In vitro anti-yeast activity, kinetics and mechanism of action of essential oils from two cameroonian medicinal plants

Background Treatment of Candida infections have become increasingly difficult due to antifungal drug resistance, which has drawn attention toward the search for innovative and effective drugs. This study aimed to assess the activity of essential oils (EOs) from Pentadiplandra brazzeana Baillon (PB) root and Drypetes gossweileri S. Moore (DG) stem bark against Candida albicans and Candida parapsilopsis strains, and determine their antifungal mechanism when tested alone or combined. Methods The anticandidal activity of the EOs using the checkerboard format was assessed using the broth micro-dilution technique. The checkerboard microtiter test was performed to evaluate the interaction of the EOs. The in vitro pharmacodynamics of the EOs alone or combined, using time-kill assays, following the chequerboard technique were evaluated. The anticandidal mode of action of these EOs, combined or not, was investigated using the sorbitol protection assay, and the ergosterol binding assay. Differences (p < 0.05) between the experimental and the control groups were evaluated using one way analysis of variance (ANOVA) followed by Tukey’s test for multiple comparisons. Results Essential oils (EOs) from Drypetes gossweileri (DG) stem bark showed activity with MIC value of 62.5 µg/mL against Candida albicans and Candida parapsilopsis, whereas EOs from Pentadiplandra brazzeana (PB) root exhibited MICs of 125 µg/mL and 250 µg/mL against the respective yeasts. The EOs were fungicidal with synergism on C. parapsilopsis and additivity on C. albicans, with 2 to 64-fold drop in MIC values. The MIC combination of 31.25/7.81 µg/mL and 1.95/31.25 µg/mL (DG/PB EOs) required 20 and 18 h of exposure, respectively to effectively kill 99.9% of the inoculum. This fungicidal effect was accompanied by alteration of the cell walls and membranes of yeasts. Conclusion The potency of the EOs combinations indicates further directions in their investigation as potential anticandidal agents.

they can cause severe systemic infections, thus contributing significantly to morbidity and mortality. Risk factors for patients include infection by the human immunodeficiency virus (HIV), anticancer therapy, organ transplantation, abdominal surgery, catheters, diabetes, and the prolonged use of broad-spectrum antibiotics [1][2][3][4]. Although C. albicans (CA) is the prevalent species in candidemia, other species, such as C. krusei, C. glabrata, C. tropicalis, and C. parapsilosis (CP), have been observed [5].
The resistance to available antifungals, apart from their severe side effects and lesser efficiency highlight the great need for innovative and effective medicines to treat yeasts infections. An important aspect comprises the search for new compounds that have anti-yeast properties and synergism or additive effect when combined. The advantage of combinational therapy is the greater effect (synergy or additive) of the drugs compared to an individual drug. Similarly, combination of drugs/compounds may result in antagonistic effect. Hence, medicinal plants are used as primary health care in many parts of the world for numerous diseases including candidiasis. Therefore, they can serve as the natural source for the discovery and development of new bioactive medicines. Indeed, the essential oils (EOs) of Drypetes gossweileri S. Moore (Euphorbiaceae) stem barks and Pentadiplandra brazzeana Baillon (Capparidaceae) roots have been shown to have antioxidant, anti-inflammatory and antimicrobial properties [6][7][8][9]. However, to our knowledge, there are no available reports on the underlying kinetics of cell death and mechanism of anticandidal action of D. gossweileri and P. brazzeana EOs against C. albicans and C. parapsilosis. Thus, this study is aimed at evaluating the in vitro growth inhibition of D. gossweileri and P. brazzeana EOs both alone and in combination, against C. albicans ATCC P37037 and C. parapsilopsis ATCC 22,019 strains, and their effects in their growth kinetics, cell wall formation, and ergosterol interactions.

