Optimized Subcritical Water Extraction of β-D-Glucan from Submerged-Cultured Ophiocordyceps sinensis OS8 Mycelium and Its Antiproliferative and Antimigratory Activity Against HepG2 Liver Cancer Cells

Background/Objectives: β-D-Glucan accounts for much of the immunomodulatory and anticancer activity of Ophiocordyceps sinensis, yet there is still no green, efficient way to recover it from cultured mycelium, and subcritical water extraction (SWE) had not been tried on this fungus. We optimized SWE of β-D-glucan from submerged-cultured O. sinensis OS8 mycelium and tested the resulting extract on HepG2 liver cancer cells. Methods: Mycelium was grown in static submerged culture. A Box–Behnken design (BBD) with response surface methodology (RSM) was used to model how pH, extraction time and temperature affect the β-D-glucan content of the extract. The extract from the selected conditions was assessed for antiproliferative (MTT, trypan blue exclusion) and antimigratory (scratch) activity in HepG2 cells. Results: Cultures yielded 18.27 ± 2.38 g/L dry biomass. The quadratic model was highly significant (p < 0.0001; R2 = 0.99), with temperature as the dominant factor. At pH 7, 20 min and 170 °C, crude polysaccharide (crude extract) yield reached 14.80% of dry biomass, 4.60 times that of sonication, and β-D-glucan made up 68.92% (w/w) of the extract, or about 10.2% of dry biomass; the predicted maximum was 72.47%. At 250 µg/mL of total extract, HepG2 growth fell by 76.11% (MTT) and 70.77% (trypan blue), and 125–500 µg/mL significantly reduced migration at 24, 48 and 72 h (p < 0.05). Conclusions: RSM-optimized SWE is a solvent-free first step for obtaining a β-D-glucan-rich extract from cultured O. sinensis OS8 mycelium. The in vitro activity against HepG2 cells justifies mechanistic, in vivo and scale-up work.