A traditional Chinese medicine composition for treating prostate cancer and a preparation method thereof

Abstract

The present application belongs to the technical field of traditional Chinese medicine, and particularly relates to a traditional Chinese medicine composition for treating prostate cancer and a preparation method thereof. The traditional Chinese medicine composition is prepared from raw materials including the following components by weight: cassia twig 5-50 parts, poria 5-50 parts, cortex moutan 3-30 parts, red peony root 3-30 parts, persicae semen 3-30 parts, fructus psoraleae 1-20 parts, and drynaria 1-20 parts. The present application proves through experiments that the traditional Chinese medicine composition can effectively inhibit the proliferation, migration and invasion of prostate cancer cells. Through extensive transcriptome analysis, it is found that the traditional Chinese medicine composition inhibits the expression of key genes such as HSP90AA1 and CDK1, and regulates the PI3K signal pathway and AR signal transduction in the prostate cancer pathway to achieve inhibition of prostate cancer. Therefore, the traditional Chinese medicine composition has a good application prospect.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine technology, specifically relating to a traditional Chinese medicine composition for treating prostate cancer and its preparation method. Background Technology

[0002] Prostate cancer is one of the most common malignant tumors in men, primarily occurring in the prostate gland, a gland in the male reproductive system. The disease is characterized by the abnormal proliferation of prostate epithelial cells, with over 95% of cases being adenocarcinoma. Prostate cancer is more common in older men, with increasing age being a significant risk factor, especially after age 55 when the incidence rate rises significantly.

[0003] Guizhi Fuling Wan is an ancient formula derived from the classic Chinese medicine text *Jinkui Yaolue*. This herbal compound mainly contains five medicinal herbs: cinnamon twig, poria cocos, peony bark, red peony root (or white peony root), and peach kernel. It is primarily used to treat gynecological diseases, especially those related to blood stasis and abdominal masses. In recent years, research has also explored its potential application in diseases such as prostate cancer. Studies have shown that Guizhi Fuling Wan may alleviate some symptoms of prostatitis caused by qi stagnation and blood stasis, and may influence the tumor microenvironment through its blood-activating and stasis-removing mechanism, thus providing some adjuvant therapeutic effects for certain types of benign prostatic hyperplasia or early-stage prostate cancer.

[0004] However, there is still a lack of comprehensive research on the mechanism of Guizhi Fuling Pill in treating prostate cancer. How to improve this traditional Chinese medicine formula to enhance its therapeutic effect on prostate cancer is an important research topic in this field. Summary of the Invention

[0005] In view of the problems of the prior art, the present invention provides a traditional Chinese medicine composition for treating prostate cancer and a method for preparing the same.

[0006] A traditional Chinese medicine composition, which is made from raw materials comprising the following components in parts by weight:

[0007] Cinnamon twig 5-50 parts, Poria cocos 5-50 parts, Moutan bark 3-30 parts, Red peony root 3-30 parts, Peach kernel 3-30 parts, Psoralea corylifolia 1-20 parts, Drynaria fortunei 1-20 parts.

[0008] Preferably, it is made from raw materials comprising the following components in parts by weight:

[0009] Cinnamon twig 10 parts, Poria cocos 10 parts, Moutan bark 5 parts, Red peony root 5 parts, Peach kernel 5 parts, Psoralea corylifolia 3 parts, Drynaria fortunei 3 parts.

[0010] Preferably, it is a preparation made by using raw herbs or extracts of cinnamon twig, poria cocos, peony bark, red peony root, peach kernel, psoralea corylifolia, and drynaria fortunei as active ingredients, and adding pharmaceutically acceptable excipients.

[0011] Preferably, the preparation is a granule, pill, powder, decoction extract, tablet, capsule, solution or dry powder spray.

[0012] The present invention also provides a method for preparing the above traditional Chinese medicine composition, comprising the following steps:

[0013] Step 1, extracting the raw materials with an ethanol aqueous solution;

[0014] Step 2, concentrating and drying the extract to obtain the product.

[0015] Preferably, the concentration of the ethanol aqueous solution is 75% v / v; the extraction process is heating under reflux, the extraction time is 1 h, and the extraction is carried out 2 - 3 times, with the amount of the ethanol aqueous solution used each time being 5 - 8 times the amount.

[0016] Preferably, the concentration of the ethanol aqueous solution is 75% v / v; the extraction process is heating under reflux, the extraction time is 1 h, and the extraction is carried out 3 times, with the amount of the ethanol aqueous solution used each time being 6 times the amount.

[0017] The present invention also provides the use of the above traditional Chinese medicine composition in the preparation of a drug for preventing and / or treating prostate cancer.

[0018] Preferably, the drug is used for inhibiting the proliferation and / or migration and / or invasion of prostate cancer cells.

