Evaluation of Clinical Efficacy of Canmei Formula in Preventing and Treating Recurrence of Colorectal Adenoma after Endoscopic ResectionCase Screening

This randomized controlled single-center clinical trial was conducted at Yueyang Integrated Traditional Chinese and Western Medicine Hospital affiliated with Shanghai University of Traditional Chinese Medicine. The study was reviewed by the Yueyang Hospital Ethics Committee (Ethical Approval Number: 2021 − 124) and was registered in the Chinese Clinical Trial Registration Center (www.chictr.org.cn) (Registration No. ChiCTR210054824). All enrolled patients signed an informed consent form, which complies with the Helsinki Declaration and was designed and implemented in accordance with the clinical trial requirements of the CONSORT guidelines. Eligible subjects were determined by the staff during routine clinical diagnosis and treatment, and their clinical information was collected.

(1) Inclusion Criteria:

① Age ≥ 18 and ≤ 80 years old, regardless of gender;

② Within 6 months before enrollment, endoscopic or surgical polypectomy has been performed, and pathological confirmation indicates adenomatous polyps in the colon and rectum. The TCM syndrome belongs to “phlegm-blood stasis” type.

③ Patients understand and agree to participate in this study and sign an informed consent form.

(2) Exclusion Criteria:

① During colonoscopy, the colorectal adenoma was not completely removed;

② Inability to undergo colonoscopy or poor bowel preparation (evaluated as “poor” and “insufficient” according to the Aronchik scale), with a short colonoscopy time and a stop time of less than 6 min;

③ Pregnant women or lactating women;

④ Having uncontrollable mental disorders and unable to cooperate;

⑤ Complicated with active tuberculosis or severe heart, liver or kidney diseases, or other serious infectious diseases;

⑥ Long-term use of drugs such as aspirin, NSAIDs, calcium, or vitamin D for at least three months (e.g., aspirin ≥ 100 mg/day or calcium ≥ 1200 mg/day);

⑦ Long-term (at least 6 months) use of Chinese herbal medicine or traditional Chinese patent medicines and simple preparations with the effect of clearing heat and detoxification, resolving phlegm and resolving stagnation, especially Chinese herbal prescriptions including fried Coptidis Rhizoma, Picrorhizae, and other drugs containing berberine;

⑧ Patients who cannot take oral medication or vomit frequently or suffer from severe constipation.

(3) Cases that have been included in the group but meet one of the following criteria for suspension or exclusion should be excluded:

① Misdiagnosis;

② Those who have not taken medicine, tested, or had poor compliance;

③ The patient was lost during follow-up or voluntarily gave up treatment;

④ During the experiment, there were serious cases of other concurrent diseases, deterioration of the condition, and the need to take emergency measures;

⑤ During the experiment, those who changed their medication or added non prescribed therapeutic medication (violating the protocol), which affected the effectiveness and safety judgment of treatment. For example, the use of certain prohibited drugs (such as aspirin, celecoxib, berberine, probiotics, etc.) makes it impossible to evaluate the efficacy.

Withdrawal cases must undergo corresponding clinical evaluation when discontinuing the clinical trial. For patients who request to withdraw from the clinical trial midway, the reasons should be clearly recorded. If the patient does not come to the hospital for follow-up on time, they should be inquired about the reasons through phone calls and letters, and be investigated about the subsequent process, and conduct a clinical evaluation of the patient’s withdrawal from clinical trials midway.

Intervention Measures

A randomized controlled design was adopted in this study. SPSS 26.0 statistical software was used to realize random grouping and generate a group of random numbers ranging from 1 to 228. 228 subjects meeting the standard were divided into treatment group or control group according to random numbers.

The treatment group received intervention with CMF granules, while the control group received placebo granulesberberine hydrochloride tablets. The CMF granules and placebo were prepared by Jiangyin Tianjiang Pharmaceutical Co., Ltd. (Jiangsu, China, GMP number: JS20191115). CMF consists of 20 g black plum, 20 g stiff silkworm, and 10 g locust horn (ratio 2:2:1), which are equivalent to their traditional Chinese medicine decoction constituents. The granules have no additives or pigments, with one dose packaged into two small bags, taken once daily (one bag each in the morning and evening) with warm water.

Subjects assigned to the control group take a total of 0.6 g of berberine (0.2 g per dose, three times a day). The treatment period was 24 weeks, with 4 weeks as a course of treatment and 6 consecutive courses, which means the treatment period is half a year. During the experiment, all subjects received dietary and lifestyle adjustments, and maintained stable lifestyle and dietary habits throughout the entire experiment.