Plant materials and extraction procedure
This study is the continuation of a previous one conducted by Ndoyé Foé et al. in 2016 [9]. The origin of D. gossweileri and P. brazzeana was indicated and the extraction method was described in that study.In August 2013, Drypetes gossweileri stem barks and Pentadiplandra brazzeana roots were collected at Awae and Ngomedzap (Center Region of Cameroon), respectively by the sellers. The botanical identification and authentication were carried by Mr. Nana (plant taxonomist) of the National Herbarium of Cameroon (Yaoundé), where voucher specimens were kept: 25,749/SRF/Cam and 42,918/SRF/ Cam for D. gossweileri and P. brazzeana respectively. The EOs were obtained by hydrodistillation based as in the previous study of Ndoyé Foé et al., 2016, [9]. Briefly, the EOs were extracted by hydrodistillation using a Clevenger-type apparatus for 5 h, dried over anhydrous sodium sulfate and then stored at 4 °C until bioassay. The extraction yields were calculated as the ratio of the mass of EO to the mass of the starting plant material and expressed as a percentage (w/w).
The EOs were analyzed by gas chromatography and gas chromatography coupled to mass spectrometry as described by Agnaniet et al. [10].

Culture media and microplates
Sabouraud dextrose broth (SDB) and Sabouraud chloramphenicol agar (SDA) were purchased from Titan Biotech Ltd, India, and Fortress Diagnostics Ltd, United Kingdom, respectively. They were prepared and used according to the manufacturers' instructions. Polystyrene microplates containing 96 wells were purchased from Becton Dickinson and Company, USA.

Yeast reference strains
The strains used for the study were from the American Type Culture Collection (ATCC), especially Candida albicans ATCC P37037 and Candida parapsilopsis ATCC 22,019 reference strains provided by BEI Resources NIAID, NIH (Manassas, VA, USA). These yeasts were maintained at room temperature and cultured at 35 °C for 48 h on Sabouraud Dextrose Agar (Oxoid) slants prior to use.

Anti-yeast activity assay
The Minimum inhibitory concentrations (MIC) were determined by broth microdilution method using the M27-A3 protocol of the Clinical and Laboratory Standards Institute [11], with minor modification: the EOs have not been dissolved in a solvent. Initially, 100 µL of Sabouraud Dextrose Broth (SDB) (Titan Biotech Ltd., India) supplemented with chloramphenicol was distributed in the 96-wells microtiter plates. Then, 100 µL of EO (2000 µg/mL) was transferred to the first well and serially diluted by transferring an aliquot of 100 µL from the first well to the next with EO concentrations ranging from 1000 µg/mL to 7.81 µg/mL, with a geometric connection of ratio 1/2: to pass from a strong concentration The type of antifungal effect of EOs was deduced from the calculated MFC/MIC ratio, and identified as fungicidal when MFC/MIC ≤ 4, or fungistatic when MFC/ MIC > 4 [12].
Based on the anti-yeast activities (MIC, MFC) of D. gossweileri and P. brazzeana EOs alone and combined, nature of interaction between these EOs was determined by the checkerboard microdilution technique for derivation of the Fractional Inhibitory Concentration Index (FICI) as described below.

Determination of the interaction of Drypetes gossweileri essential oil with Pentadiplandra brazzeana essential oil using checkerboard method
A checkerboard microtiter test based on CLSI guideline [11] was performed to evaluate the type of interaction between D. gossweileri EO and P. brazzeana EO against C. albicans ATCC P37037 and C. parapsilopsis ATCC 22,019 strains [13]. The series of two-fold dilutions of each oil were made in SDB in the microtiter well. Mixed concentrations in wells ranged from 1/256 × MIC to 2 × MIC for the two EOs. Furthermore, 50 µL of dilution of D. gossweileri EO was added to the 96 well microtiter plates in the vertical direction, while 50 µL of dilution of P. brazzeana EO was added in the horizontal direction, so that various combinations of EOs could be achieved. Also, 100 µL of yeast inoculum (2.50 × 10 3 CFU/mL) were added to each well and plates were incubated at 35 °C for 48 h in a laboratory incubator. Each test was performed in triplicate. The nature of interaction was defined quantitatively by means of Fractional Inhibitory Concentrations (FIC) that were calculated as follow: the MIC of the combination of D. gossweileri EO with P. brazzeana EO divided by the MIC of EO alone. A FIC index (FICI) was obtained by adding both FICs. The combination result was interpreted as follows as described by Van Vuuren and Viljoen [14]: FICI ≤ 0.50, synergistic; > 0.50 to ≤ 1, additive; > 1.00 to ≤ 4.00, indifferent; and > 4.00, antagonistic.
Based on the anticandidal potential of combination of D. gossweileri and P. brazzeana EOs (synergism and additivity), an insight into the mechanism of anticandidal action was assessed on growth profile, cell wall and cell membrane of C. albicans and C. parapsilopsis strains, in the presence of EOs alone and in combination.

