Chemical Characterization and Antifungal Activity of Blue Tansy (Tanacetum annuum) Essential Oil and Crude Extracts against Fusarium oxysporum f. sp. albedinis, an Agent Causing Bayoud Disease of Date Palm

1. Introduction

The date palm (Phoenix dactylifera L.) is an important food source and commercial perennial crop in the Sahara and North Africa [1]. It plays essential economic and environmental roles in the oasis regions by creating a suitable microclimate for growing other food crops and protecting oases from silting and desertification [2]. In Morocco, the date palm has been cultivated on over 48,000 ha of traditional and genetically diverse oases and 20,000 ha of modern plantations cultivating commercial varieties in the southern part of the country [1,2]. One of the biggest destructive diseases of palm trees and a higher risk for the future of the date industrial sector in Morocco, as well as other countries producing dates, is commonly known in Morocco as “Bayoud”, which is a vascular disease, caused by a soil fungus, Fusarium oxysporum f. sp. albedinis (Foa), that blocks the circulation of raw sap in plants [2,3,4]. Bayoud was reported in Morocco for the first time in 1870, and it has been estimated that this fungus destroyed more than 10 million palm trees during the last 100 years [3,4]. Numerous strategies such as renewal of palm groves, genetic selection and fungicidal treatments have been applied to control Bayoud; however, they remain inefficient and difficult to apply [3,4]. Thus, there is an urgent requirement for discovering and developing new compounds that can control and combat Bayoud. Therefore, plants are an important natural source of secondary metabolites, such as alkaloids, terpenoids, phenolic compounds, glycosides, quinones and saponins, which play roles in the plant defence system against pathogen attacks during which their concentration in plants depends on pathosystem and environmental conditions [2].

Morocco is a country well-known for its richness and biodiversity of aromatic and medicinal plants, which are used in traditional medicine, by Moroccan populations, in a random way and with unidentified biomolecules and unknown concentrations. Among these plants, the Tanacetum species have been used for medicinal functions for decades due to their wide range of chemical, pharmacological and medicinal properties [5]. Accordingly, the species Tanacetum annum L. was chosen to conduct the present research.

T. annuum L. known as Blue Tansy, or as Moroccan blue chamomile, is an herbaceous plant that grows spontaneously in Mediterranean Bassan, and in particular in northwestern Morocco, Portugal, Spain and southern France. It belongs to the Compositae (or Asteraceae) family, which is considered the largest and within the most 20 prolific flowering plant families in the world [6]. T. annuum is used in traditional medicine as an anti-inflammatory, anti-histamine, anti-flogistic, and anti-pruriginic plant [7]; in addition, this plant has been tested for cytotoxic activity [7,8] and antifungal activity against a large spectrum of phytopathogenic fungi [9]. Nonetheless, to the best of our knowledge, and except for the essential oil, no research has been conducted on the chemical characterization of T. annuum, the antifungal activity against Fusarium oxysporum f. sp. albedinis (Foa), and the antioxidant activity. Herein, the objective of this study was first to investigate the chemical composition of essential oil and crude extracts of T. annuum collected from Northwestern Morocco, and secondly to evaluate the antioxidant (DPPH test) and antifungal activity (microdilution method) against Fusarium oxysporum f. sp. albedinis, an agent causing Bayoud disease of the Moroccan date palm, by using different extracts.

2. Results and Discussion

2.1. Extraction Yield

The extraction yield of essential oil (TaEO), crude methanolic (TaME) and hydro-methanolic (TaHME) extracts of T. annuum aerial parts are presented in . The obtained TaEO has a very dark blue color with a fruity and herbaceous aroma. Therein, the extraction yield of TaEO was on average 0.7% (wt/wt) of dry material, which was higher than the ones found previously with a percentage ranging between 0.23% and 0.5% [6,10,11], while our yield was noted to be lower than the studies by Greche et al. [12] and Zaim et al. [13] with the values of 0.8% and 1.2%, respectively.

Regarding the crude extracts, six extracts were prepared using three parts, including the stems, leaves and flowers, and two solvents, which were methanol (100%) and hydro-methanol (80:20, v/v). Generally, the results showed that hydro-methanol was the better solvent to obtain a high extraction yield in the three parts; in addition, the extraction yields of TaME and TaHME were closely similar to each other in the cases of flowers (26.66% and 28.90%) and leaves (20.07% and 21.89%), while in case of stems, the extraction yield of TaME was significantly lower than TaHME with values of 8.87% and 16.89%, respectively. These results demonstrated that extraction yields were affected by the plant part and solvent polarity used . The polarity of solvents plays an important role in increasing extraction yield; whereas, the solvent with a higher polarity, such as water, has the ability of solubility and absorbance of bioactive compounds more than solvents with less polarity such as methanol, ethanol, etc. [14,15,16]. These confirmed the present results that showed a higher extraction yield using a combination of methanol with water than with methanol alone.