Therapeutic Indicator and Follow-up

After the intervention treatment, the subjects underwent a one-year follow-up colonoscopy to record the location, number, size, and histopathological type of adenomas. Baseline laboratory tests, including serum alanine aminotransferase (ALT), blood urea nitrogen (BUN), and creatinine, were conducted to evaluate the liver and kidney function.

The one-year recurrence rate of colorectal adenomas is the main indicator, polyp recurrence rate in each group = number of recurrence cases / number of cases in each group × 100%.

Follow-up was conducted through phone calls and clinical visits. Researchers made regular monthly phone calls to monitor compliance, side effects, and efficacy, and confirm that they have not used aspirin, non-steroidal anti-inflammatory drug (NSAID), or other prohibited drugs until the end of the trial.

Optimization Combination of Canmei Formula and Colorectal Adenoma Target Based on Data Mining and Algorithm PredictionIdentification of Chemical Constituents of Canmei Formula Granules

(1) Reagents.

CMF granules were provided by Jiangyin Tianjiang Pharmaceutical Co., Ltd. (Jiangsu, China, GMP number: JS20191115) and stored in the laboratory of Shanghai University of Traditional Chinese Medicine. HPLC grade acetonitrile was purchased from Merck Company (Darmstadt, Germany), HPLC grade methyl tert-butyl ether was purchased from CNW, AR grade acetic acid and GR grade ethanol were purchased from China National Pharmaceutical Group Chemical Reagents (Shanghai, China), and Cremophor EL (BR grade) was purchased from Shanghai Yuanye (Shanghai, China). The purified used throughout the entire study was prepared using a 611VP purified water system (Sartorius, Germany).

(2) UPLC-MS analysis.

The equipment used in the analysis included the CBM-20 A system controller, LC-20ADXRpump, SIL-20ACXRautosampler, CTO-10Avp column box and equipped with protective columns (C18, 4 mm × 3.0 mm, Phenomenex Co., Ltd., Torrance, CA, United States) and Shiseido C18 column (2.1 × 150 mm, 2.5 μm, Shiseido Co., Ltd., Tokyo, Japan). The CMF granules were analyzed using the Shimadzu HPLC coupled with mass spectrometry. The conditions of UHPLC were as follows: The LC system is thermo Dionex Ultimate 3000, Column is XBridge BEH C18 (2.1 × 150 mm, 2.5 μm), Column Temperature 40 °C, Flow Rate 0. 3mL/min, and a linear gradient procedure (Table 1), the mobile phase system consisted of solvent a (0.1% formic acid + H2O, V/V) and solvent B (0.1% formic acid + acetonitrile, V/V) .

Table 1 Mobile Phase Gradient

The MS conditions are as follows: the MS system used was the Q Exactive, Ion mode was ESI– & ESI+, the Spray Voltage was 3500/3200V(+/-), the Vaporiser Temp was 350℃, with a sheath gas flow rate of 40 arb, and an auxiliary gas flow rate of 10 arb, the Capillary Temp was set to 320℃, the Scan Range was m/z 120–1500, Resolution was 70,000FWHM. The Top 3 ddms were selected for identification.

Approximately 500 mg of traditional CMF (which included stir fried silkworm 6.0 g, locust horn 6.0 g, black plum 6.0 g) was weighted. The sample was sonicated with 50% methanol for 20 min, then centrifuged at 12,000 r for 5 min. The supernatant was diluted and filtered through a 0.22 µ M microporous membrane. The filtrate sample 1 µ L was taken for analysis.

(3) Data processing.

The high-resolution information of quasimolecular ions and secondary fragments was obtained by HPLC-Q Exactive liquid-mass spectrometry. The data were analyzed by Compound Discoverer 2.1 software, Chromatogram peaks were inferred and identified by comparison of reference materials, MZ Vault, MZ clould, Chemspider and Pubchem.

Establishing a Database of Chemical Constituents of Canmei Formula

Literature searches were performed across three publicly available databases: TCMSP (https://old.tcmsp-e.com), ETCM (http://www.tcmip.cn/ETCM/) and HERB (http://herb.ac.cn/), to identify the chemical components of the three traditional Chinese medicines, black plum, stiff silkworm, and locust horn (https://pubchem.ncbi.nlm.nih.gov/). The standard SMILES format structures of compounds obtained from mass spectrometry and known compounds in the database were collected, and a total of 220 compounds with SMILES structures were obtained using SwissADME (http://www.swissadme.ch/). A network-based approach was used to filiter the ingredients based on the Lipinski “Rule of Five” [29], 28 compounds that did not meet the standards were removed, and ultimately 192 active ingredients were obtained. A CMF ingredient database was established, which includes basic information such as ingredient names, PubChem CID, SMILES structural formulas, and compliance with the Lipinski “Rule of Five”.

wSDTNBI Algorithm for Predicting the Target Points of Canmei Formula Action

Using the wSDTNBI (http://lmmd.ecust.edu.cn/netinfer/) algorithm to predict the target of 192 active compounds in CMF. For each compound, a target prediction set of 100 was generated, and prediction targets were screened based on a prediction score threshold of > 0.5. This approach identified ultimately 1044 potential for CMF. Compared with most current network-based methods, this prediction method can output prediction scores related to binding affinity, improving the accuracy of prediction results [28].