Time-kill kinetic assay
The in vitro pharmacodynamics of D. gossweileri and P. brazzeana EOs on C. albicans ATCC P37037 and C. parapsilopsis ATCC 22,019 strains was performed as described by Klepser et al. [15] with some modifications. A volume of 500 µL of an initial inoculum of 1 × 10 5 CFU/ mL prepared in 0.90% NaCl was seeded onto flat-bottomed 24-well microtitration plates, with 400 μL of SDB and 100 μL of each EO alone. For combination of EOs, same volume and concentration of inoculum was seeded, with 300 μL of SDB and 100 μL of each sample of EO of the combination. The plate was incubated at 35 °C under orbital shaking, 32 × g (IKA-Vibrax-VXR, Radnor, PA, USA) at various time periods (0, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20 and 24 h). At predetermined time points, 10 μL of the mixture was pipetted and diluted in 40 μL normal saline onto flat-bottomed 96-well microtitration plates. The dilutions were homogenized for the determination of viable colony counts using a Malassez counting cells (Thermo Fisher Scientific, Darmstadt, Germany) and expressed in log 10 CFU/mL. The experiment was performed in duplicate. Time-kill curves were constructed by plotting mean of colony count (log 10 CFU/ mL) as a function of time (hours) for each time point. The effect of the EOs was considered fungicidal when there was a decrease greater than or equal to 3 log 10 CFU/mL of the initial inoculum, resulting in reduction of 99.9% or more CFU/mL in 24 h compared with the initial inoculum. Fungistatic activity was considered as reduction in growth lower than 99.9% or < 3 log 10 in CFU/mL from the initial inoculum [14]. The criteria used to interpret the interactions between EOs: Synergism was obtained when the fungicidal effect led to ≥ 2 log 10 decrease in cells/mL for the combination compared to the most active EO; additivity was defined as < 2log 10 decrease in cells/mL for the combination compared to the most active EO; indifference as < 2 log 10 increase in cells/mL for the combination compared to the least active EO; and antagonism as ≥ 2log 10 increase in cells/mL for the combination compared to the least active EO [15]. The mode of action of EOs alone and in combination was also performed to determine whether the anticandidal activity found is the result of a direct interaction with the cell wall structure of Candida strains (sorbitol protection assay) or the ion permeability of their membrane (ergosterol effect assay).

Sorbitol protection assay
The MICs of Drypetes gossweileri and Pentadiplandra brazzeana EOs in the presence sorbitol (an osmotic protector) against Candida strains were determined using the microdilution technique [11] in triplicate.
Initially, 100 µL of SDB was introduced into each well of the microplate. Subsequently, 100 µL of EOs solutions were transferred to the first well and serially diluted twofold dilution. D. gossweileri and P. brazzeana EOs concentrations ranged from 1000 µg/mL to 15.63 µg/mL and from 2000 µg/mL to 31.25 µg/mL respectively. For EOs in combination, concentrations ranged from 500 µg/mL to 7.81 µg/mL were obtained. Then, 50 µL of yeast inoculum (2.50 × 10 3 CFU/mL) prepared in SDB and 50 µL of sorbitol (Sigma-Aldrich, USA) were transferred to the wells for a final concentration of 0.80 M sorbitol in each well [16,17]. The negative control included 100 µL of SDB and 50 µL of the inoculum with 50 µL of sorbitol (0.80 M) in each cavity. Sterility control was also performed: 100 µL SDB with 50 µL of sorbitol (0.80 M) was placed in a plate column without fungal suspension. The plates were incubated at 35 °C in a laboratory incubator, and the results were read after 48 h [16,17]. MIC was determined as the lowest concentration of test EOs inhibiting the visible growth. Each experiment was repeated three times and mean values were calculated for MICs.

