Application of rhamnus utilis in preparation of anti-pancreatic cancer drugs

Abstract

The application discloses application of Rhamnus hemisphaerica in preparation of anti-pancreatic cancer drugs, wherein the Rhamnus hemisphaerica is dry root of Semiliquidam bar cathayensis Chang of Rhamnaceae. Researches show that the Rhamnus hemisphaerica can inhibit proliferation, invasion and migration of pancreatic cancer cells Pan-1, promote apoptosis, has anti-pancreatic cancer activity, can significantly inhibit growth of tumor of a subcutaneous nude mouse xenograft model of pancreatic cancer cells Panc-1, significantly inhibit expression of a tumor proliferation related factor Ki67, and has no obvious toxic side effect on mice. The application firstly discloses that the Rhamnus hemisphaerica has obvious anti-pancreatic cancer activity, can be used for preparation of anti-pancreatic cancer drugs, provides a new efficient and safe drug source for treatment of pancreatic cancer, and provides a basis for further development and utilization of the Rhamnus hemisphaerica.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine technology, specifically relating to the application of *Symplocos henryi* in the preparation of anti-pancreatic cancer drugs. Background Technology

[0002] Currently, surgical resection is the best treatment for pancreatic cancer. For patients with mid-to-late stage pancreatic cancer who are not candidates for surgery, the main treatment methods are chemotherapy, radiotherapy, and chemotherapy drugs. Due to the pancreas's unique location and the development of drug resistance in patients, most patients are not sensitive to chemotherapy. Moreover, radiotherapy and chemotherapy can produce many adverse reactions, which are difficult for patients to tolerate, thus affecting the overall treatment effect of pancreatic cancer. Chemotherapy is a commonly used method for treating pancreatic cancer in clinical practice, and it is the main method in the comprehensive treatment of advanced pancreatic cancer. However, as the drug dose slowly accumulates in the body, eventually approaching or even exceeding the toxic dose, relatively strong toxic side effects will occur, including bone marrow suppression, nausea and vomiting, and liver and kidney damage. Therefore, finding a highly effective, safe, low-toxicity, and inexpensive treatment plan is currently a hot research topic both domestically and internationally.

[0003] Clinical manifestations of pancreatic cancer include abdominal pain, unexplained weight loss, jaundice, and itching, which in Traditional Chinese Medicine (TCM) correspond to conditions such as "abdominal masses," "massage," "jaundice," and "fuliang" (a TCM concept). TCM considers the pancreas to be located in the middle jiao (middle burner), belonging to the Taiyin Spleen Meridian. The *Introduction to Medicine* states that "all five accumulations and six masses belong to the spleen." Modern physicians generally believe that the pathogenesis of pancreatic cancer is due to external damp-heat, emotional distress, and improper diet leading to spleen and stomach dysfunction in the middle jiao, with spleen deficiency as the key factor, and heat, dampness obstruction, phlegm accumulation, qi stagnation, and blood stasis as secondary manifestations. The lesion site is in the liver and spleen, and the pathological nature is one of deficiency in the root and excess in the branches, a complex mixture of deficiency and excess. In the field of oncology, traditional Chinese medicine is increasingly accepted as a supplementary or alternative treatment strategy. Traditional Chinese medicine can regulate tumor immunity through multi-level and multi-link interactions, inhibiting malignant proliferation, angiogenesis, and metastasis of tumor cells. Traditional Chinese medicine (TCM) emphasizes a holistic approach, using syndrome differentiation and treatment to address the systemic nature of pancreatic cancer, going beyond simply targeting the lesion itself. Through herbal remedies, TCM can alleviate clinical symptoms, alter the tumor microenvironment, eliminate factors contributing to tumor recurrence, reduce metastasis, improve clinical symptoms, reduce adverse reactions from radiotherapy and chemotherapy, enhance patients' quality of life, and prolong their survival. TCM plays a unique role in the comprehensive treatment of pancreatic cancer and shows promising prospects; therefore, seeking safe and effective TCM treatments for pancreatic cancer is an important research direction.

[0004] *Semiliquidambar cathayensis* Chang, a plant belonging to the genus *Semiliquidambar cathayensis* in the family Hamamelidaceae, is also known as half-maple lotus, wood lotus, and small-leaved half-maple lotus. It is a plant endemic to my country and a national second-class protected plant. Its medicinal use was first recorded in *Lingnan Caiyao Lu*, and it is mainly used for rheumatic pain, skin itching, traumatic bleeding, and postpartum paralysis. Modern pharmacological studies have shown that *Semiliquidambar cathayensis* Chang contains chemical components such as terpenes, flavonoids, phenylpropanoids, and alkaloids, which have anti-inflammatory, analgesic, and blood-activating effects. For example, studies using the hot plate test, capillary permeability test, and rat paw edema test have found that the water and ethanol extracts of the root of *Semiliquidambar cathayensis* have analgesic and anti-inflammatory effects (Jiang Yang, Qiu Xiaohua, Chen Bo, et al. Analgesic and anti-inflammatory effects of water and ethanol extracts of *Semiliquidambar cathayensis* on mice [J]. Journal of Guangdong Medical University, 2021, 39(06): 672-5.). *Senecio scandens* possesses abundant pharmacological activities, but research on its anti-tumor effects is relatively limited. Chinese patent application number 202011506976.8 discloses the use of *Senecio scandens* or its extracts in the preparation of anti-tumor drugs, specifically for glioma, breast cancer, colon cancer, or lung cancer. However, no reports have been found regarding its anti-pancreatic cancer effects. Summary of the Invention

[0005] The purpose of this invention is to provide an application of *Liriope muscari* in the preparation of anti-pancreatic cancer drugs, providing a new, highly effective and safe drug source for the treatment of pancreatic cancer, opening up new avenues for the treatment of pancreatic cancer, and providing a basis for the further development and utilization of *Liriope muscari*.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] Application of *Senecio scandens* in the preparation of anti-pancreatic cancer drugs.