[0019] The present invention provides a new traditional Chinese medicine composition, which is obtained by adding bone - targeting traditional Chinese medicines Psoralea corylifolia and Drynaria fortunei to Cinnamon Twig and Poria Pill, thus forming the modified Cinnamon Twig and Poria Pill. The modified Cinnamon Twig and Poria Pill provided by the present invention has a better therapeutic effect on prostate cancer and can effectively inhibit the proliferation, migration and invasion of prostate cancer cells. The present invention also studies the mechanism of the modified Cinnamon Twig and Poria Pill in treating prostate cancer. Through extensive transcriptome analysis, we found that the modified Cinnamon Twig and Poria Pill inhibits the expression of key genes such as HSP90AA1 and CDK1 and regulates the PI3K signaling pathway and AR signal transduction in the prostate cancer pathway to achieve the inhibition of prostate cancer. In addition, we also revealed that Psoralea corylifolia and Cinnamon Twig play a key role in the modified Cinnamon Twig and Poria Pill. Therefore, the traditional Chinese medicine composition modified Cinnamon Twig and Poria Pill provided by the present invention has good application prospects.

[0020] Obviously, based on the above content of the present invention, according to the common technical knowledge and conventional means in the art, without departing from the above basic technical idea of the present invention, various other forms of modification, substitution or change can be made.

[0021] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0022] Figure 1 (A) and (B) show the results of CCK8 assays of cell viability of DU145 and PC3 cell lines at different concentrations (100 μg / ml, 75 μg / ml, 50 μg / ml), respectively.

[0023] Figure 2 (A) and (B) demonstrate wound healing assays to evaluate the inhibitory effects of three different concentrations of Jiawei Guizhi Fuling Wan on the migration of DU145 and PC3 cell lines. (C) and (D) show the effects of three different concentrations (100 μg / ml, 75 μg / ml, 50 μg / ml) of Jiawei Guizhi Fuling Wan on the migration and invasion of DU145 and PC3 cell lines, as detected by Transwell migration and invasion assays, respectively.

[0024] Figure 3 Using HTS 2 Gene expression profiles of experimental treatments and heatmaps showing changes in the number of differentially expressed genes at different concentrations. (A)HTS 2 Workflow diagram. (B) Through HTS 2 Heatmaps of gene expression profiles for seven drugs obtained in two cell lines, DU145 and PC3, and four drug concentrations in the control group (DMSO group). (C) Changes in the number of differentially expressed genes after treatment with four different drug concentrations of the seven drugs in the DU145 and PC3 cell lines, as shown in the line graph.

[0025] Figure 4 HTS2 analysis was used to analyze heatmaps and volcano plots of differentially expressed genes (DEGs) at 100 μg / ml. (A) and (C) are volcano plots of DEGs in DU145 and PC3 cell lines treated with a drug concentration of 100 μg / ml. The criteria for differentially expressed genes were |Foldchange|≥2 and P-value <0.05. Red dots represent upregulated DEGs, while green dots represent downregulated DEGs. (B) and (D) are heatmaps of DEGs in DU145 and PC3 cell lines treated with a drug concentration of 100 μg / ml, respectively. Data clustering was used to cluster the seven herbs based on the similarity or difference in gene expression profiles.

[0026] Figure 5 GSEA enrichment analysis. Sankey diagrams were used to visualize the single herb-gene set-biological function network (A) and (B).

[0027] Figure 6 KEGG enrichment and GO functional analysis of differentially expressed genes in DU145 cells (A,C) and PC3 cells (B,D).

[0028] Figure 7 : Active ingredient-target network and functional enrichment analysis of Jiawei Guizhi Fuling Pill. (A) Venn diagram of potential therapeutic targets for prostate cancer. (B) Venn diagram of potential targets of Jiawei Guizhi Fuling Pill and prostate cancer therapeutic targets. (C) Active ingredient-target network of Jiawei Guizhi Fuling Pill, where blue nodes represent prostate cancer therapeutic targets and green nodes represent active ingredients of Jiawei Guizhi Fuling Pill. (D) and (E) KEGG pathway enrichment analysis and GO enrichment analysis were performed on 84 potential anti-prostate cancer targets in Jiawei Guizhi Fuling Pill, respectively.

[0029] Figure 8 Genetic alteration and survival analysis of 84 potential anti-prostate cancer targets in Jiawei Guizhi Fuling Wan (a traditional Chinese medicine formula) was performed in the cBioPortal database. (A) Overview of alterations of 84 potential therapeutic targets in 10 different prostate cancer studies. (B) Kaplan-Meier survival analysis of the mutated and non-mutated groups of the 84 potential targets. P < 0.05 was considered statistically significant.

[0030] Figure 9 PPI networks were constructed on differentially expressed genes in DU145 cell lines (A) and PC3 (B), with node size and color proportional to their degree in the network. (C) A Venn diagram of the top ten core targets and 84 potential therapeutic targets in the PPI network structure was constructed.

