Supplementary Material for: Supportive Evidence for the Anticancerous Potential of Alternative Medicine against Hepatocarcinogenesis in Mice

The present study examines if Lycopodium 200 (Lyco-200) has demonstrable anti-cancer activities in mice which are chronically fed carcinogens, p-dimethylaminoazobenzene (p-DAB) and phenobarbital (PB) to induce liver cancer. Materials and Methods: Mice in 5 different groups were chronically fed for varying periods of time: group I: normal diet; group II: normal diet + alcohol 200); group III: p-DAB + PB; group IV: p-DAB + PB + alcohol 200 (vehicle of Lyco-200 being ethyl alcohol); group V: p-DAB + PB + Lyco-200. They were sacrificed at day 7, 15, 30, 60, 90 or 120, and the following parameters were assessed: cytogenetic endpoints like chromosome aberrations, micronuclei, mitotic index and sperm-head anomaly; toxicity biomarkers like acid and alkaline phosphatases, alanine and aspartate amino transferase, glutathione reductase, succinate dehydrogenase and catalase activities, lipid peroxidation and reduced glutathione content. Additionally, scanning and transmission electron microscopic analyses of liver tissues were made at day 90 and 120, and immunodetection of p53 protein as well as gelatin zymography for matrix metalloproteinases in liver tissue were performed. Furthermore, studies were conducted on blood glucose, hemoglobin and cholesterol, estradiol, testosterone and cortisol, and lymphocyte and hepatic cell viabilities. Physical properties of Lyco-200 and potentized alcohol 200 were analyzed by using methods such as UV, Fourier Transform Infrared Spectroscopy (FTIR), Fluorescence Spectroscopy, ¹H-NMR and¹³C-NMR (Nuclear Magnetic Resonance Spectroscopy). Results: Lyco-200 reduced cytogenetic damages yielding positive modulations of all biochemical, pathological and other risk factors, cell viability and expression of p53 protein and matrix metalloproteinases as compared to controls. Conclusion: Studies on other mammals are recommended to further investigate the potential of Lyco-200 in liver cancer.

本研究旨在探讨石松200（Lycopodium 200，Lyco-200）对长期喂食致癌剂对二甲基氨基偶氮苯（p-dimethylaminoazobenzene，p-DAB）与苯巴比妥（phenobarbital，PB）以诱发肝癌的小鼠，是否具有明确的抗癌活性。
材料与方法：将小鼠分为5组，分别接受不同时长的长期喂食处理：第I组：正常膳食；第II组：正常膳食+200浓度乙醇；第III组：对二甲基氨基偶氮苯+苯巴比妥；第IV组：对二甲基氨基偶氮苯+苯巴比妥+200浓度乙醇（石松200的赋形剂为乙醇）；第V组：对二甲基氨基偶氮苯+苯巴比妥+石松200。分别在第7、15、30、60、90或120天处死小鼠，评估以下指标：细胞遗传学终点，包括染色体畸变、微核、有丝分裂指数与精子头部畸形；毒性生物标志物，包括酸性磷酸酶与碱性磷酸酶、丙氨酸氨基转移酶与天冬氨酸氨基转移酶、谷胱甘肽还原酶、琥珀酸脱氢酶与过氧化氢酶活性、脂质过氧化水平与还原型谷胱甘肽含量。此外，于第90天与第120天对肝组织开展扫描电镜与透射电镜分析，并进行肝组织中p53蛋白的免疫检测以及基质金属蛋白酶（matrix metalloproteinases）的明胶酶谱实验。同时，检测了血糖、血红蛋白、胆固醇、雌二醇、睾酮与皮质醇水平，以及淋巴细胞与肝细胞活力。此外，采用紫外光谱、傅里叶变换红外光谱（Fourier Transform Infrared Spectroscopy，FTIR）、荧光光谱、¹H-核磁共振波谱（¹H-NMR）与¹³C-核磁共振波谱（¹³C-NMR）等方法，分析了石松200与活化200浓度乙醇的物理性质。
结果：与对照组相比，石松200可减轻细胞遗传学损伤，对所有生化、病理及其他风险因子、细胞活力以及p53蛋白与基质金属蛋白酶的表达产生正向调控作用。
结论：建议开展其他哺乳动物的相关研究，以进一步探究石松200在肝癌治疗中的应用潜力。