The pellet morphology and diameter range (DR) of Ganoderma lucidum were observed in a repeated-batch fermentation (RBF) for the trio total production of biomass, exopolysaccharide (EPS) and endopolysaccharide (ENS). Two factors were involved in RBF; broth replacement ratio (BRR: 60%, 75% and 90%) and broth replacement time point (BRTP: log, transition and stationary phase) in days. In RBF, 34.31 g/L of biomass favoured small-compact pellets with DR of 20.67 µm< d < 24.00 µm (75% BRR, day 11 of BRTP). EPS production of 4.34 g/L was prone to ovoid-starburst pellets with DR of 34.33 µm< d <35.67 µm (75% BRR, day 13 of BRTP). Meanwhile, the highest 2.43 g/L of ENS production favoured large-hollow pellets with DR of 34.00 µm< d < 38.67 µm (90% BRR, day 13 of BRTP). In addition, RBF successfully shortened the biomass-EPS–ENS fermentation period (31, 33 and 35 days) from batch to 5 days, in seven consecutive cycles of RBF. In a FTIR detection, β-glucan (BG) from EPS and ENS extracts were associated with β-glycosidic linkages (2925 cm −1 , 1635 cm −1 , 1077 cm −1 , 920 cm −1 and 800 cm −1 wavelengths) with similar 1 H NMR spectral behaviour (4.58, 3.87 and 3.81 ppm). Meanwhile, 4 mg/L of BG gave negative cytotoxic effects on normal gingival cell line (hGF) but induced antiproliferation (IC 50 = 0.23 mg/mL) against cancerous oral Asian cellosaurus cell line (ORL-48). Together, this study proved that G. lucidum mycelial pellets could withstand seven cycles of long fermentation condition and possessed anti-oral cancer beta-glucan, which suits large-scale natural drug fermentation.
This study aims to identify the roles of exo-β-glucan (EPS-BG) and endo-β-glucan (ENS-BG) extracted from Ganoderma lucidum (GL) in inhibiting the alpha-glucosidase enzyme, a target mechanism for postprandial hyperglycaemia regulation. Upscale production of GL was carried out using a 10 L bioreactor. The zebrafish embryo toxicity test (ZFET) was carried out based on OECD guidelines. The hatching rate, survival rate, heart rate, morphological malformation, and teratogenic defects were observed and determined every 24 h from 0–120 h of post-exposure (hpe). For diabetes induction, adult zebrafish (3–4 months of age) were overfed and induced with three doses of 350 mg/kg streptozotocin (STZ) by intraperitoneal injection (IP) on three different days (days 1, 3, and 5). The oral sucrose tolerance test (OSTT) and anti-diabetic activity of EPS-BG and ENS-BG were evaluated (day 7) using the developed model (n = 15). This study showed that EPS is the most potent compound with the highest inhibitory effect toward the alpha-glucosidase enzyme with an IC50 value of 0.1575 mg/ml compared to ENS extracts (IC50 = 0.3479 mg/ml). Both EPS-BG and ENS-BG demonstrated a strong inhibition of alpha-glucosidase activity similar to the clinically approved alpha-glucosidase inhibitor, acarbose (IC50 = 0.8107 mg/ml). ENS-BG is non-toxic toward zebrafish embryos with LC50 of 0.92 mg/ml and showed no significant changes in ZE hatching and normal heart rate as compared to untreated embryos (161 beats/min). Teratogenic effects of ENS-BG (<1.0 mg/ml) on zebrafish embryonic development were not observed. The DM model of zebrafish was acquired after the third dose of STZ with a fasting BGL of 8.98 ± 0.28 mmol/L compared to the normal healthy group (4.23 ± 0.62 mmol/L). The BGL of DM zebrafish after 30 min treated with EPS-BG and ENS-BG showed a significant reduction (p < 0.0001). Both EPS-BG and ENS-BG significantly reduced DM zebrafish’s peak blood glucose and the area under the curve (AUC) in OSTT. Hence, EPS-BG and ENS-BG extracted from GL showed promising inhibition of the alpha-glucosidase enzyme and are considered non-toxic in ZE. Moreover, EPS-BG and ENS-BG reduced blood glucose levels and inhibited hyperglycemia in DM zebrafish.
Molecular docking was done to assess the binding affinity of 6-paradol (6PRD), Zingerone (ZGR) and Zerumbone (ZRB) ligand-ERα complex in comparison to Hydroxytamoxifen (HTMX). Docking results showed that Glu353 and Arg394 active residues forms hydrogen bonding with 6PRD and ZGR. Glu353, Leu387 and Arg394 were the three identical residues found to formed hydrophobic interaction in HTMX-ERα, 6PRD-ERα and ZGR-ERα. HTMX showed lowest binding energy (-10.71 ± 0.43 kcal/mol) followed by ZRB (-8.66 ± 0.04 kcal/mol), 6PRD (-6.92 ± 0.14 kcal/mol) and ZGR (-5.93 ± 0.31 kcal/mol). Inhibition constant (Ki) range of 6PRD-ERα was found to be drastically lower than HTMX-ERα, ZGR-ERα and ZRB-ERα. Based on the docking analysis, the three bioactive compounds were showed to poses low potential as substitute towards tamoxifen. Future study is recommended for analysing 6PRD potential in substituting estradiol as Hormone Replacement Therapy (HRT) for breast cancer.
Simulation and computational analysis of 6-gingerol and 6-shogaol is done to evaluate their binding affinity against ERα. Active site prediction was done using Computed Atlas of Surface Topography of Proteins (CASTp) to determine the binding pocket of ERα. Molecular docking and molecular dynamics (MD) simulation were done to assess the binding affinity and stability of the ligand-ERα complexes formed. Results showed that Tamoxifen have lowest binding energy (-9.61 ± 0.39 kcal/mol) followed by 6-gingerol (-6.59 ± 0.29 kcal/mol) and 6-shogaol (-5.70 ± 0.36 kcal/mol). Inhibition constant (Ki) range of TMX-ERα was found to be drastically lower than both 6GN-ERα and 6SG-ERα. Based on the difference in the binding energy range and inhibition constant, 6gingerol and 6-shogaol showed less potential in substituting tamoxifen for the inhibition of ERɑ. Docking complexes formed was supported with stability in root mean square deviation (RMSD) and total binding energy of the complexes. The study is concluded that 6-gingerol have high level of interactions with the ERα active site in terms of hydrogen bonding whereas hydrophobic interactions are observed with both 6-gingerol and 6-shogaol. However, both ginger bioactive compounds poses low potential as substitute in comparison with tamoxifen against ERα.
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