2.2. Bioactive Compounds Contents

The present work was the first study to estimate the bioactive compounds including total phenolic content (TPC), total flavonoid content (TFC) and carotenoid contents (lycopene (TLC) and β-carotene (Tβ-CC)) of the medicinal plant T. annuum. As presented in , the three parts of T. annuum contained important bioactive compounds, in particular, TPC and TFC in both crude extracts TaME and TaHME with values varying between 51.32 and 116.32 mg/g of dry crude extract (DCE). Similar to the extraction yield, the hydro-methanolic extracted higher bioactive compounds than the absolute methanol, with a significant difference between the values of the two solvent extracts in each part.

The TPC of T. annuum was observed to be significantly higher in the leaves than in the flowers and stems. The highest amount was found in the leaves TaHME and the lowest was in stems TaME with values of 116.32 and 51.32 mg of gallic acid equivalents (GAEs)/g of DCE, respectively. The TPC value in leaves TaME in the present work was higher than the one in Tanacetum erzincanense (64.4 mg GAEs/g DCE) previously reported by Yapıcı et al. [17]; in addition, the TPCs of the present study were higher than several Tanacetum species methanolic extracts, with their values varying from 32.15 to 47.11 mg GAEs/mg DCE [18]. However, Devrnja et al. [19] reported that methanolic extracts of Tanacetum vulgare from Serbia contained higher TPC than our finding in the three parts stems, leaves and flowers with the values of 83.60, 112.60 and 96.20 mg GAEs/g of DCE, respectively.

Concerning TFC, the highest content was observed in the leaves TaHME with the value of 91.54 mg of catechin equivalents (CEs)/g of DCE, and the lowest content was in the leaves and stems TaME with the amounts 58.81 and 58.63 mg/g of DCE, respectively. Flowers contained significantly higher TFC value in the case of TaME (64.04 mg CEs/g DCE) and the leaves in the case of TaHME (91.54 mg CEs/g DCE). In addition, there was no significant difference in the comparison between stems and leaves in the case of TaME and between stems and flowers in the case of TaHME. In comparison with the previous studies, our TFC values in T. annuum were observed to be higher than several other Tanacetum species that were reported to contain TFC values ranging from 18.54 to 55.40 mg/g of DCE, using different parts of the plants [18,20]. Yapıcı et al. reported that the TFC value of the methanolic extract of T. erzincanense was 62.20 mg of quercetin equivalents per g of DCE, which was higher than TaME and lower than TaHME of the leaves TFC of the current study [17].

Regarding the carotenoids , lycopene was determined in the three parts of T. annuum with significantly different amounts from each other; whereas, stems contained the highest TLC, followed by leaves and flowers with the contents of 0.74, 0.69 and 0.19 µg/g of dry weight (DW), respectively. On the contrary, β-carotene was determined only in flowers with the value of 0.61 µg/g of DW.

Overall, the obtained results demonstrated that all parts of T. annuum contain an important amount of bioactive compounds, particularly total phenolic and total flavonoid, which possess a strong antioxidant activity and are responsible for many other biological activities. Moreover, the results demonstrated that bioactive compound contents were affected by solvent polarity and varied from one part to another. This verified what is described in the literature, the solvent with higher polarity extracted more bioactive compounds that are responsible for several bioactivities [14,15,16].

2.3. Chemical Composition of Essential Oil by GC-MS

The chemical composition of TaEO of mixed aerial parts was analysed by GC-MS, and the results are illustrated in the chromatogram in Figure 1 containing 39 peaks which correspond to 39 identified compounds, representing 99.92% of total volatile essential oil compounds in T. annuum. These 39 biologically active molecules are divided into three classes of terpenoids: namely, monoterpenoids (85.22%), sesquiterpenoids (14.21%) and diterpenoids (0.49%). The main compounds identified were camphor (16.69%), α-pinene (12.37%), bornyl acetate (11.97%), limonene (11.10%), borneol (6.33%), α-terpinyl acetate (4.62%) and chamazulene (3.49%). The GC-MS results of TaEO are listed in .

Figure 1. Chromatogram of T. annuum essential oil by GC–MS.

