Effect of seasonal variation on the chemical composition and antioxidant and antifungal activities of Convolvulus althaeoides L. leaf extracts

2.5.1 Polyphenol analysis by LC-PDA-ESI/MS

Polyphenol analysis was preceded by a sample preparation, in which the dichloromethane and the chloroform extracts were redissolved in 1 mL chloroform (10 mg), the ethyl acetate and ethanol extracts were redissolved in 1 mL methanol (10 mg). The obtained solutions were subsequently filtered through a 0.45 μm Acrodisc nylon membrane (Merck Life Science, Merck KGaA, Darmstadt, Germany) prior to HPLC-PDA-MS analysis.

Calibration curves for polyphenol quantification were recently reported by Haoujar et al., 2019.

LC analyses were performed on a Nexera-e liquid chromatograph (Shimadzu, Kyoto, Japan), consisting of a CBM-20A controller, two LC-30AD dual-plunger parallel-flow pumps, a DGU-20A5R degasser, a CTO-20AC column oven, a SIL-30AC autosampler, an SPD-M30A photo diode array detector and an LCMS-8050 triple quadrupole mass spectrometer, through an ESI source (Shimadzu, Kyoto, Japan). Separations were carried out on an Ascentis Express RP C18 column (2.7 µm, 150 mm, and 4.6 mm) (Merck Life Science, Merck KGaA, Darmstadt, Germany). The mobile phase was consisted of water/acetic acid (99.85/0.15 v/v, solvent A) and acetonitrile (solvent B), with the following gradient elution: 0–5 min, 5% B, 5–15 min, 10% B, 15–30 min, 20% B, 30–60 min, 50% B, 60 min, 100% B. Photodiode array detector was applied in the range of λ = 200–400 nm, where polyphenols were detected at λ = 280 nm (sampling frequency: 40 Hz, time constant: 0.08 s). MS analysis was performed in negative and positive ion mode and scan range was set at m/z 100–800; scan speed of 2500 u sec^-1; the conditions of ESI were as follows: event time 0.3 sec; nebulizing gas (N₂) flow rate 1.5 L min⁻¹; drying gas (N₂) flow rate, 15 L min⁻¹; interface temperature: 350 °C; heat block temperature: 300 °C; DL (desolvation line) temperature: 300 °C; DL voltage: 1 V; interface voltage: −4.5 kV; Qarray DL voltage 0 V. Data acquisition and processing were performed by means of the LabSolution software, version 5.65 (Shimadzu, Kyoto, Japan).

2.5.2 Carotenoid analysis by LC-PDA-APCI/MS

In order to determine the carotenoid content, 200 mg of each extract were dissolved in 1 mL of methanol/methyl tert-butyl ether (v/v) mixture solution. The obtained solutions were filtered through a 0.45 µm Acrodisc nylon membrane (Merck Life Science, Merck KGaA, Darmstadt, Germany) prior to be analyzed by HPLC-PDA-APCI/MS.

For the characterization of the native carotenoid composition, a Nexera X2 liquid chromatography system (Shimadzu, Milan, Italy), consisting of a CBM-20A controller, two LC-30AD dual-plunger parallel-flow pumps, a DGU-20 A_5R degasser, a CTO-20AC column oven, and a SIL-30AC autosampler was employed. A 0.1 mm I.D. stainless steel tube (zero dead volume) was used for the linkage between the column connection and the SPD-M30A diode array detector. The LC system was coupled to an LC-qMS-2020 mass spectrometer through an APCI source (Shimadzu, Kyoto, Japan). Separations were carried out on a 250 mm × 4.6 mm i.d., 5 μm, YMC C₃₀ column. The mobile phases were: A (MeOH/MTBE/H₂O, 81:15:4, v/v/v) and phase B (MeOH/MTBE/H₂O, 15:81:4, v/v/v); a linear gradient program was used as follows: 0–20 min, 0% B, 20–140 min, 100% B, 141–150 min, 0% B. The flow rate was set at 0.8 mL/min, the injection volume was 20 µL, the column temperature was maintained at 35 °C, the UV/vis spectra were acquired between 220 and 700 nm, and the chromatograms were processed at 450 nm. The MS detection was achieved through an APCI interface operated in both positive and negative ion modes; detector voltage: 1.05 kV; interface temperature: 350 °C; DL temperature: 300 °C; heat block temperature: 300 °C; nebulizing gas flow (N₂): 2.0 L/min; drying gas flow (N₂): 5.0 L/min; full scan range (positive and negative ion mode): 300–1200 m/z; event time: 0.2 s. Data acquisition and processing were performed by means of the LabSolution software, version 5.93 (Shimadzu, Kyoto, Japan).