[0008] The term "golden-haired half-maple" refers to the dried root of *Semiliquidam bar cathayensis* Chang, a species belonging to the Hamamelidaceae family.

[0009] Furthermore, in the aforementioned application, *Hypericum perforatum* is extracted with water to obtain its aqueous extract, which is then used to prepare an anti-pancreatic cancer drug. That is, the application of *Hypericum perforatum* aqueous extract in the preparation of an anti-pancreatic cancer drug.

[0010] Furthermore, the extraction method of the aqueous extract of *Hylocereus undatus* is as follows: take *Hylocereus undatus*, add 5-8 times its mass of distilled water and soak for 50-70 minutes, then heat to 80-100℃ and extract for 50-80 minutes, filter, and collect the filtrate; add 5-8 times its mass of distilled water to the filter residue and heat to 80-100℃ and extract for 50-80 minutes, filter, and collect the filtrate; repeat the extraction of the filter residue 1-3 times, combine the filtrates obtained from multiple extractions and concentrate to obtain the final product.

[0011] The aforementioned anti-pancreatic cancer drugs are drugs that inhibit the proliferation, migration, repair, and invasion of pancreatic cancer cells and / or drugs that induce apoptosis in pancreatic cancer cells.

[0012] In the above applications, the drug comprises a clinically acceptable pharmaceutical preparation for the prevention or treatment of pancreatic cancer, prepared alone or in combination with other drugs, consisting of *Hypericum sarmentosum* or its aqueous extract.

[0013] The aforementioned *Hypericum perforatum* or its water extract, with the addition of conventional excipients (such as starch, lactose, microcrystalline cellulose, dextrin, calcium phosphate, etc.), is processed according to conventional processes to produce a clinically acceptable pharmaceutical preparation for the prevention or treatment of pancreatic cancer. The preparation includes tablets, capsules, pellets, or granules.

[0014] This invention, through research, discovered that the inhibitory effect of *Hypericum perforatum* on the proliferation of pancreatic cancer cells exhibits a dose-dependent effect. The inhibitory effect on pancreatic cancer cell activity becomes increasingly significant with increasing concentrations of *Hypericum perforatum*. Cloning experiments, scratch assays, and Transwell assays revealed that *Hypericum perforatum* can inhibit the proliferation, migration, repair, and invasion of pancreatic cancer cells (Panc-1). Flow cytometry analysis showed that *Hypericum perforatum* can promote apoptosis in Panc-1 cells. These experimental results indicate that *Hypericum perforatum* possesses the ability to inhibit the proliferation, migration, and invasion of pancreatic cancer cells (Panc-1) and can promote apoptosis in these cells.

[0015] This invention established a subcutaneous transplantation model of Panc-1 pancreatic cancer cells in nude mice. After 21 days of gavage administration of *Hypericum perforatum*, changes in tumor volume and mouse weight were measured. Immunohistochemistry was used to detect changes in the tumor proliferation-related factor Ki67, and HE staining was used to observe changes in the liver tissue of the nude mice. The results showed that after successful establishment of the Panc-1 pancreatic cancer cell xenograft model in nude mice, after 21 days of gavage administration of *Hypericum perforatum*, the average subcutaneous tumor volume of nude mice treated with low- and high-dose *Hypericum perforatum* and 5-fluorouracil was lower than that of the control tumor-bearing group. However, there was no significant difference between the low-dose *Hypericum perforatum* group and the control group (p>0.05). The average tumor volume of the high-dose *Hypericum perforatum* group and the 5-fluorouracil group was significantly different from that of the control group (p<0.05), and the number of Ki67-positive cells was significantly reduced, indicating that *Hypericum perforatum* inhibits tumor growth. This invention evaluated the effect of *Hypericum perforatum* on the in vivo growth of pancreatic cancer using a nude mouse subcutaneous tumor-bearing model. The results showed that *Hypericum perforatum* significantly inhibited the growth of pancreatic cancer in vivo and significantly suppressed the expression of the tumor proliferation-related factor Ki67. TUNEL staining results indicated that *Hypericum perforatum* promoted apoptosis of tumor cells in nude mice. HE staining revealed that the liver tissue cells in the control group were intact and neatly arranged, while the liver tissue in the *Hypericum perforatum* treatment group had abundant cytoplasm, uniform color, and good development, indicating that *Hypericum perforatum* has no hepatotoxicity in mice.

[0016] The beneficial effects of this invention are as follows:

[0017] This invention demonstrates that *Hypericum perforatum* can inhibit the proliferation, invasion, and migration of pancreatic cancer cells (Pan-1), promote apoptosis, and possess anti-pancreatic cancer activity. *Hypericum perforatum* significantly inhibits tumor growth in a subcutaneous nude mouse xenograft model of Panc-1 cancer cells, significantly inhibits the expression of the tumor proliferation-related factor Ki67, and has no significant toxic side effects in mice. This invention discloses for the first time that *Hypericum perforatum* and its aqueous extract have significant anti-pancreatic cancer activity without significant toxic side effects, and can be used to prepare anti-pancreatic cancer drugs, providing a new, highly effective, and safe drug source for the treatment of pancreatic cancer, opening up new avenues for treatment, and providing a basis for the further development and utilization of *Hypericum perforatum*. This invention discloses the pharmacodynamics and mechanism of *Hypericum perforatum* in inhibiting pancreatic cancer, providing a theoretical basis for developing *Hypericum perforatum* for the treatment of pancreatic cancer, and providing new ideas for the discovery of new anti-pancreatic cancer drugs. Moreover, *Hypericum perforatum* resources are abundant and widely available, and the extraction and preparation methods of its aqueous extract are simple and easy to implement, showing good application prospects. Golden thread lily or its water extract can be used alone or in combination with other drugs to prepare clinically acceptable pharmaceutical preparations for the prevention or treatment of pancreatic cancer. Attached Figure Description