Ergosterol effect assay
First, 100 µL of SDB was added to each well of the microplate. Then, 100 µL of EOs solutions were transferred to the first well and serially diluted by transferring a 100 µL aliquot from the most concentrated well to the next well with D. gossweileri and P. brazzeana EOs concentrations ranged from 1000 µg/mL to 15.63 µg/mL and 2000 µg/mL to 31.25 µg/mLrespectively Concerning EOs in combination, EOs concentrations ranged from 500 µg/ mL to 7.81 µg/mL. A volume of 50 µL of yeast inoculum (2.50 × 10 3 CFU/mL) prepared in SDB and 50 µL of ergosterol (Sigma-Aldrich, China) were transferred to the wells for a final concentration of 250 µg/mL ergosterol in each well. The plates were incubated at 35 °C in a laboratory incubator, and the results were read after 48 h [17,18]. Yeast growth and sterility were also controlled. Fluconazole was tested as a positive control. MIC was determined as the lowest concentration of test EOs inhibiting the visible growth. Each experiment was repeated three times and mean values were calculated for MICs.

Statistical analysis
The results were the means of concentrations ± standard deviations from triplicate values obtained from three independent experiments.The data were statistically analysed using the software SPSS 17.0 for Windows and analysis of variance (ANOVA) coupled with Tukey test. A p < 0.05 was considered as statistically significant.  Table 1.

Anticandidal activity of essential oils alone and in combination against
The results of the study by broth microdilution showed that the MICs of D. gossweileri EO were obtained at 62.50 µg/mL for C. albicans and 125 µg/mL for C. parapsilopsis. The MICs of P. brazzeana EO were 62.50 µg/mL for C. albicans and 250 µg/mL for C. parapsilopsis. The MFC values of each EO, except for fluconazole were the same with MICs against Candida strains. The ratio MFC/ MIC values were 1 and 2 for both essential oils and fluconazole, respectively. Having established the individual MIC and MFC values, the MIC and Fractional inhibitory concentrations index (FICI) values of D. gossweileri stem barks EO and P. brazzeana roots EO in combination were determined using checkerboard assays against the same yeast strains, in the aim of the detection of synergism, additivity or antagonism between these EOs. The results of the FICI are shown in Table 2.
Based on the anticandidal potential of combination of D. gossweileri EO with P. brazzeana EO (synergistic and additive), an insight into the growth profile of C. albicans ATCC P37037 and C. parapsilopsis ATCC 22,019 strains, through the time-kill studies were performed over a period of 24 h.

Time-kill kinetics of essential oils alone and in combination for Candida strains
The results of the time-kill curves for C. parapsilopsis ATCC 22,019 and C. albicans ATCC P37037 being exposed to MIC values (EOs combination) and sub-MIC values (EO alone) of D. gossweileri and P. brazzeana are shown in Figs. 1 and 2.
The growth profile of Candida strains in the presence of D. gossweileri and P. brazzeana EOs was assessed to further corroborate the in vitro anticandidal activity results observed above.
For the combination of D. gossweileri EO with P. brazzeana EO against C. albicans strains, a significant reduction in the colony count was recorded between 18 and 24 h, whereas for C. parapsilopsis strains, it was between 16 and 24 h. The analysis of the log 10  The combination of EOs studied in this paper demonstrated fungicidal potential within 24 h, emphasizing their potential as source of anti-yeast agents with mode of action to be investigated.