2.7.1 Strains and culture media

Antifungal activity of the different extracts was evaluated against yeast, dermatophyte and Aspergillus strains: two Candida spp. strains from the American type Culture Collection ATCC (Candida albicans ATCC® 90028™ and Candida glabrata ATTC® 64677™) and one clinical Candida strain isolated from a clinical hand nail sample (Candida tropicalis 988); three dermatophyte Microsporum canis MS 8972, Trichophyton metagrophytes MS45 and Trichophyton rubrum MS 7793.1 isolated from clinical scalp samples, and four Aspergillus isolated from clinical nail samples (A. niger, A. flavus, A. fumigates and A. terreus). The fungal clinical isolates were identified by the professional team in the laboratory of parasitology and mycology of the teaching hospital Fattouma Bourguiba (Monastir-Tunisia). Before the antifungal sensitivity tests, each fungal strain was maintained on the Sabouraud dextrose agar (SDA) medium.

2.7.2 Antifungal toxicity screening assay

The evaluation of the potential antifungal activity was performed based on the food poisoning method. Extracts and plant powder were directly incorporated in SDA at a final concentration of 100 mg mL⁻¹ and 10%, respectively, then cooled in 24 wells culture plates at 45 °C, and let to solidify at room temperature before to be incubated overnight at 30 °C for sterility test. Fungal inoculums were prepared from fresh cultures of Candida spp. (24 h) and Aspergillus spp. (72 h) in sterile distilled water, and adjusted to 10⁴ UFC mL⁻¹ using a Malassez Counting Chambe. Plates were inoculated with 10 µL of each inoculum and incubated in 30–35 °C for 24 h-72 h, depending on the strain. Two millimeter plug from 7 days culture of dermatophytes were cut and deposit on each extract-SDA well to control the mycelial growth. Controls consisted of free extract/antifungal SDA medium wells. In order to control the sensitivity of the Candida spp. and Aspergillus spp. strains, the antifungal drug Amphotericin B was incorporated to a concentration of 16 µg mL⁻¹. The experiments were performed twice. Fungal growth was visibly controlled and the percentage of inhibition (I%) was estimated in relation to the number of colonies/radial mycelium growth counted in the growth control well and calculated according to the following formula: I%=[(NCC-NCE)/NCC] × 100, NCC: Number of Colonies/radial growth on the control-SDA, NCE: Number of Colonies /radial growth on the extract-SDA.

2.7.3 Determination of minimum inhibitory concentration (MIC)

2.7.4 Inhibition of hyphae formation in Candida albicans

In the present work, we aimed to estimate the effect of our extracts on C. albicans morphology. Initially, 3 to 5 colonies of 24 h C. albicans growing on SDA media were transferred to a test tube containing 10 mL of YDP (Yeast extract-peptone-dextrose) medium and incubated at 30 °C during 24 h. In sequence, the solution was diluted 1:30 in YDP medium, containing 10% of foetal calf serum (Eurobio) to obtain a final concentration of strain 1 × 10⁶ CFU mL⁻¹. Then, 100 mg of each extract were diluted in 1 mL of 10% DMSO solution. Then, 200 µL of each sample were twofold diluted in YDP medium resulting in concentrations from 100 to 0.195 mg mL⁻¹. Finally, 100 µL of the inoculum were add to all the plat wells and incubated for 3 h in 37 °C. The minimal inhibitory filamentation concentration (MIFC) was then determined by estimating the number of hyphae formes of C. albicans compared to simple cell forms, using light microscopy. Plats were re-incubated for 24 h and MIFC were checked again.

