Chemical Composition, Antifungal and Anti-Biofilm Activities of Volatile Fractions of Convolvulus althaeoides L. Roots from Tunisia

The antifungal drugs currently available and mostly used for the treatment of candidiasis exhibit the phenomena of toxicity and increasing resistance. In this context, plant materials might represent promising sources of antifungal agents. The aim of this study is to evaluate for the first time the chemical content of the volatile fractions (VFs) along with the antifungal and anti-biofilm of Convolvulus althaeoides L. roots. The chemical composition was determined by gas chromatography coupled to a flame ionization detector and mass spectrometry. In total, 73 and 86 chemical compounds were detected in the n-hexane (VF1) and chloroform (VF2) fractions, respectively. Analysis revealed the presence of four main compounds: n-hexadecenoic acid (29.77%), 4-vinyl guaiacol (12.2%), bis(2-ethylhexyl)-adipate (9.69%) and eicosane (3.98%) in the VF extracted by hexane (VF1). n-hexadecenoic acid (34.04%), benzyl alcohol (7.86%) and linoleic acid (7.30%) were the main compounds found in the VF extracted with chloroform (VF2). The antifungal minimum inhibitory concentrations (MICs) of the obtained fractions against Candida albicans, Candida glabrata and Candida tropicalis were determined by the micro-dilution technique and values against Candida spp. ranged from 0.87 to 3.5 mg/mL. The biofilm inhibitory concentrations (IBF) and sustained inhibition (BSI) assays on C. albicans, C. glabrata and C. tropicalis were also investigated. The VFs inhibited biofilm formation up to 0.87 mg/mL for C. albicans, up to 1.75 mg/mL against C. glabrata and up to 0.87 mg/mL against C. tropicalis. The obtained results highlighted the synergistic mechanism of the detected molecules in the prevention of candidosic biofilm formation.

n-Hexadecanoic (palmitic) acid (C 16 H 32 O 2 ) was the most abundant compound, which has several biological activities: antimicrobial, antieczematous, antiseborrheic, sclerosing, antihypoxic, antimutagenic, fibrinolytic, anti-inflammatory, antisecretory, cytoprotective and anesthetic [20]. In fact, n-hexadecanoic acid is one of the primary metabolites produced during microbial degradation because of the oxidized end of the molecule is used as the initial site for the β-oxidation process, whereas microbial oxidation of saturated hydrocarbons (n-hexadecane) cannot be initiated as easily as for n-hexadecanoic acid [21]. 4-vinyl guaiacol is a vinyl phenol produced by the decarboxylation of ferulic acids. Its phenolic structure allows it to play the role of a natural antioxidant. Indeed, the position of the methoxy group on the electron-donating phenolic ring is also a factor that increases the stability of the phenoxy radical and thus its antioxidant effectiveness [22].
The chemical composition of the VFs of the roots of C. althaeoides L. are herein reported for the first time. Only one study on the essential oil of the flower of C. althaeoides L. grown in Tunisia has been carried out [11], which reveled 24 compounds, representing 95.50% of the total compositions. The oil was characterized by a high proportion of sesquiterpene hydrocarbons (36.30%), followed by oxygenated sesquiterpenes (34.70%) and oxygenated monoterpenes (24.50%). The main compounds are germacrene D (12.50%), T-cadinol (11.80%) and verbenone (6.90%).

