[Screening of pharmacodynamic substances for modified-zengshengping in blocking the progression of oral squamous cell carcinoma based on salivary metabolomics].

Objective: To identify the active ingredients of modified-Zengshengping (ZSP-M) that inhibit the progression of oral squamous cell carcinoma (OSCC) through salivary metabolomics and computer virtual screening, and to screen potential key pharmacodynamic substances. Methods: An experimental tongue OSCC model induced by 4-nitroquinoline-1-oxide (4NQO) was established in 20 C57BL/6 mice. The experiment was divided into a normal group, a 4NQO group, a Zengshengping-original (ZSP-O) group, and a ZSP-M group (n=5). After 24 weeks, the number and volume of tongue tumors were recorded, and the tongue tissue was subjected to HE staining to calculate epithelial structure and cellular characteristic scores. Twelve SD rats were divided into a treatment group and a control group. They were administered ZSP-M or distilled water via gavage, and saliva samples were collected three days later. The prototype components of ZSP-M in saliva were analyzed and identified using ultra-high-performance liquid chromatography-tandem mass spectrometry. The prototype components were further subjected to computer-based absorption, distribution, metabolism, excretion, toxicity prediction, and molecular docking to screen potential key pharmacodynamic substances of interest. The impact of these potential key pharmacodynamic substances on the proliferation of human tongue squamous cell carcinoma cell lines CAL27 and SCC25 was evaluated using cell counting kit-8 assay. Results: The tumor incidence of OSCC in the ZSP-M group of C57BL/6 mice [(1.60±0.24) tumors] was significantly lower than that in the 4NQO group [(2.60±0.24) tumors] (P<0.05), and the pathological score of cytological features [(5.40±0.55) points] was significantly lower than that in the 4NQO group [(7.40±0.55) points] (P<0.05). A total of 16 prototype components entered the saliva, with 10 in positive ion mode and 6 in negative ion mode. Through computer-aided virtual screening, maackiain (MA), scutellarin (SC), chlorogenic acid (CGA), and vitexin-2-O-rhamnoside (VOR) were identified as potential key pharmacodynamic substances with strong binding potential to target proteins. In vitro experimental results showed that all four key pharmacodynamic substances could inhibit the proliferation of CAL27 and SCC25 at 48 and 72 hours (all P<0.05). Compared to the control group, the most significant proliferation inhibition occurred at a high concentration of 100 μmol/L (P<0.01), with the inhibitory effect weakened when the concentration was reduced to 10 μmol/L. Among them, MA demonstrated the most outstanding effect, with an SCC25 cell inhibition rate of (15.16±0.28)% after 72 hours of treatment with 100 μmol/L concentration (P<0.001). Conclusions: ZSP-M can inhibit the development of experimental OSCC, and MA, SC, VOR, and CGA have been identified as potential key pharmacological substances for ZSP-M to enter saliva and exert anticancer activity, reflecting the characteristics of multi-component and multi-target treatment in traditional Chinese medicine formulas.