The main compounds identified in T. annuum in the present study are already known for several biological activities. The predominant compound camphor, which possesses anti-inflammatory and antimicrobial activities is widely used in the cosmetic and pharmaceutical industries [6]; in addition, this compound showed strong activity against several phytopathogens of Fusarium species [21]. The second main substance, α-pinene, was noted to have antioxidant [22], anticancer, antimicrobial (including against F. oxysporum [23]) and anti-inflammatory activities [24]. Bornyl acetate has also been recorded to have antioxidant, antimicrobial, anti-inflammatory, anti-tumour and insecticidal properties [25]. Furthermore, the compound limonene is widely used in cosmetics, medicine and agriculture due to its activities such as antioxidant, anti-inflammatory, anticancer, insecticidal, antibacterial and herbicidal activity, and it is registered as a pesticide and a fungicide in China [26]. Moreover, borneol, α-terpinyl acetate and chamazulene were reported to possess antioxidant, anti-inflammatory and antifungal properties [6,24,25,27]. Additionally, in essential oils, even minor compounds can exert biological effects due to synergistic effects between chemical classes [25].

Several research works have investigated the chemical compositions of T. annuum essential oils using GC-MS analysis, and the majority of these works were from the northern region of Morocco, such as a study conducted by Greche et al., which indicated that sabinene (22.3%), camphor (13.2%) and β-pinene (10.1%) were the major components obtained from T. annuum aerial parts essential oil [9]. Zaim et al. reported that TaEO of aerial parts (leaves and flowers) collected from the same area of the study by Greche et al. [9] (Larache, northern Morocco) was dominated by the substances myrcene (13.67%), camphor (12.67%), sabinene (9.49%) and β-pinene (7.70%) [13]. In addition, chamazulene (17.74%), sabinene (14.39%) and camphor (14.21%) were the main compounds identified in TaEO by Belcadi et al. [6]. Moreover, a study using industrial TaEO from Germany found that the essential oil was rich in sabinene (14%), camphor (13.6%), myrcene (8%), β-pinene (7.7%), chamazulene (6.9%), α-phellandrene (6.5%) and p-cymene (5%) [5]. Similar to the GC-MS results of this study, all of the previous studies reported that camphor was one of the main compounds identified in TaEO. Overall, many factors can typically affect the quantity and chemical composition of the essential oil, including site and period of collection, stage of growth, and methods and conditions of drying, storage and extraction [28].

2.4. Characterization of Phenolic Compounds by HPLC-MS

Phenolic compounds are natural bioactive compounds extracted mainly from plants that have demonstrated interesting biological activities, including antioxidant, anti-inflammatory, antimicrobial, and antiproliferative activities, among others, which has led to great interest in their use by numerous industries [29]. Therefore, the current study investigated, for the first time, the phenolic composition of the T. annuum plant using HPLC-MS. The individual phenolic compounds in the samples were identified and quantified based on their UV-Vis spectra, mass spectra, retention times and their areas by commercial standards [30]. Figure 2 demonstrates the HPLC-MS chromatogram illustrating the peaks of phenolic compounds present in the T. annuum aerial part, which were identified, quantified and presented in . From 19 identified compounds, 10 were phenolic acids (gallic, protocatechuic, chlorogenic, p-hydroxybenzoic, caffeic, syringic, ferulic acid, methylparaben, rosmarinic and salicylic acids) and 9 were flavonoids (catechin, rutin, luteolin 7-glucoside, apigenin 7-glucoside, luteolin, quercetin, apigenin and kaempferol). T. annum extract showed great content of individual polyphenols, which were mainly predominated by apigenin 7-glucoside (4540 µg/g DW), luteolin 7-glucoside (2804 µg/g), salicylic acid (1878 µg/g), rutin (786.40 µg/g), kaempferol (592.30 µg/g) and rosmarinic acid (579.9 µg/g), while gallic and caffeic acids were detected in the lowest amounts in the plant extract with the values of 11.29 and 7.46 µg/g, respectively. However, vanillic acid, ellagic acid, p-coumaric acid, vanillin, naringin and cinnamic acid were not detected in T. annuum hydro-methanolic extract.

Figure 2. HPLC-MS chromatogram of individual phenolic compounds in the hydro-methanolic extract of T. annuum aerial part, as detected at 280 nm.

The main compounds detected in the Blue Tansy (Moroccan Blue Chamomile) are well-known for several biological effects; for instance, the major flavonoid identified, apigenin 7-glucoside, a widely distributed food flavone in the genus of Tanacetum, has shown numerous biological properties, including antioxidants, anti-inflammatory, antiviral, antifungal and hepatoprotective [31,32]. The following flavonoid luteolin 7-glucoside, a primary active component of luteolin, has demonstrated multiple biological activities such as antimicrobial, antioxidant, anti-inflammatory and antitumor [33,34]. In addition, the third main identified compound salicylic acid is a phenolic acid well-known for its medicinal impact on a variety of skin disorders and contributes to skin exfoliation and removal of dead cells [35,36].