Antifungal Activity
The outputs of the microdilution test of VF1 and VF2 from C. althaeoides L. roots against Candida spp. at different concentrations, read with Multiskan™ FC Microplate Photometer (Thermo Scientific, Waltham, MA, USA), are represented as heatmaps ( Figure 3). These illustrations demonstrated a good level of reproducibility in our repeats and a difference of inhibition for both VF1 and VF2 fractions against each fungal strain. C. albicans and C. tropicalis demonstrated the same susceptibility level to VF1 and VF2 fractions with MICs at 0.87 and 1.75 mg/mL, respectively. The MICs for the Amphotericin B were 0.125 × 10 −3 mg/mL, 0. 062 × 10 −3 mg/mL and 0.250 × 10 −3 mg/mL for C. albicans, C. glabrata and C. tropicalis, respectively (data not shown). The MFCs against these strains were reported at 1.75 mg/mL for both VF1 and VF2 fractions of C. albicans and C. tropicalis. However, C. glabrata seems to be less susceptible to VF1 and VF2 fractions with MICs/MFCs at 3.5 mg/mL and 7 mg/mL, respectively. In agreement with the photochemical results of the two VFs, the diversity and the richness of the two VFs in volatile compounds can explain their antifungal potential. Indeed, the main active compounds of the VFs of C. althaeoides L. roots were n-hexadecenoic acid [23], bis (2-ethylhexyl) adipate [24], benzyl alcohol [25] and linoleic acid [26] which are known as antifungal agents. In fact, it was reported previously that the n-hexadecanoic acid or palmitic acid, a saturated fatty acid, abundant compound in VF1 and VF2, showed antifungal activities against mycelial growth and spore production reaching 37.20% and 71.70% by 2 mM concentration [27], respectively. Eicosane (C 20 H 42 ), a straight chain alkane composed of 20 carbon atoms [28]; carvone (C 10 H 14 O), a p-menthane monoterpenoid that consists of cyclohex-2-enone having methyl and isopropenyl substituents at positions 2 and 5, respectively [29], vanillin (C 8 H 8 O 3 ) a phenolic aldehyde [30] and guaiacol (C 7 H 8 O 2 ), a monomethoxybenzene that consists of phenol with a methoxy substituent at the ortho position [31], were proven to have high antifungal potential at a limited concentration.
Furthermore, it has been demonstrated by several studies that extracts, VFs and essential oils extracted from plants of the Convolvulus genus strongly inhibit the growth of various fungal pathogens [7,[9][10][11]. A previous study showed an inhibitory effect of up to 0.31 ± 0.10 mg/mL for C. althaeoides oil against Pseudomonas aeruginosa and Enterococcus faecalis [11].
Columns A, B and C: testing the susceptibility of C. albicans to VFs different concentrations (0.05-7 mg/mL). Columns D, E and F: testing the susceptibility of C. glabrata to different concentrations (0.05-7 mg/mL). Columns G, H and I: testing the susceptibility of C. tropicalis to VFs different concentrations (0.05-7 mg/mL). J, K and L: upper C. albicans, C. glabrata and C. tropicalis positive control wells, respectively; down: negative control wells (RPMI 1640-2% glucose). s 2022, 27, x FOR PEER REVIEW 7 of 15   Microscopic observation of biofilm plates permitted us to evaluate the IBF potential of VF1 and VF2 at different concentrations on Candida spp. (Figures 4 and 5). Compared to the positive controls of growth; both VFs demonstrated a high inhibition potential on Candida Biofilm formation. In fact, VF1 and VF2 induced 80% of reduction of C. albicans biofilm biomass up to 0.21 mg/mL and 0.43 mg/mL, respectively. The filamentation was inhibited by both of VFs at concentration of 1.75 mg/mL. Thymus capitatus (0.12 mg/mL) and Cinnamomum verum (0.62 mg/mL) essential oils were efficient against C. albicans biofilm formation at a similar level with 80.60% and 85.57% of inhibition, respectively. Moreover, micrographs of biofilm formed after 24 h incubation demonstrated a filamentation inhibition at half MICs for both of them [32]. Candida albicans biofilms are inherently resistant to antifungal drugs, mainly azoles and polyenes [33]. Thus, essential oils can offer new perspectives as an alternative treatment.
Candida Biofilm formation. In fact, VF1 and VF2 induced 80% of reduction of C. albicans biofilm biomass up to 0.21 mg/mL and 0.43 mg/mL, respectively. The filamentation was inhibited by both of VFs at concentration of 1.75 mg/mL. Thymus capitatus (0.12 mg/mL) and Cinnamomum verum (0.62 mg/mL) essential oils were efficient against C. albicans biofilm formation at a similar level with 80.60% and 85.57% of inhibition, respectively. Moreover, micrographs of biofilm formed after 24 h incubation demonstrated a filamentation inhibition at half MICs for both of them [32]. Candida albicans biofilms are inherently resistant to antifungal drugs, mainly azoles and polyenes [33]. Thus, essential oils can offer new perspectives as an alternative treatment.  A higher potential of IBFs has been observed on C. tropicalis for both VF1 and VF2 fractions with biomass inhibitions of 100% and 90% up to 3.5 mg/mL and 0.87 mg/mL, respectively. A total filamentation inhibition on C. tropicalis was observed for VF1 and VF2 to the positive controls of growth; both VFs demonstrated a high inhibition potential on Candida Biofilm formation. In fact, VF1 and VF2 induced 80% of reduction of C. albicans biofilm biomass up to 0.21 mg/mL and 0.43 mg/mL, respectively. The filamentation was inhibited by both of VFs at concentration of 1.75 mg/mL. Thymus capitatus (0.12 mg/mL) and Cinnamomum verum (0.62 mg/mL) essential oils were efficient against C. albicans biofilm formation at a similar level with 80.60% and 85.57% of inhibition, respectively. Moreover, micrographs of biofilm formed after 24 h incubation demonstrated a filamentation inhibition at half MICs for both of them [32]. Candida albicans biofilms are inherently resistant to antifungal drugs, mainly azoles and polyenes [33]. Thus, essential oils can offer new perspectives as an alternative treatment.  A higher potential of IBFs has been observed on C. tropicalis for both VF1 and VF2 fractions with biomass inhibitions of 100% and 90% up to 3.5 mg/mL and 0.87 mg/mL, respectively. A total filamentation inhibition on C. tropicalis was observed for VF1 and VF2 A higher potential of IBFs has been observed on C. tropicalis for both VF1 and VF2 fractions with biomass inhibitions of 100% and 90% up to 3.5 mg/mL and 0.87 mg/mL, respectively. A total filamentation inhibition on C. tropicalis was observed for VF1 and VF2 at 0.87 mg/mL and 1.5 mg/mL, respectively. C. tropicalis have been demonstrated sensitive to treatment with essential oils of Thymbra capitata (0.62 µL/ml) with percentages of reduction of biofilm biomass and reduction of biofilm metabolism of 67.82% and 97.47%, respectively [34]. Essential oils from Pelargonium graveolens have been described to possess a high continence in geraniol and linalool, which are α-terpineol present in both VF1 and VF2 fractions from this study and were 90% inhibitory to C. tropicalis biofilm formation at 8 mg/mL and reduced only 25% of the biofilm at a concentration similar to our effective concentration (0.87 mg/mL).
Both VF1 and VF2 demonstrated a reduced inhibition potential with a reduction of 50% of C. glabrata biofilm biomass up to 3.5 mg/mL concentration. With the exception of Candida spp., Candida glabrata biofilms were more resistant as the reduction in biomass by both VF1 and VF2 remained below 60%. This limited effect can be explained by the particularity of this yeast, which possesses a structure in different multilayers making its biofilm more compact than other Candida strains [35].