Data Mining from TCGA Database

The known CRA-related genes were retrieved by searching for “COAD” (Colon Adenocarcinoma) in the TCGA database, identified according to the standards of P-value < 0.05 and Fold Change (FC) > 2 or < 0.5. R programming language was used to screen for differentially expressed genes (DEGs) between the CRA model group and the normal control group.

Drug Efficacy Evaluation Based on Network and Algorithm Prediction

Aspirin and berberine were used as positive drug controls. From the DrugBank database (https://go.drugbank.com/), 141 control drug targets were obtained from three databases: PubChem, ETCM, and HERB. Using two human genome protein-protein interaction (PPI) networks as the background network, one was the STRING network with a confidence level of ≥ 0.9 for human interactions, and the other was the BNet dataset, collected by Professor Barabasi’s team. The Random Walk with Restart (RWR) algorithm was applied to the STRING network, and the correlation between drug targets and disease-related genes was evaluated using Z-score, and the proximity between drug targets and contrast agent targets was calculated.

Network Construction and Analysis

The overlapping targets of CMF prediction targets and CRA-related DEGs were obtained through STRING11.0 (https://string-db.org/). An interaction network of “CRA-related genes and CMF potential targets” was constructed by using the screening condition of “Homo sapiens”. The results were saved, and the generated file was imported into Cytoscape v3.7.1 for visualization. This allowed for the identification of core targets within the protein-protein interaction (PPI) network.

Pathway Enrichment Analysis

The “clusterProfiler” package in R was used to perform Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis on the interacting targets. This analysis aimed to elucidate the roles of core targets in gene function and associated signaling pathways.

Molecular Docking Analysis

We screen core targets based on the degree values of nodes in the protein-protein interaction (PPI) network, and perform molecular docking analysis with chemical components that comply with the five principles of Lipinski class drugs in mass spectrometry analysis. From Pubchem (https://pubchem.ncbi.nlm.nih.gov/), download the SDF file of the 2D structure of the core compound, utilize ChemBio3D software to optimize its mechanical structure and save it as a Mol2 file, import it into AutoDockTools1.5.6 software, and save it in pdbqt format. The 3D structure of key target proteins was downloaded from the PDB database (https://www.rcsb.org). The PyMOL software was used to remove water molecules and excess inactive ligands. The AutoDockTools 1.5.6 software was imported and saved in pdbqt format. Molecular docking simulations were conducted on potential targets and their corresponding components using AutoDock vina software. The global optimal binding conformation was obtained through DiscoveryStudio 4.5 software and saved as a PDB file. The docking results were visualized using the PyMOL software.

In vitro Experimental VerificationCell Culture

The IH-CRA cells were obtained from the China Center for Type Culture Collection(CCTCC) with the cell line number C2019307. We filed a national invention patent for IH-CRA cells, patent number 201911261397.9 (Wuhan, China). Cells were cultured at 37 ° C and 5% CO2 in a medium containing 10% fetal bovine serum (FBS) (F12 medium from Shanghai Beyonce Biotechnology Co., Ltd.). The cultured cells were washed twice in PBS and digested with trypsin. Cells were centrifuged at 800 rpm for 3 min, mixed in culture medium and inoculated into a culture dish. After 24 h of cell adhesion, the drug was administered, and the cells were collected for subsequent experiments.

qPCR

The total RNA was extracted using Trizol reagent, then treated with deoxyribonuclease without RNA enzyme, and reversely transcribed with oligomeric DT using MMLV reverse transcriptase according to the instructions of the reverse transcriptase kit. The conditions for reverse transcription reaction are: reaction at 25 °C for 5 min, reaction at 42 °C for 30 min, and reaction at 85 °C for 5 s. The reaction is carried out in a PCR machine. The primer sequence is shown in Table 2.

Statistic Analysis

All data were statistically processed using SPSS 26.0 software. Measurement data adopts mean plus minus standard deviation \(\:\left(\stackrel\pm\:s\right).\:\) If the data follows a normal distribution and satisfies homogeneity of variance, paired sample t-tests are used for intra group comparisons before and after, and independent sample t-tests are used for inter group comparisons; For data with skewed distribution or uneven variance, non parametric testing is used; The counting data adopts chi square test. P < 0.05 was considered to indicate a statistically significant difference.