QUANTITATIVE ANALYSIS OF BERGAPTEN 253 0.20- Pure 5-MOP Bergamot off Y.S.L. Todet water Brut aftershave 0.18. 0.16. 0.14. • 0.12. _ • 0.10, • 0.08, 0.06, 0.02. 0 i i i I i i 225 250 275 300 325 350 400 WAVELENGTH nm Figure 2. The ultraviolet absorption spectrum of the bergapten TLC fraction of bergamot oil, Yves Saint Laurent toilet water and Brut aftershave lotion compared with that of pure 5-MOP. The absorption spectra of bergamot oil and the toilet water are similar and resemble that of pure 5-MOP, whereas the absorption spectrum of Brut aftershave is different. The apparent lack of absorption characteristics below 240 nm is due to absorption of these wavelengths by chloroform itself. It should be noted that the absorption scale is from 0 to 0.20 rather than the usual 0 to 1.00 bergamot oil but did not significantly alter the absorption spectrum of the 5-MOP frac- tion. Dose-Response Curves.' Bergamot Oil. Using 2/xl aliquots and TLC, the minimal effective concentration of bergamot oil on the yeast was 0.25% (0.005/xl). Normal growth was obtained in all control plates. The dose-response curve showed a sharp rise with the smaller concentrations but tended to flatten out with higher concentrations (Figure 3). The results of 20 and 40% concentrations were not significantly different at 5% level when the Student's "t" was applied for comparing adjacent means. A significant dif- ference, however, was obtained between the other adjacent means, the level of significance increasing when the means on the steeper part of the curve were com- pared. For example, the results of 10 and 20% concentrations were different at 5% level: 5 and 10% at 1% 2.5 and 5% at 0.1% level. Dose-Response Curves.' 5-MOP. The dose-response curve of pure 5-MOP showed a pat- tern similar to that of bergamot oil (Figure 4). The concentrations of pure 5-MOP used for deriving the dose-response curve were 0.00025, 0.0005, 0.001, 0.0025, 0.005, 0.01, 0.025 and 0.1%. The concentrations of bergamot oil containing an equivalent amount of 5-MOP would therefore be 0.09, 0.19, 0.37, 0.93, 1.85, 3.71, 9.26, 18.52 and 37.04% (v/v) respectively. When the diameters of the killing zones produced by the various dilutions of pure 5-MOP shown in Figure 4 were plotted against the calcu- lated concentrations of bergamot oil, a curve similar to that of bergamot oil was oh-

254 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS 40 i• 20 z o N 10 lb •o •o 4'0 CONCENTRATION OF BERGAMOT OIL ml/100 ml Figure 3. The effect of increasing concentrations of bergamot oil (Coty) on the diameter of the killing zone of the yeast, presented as the mean + S.E.M. A sharp increase in the zone diameter is noted with the smaller concentrations, and a levelling-off of the response with the higher concentrations. The curve with the inter- rupted lines shows the relationship of the diameters of killing zones produced by known concentrations of pure 5-MOP (shown in Figure 4) to the concentrations of bergamot oil calcalulated from these concentra- tions, assuming the 5-MOP content of bergamot oil to be 0.27 g/100 ml tained (Figure 3). The killing zones obtained with the calculated concentrations were, however, slightly larger than those produced by known concentrations of bergamot oil. Dose-Response Curves.' 8-MOP. The minimal effective concentration of 8-MOP to produce killing of the yeast was 0.0025%, concentrations of 0.00025, 0.0005 and 0.001% being ineffective. Higher concentrations were much less effective than similar concentrations of 5-MOP (Figure 4). Furthermore, the dose-response curve did not show a steep rise with the smaller concentrations as it did with 5-MOP. The results of the turbidimetric test are shown in Table III. Marked inhibition of yeast growth was Table III The Optical Density at 600 nm of Yeast Growth in Sabouraud's Liquid Medium 24 hr After Incubation with 0.1 ml of 0.005% Ethanolic Solutions of 5-MOP and 8-MOP. The Values in the Irradiated Petri Dishes Differ Significantly With a p Value of 0.001 Using Student's "t" Test Irradiated Control 5-MOP 8-MOP 5-MOP 8-MOP OPTICAL Mean 0.114 0.282 0.830 0.860 DEN S ITY S.E.M. 0.004 0.004 0.011 0.006