[0018] Figure 1IC50 (AH) of *Liriope muscari* against T98G, MCF-7, HePG2, Panc-1, A549, HCT116, A375, and NCI-H1299 cancer cells; IC50 (I) of 5-FU against Panc-1;

[0019] Figure 2 Golden-haired half-maple leaf inhibited the migration rate of pancreatic cancer cells Panc-1, ***p<0.001 vs control group, n=5;

[0020] Figure 3 Golden-haired half-maple leaf inhibited the proliferation rate of pancreatic cancer cells Panc-1, ***p<0.001 vs control group, n=5;

[0021] Figure 4 Golden-haired half-maple leaf inhibited the invasion rate of pancreatic cancer cells Panc-1, ***p<0.001 vs control group, n=5;

[0022] Figure 5 Golden-haired half-maple leaf inhibited the apoptosis rate of pancreatic cancer cells Panc-1, * p<0.05, *** p<0.001 vs control group, n=5;

[0023] Figure 6 *Hymenochloa chinensis* can inhibit the growth of pancreatic cancer Panc-1 cells in tumor-bearing mice. In the figure, (A)(B): Effect of *Hymenochloa chinensis* on tumor size, (C) Effect of *Hymenochloa chinensis* on body weight of nude mice, ns p>0.05, * p<0.05 vs control group;

[0024] Figure 7 Immunohistochemical detection of the effect of *Hypericum perforatum* on Ki67, a tumor proliferation marker in Panc-1 cell-bearing mice, ns p>0.05, * p<0.05, *** p<0.001 vs control group, n=5;

[0025] Figure 8 HE staining was used to detect the effect of *Symplocos henryi* on liver tissue structure in Panc-1 cell tumor-bearing mice, n=5;

[0026] Figure 9 Golden-haired half-maple promotes tumor apoptosis in nude mice** p<0.01, *** p<0.001 vs control group. Detailed Implementation

[0027] To provide a more detailed description of the present invention, the following embodiments are provided for further explanation. Example 1

[0028] Preparation of aqueous extract of *Hymenochloa crus-galli*: Take 1 kg of *Hymenochloa crus-galli*, add 6 L of distilled water and soak for 60 min, then heat to 98℃ and extract for 60 min, filter, and collect the filtrate; add 6 L of distilled water to the residue and heat to 98℃ and extract for 60 min, filter, and collect the filtrate; add 6 L of distilled water to the residue and heat to 98℃ and extract for 60 min, filter, and collect the filtrate; combine the three extracts and concentrate to 1.0–1.2 g / mL to obtain the final product. Application of the aqueous extract of *Hymenochloa crus-galli* in the preparation of anti-pancreatic cancer drugs. Example 2

[0029] Preparation of aqueous extract of *Symplocos rubra*: Take *Symplocos rubra*, add 5 times its mass of distilled water and soak for 50 min, then heat to 80℃ and extract for 50 min, filter and collect the filtrate; add 5 times its mass of distilled water to the filter residue and heat to 80℃ and extract for 50 min, filter and collect the filtrate; repeat the extraction of the filter residue 3 times, combine the filtrates obtained from multiple extractions and concentrate to 1.0-1.2 g / mL to obtain the extract. Example 3

[0030] Preparation of aqueous extract of *Symplocos rubra*: Take *Symplocos rubra*, add 8 times its mass of distilled water and soak for 70 min, then heat to 90℃ and extract for 80 min, filter and collect the filtrate; add 8 times its mass of distilled water to the filter residue and heat to 90℃ and extract for 80 min, filter and collect the filtrate; repeat the extraction of the filter residue twice, combine the filtrates obtained from multiple extractions and concentrate to 1.0-1.2 g / mL to obtain the extract.

[0031] Example 4: Efficacy test of *Syzygium aromaticum* against pancreatic cancer

[0032] 1. Activity analysis of *Leucaena fragrans* against pancreatic cancer cells Panc-1

[0033] The IC50 of *Hypericum perforatum* against seven cancer types was determined using the CCK-8 assay, revealing that pancreatic cancer cells (Panc-1) were most sensitive to *Hypericum perforatum*. Flow cytometry was then used to investigate the effect of *Hypericum perforatum* on Panc-1 cell apoptosis. Cloning assays were used to observe the effect of *Hypericum perforatum* on Panc-1 cell proliferation. Scratch assays were used to observe the effect of *Hypericum perforatum* on Panc-1 cell migration and repair. Finally, Transwell assays were used to observe the invasive ability of *Hypericum perforatum* on Panc-1 cells.

[0034] 1.1 Experimental Materials

[0035] 1.1.1 Cell line source: The pancreatic cancer cell line Panc-1 was kindly donated by the experimental groups of Professor Huang Min and Professor Wang Xueding of the North Campus of Sun Yat-sen University.

[0036] 1.1.2 Experimental Drugs

[0037] The root of *Semiliquidam bar cathayensis* Chang (batch number 20211005), purchased from the Nanning Traditional Chinese Medicine Market in Guangxi, was identified by Associate Researcher Huang Yunfeng of the Guangxi Zhuang Autonomous Region Academy of Traditional Chinese Medicine as the dried root of *Semiliquidam bar cathayensis* Chang (Hamamellaceae family). Preparation of the aqueous extract of *Semiliquidam bar cathayensis* Chang: 1 kg of *Semiliquidam bar cathayensis* Chang was soaked in 6 L of distilled water for 60 min, then heated to 98℃ for 60 min, filtered, and the filtrate was collected. The residue was then added to 6 L of distilled water and heated to 98℃ for 60 min, filtered, and the filtrate was collected. The residue was then added to 6 L of distilled water and heated to 98℃ for 60 min, filtered, and the filtrate was collected. The three extracts were combined and concentrated to 1.0–1.2 g / mL to obtain the final product.