3.2.1 Identification of polyphenolic compounds of C. Althaeoides L. Leaves from different seasons

LC-PDA-ESI/MS analyses were carried out on each extract of C. althaoides L. leaves from different seasons to compare its polyphenol composition. The identification of twenty two polyphenol compounds was based on the comparison of UV–Visible absorption (λ_max) and specific mass-to-charge ratio (m/z) with data reported by previous literature (Table 1).

The two species C. austroaegyptiacus and C. pilosellifolius Desr. demonstrated fewer compounds number with thirteen and fifteen polyphenolic compounds, respectively, by using the DART-ToF-MS technique (Al-Rifai et al., 2017). In contrast, LC-PDA-ESI/MS detects more than twenty individual compounds in C. althaeoides L. leaves (chromatograms) (Fig. 2), showing different composition between the extracts with increasing polarity. Ethyl acetate and the ethanol were the richest polyphenolic extracts compared to dichloromethane and chloroformic extracts. This difference could be explained by the factor of polarity, since polyphenols are polar compounds. Therefore, the identified polyphenolic compounds were classified according to their chemical structure (Table 1). Major polyphenolic compounds identified in our study belonged to phenolic acid and flavonoid subfamilies.

3.2.2 Quantification of polyphenolic compounds of C. althaeoides L. Leaves from different seasons

The distribution of polyphenolic compounds showed a notable variation in the quality, but also in quantity between the studied extracts, and seasonality seems to affect the composition of the extracts (Table S1). In fact, ethanol extracts demonstrated the presence of 14 compounds, and the same profiling was observed through chromatograms for both winter and spring collects. The total polyphenolic content of compounds was more important in winter (3904.55 µg g⁻¹) than in spring (2948.04 µg g⁻¹) for the ethanol extract. The ethanol solvent seemed to be the most appropriate solvent for a maximum extraction of polyphenolic compounds (Table S1). Higher total polyphenolic contents of the ethanol extracts deriving from the sister species Convolvulus austroaegyptiacus (125440 µg g⁻¹) and Convolvulus pilosellifolius (129970 µg g⁻¹) have been recorded using Folin-Ciocalteu method (Sousa et al., 2007) whereas ethyl acetate extract seemed to be the most effective solvent to dissolve polyphenolic compounds for C. austroaegyptiacus (146830 µg g⁻¹) and C. pilosellifolius (158140 µg.g⁻¹) (Al-Rifai et al., 2017). However, the used method  overestimated the quantities of phenolic compounds compared to chromatography LC-PDA-ESI/MS and LC-PDA-APCI/MS methods. Chlorogenic acid was the predominant polyphenolic compound with a maximum concentration for the ethanol winter extract (1299.4 µg g⁻¹) (Table S1). Caffeic acid hexoside isomers and quercetin 3-O-arabinoside were positively correlated (r = 0.30) to spring ethanol extract (Fig. 2). These compounds were also positively correlated to winter ethyl acetate extract (r = 0.30) (Fig. 2). The highest concentration of polyphenolic compounds in ethyl acetate extracts was caffeic acid, with 1951.7 µg g⁻¹ for winter and 1822.21 µg g⁻¹ for spring collects. This compound was less abundant in the ethanol extracts and no difference due to seasonality was observed. The distribution of the two isomers of dicaffeoylquinic acid was also independent form season fluctuation and was positively correlated to the ethanol extract (r = 0.36) (Fig. 1, Fig. 2). Naringin glucoside, p-coumaric acid and quercetin were correlated to the ethyl acetate extract with maximum abundance in the winter collect (Fig. 2). The dichloromethane and chloroformic extracts had similar polyphenol profiles with a few difference the detected compounds (Fig. 1, Table S1). The flavonoid compound umbelliferone was detected only in the winter dicloromethane and chloroformic extracts. Quercetin and quercetin-3-O-glucosyl-xylose distribution was positively correlated to spring dichloromethane and chloroformic extracts. Finally, luteolin diglucuronide was a major flavonoid of dichloromethane extract of winter collect (63 µg g⁻¹, r = 0.31) (Table S1, Fig. 2).