Biofilm Sustained Inhibition (BSI)
The evaluation of the effect of the two VFs of C. althaeoides L. roots on the biofilm formation of C. albicans, C. glabrata and C. tropicalis demonstrated that both of them had an inhibitory potential on biofilm formation ( Figure 6). One-way ANOVA computed a p-value < 0.05 (significant effect of different treatments) as percentages of the untreated control. At a concentration of 7 mg/mL, the inhibition percentage of VF1 on C. albicans biofilm formation was 87.23%, while the inhibition rate of VF2 was 65.91%. VF1 and VF2 showed 63.98% and 56.06% inhibition on the BSI of C. glabrata at a concentration of 7 mg/mL, respectively (Figure 4). Similar yields were reported against the biofilm formation of the C. tropicalis strain, which was inhibited by 79.74% and 65.91% for VF1 and VF2, respectively. The BSI on Candida spp. can be explained by several factors. Indeed, inhibition of adhesion is the first significant step in preventing biofilm formation. The anti-biofilm capability of the two VFs with a preference for VF1 can be attributed to its ability to inhibit the attachment of Candida spp. to surfaces. For pathogens, biofilm plays a key role in nullifying the effect of antifungal and anti-biofilm agents. The BSI is the key step in reducing the pathogenic effect of Candida spp. The anti-biofilm effect of these fractions can be attributed to specific components. The two VFs tested in this study have been demonstrated to harbor a wide range of phytochemical components that could be considered responsible for a more or less biological effect. The activity of these tested VFs varied according to its formulation. Indeed, the high amounts of fatty acids and terpenoids were attributed to a better anti-biofilm activity [36,37]. n-Hexadecanoic acid (C 16 H 32 O 2 ), namely, palmitic acid, a saturated long-chain fatty acid with a 16-carbon backbone [23]; cuminaldehyde (C 10 H 12 O), a benzaldehyde substituted by an isopropyl group at position 4 [38]; linoleic acid (C 18 H 32 O 2 ), an octadecadienoic acid in which the two double bonds are at positions 9 and 12 and have Z (cis) stereochemistry [39]; and benzyl alcohol (C 7 H 8 O), an aromatic alcohol that consists of benzene bearing a single hydroxymethyl substituent [25] were the main compounds present in these fractions that were associated with anti-biofilm activities. The synergistic effects of such compounds might be responsible for the observed anti-biofilm activity [40]. The antimicrobial activity of these compounds is based on different mode of action, as described for cuminaldehyde, which has the propriety to disrupt Staphylococcus aureus membranes and bind to DNA through the groove to impact normal cellular function [41]. Benzyl Alcohol has been proven to induce attack on bacterial membranes and protein denaturation, following an increase of reactive oxygen species levels [42]. Linoleic acid as fatty acid was involved in affecting membrane permeability (pore formation and membrane destabilization), reducing hydrophobicity but being able to interfere with microbial metabolism or signaling [43]. Palmitic acid was observed to enhance hyphal growth for C. tropicalis through upregulation of hyphal wall protein (HWP1) and enhanced filamentous growth (EFG1) genes [44].

