Anti-cryptococcal activity of ethanol crude extract and hexane fraction from Ocimum basilicum var. Maria bonita: mechanisms of action and synergism with amphotericin B and Ocimum basilicum essential oil

Figures & data

Figure 1. Bioautography technique and thin layer chromatography (TLC): 2 μL H.F. and solvent system (hexane and ethyl acetate, 75:25). (A) Bioautography and (B) TLC.

Table 1. Minimum inhibitory concentration of E.C.E. and H.F. against Cryptococcus sp.

	Cryptococcus neoformans T444	Cryptococcus neoformans H99	Cryptococcus gattii WM779
MIC (μg/ml)
E.C.E.	625	1250	2500
H.F.	156	312	312
E.O.	1250	–	–

E.C.E.: ethanol crude extract; H.F.: hexane fraction; E.O.: essential oil.

Figure 2. Gas chromatography coupled to mass spectrometry (GC-MS): (A) chromatogram, (B) mass spectrum of identified component – peak 1 and (C) amplified chromatogram of peak 2.

Figure 3. Gas chromatography coupled to mass spectrometry (GC-MS): (A) chromatogram and (B) mass spectrum of identified component.

Table 2. Evaluation of interaction resulting from the combination of the substances tested by determining the FIC_index using the checkerboard technique.

	Cryptococcus neoformans	T444
MIC (μg/mL)	E.C.E.	625
	H.F.	156
	E.O.	1250
	AMB	1.56
MIC in combination (μg/mL)	E.C.E.	39
	E.O.	157.2
	FIC index	0.187 (S)
	H.F.	20
	E.O.	78.72
	FIC index	0.19 (S)
	E.C.E.	78
	AMB	0.099
	FIC index	0.188 (S)
	H.F.	78
	AMB	0.396
	FIC index	0.75 (A)

E.C.E.: ethanol crude extract; H.F.: hexane fraction; E.O.: essential oil; AMB: amphotericin B; FIC index: fractional inhibitory concentration index; (S): synergistic; (A): additive.

Figure 4. Time-dependent kill-curve assay of 1250 μg/mL E.C.E. (A) and 312 μg/mL H.F. (B) against C. neoformans T₄₄₄. E.C.E.: ethanol crude extract; H.F.: hexane fraction; CFU: colony-forming unit.

Figure 5. Effect of 312 μg/mL of E.C.E., 78 μg/mL of H.F., 78 μg/mL + 19.5 μg/mL of E.O. and E.C.E., respectively (A1) and 39.36 μg/mL + 10 μg/mL of E.O. and H.F., respectively (A2) on the inhibition of ergosterol synthesis in C. neoformans. The results represent the mean ± standard error of two independent experiments in triplicate. A1: association 1; A2: association 2; E.C.E.: ethanol crude extract; H.F.: hexane fraction; E.O.: essential oil.

Figure 6. Effect of 312 μg/mL of E.C.E., 78 μg/mL of H.F., 78 μg/mL + 19.5 μg/mL of E.O. and E.C.E., respectively (A1) and 39.36 μg/mL + 10 μg/mL of E.O. and H.F., respectively (A2) on pigmentation inhibition. A1: association 1; A2: association 2; E.C.E.: ethanol crude extract; H.F.: hexane fraction; E.O.: essential oil.

Figure 7. Effect of 312 μg/mL of E.C.E., 78 μg/mL of H.F., 78 μg/mL + 19.5 μg/mL of E.O. and E.C.E., respectively (A1) and 39.36 μg/mL + 10 μg/mL of E.O. and H.F., respectively (A2) on the capsule size (μm). A1: association 1; A2: association 2; E.C.E.: ethanol crude extract; H.F.: hexane fraction; E.O.: essential oil.