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Fig. 1

Polyphenolic profiles of different extracts obtained by HPLC-PDA analysis. A; winter dichloromethane extract, B; spring dichloromethane extract, C; winter chloroformic extract, D; spring chloroformic extract, E; winter ethyl acetate extract, F; spring ethyl acetate extract, G; winter ethanol extract, H; spring ethanol extract of C. althaeoides L. leaves.

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Fig. 2

Principal Components Analysis (PCA) showing the distribution of polyphenolic composition of different extracts from C.althaeoides L. leaves from winter and spring collects.

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3.2.3 Identification of the pigment compounds of C. althaeoides L. leaves from different seasons

Chlorophylls, chlorophyll derivatives and carotenoids are pigments produced by green plants which are involved in the photosynthetic process. Moreover, important functions are attributed to those pigments, such as antioxidant properties. Both natural factors and analytical ones can affect the plant tissue composition in pigments, such as the solvent used for the tissue extraction and the yearly seasonal climate variation (Saini and Keum, 2018). In this study, pigment compounds in C. althaeoides L. leaf extracts were characterized using LC-PDA-APCI/MS analysis. Compounds were identified by comparing UV–visible and mass spectra data with those of literature (Table 2). In the eight different extracts of C. althaeoides L. leaves, 27 compounds were detected, including ten identified as carotenoids, eleven identified as chlorophyll derivatives and six unidentified compounds (Table 2). All analyzed extracts showed the same chromatographic profile (Fig. 3). Peak 1 (t_{R =} 3.3 min) showed UV maximum at 268 nm and specific spectrum mass at [M + H]⁺ m/z 409, was tentatively identified as 4,4′-diapophytoene (Kaiser et al., 2012). Peaks 2, 10, 16, 21 and 22 were identified as a chlorophyll derivatives, they showed five maximum absorbance in the UV–visible spectrum and fragment ions [M + H]⁺ at m/z 617, 593, 893 and 872 due to the loss of magnesium with the loss of a C₁₉H₃₈ molecule. Thus, based on the spectrum information and literature data of these compounds they were identified as chlorophyllide a, pheophorbide a, chlorophyll a and two pheophytins type (Milenković et al., 2012). Peaks 3, 8, 17, 19 and 20 were tentatively identified as chlorophyll b derivatives, they showed five maximum absorbance in the UV–vis spectrum and fragment ions [M + H] + at m/z 631 and 607, 908 and 885, by comparison with literature data those compounds were identified as chlorophyllide b, pheophorbide b, chlorophyll b and two pheophytins b type, respectively (Maurer et al., 2014; Milenković et al., 2012). Peak 4 (t_R 6.4 min), with five maximum UV–visible absorbance at 281, 314, 421, 434, 658 nm and fragment ion [M + H] + at m/z 613 was tentatively identified as chlorophyll c. Peak 6 (t_R 8.1 min), showed a maximum UV–vis absorbance at m/z 238, 266, 401, 498 nm and fragment ion [M + H] + at m/z 625, and accordingly to literature data, it has been identified as actinioerythrin (Leboeuf et al., 1981). Peaks 9, 11 displayed similar maximum UV–V is absorption spectral viz. 415, 437, 464 nm, and 422, 444, 473 nm and MS fragment in positive ion mode (protonated molecule) at m/z 551, which was attributed to the molecule of lutein isomer and lutei itself. Other characteristic fragment ion [M−H]^- at m/z 568 detected for peaks 9 and 11 confirmed them as lutein isomer and lutein according to literature data (Gupta et al., 2015). Peak 15 had maximum absorption at 409 nm and 507 nm and MS fragmentation in positive ion mode at m/z 535 (protonated) and in negative ion mode at 534 (deprotonated); it was tentatively identified as torulene (C₄₀H₅₄). The successive peaks 23 and 24, had a similar λ_max at 400, 422, 450 and MS fragment in positive ion mode at m/z 601, these peaks were identified by comparison with the data reported in literature (Meléndez-Martínez et al., 2007) as luteoxanthin isomers.