Mode of anticandidal action of essential oils alone and in combination
The elucidation of the antifungal mode of action of Drypetes gossweileri stem barks and Pentadiplandra brazzeana roots EOs, alone and in combination were investigated through sorbitol protection assay and ergosterol effect assay, whose results are presented in Tables 3  and 4, respectively.  23:115 In this paper, it was found that the P. brazzeana and D. gossweileri EOs MICs against C. albicans ATCC P37037 and C. parapsilopsis ATCC 22,019 strains increased by 4 to 16-fold in the presence of sorbitol. When the yeasts were treated with EOs combination in a medium supplemented with sorbitol, MIC values did shift to higher values. As can be seen, the results indicated that the mechanism of action of EOs tested alone or combined act by inhibiting fungal cell wall synthesis.
Based on the results of the present study, the MIC values of D. gossweileri EO against C. albicans and C. parapsilopsis increased eight (62.50 μg/mL to 500 μg/mL) and four times (125 μg/mL to 500 μg/mL), respectively, in the presence of exogenous ergosterol ( Table 3). The same was also observed for P. brazzeana EO against C. albicans (62.50 μg/mL to 500 μg/mL) and C. parapsilopsis (250 μg/mL to 1000 μg/mL). In addition, the MIC values of combination of P. brazzeana and D. gossweileri

Discussion
Anticandidal activity of essential oils alone and in combination against Candida strains C. albicans ATCC P37037 strains were more sensitive than C. parapsilopsis ATCC 22,019 to EOs and fluconazole. According to criteria (MFC/MIC < 4) proposed by Carbonnelle et al. [11], D. gossweileri, P. brazzeana EOs and fluconazole showed fungicidal activities against the two ATCC yeasts. To be fungicidal rather than fungistatic is an important finding since antifungal agents that kill fungi (cidal) have demonstrated to be, in most cases, clinically more useful than those that merely inhibit (static) fungal growth [18]. In literature, P. brazzeana roots EOs have been found to be active against yeast and filamentous fungal species [8; 9]. Indeed, Nyegue et al. [7] found that P. brazzeana EO was two-fold more active against C. albicans than this found in the present study, with MIC and MFC values of 31.25 µg/mL. This could be due to the difference in chemical composition within these essential oils [9]). The Fractional Inhibitory Concentrations Index (FICI) calculated from the results of the checkerboard assay revealed the following: the investigation of antifungal activity of D. gossweileri EO in combination with P. brazzeana EO against C. albicans and C. parapsilopsis caused a significant decrease in the MIC, compared to their individual MIC values. The MIC of D. gossweileri EO alone against C. parapsilopsis was lowered from 125 µg/mL to 1.95 µg/mL, so a 64-fold reduction, in the presence of P. brazzeana EO. The MIC of P. brazzeana EO alone also decreased from 250 to 31.25 mg/mL, so an eightfold reduction. Thus, for C. parapsilopsis, this combination was classified as synergetic, with a FIC index of 0.14. For the strains of C. albicans, the MIC value of D. gossweileri EO alone was 62.50 µg/mL. When associated with P. brazzeana EO, a twofold reduction in the MIC value (62.50 µg /mL to 31.25 µg/mL) of the same EO was observed. An eightfold reduction in the MIC value (62.50 µg /mL to 7.81 µg/mL) of P. brazzeana EO was also observed when associated with D. gossweileri EO. Thus, this association was classified as additive, with an FIC index of 0.63. These observations highlight antifungal potential of combination therapy using D. gossweileri EO with P. brazzeana EO against C. albicans and C. parapsilopsis strains.

