[0004] Through experiments, this invention has shown that Wantong Yankang significantly reduces the weight and size of pancreatic cancer solid tumors, exhibits a stronger growth inhibitory effect on pancreatic cancer cells, induces programmed cell death in pancreatic cancer cells, and has anti-tumor effects.

[0005] Therefore, this invention provides the application of Wantong Yankang in the preparation of anti-tumor drugs.

[0006] Preferably, the antitumor drug is an anti-pancreatic cancer drug.

[0007] The present invention also provides an anti-tumor drug containing Wantongyankang as an active ingredient.

[0008] Through experiments, this invention has found that Wantong Yankang significantly reduces the weight and size of pancreatic cancer solid tumors, exhibits a stronger growth inhibitory effect on pancreatic cancer cells, induces programmed cell death in pancreatic cancer cells, and has anti-tumor effects. Therefore, it can be used to prepare anti-tumor drugs. Attached image description:

[0009] Figure 1 The effect of WTYK on tumor growth in C57BL / 6J pancreatic cancer-bearing mice. Note: Results are expressed as a whole. This indicates that n=5; compared to the Vehicle group, * p<0.05, ** p<0.01.

[0010] Figure 2 The effects of WTYK, KUX, and ZJF on the proliferative activity of KPCY(6419c5), KPCY(2838c3), and Panc 02 pancreatic cancer cells were investigated.

[0011] Figure 3 The effect of WTYK on the clonal formation rate of KPCY(6419c5), KPCY(2838c3), and Panc 02 pancreatic cancer cells.

[0012] Figure 4 The effect of WTYK on apoptosis in KPCY(6419c5), KPCY(2838c3), and Panc 02 cells. Note: Results are expressed as a whole. This indicates that n=3; compared to the Control group, * p<0.05, ** p<0.01, *** p<0.001. Detailed Implementation

[0013] The following embodiments are further illustrations of the present invention, but not limitations thereof.

[0014] Example 1:

[0015] (I) Experimental Materials and Methods

[0016] 1. Sample preparation

[0017] Preparation of KXS and ZJF single-herb test samples:

[0018] The herbs *Scrophularia ningpoensis* (code KXS) and *Hedyotis diffusa* (code ZJF) were chopped and ground into powder. 1500g of each powder was placed separately into decoction bags, soaked in deionized water for 30 minutes, and then decocted twice, 2 hours each time. For the first decoction, the mixture was brought to a boil over high heat, then simmered over low heat. After 2 hours, the decoction was drained, and deionized water was added for a second decoction. The two decoctions were combined, filtered, and concentrated to a relative density of 1.10–1.15 (75℃). The mixture was then cooled, and three times the volume of the concentrated liquid was added to anhydrous ethanol. The mixture was stirred well, sealed with sealing film, and allowed to stand for 24 hours. The supernatant of the settled decoction was collected, and the ethanol was recovered by vacuum distillation. The concentrate was then concentrated to a relative density of 1.10–1.20 (70℃) to obtain extracts of *Scrophularia ningpoensis* and *Hedyotis diffusa*. The obtained single-herb extracts were cooled to room temperature and then freeze-dried to obtain dry extracts of the two herbs.

[0019] Wantong Yankang (code name WTYK) test sample: Wantong Yankang medicine was provided by Guangxi Wantong Pharmaceutical Co., Ltd., and was manufactured in accordance with the legal prescription. The specific production method is as follows:

[0020] [Prescription] Scrophularia ningpoensis 1500g, Rhizoma Scrophulariae rhizoma 1500g

[0021]

Preparation

[0022] 2. In vivo animal experiments

[0023] ① Establishment of a mouse pancreatic cancer model induced by in situ cell injection

[0024] Panc02 cells (Cytion, Germany) were resuspended in 10 μL of DMEM medium and mixed with an equal volume of Matrigel Matrix. The mixture was then injected into the pancreas of five-week-old female wild-type C57BL / 6J mice. One week after injection, the mice were randomly assigned to different groups to establish a mouse model of pancreatic cancer.

