does cbd oil kill candida

Does cbd oil kill candida

The membrane-disrupting activities of CBD on 24 h-old C. albicans biofilm were determined using the membrane-impermeable dye propidium iodide (PI) (Sigma-Aldrich). The biofilms were washed with PBS and incubated with 10 μg/mL PI in the dark for 20 min. PI fluorescence was measured in an Infinite M200 PRO plate reader (Tecan) (excitation 535 nm, emission 617 nm). The ratios between dead cells and metabolically active cells in biofilms was calculated as PI fluorescence values divided on values obtained from MTT assay (PI/MTT ratio). The data were presented as a fold increase in PI/MTT ratio in treated biofilms compared to PI/MTT ratio in untreated biofilms equaling 1. In parallel, the PI-stained biofilms were observed by NIKON confocal microscope using the 561 nm yellow-green laser excitation and collecting the data using the 635 nm filter. At least three random fields were observed. Three independent experiments were performed, and one set of representative data is shown.

In conclusion, we demonstrate here that CBD exhibits specific anti-biofilm activity against C. albicans without significantly affecting either planktonic fungal growth or viability. We propose that CBD exerts its anti-biofilm activity towards C. albicans biofilm through a multi-target mode of action, which differs from commonly used antimycotics. Thus, CBD should be explored for further development as an alternative treatment to combat fungal infections.

2.7. Determination of Chitin Content in Biofilms

Based on the above findings, we further studied the mode of anti-biofilm activity of CBD at sub-MBIC90. Our findings demonstrate significant alteration of mitochondrial activity upon fungal treatment with CBD. CBD induced intracellular ROS production with concomitant reduction in the antioxidant defense genes SOD (SOD1, SOD2 and SOD4) and TRR1. This might explain the inability of C. albicans to tolerate CBD-induced oxidative stress. Additionally, the anti-biofilm agent Thiazolidinedione-8 impairs C. albicans protection mechanism against oxidative stress in C. albicansStreptococcus mutans mixed biofilms [32]. In general, elevated intracellular ROS in fungi triggers mitochondria abnormal function [45,46] and decreases ATP generation [47]. Consistent with this, we observed a CBD-induced increase in MMP indicative for hyperpolarization of the mitochondrial membrane with concomitant decrease in ATP levels. Additionally, some other compounds have been shown to elevate MMP and decrease ATP production in yeast resulting in ROS accumulation which triggers mitochondrial dysfunction and apoptosis [48,49]. Another notable finding was related to the pronounced downregulation of the Ca 2+ /H + antiporter VCX1, which is located on the fungal vacuole membrane and regulates the concentration of cytosolic Ca 2+ by calcium uptake into the vacuole, while releasing H + ions into the cytosol [50]. Since Vcx1 acts to lower Ca 2+ in yeast [51], VCX1 downregulation by CBD can cause elevation of cytosolic Ca 2+ which subsequently impairs mitochondrial function. Numerous studies have demonstrated that oxidative stress induced by natural compounds leads to elevation of intracellular Ca 2+ , which in turn triggers mitochondrial dysfunction and subsequent apoptosis of C. albicans cells [52,53,54,55,56].

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1 Biofilm Research Laboratory, Faculty of Dental Medicine, Institute of Dental Sciences, The Hebrew University of Jerusalem, Jerusalem 9112002, Israel; [email protected] (R.V.S.); [email protected] (D.S.)

4. Discussion

Higher magnification analysis of cell morphology and structure of calcofluor white (CFW)-stained biofilms revealed a dose-dependent reduction in chitin fluorescence staining intensity with increasing concentrations of CBD [ Figure 3 (Ab’–Ae’)]. The CBD-treated hyphae appeared with thinner cell wall and septa [ Figure 3 (Ab’–Ae’)] than the control fungi ( Figure 3 (Aa’)). A similar pattern of CBD activity was observed after calculating the total chitin synthesis ( Figure 4 B). At sub-MBIC90 of 12.5 and 25 μg/mL, CBD reduced chitin content by 37% and 53%, respectively, while CBD at a dose of 50 μg/mL was able to diminish total chitin composition in biofilm by 90% as compared to the untreated control ( Figure 3 B). Along with the reduction in chitin content, the morphology of the fungal cell was notably altered by CBD. While control fungi [ Figure 3 (Aa,a’)] represented uniform true hyphae with true septa (red arrows), CBD-treated fungi [ Figure 3 (Ab–e,b’–e’)] appeared as pseudohyphae characterized by long elliptic branched forms with constrictions at the septal junctions (white arrows). In addition, true hyphae in the untreated control displayed parallel-sided walls [ Figure 3 (Aa,Aa’)], whereas the width of each segment of the pseudohyphae in CBD-treated biofilms was uneven, being wider at the center than at the septum [ Figure 3 (Ab–Ae,Ab’–Ae’)].

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