Postharvest Drying Techniques Regulate Secondary Metabolites and Anti-Neuroinflammatory Activities of Ganoderma lucidum

Abstract

Ganoderma lucidum extract is a potent traditional remedy for curing various ailments. Drying is the most important postharvest step during the processing of Ganoderma lucidum. The drying process mainly involves heat (36 h at 60 °C) and freeze-drying (36 h at -80 °C). We investigated the effects of different postharvest drying protocols on the metabolites profiling of Ganoderma lucidum using GC-MS, followed by an investigation of the anti-neuroinflammatory potential in LPS-treated BV2 microglial cells. A total of 109 primary metabolites were detected from heat and freeze-dried samples. Primary metabolite profiling showed higher levels of amino acids (17.4%) and monosaccharides (8.8%) in the heat-dried extracts, whereas high levels of organic acids (64.1%) were present in the freeze-dried samples. The enzymatic activity, such as ATP-citrate synthase, pyruvate kinase, glyceraldehyde-3-phosphatase dehydrogenase, glutamine synthase, fructose-bisphosphate aldolase, and D-3-phosphoglycerate dehydrogenase, related to the reverse tricarboxylic acid cycle were significantly high in the heat-dried samples. We also observed a decreased phosphorylation level of the MAP kinase (Erk1/2, p38, and JNK) and NF-κB subunit p65 in the heat-dried samples of the BV2 microglia cells. The current study suggests that heat drying improves the production of ganoderic acids by the upregulation of TCA-related pathways, which, in turn, gives a significant reduction in the inflammatory response of LPS-induced BV2 cells. This may be attributed to the inhibition of NF-κB and MAP kinase signaling pathways in cells treated with heat-dried extracts.

Keywords: BV2 cancer cells; Ganoderma lucidum; LPS-induced inflammation; MAPK; ganoderic acid; neuro-degradation.

Figure 1

A principal component analysis of the primary data detected by GC-TOF-MS was performed in the SIMCA program. For the metabolite analysis, three biological replicates per treatment sample were analyzed, and the results were presented in principal component 1 (PC1, 63.4%) and PC2 (14.1%). The differences in the primary metabolite data between freeze-drying (blue hexagon) and heat-drying (red hexagon) in the Student’s t-test are marked as green circles (p < 0.05), and the nonsignificant data are indicated by gray circles (p > 0.05).

Figure 2

Protein analysis based on SDS-PAGE-LC-MS. (A) Proteins were significantly (p < 0.05) changed between freeze-drying (Aa) and heat-drying (Ab) in the Student’s t-test and classified based on the drying methods in the PCA biplot (*: p < 0.05 and #: p < 0.005). (B) Blue hexagons represent the freeze-dried samples, red hexagons represent the head-dried samples, and the significant protein molecules are expressed by green circles. The results are expressed as the mean ± standard deviation, and the t-test was performed to confirm the significant differences. PC: principal component; ND: not detected; CAT: catalase; ATPase: ATP synthase; PGK1: phosphoglycerate kinase1; MDH: malate dehydrogenase; SOD: superoxide dismutase; PPlase: peptidyl-prolyl cis-trans isomerase; IMPDH: Inosine-5′-monophosphate dehydrogenase; SDADH: succinate semialdehyde dehydrogenase; ACS: ATP-citrate synthase; PK: pyruvate kinase; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; PC: pyruvate carboxylase; GS: glutamine synthetase; CBS: cystathionine beta-synthase; CS: chorismite synthase; PEC: proteasome endopeptidase complex; PHGDH: D-3-phosphoglycerate dehydrogenase; FBA: fructose-bisphosphate aldolase; spe-Sdh: Spermidine synthase-saccharopine dehydrogenase; IPPisomerase: Isopentenyl diphosphate isomerase.

Figure 3

The effects of the heat-dried extract (HDE) (A) and freeze-dried extract (FDE) (B) on the cell viability in microglial BV2 cells were assessed by the MTT assay. The BV2 cells were treated with various concentration (1, 10, and 100 µg/mL) of GL extracts, and the cell viability was measured. The red line indicates a 70% acceptable range of cell viability.

Figure 4

Inhibitory effect of HDE on the MAPKs in LPS-stimulated BV2 cells. (A–D) Western blot analysis on the p38, ERK1/2, and JNK phosphorylation levels in the BV2 cells. The cells were pretreated with HDE 2–10 µg/mL for 2 h prior to LPS (1 µg/mL) exposure for 24 h. (E,F) Western blot analysis on ph-p65 and protein expression in the BV2 cells when pretreated with HDE prior to LPS exposure. β-actin was used as the housekeeping protein.

Figure 5

(A–D) Effect of the freeze-dried extract (FDE) on the phosphorylation levels of ph-p65, ph-p38, and ph-JNK in LPS-treated BV2 cells pretreated with FDE 2–10 µg/mL for 2 h, followed by 24 h of exposure to LPS (1 µg/mL). The cells were lysed with a radioimmunoprecipitation assay buffer, and the phosphorylation levels were measured by performing an immunoblot analysis. (E,F) Western blot analysis on ph-p65 and the protein expression in BV2 cells when pretreated with FDE prior to LPS exposure. β-actin was used as the housekeeping protein.