Anti-hyphal properties of potential bioactive compounds for oral rinse in suppression of Candida growth

Figures & data

Table 1. Details of forward and reverse primer of ACT1, RAS1, NRG1 and HWP1 used for gene expression analysis.

Sets	Gene	Direction	Primers (5’ – 3’)	Tm (°C)	Product size (bp)
1	ACT1	F	CGATAACGGTTCTGGTATG	52.5	95
		R	CCTTGATGTCTTGGTCTAC	50.6
2	RAS1	F	TTGTTGGAGGTGGTGGTGTT	59.7	212
		R	AAACCCTTCACCGGTTCTCA	59.2
3	NRG1	F	CCCCATCCTTCCCAAGTACC	59.8	228
		R	TGGGTCTTTGCTTTGGGTGT	59.8
4	HWP1	F	TGAACCTTCCCCAGTTGCTC	59.3	237
		R	GCAGATGGTTGCATGAGTGG	58.5

Table 2. The MIC of the selected bioactive compounds against seven Candida strains.

	Minimum inhibitory concentration (MIC) (µg/mL)^a
Compounds	Candida albicans ATCC 14 053	Candida dubliniensis ATCC MYA-2975	Candida glabrata ATCC 90 030	Candida krusei ATCC 14 243	Candida lusitenaie ATCC 64 125	Candida parapsilosis ATCC 22 019	Candida tropicalis ATCC13803
Bakuchiol	125	31.3	125	62.5	62.5	62.5	62.5
Hydroxychavicol	250	250	250	500	500	500	250
Luteolin	ND^b	ND^b	ND^b	ND^b	ND^b	ND^b	ND^b
Pseudolaric acid B	3.9	3.9	250	250	125	15.6	125
Sakuranetin	ND^b	ND^b	ND^b	ND^b	ND^b	ND^b	ND^b

^Note:ND = Not determined.

^aMIC value was determined through observation of clear optical wells of sample compared to positive control.

^bMIC was unsuccessful determined at concentration ≤1 mg/mL.

Table 3. IC₅₀ with 95% CI of the antifungal bioactive compounds (compounds with antifungal activity) and CHX (comparative control) against germination of C. albicans.

	Inhibition of germ tube formation (IC50 with 95% CI) (µg/mL)
Compounds	IC50^a	95% CI^b
Bakuchiol	642.6	507.2–814.2
Hydroxychavicol	151.7	100.5–228.9
Pseudolaric acid B	502.9	354.7–713.0
^cChlorhexidine gluconate (CHX)	15.8	11.58–21.46

^aConcentration of the compounds that is required for 50% inhibition of germ tube formation.

^b95% confidence interval for the IC₅₀s concentration range determined.

^cActive compound in oral rinse used as comparative control.

Table 4. IC₅₀/MIC ratio and preferential growth inhibition of antifungal bioactive compounds and CHX against C. albicans.

	Preferential growth inhibition based on IC50/MIC ratio
Compounds	IC50 (µg/mL)	MIC (µg/mL)	IC50/MIC ratio	Preferential growth inhibition
Bakuchiol	642.6	125	5.1	Budding yeast
Hydroxychavicol	151.7	250	0.6	Hyphal growth
Pseudolaric acid B	502.9	3.9	128.6	Budding yeast
^cChlorhexidine gluconate (CHX)	15.8	9.4	1.7	Budding yeast and hyphal growth

Note: Determination is based on IC₅₀/MIC ratio. This ratio determines the relative potency of each agent to inhibit budding and hyphal growth: (1) ratio <1 (agents which preferentially inhibit hyphal growth), (2) ratio of 1–2 (those with approximately equal effects on both) and (3) ratio >2 (agents with a lower ability to inhibit the hyphal growth but exert their activity by inhibiting the budding yeast growth).

Figure 1. Absorbance reading (590 nm) for germination of hydroxychavicol-treated and untreated C. albicans.

Figure 2. SEM images of germinated cells of hydroxychavicol-treated and untreated C. albicans.

Figure 3. Absorbance reading (590 nm) for adherence of germinated C. albicans on hydroxychavicol-treated and untreated salivary pellicle.

Figure 4. Relative expression of RAS1, NRG1 and HWP1 genes of treated and untreated C. albicans.

Figure 5. Illustration of the suggested mechanism of hydroxychavicol-induced delay of the yeast–hyphae transition in C. albicans. Note: Downregulate (downward paired arrows); upregulate (upward paired arrows); suppress (cross) and promote (tick)