[0031] Figure 10 Schematic diagram of the 3D docking results of CDK1 and HS90AA1 proteins. The yellow bonds represent hydrogen bonds, while the blue bonds represent PI-PI bonds.

[0032] Figure 11 The mechanism of action of Jiawei Guizhi Fuling Wan in the treatment of prostate cancer. Arrows in the figure represent activation effects, T-shaped arrows represent inhibition effects, and deviations represent interruptions. Furthermore, targets associated with Jiawei Guizhi Fuling Wan in prostate cancer treatment are marked in red. Detailed Implementation

[0033] In the following examples and experimental cases, reagents and raw materials not specifically described are all commercially available products.

[0034] Example 1: Traditional Chinese Medicine Composition

[0035] The traditional Chinese medicine composition in this embodiment is made from the following raw materials:

[0036] Cinnamon twig 10 parts, Poria cocos 10 parts, Moutan bark 5 parts, Red peony root 5 parts, Peach kernel 5 parts, Psoralea corylifolia 3 parts, Drynaria fortunei 3 parts.

[0037] The preparation process is as follows:

[0038] The drugs were ground into powder and extracted with 75% ethanol under continuous reflux for 1 hour. The extractions were repeated three times, and the extracts were combined. Each extraction used six times the volume of the ethanol-water solution. The mixture was then evaporated to dryness under reduced pressure and stored in a refrigerator at -80°C.

[0039] Example 2 Traditional Chinese Medicine Composition

[0040] The traditional Chinese medicine composition in this embodiment is made from the following raw materials:

[0041] 50 parts cinnamon twig, 5 parts poria cocos, 30 parts peony bark, 3 parts red peony root, 30 parts peach kernel, 1 part psoralea corylifolia, and 20 parts drynaria fortunei.

[0042] The preparation process is as follows:

[0043] The drugs were ground into powder and extracted with 75% ethanol under continuous reflux for 1 hour. The extractions were repeated three times, and the extracts were combined. Each extraction used six times the volume of the ethanol-water solution. The mixture was then evaporated to dryness under reduced pressure and stored in a refrigerator at -80°C.

[0044] Example 3 Traditional Chinese Medicine Composition

[0045] The traditional Chinese medicine composition in this embodiment is made from the following raw materials:

[0046] 5 parts cinnamon twig, 50 parts poria cocos, 3 parts peony bark, 30 parts red peony root, 3 parts peach kernel, 20 parts psoralea corylifolia, and 1 part drynaria fortunei.

[0047] The preparation process is as follows:

[0048] The drugs were ground into powder and extracted with 75% ethanol under continuous reflux for 1 hour. The extractions were repeated three times, and the extracts were combined. Each extraction used six times the volume of the ethanol-water solution. The mixture was then evaporated to dryness under reduced pressure and stored in a refrigerator at -80°C.

[0049] The technical solution of the present invention will be further illustrated by the following experiments.

[0050] Experimental Example 1: The preventive and therapeutic effects and regulatory mechanisms of modified Guizhi Fuling Wan on prostate cancer metastasis, a major disease.

[0051] I. Experimental Methods

[0052] 1. Chemicals and reagents

[0053] In this experimental example, the Chinese herbal composition was prepared into a dry powder (hereinafter referred to as the extract of Jiawei Guizhi Fuling Wan) according to the method of Example 1. In addition, the seven single herbs were also prepared into dry powder using the same method. In subsequent experiments, the dry powder was dissolved in dimethyl sulfoxide (DMSO) for further experimental research.

[0054] 2. Cell Culture

[0055] The human prostate cancer cell lines used in this study included DU145 and PC3 cell lines (purchased from Bio-Tech Biotechnology). DU145 cells were cultured in RPMI 1640 medium (Gibco) containing 10% fetal bovine serum (Gemini) and 100 units / ml streptomycin and penicillin (Gibco). PC3 cells were cultured in F12K medium (Gibco) containing 10% fetal bovine serum (Gemini) and 100 units / ml streptomycin and penicillin (Gibco). All cell lines were cultured at 37°C in a 5% CO2 environment.

[0056] 3. Cell viability assay

[0057] Various prostate cancer cells were cultured in 96-well plates, with both cell lines cultured at 5 × 10⁶ cells / well. 3 / 100μL / well. Drug administration was performed 24 hours after plating. Different concentrations of drug were added to the experimental wells, while the control group received an equal volume of DMSO containing the highest concentration of drug. Five biological replicates were set up for each group. The culture plates were incubated in a constant temperature incubator for 48 hours. After incubation, 10μL of CCK-8 reagent was added to each well, and the plates were incubated for 3 hours. The absorbance at 450nm was then measured using a microplate reader. Cell viability was calculated based on the measured OD value using the formula: Cell viability = (Experimental wells - Blank wells) / (Control wells - Blank wells) × 100%.