Plant Material
The roots of C. althaeoides L. were collected in the region of Kondar (TUNISIA) in January 2017, a rural area of the Tunisian Sahel located about 30 km north-west of the governorate of Sousse. The plant material was naturally dried in the shade at room temperature for 2-3 weeks, ground into a fine powder and later used for extractions.

Chemicals and Reagents
All solvents used in the experiments (hexane and chloroform) were analytical grade (Merck Life Science, Merck KGaA, Darmstadt, Germany). Dimethyl sulfoxide (DMSO) was purchased from BIO BASIC INC (Desk, Markham, ON, Canada). Culture media were purchased from Sigma-Aldrich (CHEMIE GmBH, Riedstr, Germany). RPMI-1640 medium was purchased from Gibco and stored at 4 °C. Glucose solution 30% (Siphal, Tunisia).

Preparation of VF from Roots of C. althaeoides L.
The whole process from preparing VFs from root powder was followed according to the method described by Hrichi et al. [40]. Briefly, 100 g of root powder was subjected to hydrodistillation for 3 h. The collected hydrodistillates were subjected to a liquid-liquid extraction with hexane and chloroform, successively. The two VFs, VF1 (hexane) and VF2 (chloroform), were dried over anhydrous sodium sulphate (Na2SO4) and stored in a refrigerator at −4 °C until analysis. The yield of VFs was calculated following the next formula: % Yield of VFs = [Weight of VFs/Weight of dried roots] × 100%

Plant Material
The roots of C. althaeoides L. were collected in the region of Kondar (TUNISIA) in January 2017, a rural area of the Tunisian Sahel located about 30 km north-west of the governorate of Sousse. The plant material was naturally dried in the shade at room temperature for 2-3 weeks, ground into a fine powder and later used for extractions.