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Fig. 3

Carotenoids profiles of different extracts obtained by HPLC-PDA analysis. A; winter dichloromethane extract, B; spring dichloromethane extract, C; winter chloroformic extract, D; spring chloroformic extract, E; winter ethyl acetate extract, F; spring ethyl acetate extract, G; winter ethanolic extract, H; spring ethanolic extract of C. althaeoides L. leaves.

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Finally, peaks 25 and 26 had a similar MS fragment in positive ion mode 537 characteristic to carotene molecules and specific UV–vis absorbances at 428, 452, 478 nm (peak 25) and 333, 421, 446, 472 nm (peak 26) permitted to identify them as β carotene and 9-Z-β-carotene, respectively.

3.2.4 Quantification of carotenoid compounds in C. althaeoides L. leaves from different seasons

C. althaeoides L. leaves, harvested in different seasons were treated successively with solvents of decreasing polarity. The chromatograms showed a complex carotenoid profile and a total of 10 compounds were identified (Table S2, Fig. 4). The chloroform was the best solvent to extract carotenoids, with similar total contents for the winter (2693. 93 µg g⁻¹) and the spring (2655. 26 µg g⁻¹) collects. Interestingly, the dichloromethane extract from the spring collect of leaves was the most diversified in carotenoids, with 10 detected compounds and total concentration of 2607.96 µg g⁻¹ compared to the same extract from the winter collect of leaves, with 6 compounds and total concentration of 893.79 µg g⁻¹. On the other hand, ethanol extracts contained negligible amounts of carotenoids, for both winter (401.71 µg g⁻¹) and spring (333. 06 µg g⁻¹) collects. In view of all these findings, no important difference in total contents between seasons was observed. Nevertheless, interesting deviation were observed in the composition and concentration of each identified pigment within extracts and according to seasonality. Lutein was the major carotenoid identified in all extracts with concentrations ranging from 287.04 to 2014.7 µg g⁻¹, except for the ethanol extract from spring collect for which the most abundant compound was the 4, 4′-diapophytoene (Table S2). Furthermore, PCA analyses demonstrated that lutein and lutein isomer were correlated to dichloromethane and chloroform in both winter and spring extracts. 9-Z- β-carotene and β-carotene were positively correlated to the dichloromethane extracts with a maximum of 379.94 µg g⁻¹ and 71.7 µg g⁻¹, respectively, for the spring collect (Table S2, Fig. 4). Luteoxanthin isomer 1 and luteoxanthin isomer 2 were detected only for spring extract, with positive correlation (r = 0.65) for the chloroform and dichloromethane extracts, respectively (Fig. 4). Torulene was present in both winter and spring season extracts with a positive correlation, with the ethyl acetate (r = 0.62) for winter collect and less significant correlation (r = 0.40) for both dichloromethane and chloroform of spring leaf collect (Fig. 4). 4,4′-diapophytoene was more abundant in winter chloroformic extract, however it was the most correlated to the ethyl acetate extract in the spring collect. Finally, actinioerythrin and echinenone were positively correlated to the chloroformic extract in both winter (r = 0.40) and spring (r = 0.57) collects (Fig. 4). The high diversity of carotenoids and chlorophylls suggested that C. althaeoides L. leaves are a potential source of natural pigment. Pigment compounds were shown to have many health benefits such as antioxidant activity and proven to have an antimicrobial activity (Moreira et al., 2018). Seasonal variations in the total phenolic compounds and flavonoids were demonstrated by (Ncube et al., 2011) for four plants from South Africa. In addition, a recent research showed that the quantity of these compounds from I. helenium, greatly differs regarding the different time periods (Zlatić et al., 2019). By the way, metabolites seems to have major roles in the adaptation of plants to unfavorable conditions generated by climatic and environmental factors across season fluctuations, which could explain the difference of nature and the concentration of the identified compounds.