[0038]

[0039]

[0040] 1.1.5 Solution Preparation

[0041] Preparation of mother liquor from *Hypericum perforatum*: Accurately weigh 1g of *Hypericum perforatum* extract, dissolve it thoroughly in sterile water to a concentration of 500mg / ml, filter through a 0.45um filter, and then dispense and store at -20℃. Dilute to the required concentration before use.

[0042] DMEM complete medium preparation: 1% penicillin-streptomycin + 10% fetal bovine serum + 89% DMEM high glucose medium. Prepare 50 mL each time, use immediately, and store at 4℃.

[0043] Preparation of 5-FU stock solution: Weigh 2.460 mg of the standard and add 0.819 mL of DMSO to prepare a 10 mM stock solution, which should be stored at low temperature. Before starting the experiment, dilute according to the drug concentration settings.

[0044] 1.2 Experimental Methods

[0045] 1.2.1 Cell Culture Methods

[0046] Cell thawing: Remove frozen cells from liquid nitrogen tanks or -80°C freezers and immediately place them in a 37°C water bath. Shake the cryovials to rapidly thaw the cells. Once completely thawed, transfer them to 10mL centrifuge tubes and add twice the volume of fresh complete culture medium. Mix well with a pipette and centrifuge at 1000rpm for 5min. Discard the supernatant, gently tap the bottom of the tube to disperse the cells, add an appropriate amount of complete culture medium, mix well, and transfer to suitable culture flasks. Incubate in a 5% CO2, 37°C incubator. Panc-1 cells were cultured using DMEM complete medium, and cells in good growth condition and in the logarithmic growth phase were used for each experiment.

[0047] Panc-1 cell passage: When the cells reach 80%-90% confluence under a microscope, discard the old culture medium, rinse once with PBS and discard the PBS, then digest the cells with 0.25% trypsin. When the intercellular spaces increase and become rounded under a microscope, add fresh complete culture medium equal to the amount of trypsin to stop the digestion. Gently pipette the cells on the lower wall to completely separate them from the lower wall, transfer them to a 10mL centrifuge tube, centrifuge at 800rpm for 3min, discard the supernatant, gently tap the bottom of the tube to disperse the cells, add an appropriate amount of complete culture medium, pipette to mix, and transfer to a suitable culture flask. Incubate in a 5% CO2 incubator at 37℃.

[0048] Cell cryopreservation: After digestion and centrifugation, the supernatant of the collected cells was discarded and the pre-prepared cryopreservation solution (90% FBS + 10% DMSO) was added. After mixing with a pipette, the cells were quickly transferred to cryovials labeled with cell type, passage number and cryopreservation date. 0.8-1.2 mL of cell suspension was added to each cryovial, and the cells were cryopreserved in a gradient (4℃ for 30 min, -20℃ for 60 min, -80℃ overnight, and finally stored in liquid nitrogen).

[0049] 1.2.2 IC50 of *Leymus chinensis* in different types of cancer cells 50

[0050] Eight types of cancer cells in good condition and in the logarithmic growth phase were selected, including T98G, MCF-7, HePG2, Panc-1, A549, HCT116, A375, and NCI-H1299. The cell density was adjusted to 5*102 4 Seeds were prepared at 100 μL / well in 96-well plates at a concentration of 1 / mL. The plates were incubated at 37°C with 5% CO2 for 24 hours. The old culture medium was discarded, and the plates were washed twice with sterile PBS. Different concentrations of drug-containing culture medium were added at 100 μL / well, with three parallel wells per group. The plates were incubated for another 48 hours. The conditioned medium was discarded, and 100 μL / well of CCK8 solution diluted with DMEM was added. After incubation for 1 hour, the absorbance at OD450 nm was measured using a microplate reader. The IC50 of *Symplocos henryi* in different types of cancer cells was calculated. 50 .

[0051] 1.2.3 Cloning assay to detect cell proliferation capacity

[0052] Panc-1 cells in the logarithmic growth phase were seeded into 6-well plates at a concentration of 500 cells / well and cultured overnight in an incubator at 37°C and 5% CO2. After 48 hours, medium containing *Hydrangea macrophylla* (1 mg / ml, 2 mg / ml) and 5-FU (0.7 μM) was added. The cells were then cultured in fresh medium for 5-6 days. The presence and size of clones were observed and counted.

[0053] 1.2.4 Flow cytometry detection of apoptosis

[0054] Take Panc-1 cells in the logarithmic growth phase and administer at a concentration of 2*10-1 5 Cells were seeded at a density of 1 / mL in 6-well plates and cultured at 37°C with 5% CO2 for 24 hours. Medium containing either *Hypericum esculentum* (1 mg / mL) or 2 mg / mL, or 5-FU (0.7 μM), was added, and the cells were incubated for another 48 hours. The supernatant was collected, and the cells were washed three times with PBS. Cells were digested with trypsin without EDTA, centrifuged at 1000 rpm for 5 min, and resuspended in 195 μL of Annexin V-FITC binding solution. Immediately after resuspending, 5 μL of Annexin V-FITC was added and thoroughly mixed. Then, 10 μL of propidium iodide (PI) staining solution was added and gently mixed. The cells were incubated at room temperature in the dark for 15 min. Parallel single-stain groups (containing only Annexin V-FITC or PI dye) and a control group (without dye) were established. Apoptosis was detected by flow cytometry within half an hour.

[0055] 1.2.5 Scratch assay for cell migration

[0056] Take healthy Panc-1 cells in the logarithmic growth phase, adjust the cell density to 5*102 5 Cells were seeded at 2 mL / well in 6-well plates and incubated at 37°C with 5% CO2 for 24 hours. The old culture medium was discarded, and a straight scratch was made perpendicular to the bottom wall of the well using a 200 μL pipette tip and a ruler. The cells were washed three times with sterile PBS to remove the scratched cells. 2 mL / well of medium containing either *Hypericum esculentum* (1 mg / mL) or 5-FU (0.7 μM) was added to each well, with 3 parallel wells per group. The plates were incubated for 48 hours. The plates were then removed, and three fields of view were randomly selected from each well for photographs to observe the effects of the drugs on cell migration and scratch repair. The area of ​​the unhealed region was calculated using ImageJ software to observe the effects of the drugs on cell migration and scratch repair.