Chemicals and Reagents
All solvents used in the experiments (hexane and chloroform) were analytical grade (Merck Life Science, Merck KGaA, Darmstadt, Germany). Dimethyl sulfoxide (DMSO) was purchased from BIO BASIC INC (Desk, Markham, ON, Canada). Culture media were purchased from Sigma-Aldrich (CHEMIE GmBH, Riedstr, Germany). RPMI-1640 medium was purchased from Gibco and stored at 4 • C. Glucose solution 30% (Siphal, Tunisia).

Preparation of VF from Roots of C. althaeoides L.
The whole process from preparing VFs from root powder was followed according to the method described by Hrichi et al. [40]. Briefly, 100 g of root powder was subjected to hydrodistillation for 3 h. The collected hydrodistillates were subjected to a liquid-liquid extraction with hexane and chloroform, successively. The two VFs, VF1 (hexane) and VF2 (chloroform), were dried over anhydrous sodium sulphate (Na 2 SO 4 ) and stored in a refrigerator at −4 • C until analysis. The yield of VFs was calculated following the next formula: % Yield of VFs = [Weight of VFs/Weight of dried roots] × 100%

Gas Chromatography-Mass Spectrometry (GC-MS) Analysis
The composition of VFs of C. althaeoides L. was identified by GC-MS analysis. GC-MS analysis was carried out on a GC-MS-QP2020 system (Shimadzu, Kyoto, Japan) equipped with an "AOC-20i" system auto-injector, and separation was attained in a column SLB-5ms (30 m in length × 0.25 mm in diameter × 0.25 µm in thickness of film, Merck Life Science, Merck KGaA, Darmstadt, Germany). The temperature of the injection port was set at 50 • C and afterwards increased up to 350 • C (increase rate: 3 • C/min; holding time: 5 min). The GC parameters were as follows: injection temperature, 280 • C; injection volume, 1.0 µL (split ratio: 10:1); pure helium gas, 99.9%; linear velocity, 30.0 cm/s; inlet pressure, 26.7 KPa. The MS conditions included an interface temperature of 220 • C, a source temperature of 250 • C and a mass scan range of 40-660 amu. The peak of the samples was identified by using the "FFNSC 3.01" (Shimadzu Europa GmbH, Duisburg, Germany) and "W11N17" (Wiley11-Nist17, Wiley, Hoboken, NJ, USA; Mass Finder 3). Each compound was identified applying a MS similarity match and an LRI filter. Linear retention indices (LRI) were calculated by using a C7-C40 saturated alkanes reference mixture (49452-U, Merck Life Science, Merck KGaA, Darmstadt, Germany). A relative quantity on the basis of peak area percentages was carried out.

Determination of the Minimum Inhibitory Concentrations (MICs)
MICs of the VFs were determined using the microdilution technique, as previously described by Hrichi et al. [10], with some modification. Briefly, microtiter plates with 96 flatbottomed wells were used, and serial dilutions of the test substance (7.00, 3.50, 1.75, 0.87, 0.44, 0.22, 0.11 and 0.05 mg/mL) were prepared using RPMI-1640 media supplemented with 2% glucose. Candida spp. strains were subjected to a susceptibility test face to Amphotericin B concentrations ranging from 16 × 10 −3 mg/mL to 0.03 × 10 −3 mg/mL as drug standard. Inoculums for the assay were prepared for each strain using diluting a 24 h fresh colony from sabouraud chloramphenicol agar into RPMI-1640-2% glucose and adjusting it to 2.5-5 × 10 5 CFU mL −1 . The final concentration was confirmed using a Malassez counting chamber. Working inoculums (100 µL) were added to the 96-well plates, which were incubated at 37 • C for 24 h. A fungal suspension in the medium was used as growth positive control, and non-inoculated medium RPMI-G 2% (200 µL) was included as a negative control. All experiments were repeated in triplicate. MICs were determined by spectrophotometic lecture at 570 nm to control the growth of fungal strains and defined as the lowest concentration of the VFs produced growth inhibition compared with the growth in the untreated control well.