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Fig. 4

Principal Component Analysis PCA for quantitative of carotenoid composition of different extracts from C. althaeoides L. leaves from winter and spring collects.

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3.4.1 Anti-dermatophytic activity

The screening of the susceptibility of T. rubrum, T. mentagrophytes and M. canis clinical strains for each evaluated powder and extract from C. althaeoides L. leaves, for winter and spring collects, showed different results (Fig. 6, Fig. 7). Sterilized powder showed an important increasing inhibition percentage for both T. rubrum and T. mentagrophytes, reaching 52% for the winter collect and not exceeding 37.5% for the spring collect within 15 days of culture, and this with an initial concentration of 10%. In the same conditions, the inhibition percentage for M. canis was important in the beginning of culture evolution (46–50%), however it slightly decreased within 15 days for both winter and spring collects (12–15%) (Fig. 7). Attempting to estimate the MIC values, decreasing percentage of powder (7.5%, 5%, 2.5%, 1%, 0.5%) were tested, showing an inhibitory effect of 20% maintained up to a 0.5% of powder concentration, and this up to 15 days on T. rubrum culture, for both winter and spring collects (Fig. 7). Less important inhibition was shown for T. mentagrophytes and M. canis with an inhibitory effect not exceeding 6.2% for a concentration of 0.5% and 10% for a concentration of 2.5% within 15 days, respectively (Fig. 7). In the other hand, ethanol and ethyl acetate extracts obtained from different collects of C. althaeoides L. leaves showed 100% of inhibition for T. rubrum, T. mentagrophytes and M. canis with a concentration of 100 mg mL⁻¹, and this up to 15 days. Gallic acid, a constant composite of these extracts, have been demonstrated efficient against T. rubrum (MIC = 43.75 μg mL⁻¹) (Li. Z.-J et al., 2017). p-coumarinic acid derivatives (3-Prenyl-p-coumaric acid and 3,5-diprenyl-p-coumaric acid) were also active towards T. rubrum with MICs ranging from 100 to 125 μg mL⁻¹ (Feresin et al., 2003). Similarly, the flavonoid quercetin present in ethanol and ethyl acetate extracts, particularly the quercetin 3-O-arabinoside, have been shown to be actif against T. rubrum, and thus via the ergosterol reduction and membrane damage mechanisms (Bitencourt et al., 2013). Phenolic compounds belonging to the cinnamic acid derivative group (chlorogenic acid and caffeic acid derivatives) were proven to defend plants against the filamentous fungi Fusarium graminearum and to limit the accumulation of mycotoxins in cereals (Xu et al., 2019). Seasonality seems not to influence the antifungal capacity of the ethanol and ethyl acetate extracts towards dermatophytes (Fig. 9). Nevertheless, the chloroformic extract demonstrated a significant difference, and this with 100% of inhibition for the spring collect compared to 14.28–33.34% for the