[0057] 1.2.6 Transwell assay for cell invasion

[0058] Thaw Matrigel on ice beforehand. Dilute Matrigel with serum-free medium (Matrigel:serum-free medium = 1:8). Add 80 μL of the diluted Matrigel to the upper chamber of the Transwell chamber and incubate at 37°C with 5% CO2 for 3 hours (avoiding air bubbles). After gel formation, hydrate with serum-free medium for 30 minutes. Add 200 μL of Panc-1 cell suspension containing *Hypericum esculentum* (1 mg / ml, 2 mg / ml) and 5-FU (0.7 μM) to the upper and lower chambers of the Transwell chamber, respectively, and 750 μL of DMEM medium containing fetal bovine serum. Remove air bubbles thoroughly and incubate at 37°C with 5% CO2 for 24 hours. Remove the Transwell chamber, discard any remaining culture medium in the wells, wash twice with calcium-free PBS, and gently wipe away any unmigrated cells from the supernatant with a cotton swab. Fix with methanol for 20 minutes. Wash twice with PBS, place the chamber in 1 mL of 0.1% crystal violet solution, stain at room temperature in the dark for 15 minutes, wash the chamber with PBS, wipe the cells off the upper surface of the chamber with a cotton swab, and take a picture under an inverted microscope at 200x magnification.

[0059] 1.2.7 Statistical Analysis Methods

[0060] All experimental data were normalized and statistically analyzed using GraphPadPrism 8.0. Each experiment was repeated at least three times (N≥3). All experimental values ​​represent mean ± standard deviation (X±SD). One-way ANOVA was used for data analysis between groups, and t-tests were used for comparisons between two groups. p<0.05 was considered statistically significant.

[0061] 1.3 Results

[0062] 1.3.1 IC50 of *Leymus chinensis* on Panc-1 cells 50

[0063] IC50 of Fiberglass 'Golden Witch' against 8 types of cancer cells 50 like Figure 1 As shown, the results indicate that *Leymus chinensis* has an IC50 effect on pancreatic cancer cells Panc-1. 50 The lowest IC50 value indicates that *Ligustrum lucidum* is most sensitive to pancreatic cancer; therefore, this study focused on pancreatic cancer cells (Panc-1). The IC50 of 5-FU against Panc-1 pancreatic cancer cells... 50 =0.6603 μmol / L. Therefore, the concentration of 5-FU selected in subsequent experiments was 0.7 μmol / L.

[0064] 1.3.2 Effects of *Leucaena fragrans* on the migration ability of Panc-1 cells

[0065] Compared with the control group, the low-concentration (10 g / kg) and high-concentration (20 g / kg) groups of *Hypericum perforatum* significantly reduced the scratch healing rate of Panc-1 cells (p<0.001), indicating that *Hypericum perforatum* significantly inhibited Panc-1 migration. Simultaneously, it was found that 5-FU also inhibited the healing ability of Panc-1 cells compared with the control group, and the difference was statistically significant (p<0.001). See Figure 2 .

[0066] 1.3.3 Effects of *Leymus chinensis* on the cloning ability of Panc-1 cells

[0067] Compared with the control group, both low and high concentrations of *Hypericum perforatum* reduced the number of Panc-1 cell clones (p<0.001), indicating that *Hypericum perforatum* significantly inhibited Panc-1 proliferation. 5-FU also inhibited cell proliferation within the same timeframe (p<0.001), see [link to relevant data]. Figure 3 .

[0068] 1.3.4 Effects of *Leymus chinensis* on the invasive ability of Panc-1 cells

[0069] Compared with the control group, the low and high concentrations of *Hypericum perforatum* reduced the number of invasive Panc-1 cells (p<0.001), indicating that *Hypericum perforatum* significantly inhibited Panc-1 invasion. 5-FU also inhibited cell invasion within the same timeframe (p<0.001). See [link to relevant documentation]. Figure 4 .

[0070] 1.3.5 Effect of *Leymus chinensis* on apoptosis in Panc-1 cells

[0071] Compared with the control group, the low and high concentrations of *Hypericum perforatum* significantly increased the apoptosis rate of Panc-1 cells (p<0.001). 5-FU also inhibited cell apoptosis within the same timeframe (p<0.001), see [link to relevant documentation]. Figure 5 .

[0072] 1.4 Discussion

[0073] Apoptosis, a process of programmed cell death, plays a crucial role in human development and tissue homeostasis. Increasing evidence suggests that apoptosis is closely related to cancer survival and has become a key target for the discovery and development of novel anticancer drugs. Studies have found that promoting apoptosis can inhibit the growth of pancreatic cancer cells. Our research revealed that the inhibitory effect of *Hypericum perforatum* on the proliferation of pancreatic cancer cells is dose-dependent. The inhibitory effect on pancreatic cancer cell viability becomes more significant with increasing concentrations of *Hypericum perforatum*. Cloning assays, scratch assays, and Transwell assays showed that *Hypericum perforatum* can inhibit the proliferation, migration, repair, and invasion of pancreatic cancer cells (Panc-1). Flow cytometry analysis showed that *Hypericum perforatum* can promote apoptosis in Panc-1 cells. These results indicate that *Hypericum perforatum* possesses the ability to inhibit the proliferation, migration, and invasion of pancreatic cancer cells (Panc-1) and can promote apoptosis in these cells.

[0074] 1.5 Summary

[0075] This experiment shows that *Leontopodium album* can inhibit the proliferation, invasion, and migration of pancreatic cancer cells Panc-1 and promote apoptosis of pancreatic cancer cells Panc-1, preliminarily demonstrating anti-pancreatic cancer activity, which needs further verification in subsequent animal experiments.