[0025] ② Mouse grouping

[0026] The mice that had successfully established a pancreatic cancer model in step (1) were randomly divided into 5 groups: control group (Vehicle group), positive drug gemcitabine group, Wantongyankang group (WTYK group), Kuxuanshen group (KXS group) and Zhongjiefeng group (ZJF group), with 5 mice in each group.

[0027] ③ Animal dosage

[0028] Vehicle group: Daily oral gavage with purified water at a dose of 1322 mg (water) / kg body weight for three weeks.

[0029] WTYK group: Daily oral gavage of Wantongyankang at a dose of 1322 mg (Wantongyankang drug) / kg body weight for three weeks.

[0030] Gemcitabine: 25 mg / kg, administered via tail vein twice a week for three weeks.

[0031] KXS group: Daily oral gavage of laboratory-prepared KXS at a dose of 661 mg (dried extract of Scrophularia ningpoensis) / kg body weight for three weeks.

[0032] ZJF group: The patients were given oral gavage of laboratory-prepared ZJF at a dose of 661 mg (dried extract of ZJF) per kilogram of body weight for three weeks.

[0033] The dosage for mice in the WTYK group was determined as follows: To determine the appropriate dosage for mice, this invention adopted the equivalent dose conversion method from the appendix of "Pharmacological Experimental Methodology" (People's Medical Publishing House, 2002). The drug dosage for mice = X mg / kg × 70kg × 0.0026 / 20g = 9.1X mg / kg, where X is the human dosage. Through this conversion, the dosage of WTYK for mice was calculated to be 1322 mg / kg. For the KXS and ZJF groups, considering the 1:1 ratio of the two components in the product, the actual content of a single herb = WTYK dosage for mice × component ratio = 661 mg / kg. All of the above calculations assume the drug is dissolved in water and administered in equal volumes.

[0034] ④ Appearance of mouse pancreas

[0035] After painless euthanasia of the mice, the abdomen was disinfected to expose the abdominal cavity. Blood vessels, mucous membranes, and other tissues connected to the spleen were carefully removed or severed. The entire spleen, pancreas, and tumor were then removed and photographed against a clean background.

[0036] 3. In vitro cell experiments

[0037] ① Sample grouping

[0038] Mouse pancreatic ductal adenocarcinoma cell line (Panc02) and two mouse pancreatic cancer cell lines (KPCY) were cultured. Logarithmic growth phase cells were divided into four groups: control group (CTL group), Wantongyankang group (WTYK group), Kuxuanshen group (KXS group), and Zhongjiefeng group (ZJF group), with three replicates in each group. Cells were cultured in DMEM medium for 24 hours.

[0039] ② Cell viability detection

[0040] Cell viability was determined using the MTT assay. Two different cell lines were seeded into 96-well cell culture plates at a concentration of 1000 cells / well. After one day of culture, DMEM medium containing four different concentrations of the four drugs and a blank medium were added for 24 hours. Four hours before the end of the experiment, 10 μl of MTT solution (5 mg / mL) was added, and the cells were incubated at 37°C. The culture supernatant was discarded, and 200 μl of DMSO was added to each well. The plates were shaken for 10 minutes to dissolve any crystals. The absorbance of each well was read at 570 nm using a microplate reader.

[0041] ③ Clonogenesis assay

[0042] Different cell lines were seeded into 12-well plates at a concentration of 500 cells / well. After one day of culture, DMEM medium containing different concentrations of the four drugs and blank medium were added for 24 hours. After 24 hours, all mediums were replaced with blank medium, and the plates were placed in an incubator and allowed to stand until cells in the control wells formed sufficiently large colonies. The medium in the wells was aspirated, the plates were washed three times with PBS, and fixed with 4% paraformaldehyde for 15 minutes. The liquid in the wells was aspirated, and the plates were washed three times with PBS. Crystal violet solution was added, and the plates were stained for 30 minutes. The crystal violet solution was removed, and the plates were rinsed thoroughly with tap water. After drying, the plates were photographed.

