Extraction and Characterization of Ganoderma Triterpenes

GT samples were gifts from Professor Ruey-Shyang Hseu, Department of Biochemical Science and Technology, National Taiwan University. Dr. Hseu entrusted the Li-Kang Biotechnical Co., Ltd (I-Lan, Taiwan) to culture and collect the mycelium. He further extracted the GT from the fruiting body using hot water as described previously^{30 (link)}. Briefly, the fruiting body was homogenized in sterilized water. The collected sample was frozen at − 20 °C, vacuum-dried for 36 h, then stored at − 20 °C until further use. Dr. Hseu provided and authorized us to use the water extract for this study. The crude mixture contained 1.96% triterpenes and 3.93% polysaccharides^{31 (link)}. The dried extract was further analyzed for the total water-soluble polysaccharide, total triterpenoid, beta-d-glucan, and heavy metal contents. For total water-soluble polysaccharides, the sample was extracted, the polysaccharides were precipitated with ethanol, re-dissolved, reacted with phenol–sulfuric acid, and analyzed under a UV–Vis Spectrophotometer (UV–Vis) (ChromTech® CT-2200, Taiwan). The amount of total water-soluble polysaccharides was 45.4 g/100 g. For total triterpenoids, the sample was extracted, reacted with a vanillin-glacial acetic acid-perchloric acid solution as a color-developing agent, and analyzed under a UV–Vis. We registered the amount of total triterpenoids as 463 mg/100 g. Concerning beta-d-glucans, the sample was dissolved to remove low-molecular-weight sugar, hydrolyzed by enzyme specificity, and analyzed using a High-Performance Anion Exchange Chromatography-Pulsed Amperometric Detector (HPAEC-PAD) (Thermo Fischer Scientific, Germering, Germany). The amount of beta-D-glucans was 0.24 g/100 g. Finally, we detected no heavy metals. The GT water extraction was followed by DMSO precipitation, reserve dialysis, and protein depletion. The final triterpenoid and water-soluble polysaccharide content was 128 and 0.39 g/100 g in GT-DMSO solution, respectively. Our previous preliminary study followed a GT dosage of 200 μg/kg/day³² .