[0058] 4. Scratch test

[0059] PC3 and DU145 cells were resuspended in culture medium containing different concentrations of Jiawei Guizhi Fuling Wan extract and seeded into six-well plates. Vertical scratches were made on the surface of monolayer cells, and the cells were observed under an inverted microscope after 0 h, 24 h, and 48 h of culture to observe the effect of Jiawei Guizhi Fuling Wan on the migration ability of prostate cancer cells.

[0060] 5. Transwell experiment

[0061] PC3 and DU145 cells were transferred to the upper transwell chamber (5 × 10⁶ cells / mL). 4Cells were cultured in a polycarbonate membrane (6.5 mm in diameter, 8 μm pore size) and serum-free cell suspension, supplemented with different concentrations of Jiawei Guizhi Fuling Wan extract. Conditioned medium containing 10% fetal bovine serum was placed in the lower chamber. After 24 or 48 hours of culture, the chamber was carefully removed with forceps, the liquid in the upper chamber was aspirated, and the cells were gently washed once with PBS. The cells were then transferred to chamber wells pre-filled with 800 μl of 4% paraformaldehyde solution for 10 min for fixation. The upper surface of the Transwell membrane was then gently wiped with a cotton swab to remove non-migrating cells. Cells precipitated on the lower surface of the membrane were stained with crystal violet for 15 minutes, washed twice with PBS, and allowed to air dry before being counted under a microscope. The average values ​​were calculated based on eight different observation fields.

[0062] 6. HTS 2 Technology and data processing

[0063] HTS 2 As a high-throughput screening technique, HTS offers the opportunity to generate large-scale cellular transcriptional data in the context of herbal interference. 2 The assays mainly included cell culture, probe design, and screening. In this study, HTS was performed. 2 The study determined the effects of extracts from seven herbs in Jiawei Guizhi Fuling Wan on a total of 3407 genes in PC3 and DU145 cells.

[0064] For sample preparation, PC3 and DU145 cells were cultured in 384-well plates at a density of 2 × 10³ cells per well for 24 hours. They were then treated with a 100 μg / ml extract of seven herbal herbs for 24 hours using HTS. 2 The platform automatically detects samples and performs sequencing using an Illumina HiSeq X Ten sequencer.

[0065] Data processing began by mapping all reads to probe sequences and normalizing them relative to the expression of 18 stable housekeeping genes. Subsequently, after treatment with 12 replicates of DMSO and 4 replicates of 7 herbs in Jiawei Guizhi Fuling Wan (a traditional Chinese medicine formula), Pearson correlation coefficients between normalized transcriptional data were calculated using R software to assess the reliability and reproducibility of the transcriptional profiles. Notably, a correlation coefficient > 0.9 indicates that the HTS2 assay is reliable and reproducible. Then, gene expression values ​​with FC and P-values ​​were calculated using the R package DESeq2, and genes with |FC| > 2 and P-values ​​< 0.05 were considered differentially expressed genes. Finally, heatmaps and volcano plots of DEG were generated using the R packages pheatmap and ggplot2, respectively.

[0066] 7. GO and KEGG enrichment analysis

[0067] Key targets were imported into the DAVID database (https: / / david.ncifcrf.gov / , updated October 11, 2023) to obtain KEGG pathways and GO biological processes associated with the treatment of prostate cancer using Jiawei Guizhi Fuling Wan (a traditional Chinese medicine formula). Enrichment of GO terms and KEGG signaling pathways was still based on p-values ​​<0.05, and the 10 most significant GO terms and KEGG signaling pathways were subsequently visualized.

[0068] 8. PPI Network Interaction Analysis

[0069] The STRING database (https: / / cn.STRING-db.org / , updated November 2022) was used to obtain protein-protein interaction networks of targets (Szklarczyk et al., 2021). Subsequently, PPI networks with a total score > 0.9 were constructed. These networks were visualized using Cytoscape 3.9.0 software, and topological analysis was performed using the CytoNCA package. The initial screening used a centrality (DC) greater than twice the median of all nodes. Subsequently, proximity centrality and center centrality were used as secondary references. The top 10 key proteins obtained were selected as core targets, and a visual network was constructed.

[0070] 9. GSEA enrichment analysis

[0071] The effects of various Chinese herbal medicines in Jiawei Guizhi Fuling Wan on different genomes were evaluated using the GSEA method. This involved using differentially expressed gene data for each herb and genome from the GSEA setup (Subramanian et al., 2005), and then aligning the genes against a list of differentially expressed genes to calculate a run sum. Normalized enrichment scores (NES) were then obtained through normalized enrichment, and a calculated false discovery rate (FDR) < 0.05 was considered statistically significant. The results of the GSEA analysis were visualized using Sankey plots via the networkD3 package in R (4.2.2).