[0076] 2. Effects of *Senecio scandens* on subcutaneous pancreatic cancer xenografts in nude mice

[0077] Previous in vitro studies on the proliferation and apoptosis of Panc-1 pancreatic cancer cells (Hypericum perforatum) suggested that it could inhibit the proliferation of Panc-1 cells and induce apoptosis. However, the growth environment and growth status of cells differ significantly between in vivo and in vitro, and the composition of drug-containing serum also differs greatly from the effective components after oral administration and metabolism. Therefore, to address these issues, this part of the experiment conducted a nude mouse xenograft experiment based on the in vitro experiments. Through tumor observation, HE staining, and immunohistochemistry, the effects of Hypericum perforatum on the growth of BALB / c Panc-1 cell xenografts in nude mice and its possible mechanisms were further clarified.

[0078] 2.1 Materials and Instruments

[0079] 2.1.1 Experimental Animals: Twenty BALB / c nude mice, 4 weeks old, weighing approximately 18-22g, were purchased from Guangzhou Ruige Biotechnology Co., Ltd. The animals were housed in a clean-grade animal room, which was regularly disinfected by ultraviolet irradiation. The feed, drinking water, and equipment used by the nude mice were also regularly disinfected. The room temperature was controlled at (22±1)℃, and the relative humidity was 45%-55%.

[0080] 2.1.2 Cell line source: Same as 1.1.1.

[0081]

[0082]

[0083] 2.2 Experimental Methods

[0084] 2.2.1 Preparation of drugs and reagents

[0085] Golden-haired half-maple extract: Accurately weigh 14.48g of golden-haired half-maple extract, dissolve it fully in sterile water to 20g / kg, dilute it with sterile water to 10g / kg before use, and store it in a -20℃ refrigerator.

[0086] Preparation of 5-FU injection: Weigh 27.8 mg of the standard, add 9.26 mL of 0.9% physiological saline, and prepare an injection solution with a concentration of 30 mg / kg. Store at low temperature.

[0087] 2.2.2 Construction of a subcutaneous pancreatic cancer xenograft model

[0088] Cells in the logarithmic growth phase were collected, digested with trypsin, centrifuged, and resuspended in PBS. The cell concentration was adjusted to 1.5 × 10⁻⁶ cells / day. 7 / mouse, each nude mouse was subcutaneously (axillary) inoculated with 200 μL of cell suspension. Before inoculation, the axilla was wiped with povidone-iodine to avoid cell contamination; during inoculation, the needle was inserted at a 30° angle to ensure subcutaneous insertion, and the cell suspension was slowly injected. After inoculation, a cotton swab was gently pressed on the inoculation site to prevent leakage of the cell suspension.

[0089] Several days later, granular protrusions were observed under the armpits of nude mice, indicating successful model establishment. The mice were randomly divided into four groups using a random number table: tumor-bearing model group, 5-fluorouracil group (30 mg / kg), low-dose *Hydrangea macrophylla* group (10 g / kg), and low-dose *Hydrangea macrophylla* group (20 g / kg). The 5-fluorouracil group received intraperitoneal injections twice weekly, the low- and high-dose *Hydrangea macrophylla* groups received gavage once daily, and the tumor-bearing model group received sterile water via gavage once daily. Following the equivalent dose conversion formula between human and mouse body weight, each mouse received 0.2 ml per day. The treatment period was 21 days, during which the physiological status of the nude mice in each group was closely monitored.

[0090] 2.2.3 Plot the tumor volume change curve and measure the mouse body weight.

[0091] To investigate the therapeutic toxicity of *Liriope muscari* in animals, mice were weighed every other day. The shorter vertical axis (W) and longer vertical axis (L) of the abdominal tumor were measured, and the tumor volume was calculated using the formula: V = W 2*L*0.5, based on the mouse growth time, a tumor volume change curve was plotted with time on the horizontal axis and tumor volume change on the vertical axis. Mouse weight was measured every two days, and the experiment lasted for 3 weeks. 21 days after drug administration, nude mice in each group were weighed after drug withdrawal. All nude mice were euthanized by cervical dislocation under deep anesthesia with sodium pentobarbital, and the transplanted tumor and liver were dissected. The tumor tissue was divided into two parts: one part was frozen in liquid nitrogen and stored in an ultra-low temperature freezer at -80℃; the other part was fixed in 4% paraformaldehyde for subsequent experiments.

[0092] 2.2.4 HE staining method for observing pathological changes in tumor tissue

[0093] (1) Sample collection: Nude mouse tumor tissue and liver tissue were soaked in 4% paraformaldehyde for 24 hours, then the tissue was repaired with a blade and placed in a tissue embedding box and soaked in 70% ethanol for 24 hours.

[0094] (2) Dehydration, clearing, and paraffin infiltration: Place the embedding cassette in the fully automatic tissue dehydrator and set the program in Table 2-1.

[0095]

[0096] (3) Embedding: Place the tissue into the embedding clip, allow the paraffin to solidify completely, mark it, and store at 4°C.

[0097] (4) Slicing: Fix the wax block, and be careful not to use too much force to break the wax block. First make a rough cut and then a fine cut. Cut a slice with a thickness of 4μm, mark it, and bake at 60℃ for 4 hours.

[0098] (5) Dewaxing and HE staining: Dewaxing the sections according to the procedure in Table 2-2, then staining with hematoxylin for 3 minutes, rinsing with running water to reverse blue staining, rinsing with ultrapure water for 3 seconds, soaking in eosin staining for 1.5 minutes, soaking in 70% ethanol for 1 minute, soaking in 80% ethanol for 1 minute, soaking in 90% ethanol for 1 minute, soaking in 95% ethanol for 1 second, soaking in anhydrous ethanol I and II for 5 minutes each, and soaking in xylene I and II for 5 minutes each.

