HPLC quantitative analysis of protocatechuic acid contents in 11 Phellinus mushroom species collected in Thailand

Abstract Phellinus mushrooms have been traditionally used for various medicinal purposes. Protocatechuic acid, which was previously reported to be a component in some Phellinus mushrooms, has some pharmacological effects. This study aimed to validate a HPLC method for the quantitative analysis of the protocatechuic acid contents in the extracts from different Phellinus mushroom species collected in Thailand. HPLC was carried out using a C18 column and the gradient mobile phases of 0.1% formic acid in water and 0.1% formic acid in acetonitrile. Method validation was performed to assure the linearity, accuracy, precision, limit of detection and limit of quantitation of the analytical method. The linearity range of protocatechuic acid was 1 - 10 µg/ml. The average recovery was 104.16%. The method was shown to be precise with the RSD of repeatability and intermediate precision at less than 3%. The protocatechuic contents in 11 Phellinus mushrooms were in the range of less than 0.0099 - 0.4121 %w/w of the extract. The developed HPLC method was reliable and suitable for the quantitative analysis of protocatechuic acid content in Phellinus mushrooms.

Cladogynos orientalis Zipp. extracts inhibit cell culture-derived hepatitis C virus genotype 2a replication in Huh-7 cells through NS5B inhibition

Objective: To evaluate the potential anti-hepatitis C virus (HCV) activities of Cladogynos orientalis Zipp. ex Span and to investigate the molecular mode of action. Methods: Ethanolic and water extracts from various parts of Cladogynos orientalis were examined for cytotoxicity by MTT assay. Sub-cytotoxic concentrations of the extracts were used for further determining anti-HCV activity using cell culture-derived HCV genotype 2a propagated in HepaRG cell line. Immunofluorescence assay was performed to observe the effect on viruses at the pre-entry step. Mode of action at the post-entry step was investigated for the viral RNA and protein expressions by real time RT-PCR and Western blotting assays, respectively. Results: Although Cladogynos orientalis water extracts exhibited lower cytotoxicity than ethanolic extracts, all ethanolic extracts from roots, stems, and leaves of Cladogynos orientalis exhibited higher anti-HCV activities than water extracts. The highest anti-HCV activity was observed in infected cells treated with the extracts 5 h after absorption. No extracts showed pre-viral entry effect. At the post-viral entry step, only leaf ethanolic extracts inhibited NS5B expression, while all extracts did not inhibit HCV NS3 expression. Conclusions: Cladogynos orientalis ethanolic extracts could be further studied and the major active compound needs to be identified as a promising source for anti-HCV agents.