[0072] 10. Acquisition of active ingredients and targets of Jiawei Guizhi Fuling Wan (a traditional Chinese medicine formula).

[0073] From Traditional Chinese Medicine System Pharmacology Data

[0074] The active ingredients of Jiawei Guizhi Fuling Pill were extracted from the TCMSP (https: / / old.tcmsp-e.com / TCMSP.php) and the chemical professional database (http: / / www.organchem.csdb.cn / scdb / default.asp). Oral bioavailability (OB) ≥ 30% and drug substance similarity (DL) ≥ 0.18 were used as screening criteria. The 2D structures of the active ingredients were collected by searching the PubChem database (https: / / pubchem.ncbi.nlm.nih.gov / ). Subsequently, using these 2D structures, the Swiss Target Prediction database (https: / / www.swisstargetprediction.ch / ) was used to identify targets predicted to be associated with compounds in Jiawei Guizhi Fuling Pill.

[0075] 11. Acquisition and network construction of prostate cancer disease targets

[0076] Using "cancer" or "metastatic castration-resistant cancer" as search criteria, the OMIM (https: / / omim.org / , updated November 17, 2023), GeneCards (http: / / www.genecards.org / , updated October 5, 2023), and DisGeNET (https: / / disgenetplus.com / , updated February 3, 2021) databases were searched to identify potential therapeutic targets for PRAD. The overlapping portions of the three databases were used as the disease target set. VennOnline software (http: / / bioinformatics.psb.ugent.be / ) was then used to generate Venn diagrams illustrating the intersections of diseases and drug targets. Finally, a visualization network of therapeutic targets for herbal active ingredients was constructed using Cytoscape 3.9.0 software.

[0077] 12. Gene alteration analysis

[0078] Using cBioPortal (https: / / www.cbioportal.org / , updated August 21, 2023), a web-based comprehensive data mining system, we performed genetic variation analysis and survival analysis on key targets.

[0079] 13. Molecular docking

[0080] First, the crystal structures of key anti - prostate cancer targets (CDK1, HSP90AA1) were retrieved from the Protein Data Bank (PDB). At the same time, the most suitable protein crystal structure was selected according to the resolution, and then each protein structure ( Release, 2019 - 1) was prepared. On this basis, the three - dimensional structure of this module was obtained using the LigPrep module in Maestro, and the energy minimization of the active ingredients in Polygonum aviculare L. was achieved. All molecular docking was simulated and calculated using Glide software. Finally, ligand - target interactions were visualized through the ligand interaction map module.

[0081] 14. Statistical analysis

[0082] GraphPad Prism (9.0) software was used for statistical analysis of the mean values. Based on the unpaired t - test, the significance of differences between groups was determined as *p < 0.05, **p < 0.01, ***p < 0.001.

[0083] II. Experimental results

[0084] 1. CCK8 experiment verified that Jiawei Guizhi Fuling Pills can inhibit the proliferation of prostate cancer in vitro

[0085] After treating DU145 and PC3 cell lines with extracts of Jiawei Guizhi Fuling Pills at different concentrations for 48 hours, cell viability was then measured by CCK - 8 assay. The results showed that the calculated IC50 values for the two cell lines were 118.2 μg / mL and 3104.1 μmg / mL respectively ( Figure 1 A, B). This indicates that Jiawei Guizhi Fuling Pills have obvious inhibitory effects on the proliferation of DU145 and PC3 cell lines.

[0086] 2. Scratch repair test and Transwell experiment verified that Jiawei Guizhi Fuling Pills can inhibit the migration and invasion of prostate cancer in vitro

[0087] First, the ability of Jiawei Guizhi Fuling Pills to inhibit the migration of DU145 and PC3 cell lines at high (100 μg / ml), medium (75 μg / ml), and low (50 μg / ml) concentrations at 12 h and 24 h was verified through the wound repair experiment ([[ID=**********]] Figure 2 A, B). Subsequently, through the Transwell invasion and migration experiments, it was verified that Jiawei Guizhi Fuling Pills can inhibit the migration and invasion abilities of DU145 and PC3 cells under three different concentrations of high, medium, and low, and inhibit them in a concentration - dependent manner ( Figure 2 C, D).

[0088] 3. Constructing the gene expression profile of Jiawei Guizhi Fuling Wan (a traditional Chinese medicine formula) for the treatment of prostate cancer using HTS2 technology.