[0099]

[0100] 2.2.5 Immunohistochemical detection of Ki-67

[0101] (1) The experimental steps such as tissue fixation, embedding, and dewaxing are consistent with HE staining.

[0102] (2) Antigen retrieval: High-temperature retrieval. Place the tissue sections in a beaker and add 20× citrate antigen retrieval solution, ensuring the solution completely submerges the tissue sections. Retrieve for approximately 12 minutes, being careful not to allow the tissue sections to dry. After completion, remove the beaker and allow it to cool naturally to room temperature. Buffer the sections with PBS three times. Do not rinse the tissue directly during rinsing to avoid damaging it.

[0103] (3) Inactivation: that is, the blocking of endogenous catalase. Add 3% hydrogen peroxide solution to the tissue section, incubate for 5 minutes at room temperature, and then rinse with PBS 3 times, each time for 3-5 minutes.

[0104] (4) Blocking: Before blocking, wipe off the PBS on the slide with absorbent paper, and apply 1-2 drops of 5% BSA to each tissue. Block at 37°C for 30 minutes. Draw an appropriately sized circle around the tissue with a histochemical pen.

[0105] (5) Incubation: Add 100 μL of primary antibody (diluted at a certain ratio) to the circle of the tissue section, making sure to completely cover the tissue. Place the slides in a humidifier and incubate overnight at 4°C. Rinse with PBS 2-3 times, 5 minutes each time. After drying the sections, add HRP-labeled goat anti-rabbit secondary antibody and incubate at room temperature for 30 minutes.

[0106] (6) DAB staining and counterstaining: After drying the secondary antibody solution, rinse the sections 5 times with PBS for 3 minutes each time. After drying the PBS, add DAB developing solution and observe under a microscope. If positive expression is observed, immediately rinse off the developing solution gently with distilled water. Counterstain with hematoxylin solution for 1-2 minutes. After rinsing with water, differentiate with 1% hydrochloric acid alcohol and rinse with ultrapure water to restore blue color.

[0107] (7) Dehydration: After rinsing the sections under running water, the sections are placed in different concentration gradients of ethanol (70%, 80%, 90%, 95%, anhydrous ethanol I, anhydrous ethanol II) and xylene for dehydration, and then the sections are air-dried.

[0108] (8) Mounting and observation: Mount the slides with neutral resin, air dry at room temperature, observe under a microscope, take images, and calculate the positive cell ratio using ImageJ. Ki-67 positive expression is located in the cell nucleus and appears as brownish-yellow granules.

[0109] 2.2.6 TUNEL staining of tumor tissue:

[0110] (1) Routine dewaxing and hydration of liver tissue slides;

[0111] (2) Soak the slide in 1×PBS and wash it. Carefully blot the excess liquid around the sample on the slide with filter paper. Then use a wax pen to outline the distribution of the sample around the sample.

[0112] (3) Dilute 2 mg / ml Proteinase K solution with deionized water at a ratio of 1:100 to make the final concentration 20 μg / ml. Add 100 μl of Proteinase K solution with a concentration of 20 μg / ml to each sample to cover it completely, and incubate at room temperature for 20 min.

[0113] (4) Soak the sample in 1×PBS 2-3 times, 5 min each time; gently remove excess liquid and blot the liquid around the sample on the slide with filter paper; keep the treated slide moist in a humidifier.

[0114] (5) Dilute 5× Equilibration Buffer with ddH2O at a ratio of 1:5;

[0115] (6) Add 100 μl of 1×Equilibration Buffer to each sample to completely cover the sample area to be tested, and incubate at room temperature for 30 minutes;

[0116] (7) Prepare TdT incubation buffer under light-protected conditions during equilibration.

[0117] (8) After equilibration, wash off 1×Equilibration Buffer with absorbent paper, and then add 50ul TdT incubation buffer to the sample under light-protected conditions;

[0118] (9) Cover the tissue with a plastic coverslip to ensure even distribution of the reagents, and place a water-moistened paper towel at the bottom of the humidified chamber. Place the slide in the humidified chamber and incubate at 37°C for 60 min. Wrap the humidified chamber with aluminum foil to protect it from light. Then wash three times with PBS, 5 min each time;

[0119] (10) Counterstaining the nucleus: Add DAPI and incubate in the dark for 5 min to stain the nucleus. Wash away excess DAPI with PBST for 5 min × 4 times.

[0120] (11) Mount the slide with a mounting solution containing an anti-fluorescence quencher, and then observe the acquired image under a fluorescence microscope.

[0121] 2.2.7 Statistical Analysis

[0122] Statistical data were processed using GraphPadPrism 8.0 software. Results are expressed as mean ± standard deviation. T-tests were used for comparisons between groups, and one-way ANOVA was used for comparisons among multiple groups. A p-value < 0.05 was considered statistically significant.

[0123] 2.3 Results

[0124] 2.3.1 *Senecio scandens* can inhibit the growth of pancreatic cancer Panc-1 cells in tumor-bearing mice.

[0125] Five days after axillary inoculation of pancreatic cancer Panc-1 cells, small, raised masses were observed in the axilla, with a tumor formation rate of approximately 95%. To evaluate the function of *Hypericum perforatum* in nude mice against pancreatic cancer Panc-1 cells, we treated tumor-bearing mice with different concentrations of *Hypericum perforatum* and observed its effect on tumor growth. Figure 6 As shown, the results indicated that the average subcutaneous tumor volume in nude mice treated with low-dose (10 g / kg), high-dose (20 g / kg), and 5-fluorouracil (30 mg / kg) groups of *Hydrangea macrophylla* was lower than that in the control group, but there was no significant difference in tumor growth between the low-dose group and the control group (p>0.05). Tumor growth was significantly inhibited in the high-dose group and the 5-fluorouracil group (p<0.05).