[0043] ④ Apoptosis experiment

[0044] Cells were seeded in 6-well plates at a concentration of 2.5 × 10⁵ cells / well and cultured for one day. The next day, DMEM medium containing four different concentrations of the drugs and a blank medium were added for 24 hours. All cells (including floating cells in the medium) were collected and stained with 5 μL FITC Annexin V and 5 μL 7-Aminoactinomycin D for 15 minutes in the dark at room temperature. The samples were then analyzed by flow cytometry.

[0045] (II) Experimental Results

[0046] 1. Effects of WTYK on tumor growth in C57BL / 6J pancreatic cancer-bearing mice

[0047] After 21 days of treatment with Vancomycin, C57BL / 6J pancreatic cancer-bearing mice were painlessly euthanized. The abdomens were disinfected to expose the abdominal cavity. Blood vessels, mucosa, and other tissues connected to the spleen were carefully removed or severed. The entire spleen, pancreas, and tumor were then removed and photographed against a clean background to measure tumor weight and volume. Figure 1 As shown, compared with the Vehicle group, the gemcitabine and Wantongyankang groups significantly reduced the weight and size of solid tumors, indicating that Wantongyankang has anti-tumor activity.

[0048] 2. WTYK vs. KPCY(6419c5), KPCY(2838c3)

[0049] like Figure 2As shown in Table 1, the inhibitory effect of WTYK on pancreatic cancer cell proliferation was dose-dependent, with the survival rate of pancreatic cancer cells gradually decreasing with increasing drug dosage. The IC50 values ​​of WTYK against KPCY(6419c5), KPCY(2838c3), and Panc02 pancreatic cancer cell lines were 0.4873 mg / ml, 0.3423 mg / ml, and 2.175 mg / ml, respectively. Under ZJF treatment, the survival rates of the three pancreatic cancer cell lines gradually decreased to 1.528 mg / ml, 1.012 mg / ml, and 0.8549 mg / ml, respectively; under KXS treatment, the survival rates were 0.4768 mg / ml, 3.119 mg / ml, and 7.613 mg / ml, respectively. These results indicate that there are differences in cell proliferation inhibition rates among different drug treatment groups. Compared with single extracts of *Scrophularia ningpoensis* and *Scrophularia ningpoensis*, the Wan Tong Yan Kang Fu Fang preparation showed a stronger inhibitory effect on pancreatic cancer cell growth.

[0050] Table 1. IC50 of pancreatic cancer cells under different drug treatments

[0051]

[0052]

[0053] 3. Effects of WTYK on the clonogenic rate of KPCY(6419c5), KPCY(2838c3), and Panc 02 pancreatic cancer cells.

[0054] like Figure 3 As shown, after 24 hours of drug intervention, all experimental groups showed a significant inhibitory effect on the clonogenic ability of pancreatic cancer cells. Notably, the Wantongyan Kangfufang preparation exhibited a dose-dependent inhibitory effect, demonstrating anti-tumor efficacy.

[0055] 4. Effects of WTYK on apoptosis in pancreatic cancer cells

[0056] like Figure 4 As shown, after 24 hours of drug intervention, compared with the control group, the KXS and WTYK groups significantly induced apoptosis in KPCY (6419c5), KPCY (2838c3), and Panc 02 pancreatic cancer cells, with the apoptosis rate gradually increasing. * p<0.05, ** p<0.01, *** The results (p<0.001) suggest that KXS and WTYK may exert their anti-tumor properties by inducing programmed cell death.

Claims

1. Application of Wantong Yankang in the preparation of anti-tumor drugs.

2. The application according to claim 1, characterized in that, The anti-tumor drug mentioned is a drug for treating pancreatic cancer.

3. An antitumor drug, characterized in that, It contains Wantong Yankang as an active ingredient.

4. The antitumor drug according to claim 3, characterized in that, The anti-tumor drug mentioned is a drug for treating pancreatic cancer.

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