[0089] Based on HTS2 technology, expression profiles of 3407 cancer-related genes after treatment with each single drug of Jiawei Guizhi Fuling Wan were constructed in the DU145 and PC3 prostate cancer cell lines. These data included a blank control group (DMSO group), a positive control group (JQ1 group), and four drug concentrations (25 μg / ml, 50 μg / ml, 75 μg / ml, 100 μg / ml) of the seven traditional Chinese medicines (Psoralea corylifolia, Cinnamomum cassia, Drynaria fortunei, Paeonia suffruticosa, Paeonia lactiflora, Poria cocos, and Prunus persica). Four replicates were designed for each treatment group—135 gene expression profiles were obtained from each cell line, for a total of 270 gene expression profiles. Figure 3 A).

[0090] Next, we took the average value of each group of these gene expression profile data and then displayed it as a heatmap. Figure 3 B). Furthermore, by observing the changes in differentially expressed genes (DEGs) in the drug treatment groups at different concentrations ( Figure 3 C) We found that the number of differentially expressed genes increased with increasing drug concentration, suggesting a potentially better anti-tumor effect. We then selected the treatment group with a drug concentration of 100 μg / ml for further analysis.

[0091] 4. Screening differentially expressed genes (DEGs) for seven herbal treatments in DU145 and PC3 cells based on gene expression profiling data.

[0092] DU145 and PC3 cells were treated with each herb from the modified Guizhi Fuling Wan formula at a concentration of 100 μg / ml, and the expression changes of 3407 cancer-related genes were detected using HTS2. The results showed that the DU145 cell line had the highest number of DEGs (determined gene expression groups) from Psoralea corylifolia, including 107 upregulated and 68 downregulated. Paeonia lactiflora had the fewest DEGs, with 20 in total, including 13 upregulated and 7 downregulated. Furthermore, 102, 48, 67, 44, and 38 DEGs were identified in Cinnamomum cassia, Drynaria fortunei, Paeonia suffruticosa, Poria cocos, and Prunus persica, respectively. Figure 4 A). Similarly, the PC3 cell line showed the highest number of DEGs in Psoralea corylifolia, including 127 upregulated and 154 downregulated. Paeonia lactiflora had the fewest DEGs, with 23 in total, of which 15 were upregulated and 8 were downregulated. Furthermore, 85, 35, 42, 35, and 26 DEGs were identified in Cinnamomum cassia, Drynaria fortunei, Paeonia suffruticosa, Poria cocos, and Prunus persica, respectively. Figure 4 B).

[0093] To study the relevance of each herbal medicine, seven herbal medicines were subjected to cluster analysis based on the similarity or difference in gene expression profiles. The results showed that by integrating the differential gene expression profile data of the DU145 cell line and the PC3 cell line, the seven herbal medicines of the modified Guizhi Fuling Pills were divided into three major categories: the first group was Psoralea corylifolia and Cinnamomum cassia, the second group was Poria cocos and Drynaria fortunei, and the third group was Moutan cortex, Paeonia lactiflora, and Prunus persica( Figure 4 C, D).

[0094] 5. To analyze the impact of each pair of herbs in the modified Guizhi Fuling Pills on gene sets through GSEA enrichment

[0095] To further study the role of each herbal medicine in prostate cancer, GSEA was used to evaluate the overall impact of each herbal medicine on different gene sets. The results showed that in both cell lines, Psoralea corylifolia and Cinnamomum cassia after modification played a major role and participated in most signaling pathways. In the DU-145 cell line( Figure 5 A), Moutan cortex, Drynaria fortunei, Poria cocos, and Paeonia lactiflora exerted anti-tumor effects by synergistically participating in related pathways such as the inflammatory pathway, apoptosis pathway, and KRAS pathway. In addition, Prunus persica mainly participated in the coagulation pathway and was closely related to tumor metastasis; furthermore, in the PC3 cell line( Figure 5 B), Prunus persica, Moutan cortex, and Paeonia lactiflora mainly exerted synergistic anti-tumor effects through pathways such as endocrine metabolism and inflammatory response. It is worth noting that the biological phenotypes of the seven Chinese herbs mainly focused on aspects such as proliferation, apoptosis, cell cycle, and invasion / migration.

[0096] 6. To perform KEGG analysis and GO analysis on the differentially expressed genes of the modified Guizhi Fuling Pills

[0097] The DEGs of the two cell lines treated with the modified Guizhi Fuling Pills were respectively aggregated and duplicate removed, and then KEGG pathway analysis and GO analysis were performed. As we can see( Figure 6 A, B), in the DU145 cell line, the DEGs mainly participated in pathways such as the cancer pathway, apoptosis, and cell cycle, while in the PC3 cell line, the DEGs mainly participated in pathways such as the cancer pathway, lipid metabolism pathway, and PI3K-AKT pathway. These pathways are closely related to prostate cancer. In addition, through GO analysis( Figure 6 C, D), we found that the modified Guizhi Fuling Pills mainly participated in regulating biological functions such as DNA transcription, apoptosis process, and cell cycle. The above results indicate that the modified Guizhi Fuling Pills may mainly exert its anti-prostate cancer therapeutic effect by its anti-proliferation, pro-apoptosis, regulation of cell cycle, and inhibition of tumor invasion / migration.

