Lien Hsiu-Man, Chiu Chun-Hung, Chen Chia-Chang, Chang Wan-Lin, Chyau Charng-Cherng, Peng Robert Y
Department of Chemistry, Tunghai University, No.1727, Sec. 4, Taiwan Boulevard, Xitun District, Taichung 40704, Taiwan.
Research Institute of Biotechnology, Hungkuang University, No. 1018, Sec. 6, Taiwan Boulevard, Shalu District, Taichung 43302, Taiwan.
Molecules. 2014 Jun 27;19(7):9033-50. doi: 10.3390/molecules19079033.
Antrodia cinnamomea (AC) has been widely used as a folk medicine in the prevention and treatment of liver diseases, such as hepatitis, hepatic fibrosis, and hepatocellular carcinoma. Previous studies have indicated that triterpenoids and benzenoids show selective cytotoxicity against human hepatoma cell lines. The aim of the study was to compare the triterpenoid content of extract and the extract-induced cytotoxicity in HepG2 cells from mycelia extracts of solid state cultured AC obtained by supercritical fluid extraction (SFE) and the conventional solvent extraction method. SFE with CO2 mixed with a constant amount of ethanol co-solvent (10% of CO2 volume) applied at different temperatures and pressures (40, 60 and 80 °C and, 20.7, 27.6 and 34.5 Mpa) was also compared in the study. Although the extraction yield of triterpenoids (59.7 mg/g) under the optimal extraction conditions of 34.5 MPa (5000 psi)/60 °C (designated as sample S-5000-60) was equivalent to the extraction yield using conventional liquid solvent extraction with ethanol (ETOH-E) at room temperature (60.33 mg/g), the cytotoxicity of the former against the proliferation of HepG2 cell line measured as the inhibition of 50% of cell growth activity (IC50) at dosages of 116.15, 57.82 and 43.96 µg/mL was superior to that of EtOH-E at 131.09, 80.04 and 48.30 µg/mL at 24, 48 and 72 h, respectively. Additionally, we further proved that the apoptotic effect of S-5000-60 presented a higher apoptosis ratio (21.5%) than ETOH-E (10.5%) according to annexin V-FITC and propidium iodide double staining assay results. The high affinity and selectivity of SFE on bioactive components resulted in a higher extraction efficiency than conventional solvent extraction. The chemical profile of the obtained extracts from solid state cultivated mycelium of AC was also determined by high-performance liquid chromatography electrospray ionization tandem mass spectrometry (LC-MS/MS), whereby three benzenoids and four triterpenoids were found for the first time in SFE extracts with 4,7-dimethoxy-5-methyl-l,3-benzodioxole (5.78 mg/g) being the most abundant component, followed by 2,4-dimethoxy-6-methylbenzene-1,3-diol (3.03 mg/g) and dehydroeburicoic acid (0.40 mg/g).
樟芝（Antrodia cinnamomea，AC）作为一种民间药物，已被广泛用于预防和治疗肝脏疾病，如肝炎、肝纤维化和肝细胞癌。先前的研究表明，三萜类化合物和苯类化合物对人肝癌细胞系具有选择性细胞毒性。本研究的目的是比较超临界流体萃取（SFE）和传统溶剂萃取法从固态培养的AC菌丝体提取物中获得的提取物中三萜类化合物的含量以及提取物对HepG2细胞的细胞毒性。该研究还比较了在不同温度和压力（40、60和80℃以及20.7、27.6和34.5MPa）下，用二氧化碳与恒定含量的乙醇共溶剂（占二氧化碳体积的10%）混合进行的SFE。尽管在34.5MPa（5000psi）/60℃的最佳萃取条件下（指定为样品S-5000-60）三萜类化合物的萃取产率（59.7mg/g）与室温下用乙醇进行传统液体溶剂萃取（ETOH-E）的萃取产率（60.33mg/g）相当，但前者对HepG2细胞系增殖的细胞毒性，在24、48和72小时时，以116.15、57.82和43.96μg/mL的剂量抑制50%细胞生长活性（IC50）来衡量，优于ETOH-E在131.09、80.04和48.30μg/mL时的细胞毒性。此外，根据膜联蛋白V-异硫氰酸荧光素和碘化丙啶双染色分析结果，我们进一步证明S-5000-60的凋亡作用呈现出比ETOH-E更高的凋亡率（21.5%）（10.5%）。SFE对生物活性成分的高亲和力和选择性导致其萃取效率高于传统溶剂萃取。还通过高效液相色谱-电喷雾电离串联质谱（LC-MS/MS）测定了从固态培养的AC菌丝体中获得的提取物的化学图谱，由此在SFE提取物中首次发现了三种苯类化合物和四种三萜类化合物，其中4,7-二甲氧基-5-甲基-1,3-苯并二恶唑（5.78mg/g）是含量最丰富的成分，其次是2,4-二甲氧基-6-甲基苯-1,3-二醇（3.03mg/g）和脱氢齿孔酸（0.40mg/g）。
