Ganoderma Isolates from the Lower Volta River Basin of Ghana: Molecular Identification and Phylogenetic Analysis, Metabolomics and Biological Activity Evaluation

Abstract

Ganoderma, a cosmopolitan genus of polypore mushroom, is known to have a number of interesting medicinal properties. The Lower Volta River Basin is reportedly rich in several species of polypore mushroomsresembling Ganoderma. Despite the medicinal importance, the Ganoderma mushroom isolates obtained from this river basin have not been well studied. In this present research study, sequence analysis of the internal transcribed spacer 2 (ITS 2), complete internal transcribed spacer (ITS) and the nuclear large subunit (nLSU) was used to identify collected Ganoderma from this riverine Lower Volta Basin. Ultra performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS) was used to study the chemical constituents of the mycelial biomass of these Ganoderma mushrooms and the effect of their extracts and fractions on the human carcinoma cell line PC-3 and two human lymphoma cell lines; Jurkat, derived from a T cell leukemia and plasmacytoid dendritic cell (pDC) derived from acute leukemia evaluated using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. The result of the sequence analysis revealed that the Ganoderma sample designated Ganoderma sample 2 belongs G. mbrekobenum species whereas three of the Ganoderma muhrooms belong to the species G. enigmaticum. The sequence analysis further demonstrated that Ganoderma sample coded Ganoderma sample 17 belongs to the species G. resinaceum whereas the sample designated Ganoderma sample 9 belongs to G. weberianum-sichuanese species complex. Thus, the native Ganoderma mushrooms collected in the present study belong to four mushroom species, namely G. mbrekobenum, G. enigmaticum, G. resinaceum and G. weberianum-sichuanese species complex. The current data on molecular identity and phylogeny of Ganoderma mushrooms from Ghana would be helpful in future studies relating to molecular evolution and medical implications of Ganoderma isolates from different regions of Ghana and other part of the world. The total ion chromatogram (TIC) data demonstrated an interesting metabolic profile difference, suggesting UPLC-Q-TOF-MS could be used to differentiate between Lower Volta River Basin Ganoderma isolates based on their mass spectra. The PLS-DA score plot of the mycelial biomass was separated into three distinct clusters, consistent with the phylogenetic analysis in the current study which showed that the Ganoderma mushrooms used in the current metabolomic study belong to three different species. UPLC-Q-TOF-MS analysis revealed the presence of six lanostane-triterpenoids in the mycelia biomas of three Ganoderma mushrooms. Ganoderenic acid A, Ganoderenic acid D, Ganoderic C6 and Ganoderic acid G were identified in the mycelia biomass by comparing their mass spectra with pure reference compounds. The remaining two (Ganoderenic acid K and Ganoderic acid AM1), due to absence of reference pure compounds, were annotated by comparing their mass spectra with Ganoderma lanostane triterpenoids previously reported in literature. The result of the biological activity evaluation showed the fraction GL-C2 significantly (≤ 0.05%) inhibited the proliferation and survival of the three cancer cell lines, PC-3, pDC and Jurkat with increasing concentrations and with IC50 values of 27.73±5.25, 21.31±2.40 and 17.09±0.86 μg/mL, respectively compared to Chang liver cells (CVCL_0238) with an IC50 value of 75.41±1.95 μg/mL. The study further demonstrated that the subfraction GL-C2-C1 from GL-C2 demonstrated a potent cytotoxic effect against PC-3 with IC50 value of 3.24± 0.10 μg/mL compared to curcumin with IC50 = 5.13± 0.86 μg/mL. This finding suggests that the subfraction GL-C2-C1 could be an excellent candiadate for developing new treatment option for prostate cancer prevention or treatment. The results also revealed that the subfractions GL-C2-C4 and GL-C2-C5 potently inhibited the growth and survivl of pDC with IC50 values of 19.95±0.50 and 13.57±2.14 μg/mL, respectively, suggesting GL-C2-C4 and GL-C2-C5 may be useful in modulating the production of type I interferon (IFN-1) by suppressing the viability of pDCs and may thereby be useful in developing biopharmaceuticals for treating disorders associated with pDCs. Thus, the current findings demonstrated that specific mycelial fractions are selectively cytotoxic to the three human cancer cell lines suggesting their potential efficacy in the treatment of malignancies. Future study with other cancer cell lines, primary pDCs, T cells, B cell and macrophages as well as animal models is worthy of investigation. The isolation of the bioactive compounds in GL-C2- C1 may lead to a novel bioactive compound that can be used in developing new treatment for prostate cancer whereas novel bioactive compounds from GL-C2-C4 and GL-C2-C5 may lead to a novel compound for developing new treatment for disorders associated with pDC.