[0098] 7. To construct an active ingredient-target network and perform its enrichment analysis through systems pharmacology

[0099] The above experiments demonstrate that Jiawei Guizhi Fuling Wan (a traditional Chinese medicine formula) can inhibit the proliferation and invasion of prostate cancer. This was achieved using systems pharmacology combined with HTS (Heat-Sensitive Therapy). 2 To further verify and explore the potential mechanism of the modified Guizhi Fuling Pill in treating prostate cancer.

[0100] First, 64 active ingredients related to Jiawei Guizhi Fuling Pill were retrieved from the TCMSP, TCMID, and Chemistry Databases based on the screening criteria of OB≥30% and DL≥0.18. These included Poria (12), Prunus persica (12), Paeonia lactiflora (13), Psoralea corylifolia (18), Drynaria fortunei (13), Paeonia suffruticosa (3), and Cinnamomum cassia (3) (see Table 1). Then, SwissTargetPrediction was used to predict drug targets using all active ingredients present in Jiawei Guizhi Fuling Pill, and targets with a probability score >0 were selected. After deduplication, a total of 672 drug targets were obtained. Next, known prostate cancer-related targets were retrieved from databases such as DisGeNET, OMIM, and Genecards. The intersecting parts were selected as disease targets for prostate cancer (see Table 1). Figure 7 A) A total of 418 targets were identified. After removing the intersection of drug targets and disease targets, 84 potential therapeutic targets were obtained. (See A) Figure 7 B)

[0101] Next, an active ingredient-target network for Jiawei Guizhi Fuling Wan was constructed using Cytoscape to demonstrate the relationship between active ingredients and potential therapeutic targets. Specifically, the network consists of 84 prostate cancer-related targets and 64 active ingredients. Figure 7 C). To explore the biological mechanisms of the 84 potential anti-prostate cancer targets identified by Jiawei Guizhi Fuling Wan, GO / KEGG enrichment analysis was performed using the David website (https: / / david.ncifcrf.gov / ). The KEGG enrichment analysis results showed that the role of Jiawei Guizhi Fuling Wan in the treatment of prostate cancer was closely related to the KEGG terms "cancer pathway," "prostate cancer pathway," and "P endocrine resistance pathway." Figure 7 D). Furthermore, GO enrichment analysis revealed that these 84 prostate cancer-related targets are significantly involved in multiple biological processes, including positive and negative regulation of signal transduction, apoptosis, and RNA polymerase II promoter transcription. Figure 7 E).

[0102] Table 1. Active Ingredients of Jiawei Guizhi Fuling Pill

[0103]

[0104]

[0105]

[0106]

[0107] 8. Genetic variation analysis and survival analysis

[0108] To further explore the molecular characteristics of 84 targets in different cohorts of prostate cancer patients, gene alteration mining and survival analysis were performed using cBioPortal. According to the results, in 10 datasets of prostate cancer patients, these 84 targets were mostly mutated in patients without metastasis, while in the cohort of patients with metastasis, these 84 targets mostly showed multiple alterations. Specifically, the gene alterations of these 84 potential anti-prostate cancer targets in Jiawei Guizhi Fuling Wan included mutations, fusions, amplifications, deep deletions, and multiple alterations, with mutations being the most common alteration across all patient types. Figure 8 A). It is noteworthy that cases with genetic alterations had a lower survival rate compared to cases without alterations. Figure 8 B). These results indicate that these 84 targets are closely related to the prognosis of prostate cancer, which also supports the clinical application of Jiawei Guizhi Fuling Wan in the treatment of prostate cancer.

[0109] 9. Combining 84 potential targets with the core targets in HTS2, the key targets for treating prostate cancer with modified Guizhi Decoction were identified.

[0110] First, a PPI network was constructed for the DEGs of DU145 and PC3 cell lines obtained from the HTS2 experiment, and the genes were scored and visualized using CYTOSCAPE software. The top 10 genes were selected as core targets. Figure 9 A, B). In the DU145 cell line, HSP90AA1, HSPA5, and PCNA ranked highly, while in the PC3 cell line, CDK1, CCND1, and PI3KR1 ranked highly. These top-ranking core genes were compared with 84 previously identified targets closely related to the treatment of prostate cancer using Jiawei Guizhi Fuling Wan (a traditional Chinese medicine formula) using a Veen plot to remove intersection. Figure 9 C) It can be seen that the DU145 cell line and 84 target sites overlapped with HSP90AA1 and HSPA5, and the PC3 cell line and 84 target sites overlapped with CDK1 and CCND1. In addition, MYC and PCNA were enriched in both DU145 and PC3. This proves that these genes are key genes in the treatment of prostate cancer with Jiawei Guizhi Fuling Pill.