Time-kill kinetics of essential oils alone and in combination for Candida strains
The growth profile of Candida strains in the presence of Drypetes gossweileri and Pentadiplandra brazzeana EOs was assessed to further corroborate the in vitro anticandidal activity results observed.
The time of death curves showed that both EOs tested alone at sub-MIC were fungistatic against the two Candida strains. On the contrary, the combination of D. gossweileri and P. brazzeana EOs at MIC exhibited fungicidal activity against the tested yeasts. The combination of D. gossweileri and P. brazzeana EOs recorded significant reduction in the CFU/mL over the time when compared to the effect of individual EO. Indeed, to promote greater efficiency of D. gossweileri and P. brazzeana EOs, when used at lower concentrations, the association of these EOs could be proposed.
Besides, synergistic and additive effects observed in checkerboard microdilution were confirmed by time-killing assay. The time kill characterization is very important because it has valuable therapeutic implications, such as adjusting the dose for a more effective treatment [18] or shorten the duration of therapy and avoid the emergence of resistance to available antifungals [19,20]. To the best of our knowledge, the kinetics of D. gossweileri and P. brazzeana EOs on C. albicans and C. parapsilosis cells death remain unknown. Also, it is noteworthy that this is the first study on the aspect of optimizing anti-Candida activity by coupling D. gossweileri and P. brazzeana EOs in combination. These anti-Candida activities of D. gossweileri and P. brazzeana EOs and their optimization in combination is probably due to the combined effect of all components of both essential oils (as indicated in a previous report by Ndoyé et al. [9]): terpenes, sulfur-and nitrogen-containing compounds, acting synergistically and additively against the targeted Candida strains.