winter extract. With a return to chemical composition, ferulic acid was the only element that was found to be in favor of this variation, regardless its limited presence in the chloroformic spring extract (6.88 µg g⁻¹). A previous study highlighted an inhibitory effect reaching 81.45% ± 0.05 for the ferulic acid against filamentous fungi from Fusarium, Penicillium and Aspergillus genus (Zabka and Pavela, 2013). For all the studied dermatophyte strains, dichloromethane extract showed a minor inhibitory effect for the spring collect of leaves (14.28–25%) and a surprising growth stimulating effect on the tested fungi cultures (-21.43 to −41.67%) for the winter collect of leaves (Fig. 6). The inhibition of dermatophyte cultures was also verified at the microscopic scale and numerous cytopathologic effects on mycelium growth were noted comparing powder agar and extract incorporated agar plates to negative control. T. rubrum demonstrated very limited mycelium abundance with a total absence of fructification elements (microconidia) for powders from both winter and spring collects (Fig. 8). However, the ethanol extract treatment enhanced the fructification of T. rubrum and the appearance of chlamydioconida was noted. The species T. mentagrophytes showed less abundant microconidia and pencil-shaped macroconidia. Moreover, the emergence of terminal chlamydoconidia, which are in general observed when fungi are under suffering conditions, was clearly detected as a result of the powder treatment (Fig. 8). For the ethanol extract micro and macroconidia were less abundant than for the negative control for T. mentagrophytes. Finally, M. canis evolution was regressive and the characteristic barrel-shaped macroconidia were less numerous, reduced in size and sometimes without septation, and this for the powder of both winter and spring collects (Fig. 8). The ethanol extract showed cultures with only filaments, and with no fructification elements. An attempt to determinate the MIC/MFC was realized with decreased concentrations of the extract by the screening approach on SDA 24 well plates. The concentration of 100 mg mL⁻¹ was proven effective against the three dermatophyte species with 100% of inhibition of radial growth, and this for the ethanol and ethyl acetate extracts from both spring and winter seasons and chloroform extract of spring collect (Fig. 9). However, this efficacy was maintained only for ethanol and ethyl acetate extracts of winter leaves collect, when the concentration was decreased up to 25 mg. The species T. mentagrophytes have been more sensitive with a MIC₅₀ of 6.25 mg mL⁻¹, followed by M. canis (MIC₅₀ = 12.5). T. rubrum was resistant to low concentrations of extracts with large interval of MIC₅₀ (12.5–50 mg/ml). All extracts from C. althaeoides L. leaves demonstrated no mycelia inhibition on the filamentous fungi from Aspergillus genus (A. flavus, A. fumigates, A. terreus and A. niger) with an initial concentration of 100 mg mL⁻¹.