[0111] 10. Molecular docking study of active ingredients with CDK1 and HSP90AA1 proteins

[0112] Most of the active ingredients bind well to the active pockets of CDK1 and HSP90AA1 proteins. Among them, the one with the smallest Glide gscore for CDK1 protein is, and the one with the smallest Glide gscore for HSP90AA1 protein is, indicating that they can have a stable binding energy with the related proteins( Figure 10 A-D, Table 2).

[0113] Table 2 Molecular docking scoring results

[0114]

[0115] 11. Mechanism diagram of Guizhi Fuling Pills with added herbs

[0116] Based on the above results, a complete mechanism diagram was drawn to more clearly reflect the anti-prostate cancer mechanism of Guizhi Fuling Pills with added herbs. As Figure 11 shown, Guizhi Fuling Pills with added herbs may mainly exert its anti-prostate cancer effect through the PI3K-AKT signaling axis and AR-HSP signaling axis in the prostate cancer pathway. When prostate cancer cells were treated with each of the seven herbs in Guizhi Fuling Pills with added herbs, many genes in the prostate cancer pathway were enriched and their expression was downregulated, such as HSP90AA1, CDK1, etc. In addition, through the PI3K signaling axis, Guizhi Fuling Pills with added herbs regulated key genes such as CDK1, MYC, CCND1, NFkB, etc., thus exerting functions of anti-proliferation, promoting apoptosis, inhibiting migration / invasion and regulating the cell cycle; while in the classical AR signaling axis of prostate cancer, it mainly inhibited its promoting effect on prostate cancer by regulating the heat shock protein family (genes such as HSP90AA1, HSPA5, etc.).

[0117] In summary, through the above experiments, it can be seen that the traditional Chinese medicine composition Guizhi Fuling Pills with added herbs provided by the present invention can effectively inhibit the proliferation, migration and invasion of prostate cancer cells. Through extensive transcriptome analysis, it was found that Guizhi Fuling Pills with added herbs inhibit the expression of key genes such as HSP90AA1, CDK1, etc. and regulate the PI3K signaling pathway and AR signal transduction in the prostate cancer pathway to achieve the inhibition of prostate cancer( Figure 11 ). In addition, the above experimental results also revealed that Psoralea corylifolia and Cinnamomum cassia play a key role in Guizhi Fuling Pills with added herbs.

[0118] Therefore, the traditional Chinese medicine composition Guizhi Fuling Pills with added herbs provided by the present invention has good application prospects in the treatment of prostate cancer.

Claims

1. A traditional Chinese medicine composition for treating prostate cancer, characterized in that, It is made from the following ingredients by weight: Cinnamon twig 5-50 parts, Poria cocos 5-50 parts, Moutan bark 3-30 parts, Red peony root 3-30 parts, Peach kernel 3-30 parts, Psoralea corylifolia 1-20 parts, Drynaria fortunei 1-20 parts.

2. The traditional Chinese medicine composition according to claim 1, characterized in that, It is made from the following ingredients by weight: Cinnamon twig 10 parts, Poria cocos 10 parts, Moutan bark 5 parts, Red peony root 5 parts, Peach kernel 5 parts, Psoralea corylifolia 3 parts, Drynaria fortunei 3 parts.

3. The traditional Chinese medicine composition according to claim 1 or 2, characterized in that, It is a preparation made by using raw herbs or extracts of cinnamon twig, poria cocos, peony bark, red peony root, peach kernel, psoralea corylifolia, and drynaria fortunei as active ingredients, and adding pharmaceutically acceptable excipients.

4. The traditional Chinese medicine composition according to claim 3, characterized in that: The preparations are granules, pills, powders, decoctions, tablets, capsules, solutions, or spray-dried powders.

5. A method for preparing the traditional Chinese medicine composition according to any one of claims 1-4, characterized in that, Includes the following steps: Step 1: Extract the raw material using an ethanol-water solution; Step 2: Concentrate and dry the extract to obtain the final product.

6. The preparation method according to claim 5, characterized in that: The concentration of the ethanol-water solution is 75% v / v; the extraction process is heating and reflux extraction, the extraction time is 1 hour, and the extraction is repeated 2-3 times, with the amount of ethanol-water solution used each time being 5-8 times the volume.

7. The preparation method according to claim 6, characterized in that: The concentration of the ethanol-water solution is 75% v / v; the extraction process is heating and reflux extraction, the extraction time is 1 hour, and the extraction is performed 3 times, with the amount of ethanol-water solution used each time being 6 times the volume.

8. Use of the traditional Chinese medicine composition according to any one of claims 1-4 in the preparation of a medicament for treating prostate cancer.

9. The use according to claim 8, characterized in that: The drug is used to inhibit the proliferation and / or migration and / or invasion of prostate cancer cells.

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