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The aim of the reported study was to optimize the extraction process for ganoderma triterpenes and to investigate the in vivo inhibitory effect of ganoderma triterpenes on the genesis and progression of oral cancer. Single-factor and orthogonal methods were used to investigate the effects of extraction solvent, solvent amount, extraction time, extraction temperature, and number of extractions, on the extraction rate for ganoderma triterpenes. A golden hamster model with cheek pouch dynamic canceration was established to receive oral treatment of ganoderma triterpenes water solution. Animals were continuously monitored, oral tissue samples were collected for histopathologic examination, and changes in the expression of VEGF (vascular endothelial growth factor) and Caspase-3 were detected by immunohistochemical methods. Optimization of the experimental conditions allowed the identification of the optimal extraction conditions: 90% ethanol as the extraction solvent, a solvent amount by the liquid-material ratio of 35 mL/g, extraction time of 2 h and extraction temperature of 80 °C. Under these conditions, the average extraction rate of ganoderma triterpenes was 1.09%. Tests in golden hamsters showed that compared with the model group during the same period, animals in the treatment group had better conditions, constantly larger number of normal cases shown by histopathologic results (P < 0.01), and consistently smaller numbers of cases with paraplasm (P < 0.05). Immunohistochemical results showed that compared with the model group, the treatment group had significantly lower (P < 0.05) rates of positive VEGF expression in the normal state, simple epithelial hyperplasia, epithelial dysplasia or squamous cell carcinoma disease stages. Caspase-3 expression showed a tendency toward a gradual increase with the worsening of disease severity in each group. Compared with the model group, the treatment group had significantly lower (P < 0.05) rates of positive Caspase-3 in the normal state, simple epithelial hyperplasia, epithelial dysplasia or squamous cell carcinoma disease grades. Using the optimized extraction process, ganoderma triterpenes could be extracted with high efficiency, and the results of animal tests showed inhibitory effects of ganoderma triterpenes on oral mucosa cancer. [ABSTRACT FROM AUTHOR]