Abstract
Ganoderma lucidum (Leyss. ex Fr.) Karst. (G. lucidum, GL) belongs to the family of Ganodermataceae (Basidiomycetes), and possesses activities including antitumor, antimicrobial, antiviral, and antiaging activities. Triterpenoids are typical chemical constituents in G. lucidum, and play an important role in the anti-cancer effects. According to the substituent group at the carbon 26 position, GL total triterpenes fraction can be divided into two types, Neutral Triterpene Fraction (NTF) and an Acidic Triterpene Fraction (ATF). The anti-cancer effects of total triterpenes fraction and total acidic triterpene fraction extracted from G. lucidum have been widely known in vivo and in vitro, whereas few have focused on total neutral triterpene fraction. The aim of this study was to evaluate the anti-cancer effects of NTF extracted from G. lucidum in vitro and in vivo and explore its anti-cancer active constituents on SW620 human colorectal cancer cells. NTF and ATF were extracted from the dry fruiting body of G. lucidum by impregnation method with 90% ethanol, and further isolated by using alkaline extraction and acid precipitation method. The total triterpenoid content of NTF and ATF was determined by using ultraviolet-visible spectrophotometry. The cytotoxic effects on human colon cancer cells SW480, SW620, SW1116, and mouse embryonic fibroblast cell line NIH3T3 were evaluated by using the MTT method. The anti-cancer activity of NTF in vivo was evaluated in Athymic nude mice against SW620 cells. An activity-guided separation and purification process were used to identify the anti-cancer active constituents of NTF by column and preparative high-performance liquid chromatography. Structures of the constituents were confirmed by 1H-NMR, 13C-NMR and MS. Protein expression was performed by Western blotting. The percentage of total triterpenoids was 46.7% and 57.6% in ATF and NTF, respectively. Both fractions could reduce the viability of SW480, SW620, and SW1116 cells in vitro, whereby NTF exhibited a stronger effect than ATF. NTF markedly inhibited the growth of SW620 cell xenografts in mice at doses (250, 500mg/kg) during the treatment. Furthermore, a new garnoderic alcohol, named as ethyl ganoderate A and eight known ganoderic alcohols were isolated and identified from NTF by a bioassay-guided separation process. All of these compounds possessed anti-cancer activities against SW620 cells in vitro. As a representative ganoderma alcohol, ganodermanondiol significantly reduced the viability of SW620 cells through the induction of apoptosis, which was associated with the upregulated the levels of cleaved-poly (ADP-ribose) polymerase (PARP), cleaved-caspase-3, and -9. In addition, ganodermanondiol showed low cytotoxic activity against normal NIH3T3 cells. NTF are potential anti-cancer agents against colon cancer and the active constituents may be ganoderic alcohols whose inhibitory mechanism of anti-cancer action may be related to the activation of a mitochondrial- dependent pathway.
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