IN VITRO TESTS

The experimental data shown below, confirm the fungicidal (lethal) effect of canosic acid together with propolis, over the fungistatic effect. In this assay, the existence of the synergistic actuation between both compounds must be emphasized, inducing a very high degree of mortality, almost complete after an hour of treatment.

Assays were carried out in liquid YPD medium (1% yeast, 2% peptone and 2% glucose) at 37ºC. The C. albicans strains used was SC5314 (wild type) and 015 (oral clinical isolated), the C. glabrata strain used was YA-1 (clinical isolated) and the C. neoformans strain used was H99 (wild type). The general procedure consisted of applying known concentrations of carnosic acid and propolis at different times on exponential cultures of C.albicans grown in YPD. The percentage of cell viability was determined by counting the number of viable cells in the solid YPD medium after incubation at 37ºC for 24-48 hours. The results in liquid medium showed good correlation with the colonial growth recorded by “drops” in solid YPD (Figures B).

The tested aliquots came from a single initial exponential culture and, therefore, the physiological state of the cells was identical.

  • C. albicans (SC5314)

Figure 1: (A) Measurement of the kinetic growth in the parental strain SC5314 to carnosic acid (CA) 50 µg/ml, propolis (P3) 200 µg/ml and the mixture of both compounds at different exposure times. (B) Assay of colony formation on plate. The results confirm the synergism and its antifungal activity.

  • C. glabrata

Figure 2: Viability assay in a liquid medium of the strain C. glabrata in response to treatment with carnosic acid (CA) 50 µg/ml, propolis (P3) 200 µg/ml and a synergistic combination of both: S (50 µg/ml of CA and 200 µg/ml of P3) and S’ (100 µg/ml of CA and 400 µg/ml) at different times. (B) Assay of colony formation on plate.

  • C. auris

It is a pathogen responsible for a global health emergency. As it can be seen, C. auris is also susceptible to VG-01:

  • C. neoformans

Figure 4: (A) shows cell viability in a liquid medium of the strain Cryptococcus neoformans in response to treatment with carnosic acid (CA) 50 μg/ml, propolis (PP) 200 μg/ml and a synergistic combination of both (S), taking as reference a control sample with no treatment. (B) Assay of colony formation on plate. The results confirm those obtained in liquid medium.

  • C. albicans (015)

Figure 5: A comparative antibiogram of the antifungal activity found in internationally renowned commercial toothpastes (D, S and C) and the toothpaste prepared with the synergic combination of rosemary and propolis (M) as active ingredients. We can easily observe how the inhibition zone diameter is much bigger with our toothpaste in all cases. The effectiveness of our preparation is noticeable even with different Candida species. (A) S. mutans, (B) E. coli, (C) C. parapsilosis, (D) C. albicans (SC5314), (E) C. albicans (025: oral clinical isolated) and (F) C. albicans (015: oral clinical isolated).

Example of form of application in oral health: a toothpaste

Considering the many possibilities and needs of application of this synergistic combination, an example of use is studied below. For oral health, toothpaste represents a mechanism with simultaneous multifactorial action against degenerations and losses of function, covering candidiasis, avoiding dental caries and gingivitis, as well as the standardization of the saliva and oral flora.

  • C. albicans (015)

Figure 6: Stability test of the antifungal activity of the toothpaste formulated with the active principles (VG-01) during a year. It was carried out in a liquid medium of the strain 015 (C. albicans) following the protocol described.