Mode of anticandidal action of essential oils alone and in combination
The sorbitol assay consisted of determining the MIC in the presence and absence of 0.8 M sorbitol, an osmotic protector used to stabilize fungi protoplasts. Cells protected with sorbitol can grow in the presence of fungal cell wall inhibitors, whereas growth would be inhibited in the absence of sorbitol. This effect is detected by increase in the MIC value as observed in medium with sorbitol as compared to the MIC value in medium without sorbitol (standard medium) [16,21].
The ergosterol effect assay consisted of determining whether D. gossweileri and P. brazzeana EOs bind to the membrane sterols of tested yeasts. If the activity of EOs was caused by binding to ergosterol, the exogenous ergosterol would prevent the binding to ergosterol in the membranes of yeasts. Consequently, MIC increase for EOs (in the presence of exogenous ergosterol in relation to the control assay) would occur because only increased EOs concentration in the growth medium might assure interaction with ergosterol in the membranes of yeasts [22,23]. Thus, the effect of exogenous ergosterol on EOs and fluconazole MIC was determined. The findings of this study suggest that Pentadiplandra brazzeana and Drypetes gossweileri EOs alone act by altering the structure of the cell wall and cell membrane of yeast. When combined, it could be expected that P. brazzeana and D. gossweileri EOs could enhance their permeability to fungi by altering fungal cell wall and membrane integrity that may intensify the fungal killing. However, the cascades of multiple secondary effects such as reactive oxygen species (ROS) accumulation, mitochondrial membrane potential dissipation, and DNA condensation and fragmentation (remain to be established in studies that are out of scope for this paper) as a result of membrane disruption action cannot be overlooked as a cause of death.
The elucidation of the action mechanisms of P. brazzeana and D. gossweileri EOs is another strategy which require further detailed investigations for limiting the emergence of resistance to the currently available antifungal agents, as well as for developing rational, safer and more potent alternative therapies against Candida infections which frequently require combinations of drugs or the use of new drugs when the first-choice agent is not effective. It is noteworthy that from our literature review, there is no scientific report on investigation of possible action of P. brazzeana and D. gossweileri EOs on cell wall and cell membrane, as primary action mechanism. Drugs that act on the cell wall cause lysis of fungal cells in the absence of an osmotic stabilizer (sorbitol), but their growth can continue in the presence of sorbitol [22]. Inhibition of growth is detected by increase in the MIC values as observed in medium with sorbitol as compared to the MIC value in medium (standard medium) without sorbitol [16]. According to Frost et al. [16], this assay is generic in nature and is of use in the search of substances that directly inhibit the synthesis of cell wall constituents such as glycans, mannans or chitin as found in this study of the effect of P. brazzeana and D. gossweileri EOs on cell wall.
The ability of P. brazzeana and D. gossweileri EOs to form complexes with ergosterol was evaluated from the perspective of investigating their action on the yeasts cell membrane. It was found that the EOs bind to exogenous ergosterol, avoiding it to ergosterol membrane binding. The results of this study suggest that P. brazzeana and D. gossweileri EOs appear to bind to the ergosterol in the membrane, promoting increased membrane permeability, or inactivated plasma membrane-ATPase (an important fungal pump which transfers substances in fungal plasma membrane), causing the depletion of components essential to fungal cell survival and ultimately cell death. It seems that antifungal mechanism of action of these EOs is similar to fluconazole. Fluconazole is a triazole which mainly act through the inhibition of lanosterol-14-alpha-demethylase, a key enzyme involved in the biosynthesis of ergosterol, an important component of the fungal cell membrane [24].
Damage to cell wall and cell membrane by P. brazzeana and D. gossweileri EOs is probably due to the combined effect of all components of both oils: terpenes, sulfurand nitrogen-containing compounds, acting synergistically and additively against tested yeasts. Indeed, it was reported that the antifungal mechanisms of action for the nitrogen containing compounds are mainly a result of cell membrane disruption via inhibition of ergosterol biosynthesis, or complexing with ergosterol [25,26]. Also, Souza et al. [26] reported that the sulfur compounds interfere with membrane integrity or associated enzyme proteins, stopping their production or activity.
Similarly, the simultaneous actions of EOs components on different targets enhance their bioactivity and might also reduce the advent of resistance by the fungi. Generally, the chemical configuration of terpenes gives them hydrophobic properties and allows them to deposit on the lipophilic structures of microorganisms such as the plasma membrane; this deposition leads to increased permeability with a consequent loss of the electrolytes essential to cell survival [27]. In line with this, Nguefack et al. [28] showed that the carbohydrates terpenes, although having a lesser activity, can allow rupture of the cell membrane, thus promoting the intracellular transport of antimicrobial compound such as sulfur derivatives present in both EOs. Indeed, isothiocyanate derivatives are known for their strong antimicrobial activity. Their isothiocyanate group (R-N = C = S), has a strongly electrophilic carbon that can easily react with a nucleophilic center, and cleaving the disulfide bonds of the proteins and degrading amino acids via oxidation reactions [29,30] with production of reactive oxygen species (ROS). Therefore, free radicals oxidize and damage lipids, proteins and DNA. Moreover, some phenolic components of essential oils are oxidized by contact with ROS producing very reactive phenoxyl radicals which add to the ROS released by mitochondria. These types of radical reactions are dependent on and enhanced by the presence of cell transition metal ions [31,32]. The anticandidal activity of combined EOs can promote greater efficiency of D. gossweileri and P. brazzeana EOs when used at lower concentrations, allowing synergistic and additive

Conclusion
This study assessed the antifungal activity, kinetics of cell death and mode of action of essential oils (EOs) from Pentadiplandra brazzeana roots and Drypetes gossweileri stem barks, alone and in combination, against C. albicans ATCC P37037 and C. parapsilopsis ATCC 22,019 strains. The findings showed that EOs from P. brazzeana roots and D. gossweileri stem barks were fungicidal, alone and combined, with synergism on C. parapsilopsis and additivity on C. albicans, with 2 to 64-fold drop in MIC values after 18 h and 20 h, respectively. Furthermore, fungicidal effect of EOs alone and in combination was accompanied by the disturbance of cell wall and ergosterol biosynthesis in C. albicans cells. The potency of the EOs combination might be mainly beneficial to treat candidiasis. Meanwhile, further investigations as potential anticandidal agents will be necessary to elaborate more knowledge about other Candida cell targets with respect to EOs from P. brazzeana roots and D. gossweileri stem barks.