ABSTRACT
Objectives Aspidin BB, a typical phloroglucinol derivative from Dryopteris fragrans, possesses significant antifungal property. This study aimed to investigate potential mechanism of antifungal activity of Aspidin BB against Trichophyton rubrum which is the most common pathogens responsible for chronic dermatophytosis. Methods The minimum inhibitory concentration (MIC) of Aspidin BB against strains was determined by broth microdilution. The effects of Aspidin BB on ergosterol biosynthesis were investigated by content determination based on UPLC method. Besides, the effects of drugs on squalene epoxidase (SE) in T. rubrum cell membrane were analyzed. Results MIC value of Aspidin BB against T. rubrum was 25.0 μg/mL. Aspidin BB reduced ergosterol content significantly, but no notable effect on squalene epoxidase activity. Conclusion The results suggested that Aspidin BB inhibited ergosterol biosynthesis. However, it was not squalene epoxidase but other components may sever as possible targets in ergosterol biosynthesis pathway.
ABSTRACT
In the present study, antibacterial activity of four kinds of phloroglucinol derivatives extracted from Dryopteris fragrans (L.) Schott against S. aureus, S. epidermidis and P. acnes has been tested. Aspidin BB exerted the strongest antibacterial activity with minimal inhibition concentration (MIC) values ranging from 7.81 to 15.63 µg/mL. The time-kill assay indicated that aspidin BB could kill P. acnes completely at 2 MIC (MBC) within 4 h. By using AFM, we demonstrated extensive cell surface alterations of aspidin BB-treated P. acnes. SDS-PAGE of supernatant proteins and lipid peroxidation results showed that aspidin BB dose-dependently affected membrane permeability of P. acnes. DNA damage and protein degradation of P. acnes were also verified. SDS-PAGE of precipitated proteins revealed possible targets of aspidin BB, i.e., heat shock proteins (26 kDa) and lipase (33 kDa) which could all cause inflammation. Aspidin BB also seriously increased the inhibition rate of lipase activity from 10.20 to 65.20 % to possibly inhibit the inflammation. In conclusions, the effective constituents of D. fragrans (L.) Schott to treat acne might be phloroglucinol derivatives including aspidin BB, aspidin PB, aspidinol and dryofragin. Among this, aspidin BB inhibited the growth of P. acnes by disrupting their membrane, DNA and proteins and finally leaded to the cell death. The obtained data highlighted the potential of using aspidin BB as an alternative treatment for acne vulgaris.
Subject(s)
Acne Vulgaris/drug therapy , Anti-Bacterial Agents/therapeutic use , Phloroglucinol/analogs & derivatives , Propionibacterium acnes/drug effects , Staphylococcus aureus/drug effects , Bacterial Proteins/metabolism , Cell Membrane Permeability/drug effects , DNA Fragmentation/drug effects , Dryopteris , Electrophoresis, Polyacrylamide Gel , Humans , Inflammation/drug therapy , Lipase/antagonists & inhibitors , Microbial Sensitivity Tests , Microscopy, Atomic Force , Phloroglucinol/therapeutic use , Plant Extracts/therapeutic use , Proteolysis/drug effectsABSTRACT
Objective:To isolate and prepare the reference substances of phloroglucinol derivatives from Dryopteris fragrans by semi-preparation HPLC. Methods:After reflux extraction of Dryopteris fragrans with petroleum ether,the extracting solution was con-centrated and separated by silica gel column chromatography,and then isolated by semi-preparation HPLC. The isocratic elution was carried out using acetonitrile as the mobile phase at 3. 0 ml·min-1 and the injection volume was 0. 5 ml. Two phloroglucinol deriva-tives were isolated. Results:The chemical structure of the two phloroglucinol derivatives respectively was aspidin BB with the purity of 98. 81% and aspidin PB with the purity of 98. 57% by ultra high performance liquid chromatography. Conclusion:The isolation of as-pidin BB and aspidin PB by semi-preparation HPLC is simple and fast with the purity over 98%,which can be used to prepare the ref-erence substances.
ABSTRACT
Aspidin BB, a phloroglucinol derivative extracted from Dryopteris fragrans (L.) Schott, has been previously reported to exert high biological activities. In the present study, antibacterial activities and mechanisms of aspidin BB against Staphylococcus aureus were investigated. Aspidin BB exhibited strong antibacterial activity against standard and three clinical S. aureus, with minimal inhibition concentration (MIC) values ranging from 15.63 µg/mL to 62.5 µg/mL. After treatment with aspidin BB for 72 h using the MTT assay, the IC50 value was 48.14 µM (22.11 µg/mL). The time-kill assay indicated that aspidin BB could kill S. aureus completely at 2 MIC (MBC) within 4 h. Aspidin BB was capable to induce an increase in NADPH oxidase activity from 4.03 U/mg to 7.48 U/mg when the concentrations were increased from 1/2 MIC to 4 MIC. Simultaneously, aspidin BB attenuated antioxidant defense by decreasing superoxide dismutase (SOD) activity and glutathione (GSH) levels. The level of reactive oxygen species (ROS) was apparently elevated when measuring OD575. This phenomenon was blocked by pretreatment of S. aureus with the antioxidant compound catalase (200 U/mL) and the survival rate of S. aureus increased from 3.95% to 73.04%. These results showed that ROS was indeed an important mediator in the antibacterial action of aspidin BB. In addition, aspidin-BB-induced peroxidation of membranes, DNA damage and protein degradation of S. aureus were all verified in a dose-dependent manner. In conclusion, aspidin BB induced generation of ROS by activating NADPH oxidase activity and inhibiting SOD activity and GSH levels, damaging the membrane, DNA and proteins and finally led to cell death.
