*2.1. Chemical Composition*

The chromatographic analysis of the essential oil of *P. caldense* identified 25 phytochemical constituents, corresponding to 94.3% of the total composition. Sesquiterpenes are the main class observed, reaching a percentage of 89.7% of the sample. Monoterpenes were found in very low concentration (4.6%). There were no major constituents (>20%) and no constituent in trace concentration (<1%). All the identified phytochemicals are secondary metabolites, with caryophyllene oxide (13.9%), spathulenol (9.1%), δ-cadinene (7.6%), and bicyclogermacrene (6.7%) as constituents with the highest concentrations (Table 1).

## *2.2. Antifungal Activity*

#### 2.2.1. Cell Viability Curve and IC50

Regarding the antifungal activity of the essential oil of *P. caldense*, it was found that it has a low antifungal e ffect against strains of *C. albicans* because the IC50 values were high, 2256.24 and 1790.24 μg/mL for the CA INCQS 40006 and CA URM 4387, respectively (Table 2) (Figures 1 and 2). However, it was observed that for the *C. albicans* URM 4387 strains, the oil potentiated the e ffect of the drug used, fluconazole, since it had an IC50 of 7.73 μg/mL alone, and when associated with the essential oil, the value of evaluated parameter dropped to 2.7 μg/mL, i.e., a significant reduction. It is noteworthy that for *C. albicans* 40006 strains, no potentiating e ffect was found for fluconazole but an antagonistic effect, since there was an increase in the IC50 of the oil associated with fluconazole. Thus, based on the behavior of the curve through non-linear regression, the IC50 values of *P. caldense* oil were clinically irrelevant when evaluated alone.


**Table 1.** Chemical composition of the essential oil of leaves from *Piper caldense*.

AIlit—Arithmetic Retention Indices from literature; MF: Molecular Formula.

**Table 2.** IC50 of the essential oil of *Piper caldense* (EOPc) against *Candida albicans*.


INCQS: National Institute for Health Quality Control. URM: University Recife Mycology.

**Figure 1.** Anti-*Candida* potential of essential oil of *Piper caldense* (EOPc) against strains of *Candida albicans* 40006 INCQS.

**Figure 2.** Anti-*Candida* potential of essential oil of *Piper caldense* (EOPc) against strains of *Candida albicans* URM 4387.

2.2.2. Cell Viability Curve and Minimum Fungicidal Concentration (MFC)

In determining MFC it was considered those samples capable of inhibiting the growth of the fungal colonies. Thus, for the CA INCQS 40006 line, none of the products alone or in combination were able to totally inhibit colony growth, since MFC was ≥ 16,384 μg/mL (Table 3). However, there was an MFC for the oil against CA URM 4387, in which fluconazole alone and combined with *P. caldense* oil showed a much lower MFC, 16 μg/mL, so that the oil did not modulate the fungicidal effect of fluconazole for this lineage (Table 3).

**Table 3.** Minimum fungicidal concentration (MFC) of essential oil of *Piper caldense* (EOPc) and fluconazole associated and isolated against strains of *Candida albicans*.


INCQS: National Institute for Health Quality Control. URM: University Recife Mycology.

#### 2.2.3. Activity of the *Piper caldense* in the Control of Virulence of *Candida albicans*

In the evaluation of the activity of the oil in the morphology of the yeasts of *C. albicans*, the effect was caused by impoverishment of the culture medium, so that the yeasts project the hyphae and pseudohyphae in search of nutrients. For the growth control of CA INCQS 40006 (Figure 3, Slides 1–7), it is possible to observe the formation of several hyphae (S1), whereas in the treatments with fluconazole, there is a significant decrease in the hyphae projection (S2–4), resulting in a complete inhibition of hyphae at concentration of 4096 μg/mL (S2). The EOPc showed a similar result, being as effective as fluconazole.

For the CA URM 4387 strain, fluconazole at a concentration as low as 8 μg/mL could diminish the virulence (Figure 4, S2). The oil at higher concentrations, such as 2048 μg/mL (S3), was able to inhibit the hyphae projection; on the other hand, at concentration of 512 μg/mL (S5), there was a significant decrease in hyphae projections.

**Figure 3.** Effects of essential oil of *Piper caldense* on the dimorphism of *Candida albicans* INCQS 40006. Slide (S1): Growth control; S2–4: Effect of fluconazole at concentrations 4096 μg/mL (S2), 2048 μg/mL (S3), 1024 μg/mL (S4). S5–7: Effect of essential oil at concentrations of 4096 μg/mL (S5), 2048 μg/mL (S6), 1024 μg/mL (S7). Display 400× increased.

**Figure 4.** Effects of essential oil of *Piper caldense* on the dimorphism of *Candida albicans* URM 4387. Slide (S1): Growth control; S2: Effect of fluconazole at the concentration of 8 μg/mL. S3–5: Effect of the essential oil at 2048 μg/mL (S3), 1024 μg/mL (S4), 512 μg/mL (S5).