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Fig. 6

Screening anti-dermatophyte activities of the different extracts from C. althaeoides L. leaves, harvested in winter and spring seasons. Signification difference, one-way ANOVA test, p < 0.05.*100% of inhibition was attributed when no mycelia growth was observed on the dermatophyte disc.

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Fig. 7

Anti-dermatophytes activitiy of leaf powders of C. althaeoides L., harvested in winter and spring seasons. Signification difference, one-way ANOVA, p < 0.05.

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Fig. 8

Microscopic observation of the dermatophyte treated with powder and extracts from C. althaeoides L. compared with negative growth control. A; T. rubrum negative control, B; T. mentagrophytes negative control, C; M. canis negative control, D; T. rubrum treated with powder, E; T. mentagrophytes treated with powder, F; M. canis treated with powder, G; T. rubrum treated with ethanol extract, H; T. mentagrophytes treated with ethanol extract, I; M. canis treated with ethanol extract.

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Fig. 9

Anti-dermatophyte activity of C. althaeoides L. leaf extracts from winter and spring seasons. A: winter ethyl acetate extract, B: winter ethanol extract, C: spring chloroformic extract, D: spring ethyl acetate extract, E: spring ethanol extract.

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3.4.2 Anti-candidal activity

The antifungal activity of C. althaeoides L. extracts against Candida spp. strains was evaluated. The food poisoning method was used for the screening of the inhibitory potential on the C. albicans cultures. Except for the chlorofomic extract who did not demonstrated effect, all other extracts showed a maximum of inhibition at 75% (Table 3). The ethanol and ethyl acetate extracts were the most effective on C. glabrata cultures (75%) followed by the spring chloroformic extract (50%). All extracts demonstrated a moderate potential of inhibition for C. tropicalis, with a maximum of 50%. The microdilution method permitted to estimate the MIC values and deduce MFC values after the re-inoculation of susceptible wells on SDA plates. The ethanol and ethyl acetate extracts showed a maximum inhibition potential with MICs/ MFCs ranging from 0.78 to 3.12 mg mL⁻¹ and 3.12–6.25 mg mL⁻¹ for C. albicans and C. glabrata, respectively (Table 3). In correlation with solid media outcomes, the dichloromethane was highly inhibitory for C. albicans (MICs/ MFCs: 3.12–12.5 mg mL⁻¹) but not for C. glabrata. Chloroformic extract had a moderate inhibition on yeast 12.5–100 mg mL⁻¹. No visible inhibition was detected for C. tropicalis using microdilution method, for all the studied extracts. In a previous study, a close Concolvulacae to C. althaeoide L., C. arvensis L. was tested for antimicrobial capacity and the recovered MICs for butanol extract were 5 and 50 mg mL^-1for stem and leaves, respectively (Khan et al., 2015). In a second study the MIC values for the ethanol extracts of Convolvulus pilosellifolius (0.25 mg mL⁻¹) and Convolvulus austroaegyptiacus (0.78 mg mL⁻¹) against the microbiological agent Bacillus subtilis were closer to our results (Bitencourt et al., 2013). As it was reported here for filamentous fungi, major polyphenolic compounds of the most efficient extracts were highlighted in previous studies as anti-candidiasis agents. Gallic acids from Buchenavia tomentosa presented MICs ranging from 2 to 12.5 mg mL⁻¹ for C. albicans, C. glabrata and C. tropicalis (Teodoro et al., 2018). Ferulic acid from ethanol extract of Teucrium arduini L. has been shown to inhibit the C. albicans at MIC 4 mg mL⁻¹ (Kremer et al., 2013). Three phenolic acid compounds (caffeic, p-coumaric and ferulic acids) from the ethanol extract of Limonuim avei were efficient at a MIC of 4 mg mL⁻¹ against C. albicans (Nostro et al., 2012). Quercetin and quercetion derivatives showed antifungal activity against C. albicans and C. tropicalis with a MIC value of 31.2 µg mL⁻¹ (Agüero et al., 2014). C. albicans is a trimorphic yeast from the Ascomycota. Serum induces the transition of C. albicans from single cell yeast (blastospores) to two filamentous growth modes: pseudohyphae and hyphae (germ tubes) (Bastidas and Heitman, 2009). Filamentous forms of C. albicans are incriminated in strains virulence and contribute to the disease aggravation in both humans and animals. Thus, filamentation is receiving the most attention and are promising as a target for new anti-virulence treatments (Vila et al., 2017). The extracts demonstrating the maximum of inhibition on C. albicans were subjected to testing their effect on yeast morphological transitions. Under filamentation conditions, the ethanol and ethyl acetic extracts of winter collect demonstrated maximum of inhibition of 90% on germ tube formation until a concentration of 3.1 mg mL⁻¹, after 3 h and 50% of inhibition after 24 h. The ethanol extract of the spring collect showed 90% of inhibition, with a concentration of 6.2 mg mL⁻¹ after 3 h and 50% of inhibition after 24 h. The ethyl acetic extract of spring collect showed a maximum effect of 50% for both 3 h and 24 h incubation times, and this with a concentration of 3.1 mg mL⁻¹. In a previous study, the effect of polyphenols was investigated whether transcription of the gene HWP1, involved in C. albicans hyphal transition, showing a significant down regulation for curcumin and pyrogallol, and microscopy analysis confirmed that both of these compounds can clearly be observed to impede filamentation and biofilm formation (Shahzad et al., 2014).