Minimum fungicidal concentrations (MFCs)
The MFCs were determined using a subculture of 10 µL from MICs wells onto sabouraud chloramphenicol agar plates. The plates were incubated at 37 • C for 24 h. The concentration that induces no visible colony growth after subsequent 24 h incubation was accepted as MFC, which is the lowest concentration without visible biomass growth and corresponding to the death of 99.90% of the original inoculum.

Inhibition of Biofilm Formation (IBF)
The VFs VF1 and VF2 were tested for their capacity to inhibit Candida spp. biofilm formation. Serial dilutions of (from 7 to 5 × 10 −2 mg/mL) were incubated with 5-2.5 × 10 5 CFU mL −1 fungal cells in RPMI 1640-2% glucose. Inhibition of biofilm formation was determined after crystal violet stain at its sub-inhibitory concentrations [45]. Briefly, serial dilutions of VFs were added to a 96-well plate (Orange Scientific, Braine-l'Alleud, Belgium) and incubated with different Candida spp. inoculums. The media with inoculums were used as positive controls of growth, and the Amphotericin B was used as drug standard at a concentration of 0.2 mg/mL. The morphology of Candida spp. was monitored using an inverted microscope (Olympus CK2) at 40× magnification. The minimum biofilm inhibitory concentration was also determined as the minimum concentration of substance without fungal development [46].

Biofilm Sustained Inhibition (BSI)
The standard optical density of biofilm sustained inhibition assay of C. althaeoides L. root VFs followed the previously reported protocol for the 96-well format of the biofilm screening assay [47] with some modification. Volatile fractions at different concentrations (7, 3.5, 1.75, 0.87, 0.44, 0.22, 0.11 and 0.05 mg/mL) were added during the 90 min adherence and 24 h growth steps of the Sustained Inhibition Biofilm Assay. Simultaneously different concentrations of Amphotericin B were tested ranging from 4 × 10 −3 mg/mL to 0.03 × 10 −3 mg/mL as standard drugs. The MIC concentrations for our fungi strains were run many times but not for this plate, if it is possible to mention it as follows. In brief, inoculums were adjusted to 2 × 10 6 CFU mL −1 from overnight Candida spp. cultures. One hundred microliters of suspension were added to the 96-well plates and shaken at 37 • C for 90 min at 350 rpm in an APELEX shaker. Media was then removed, wells were washed with PBS, and fresh media (or media with VFs) was added back to wells. Plates were then resealed and shaken for a further 24 h at 37 • C. Finally, media was removed, and the absorbance (OD) at 570 nm was determined on a Multiskan™ FC Microplate Photometer.

Statistical Analysis
Results were reported as means ± standard deviation. All assays were performed considering three analytical replications. Based on the replications, one-way analysis of variance (ANOVA, followed by Turkey's multiple range test at p < 0.05 level) was employed to assess the significant difference among the biological activities of the samples.
All statistical tests were performed by GraphPad Prism 6 software (GraphPad Software, Inc., San Diego, CA, USA). Heatmaps graphical representations were guided on OD of microdilution 96 plates at 570 nm.

Conclusions
The present research investigates for the first time the chemical profiles of the two VFs of C. althaeoides L. roots and their biological activities. A large number of volatile chemical compounds in the two VFs were identified reaching a value as high as 86 in the chloroformic extarct. These two VFs showed a good antifungal activity against three strains of Candida genus which are pathogenic years. Anti-biofilm activity in Candida spp. with up to 90% was IBF/BSI, indicating that they are promising sources of natural antifungal agents. The richness of the biologically active compounds in these two VFs may be responsible for this activity. Therefore, on the basis of the cytotoxicity study of these two VFs, such a species might be suitable for pharmaceutical applications.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/molecules27206834/s1, Table S1: List of volatile compounds detected in the hexane fraction (VF1) of C. althaeoides L. roots by GC-MS; Table S2: List of volatile compounds detected in the chloroform fraction (VF2) of C. althaeoides L. roots by GC-MS.