[0126] Simultaneously, we observed the behavior and emotional state of the tumor-bearing mice in the experimental group. The results showed that no behavioral abnormalities were observed in the mice during the experiment. We also monitored the weight of all mice to further assess the potential toxic side effects of *Hypericum esculentum*. Figure 6 As shown, after 21 days of continuous gavage administration of *Hypericum perforatum*, the experimental group of tumor-bearing mice showed a slight decrease in body weight compared to the control group, but the difference was not significant, indicating that *Hypericum perforatum* did not have significant systemic toxic side effects at the doses used in this study.

[0127] 2.3.2 Immunohistochemical detection of the effect of *Hypericum perforatum* on Ki67, a tumor proliferation marker in Panc-1 cell-bearing mice

[0128] Malignant proliferation is a characteristic of cancer. To further confirm the chemopreventive potential of *Hypericum perforatum* in mice, we observed the expression of the tumor proliferation marker Ki67 by *Hypericum perforatum* through immunohistochemistry. Figure 7 The results showed that, compared with the Control group, the proportion of Ki67 positive cells in tumor tissue was reduced in both the low and high dose groups of *Hypericum perforatum*, with a significant difference compared with the Control group (p<0.05), further confirming the inhibitory effect of *Hypericum perforatum* on tumor growth.

[0129] 2.3.3 HE staining to observe changes in liver tissue of nude mice

[0130] After three weeks of intervention with different concentrations (10, 20 g / kg) of *Hypericum perforatum* and 30 mg / kg of 5-fluorouracil, preliminary changes in liver tissue structure in tumor-bearing mice were observed. Figure 8 As shown, HE staining revealed that the liver cells in the blank control group were intact and neatly arranged. After treatment with *Hypericum perforatum* and 5-fluorouracil, microscopic examination revealed abundant cytoplasm, uniform color, and good development in the liver tissue.

[0131] 2.3.4 *Senecio scandens* promotes tumor apoptosis in nude mice

[0132] After 3 weeks of intervention with different concentrations (10, 20 g / kg) of *Hypericum perforatum* and 30 mg / kg of 5-fluorouracil, the TUNEL staining results of tumor tissues in tumor-bearing mice are as follows: Figure 9 The results showed that with increasing concentrations of *Hypericum perforatum*, the number of positive cells increased slightly. Compared with the control group, the apoptosis rate (number of apoptotic cells / total cells) was significantly increased in the low-concentration, high-concentration (10, 20 g / kg), and 5-FU (30 mg / kg) groups of *Hypericum perforatum* (p<0.01). These results suggest that *Hypericum perforatum* may promote tumor apoptosis in nude mice to combat pancreatic cancer.

[0133] 2.4 Discussion

[0134] Tumor growth is a multi-step, multi-stage process. In vitro tumor models can be used to study the etiology and mechanisms of tumors and to screen anticancer drugs. Subcutaneous xenografts involve inoculating in vitro-derived tumors subcutaneously into animals to form transplanted tumor tissue. Because these models are quick to construct, have a high tumor formation rate, are low-cost, and technically relatively simple, they can directly reflect the biological characteristics of tumors. Nude mice and humans share high similarities in genetics, therefore nude mouse models are widely used. In vitro tumor cell models are faster and simpler than in vivo models for studying gene function analysis and evaluating the anticancer effects of drugs. We evaluated the effect of *Hypericum perforatum* on in vivo pancreatic cancer growth using a nude mouse subcutaneous tumor-bearing model of pancreatic cancer, finding that *Hypericum perforatum* significantly inhibited in vivo pancreatic cancer growth. We also evaluated the toxic effects of *Hypericum perforatum* on normal mouse liver tissue, finding no significant toxic effects on the mouse liver.

[0135] Ki67 is a proliferation-associated nuclear antigen widely used in routine tumor pathology diagnosis and research. Its expression is closely related to tumor proliferation and growth, and it can be detected only in dividing cells (G1, S, G2, and M phases), but is usually undetectable in resting cells (G0 phase). Ki67 expression is significantly higher in poorly differentiated cancer tissues than in normal tissues. Experimental results show that *Syzygium serratum* can significantly inhibit the expression of the tumor proliferation-associated factor Ki67.

[0136] 2.5 Summary

[0137] This experiment aimed to further verify the anti-pancreatic cancer effect of *Hypericum perforatum*. A nude mouse axillary xenograft model was successfully established, and both *Hypericum perforatum* and 5-fluorouracil effectively inhibited the growth of xenografts in nude mice. The anti-pancreatic cancer effect of *Hypericum perforatum* was observed using HE staining, TUNEL staining, and IHC techniques, and no significant toxic side effects were observed in mice.

Claims

1. Application of *Leucaena macrantha* in the preparation of anti-pancreatic cancer drugs, wherein *Leucaena macrantha* is the dried root of *Leucaena macrantha* of the Hamamelidaceae family, and water is added to extract the water extract of *Leucaena macrantha* to obtain its water extract, which is used to prepare anti-pancreatic cancer drugs.

2. The application according to claim 1, characterized in that, The extraction method of the aqueous extract of *Hylocereus undatus* is as follows: Take *Hylocereus undatus*, add 5-8 times its mass of distilled water and soak for 50-70 minutes, then heat to 80-100℃ and extract for 50-80 minutes, filter and collect the filtrate; add 5-8 times its mass of distilled water to the filter residue and heat to 80-100℃ and extract for 50-80 minutes, filter and collect the filtrate; repeat the extraction of the filter residue 1-3 times, combine the filtrates obtained from multiple extractions and concentrate to obtain the final product.

3. The application according to claim 1 or 2, characterized in that, The aforementioned anti-pancreatic cancer drugs are drugs that inhibit the proliferation, migration, repair, and invasion of pancreatic cancer cells and / or drugs that induce apoptosis in pancreatic cancer cells.

4. The application according to claim 1 or 2, characterized in that, The water extract of *Hydrocotyle sibthorpioides* was mixed with conventional excipients and processed using conventional techniques to produce a clinically acceptable drug formulation for the treatment of pancreatic cancer.

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