As there is no literature on phenolic compounds of T. annuum, the HPLC-MS results of this work will be compared with T. vulgare, which is a species closer to ours, and also compared with other Tanacetum such as T. balsamita and T. parthenium. Herein, the studied plant was more rich and diversified in polyphenols than the Tanacetum species cited above [20,34,37]. Bączek et al. found that T. vulgare and T. balsamita contained a higher concentration of phenolic acids than flavonoids which is contrary to our findings; however, apigenin-7-O-glucoside and luteolin 7-O-glucoside was observed as the main flavonoids in T. balsamita, which agrees with this study [37]. In the study by Babich et al., chlorogenic acid, ferulic acid and rosmarinic acid were the main phenolic compounds identified in T. vulgare from Russia [34]. The leaves of Tanacetum species T. vulgare, T. macrophyllum, and T. corymbosum as well as the aerial parts of T. parthenium have recently been found to have high levels of chlorogenic acid [20,38]. Generally, the diversity and richness of phenolic compounds differ from one plant to another. Overall, the aerial part of the investigated plant T. annuum can be considered a great natural source of polyphenols, which are important for the development of several agricultural and pharmaceutical products.

2.5. Antifungal Activity and Synergistic Effect

The antifungal activity of T. annuum crude extracts and essential oil was carried out against the fungal pathogen Fusarium oxysporum f. sp. albedinis (Foa) that causes Bayoud disease, vascular wilt in date palm, which is considered a major constraint that impedes the development of the date palm sector in North Africa and Sahara, and more specifically in Morocco [39]. This research revealed that all T. annuum extracts tested effectively inhibited the growth of Foa, indicating the presence of strong antifungal compounds. The minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) values of different tested samples are listed in ; whereas the concentrations required to cause growth inhibition of Foa ranged from 3.33 to 9.17 mg/mL by crude extracts, and from 0.5 to 4 mg/mL by commercial fungicides and its combination with essential oil. Moreover, the best growth inhibition result was obtained by essential oil alone with MIC and MFC values of 3.33 and 4.58 µL/mL, respectively. Concerning the crude extracts, the leaves showed the highest anti-Foa property with an MIC of 3.33 mg/mL for both solvents and an MFC of 7.50 and 8.33 mg/mL for TaHME and TaME, respectively. The anti-Foa activity of extracts from the stems and flowers was slightly similar. In addition, generally speaking, there were no significant differences between TaME and TaHME of each part, these could be due to their similarity and no higher difference in the amounts of bioactive compounds such as total phenolic and total flavonoids or individual polyphenols, which are already known for their antimicrobial activity [29]. Therefore, as there was a highly similar amount of total phenolic and total flavonoids between the three parts and by using the two solvents, the major individual phenolic compounds (namely, apigenin 7-glucoside, luteolin 7-glucoside, salicylic acid, rutin, kaempferol, rosmarinic acid, ferulic acid, quercetin, syringic acid and catechin) found in the mixed aerial parts of T. annuum could be the main phytochemical contributors for the growth inhibition of the harmful pathogen Foa by acting alone or in synergy. In terms of mechanisms of action, phenolic acids and flavonoids can exhibit antifungal activity by disrupting cell division, hyphal formation and/or triggering severe oxidative stress leading to cell death. This may be attributed to the direct impact of active phytochemical ingredients on fungal cells or due to the induction of defense mechanisms in the host plant [40,41].

Regarding commercial fungicides which are used against Bayoud diseases, the results demonstrated that SAAF 75% inhibited the growth of Foa better than carbendazim 50% with a half dose. As presented in , the results obtained on the synergistic effects of TaEO in combination with the two used synthetic fungicides showed a synergistic interaction in the association with carbendazim 50% with an FICI value of 0.4, while showing an additive interaction in the case of the combination between TaEO and SAAF 75% with an FICI value of 0.65. The combination of TaEO (3 µL/mL) with the used fungicides (1 mg/mL) reduced the dose for inhibition of the pathogen in half for SAAF 75% and by two thirds for carbendazim 50% . The combination of individual compounds, essential oil and other antifungal agents possess particular benefits for antimicrobial activity, and their mechanism of action has multiple targets on microorganisms such as cell wall destruction, increasing permeability, damaging of the cytoplasmic membrane, membrane protein damage, and coagulation of cytoplasm resulting in metabolic damage and cell death [42]. Generally, this combination could be an interesting alternative to combat Bayoud diseases.

The strong antifungal activity of T. annuum essential oil against Foa could be attributed to its major compounds such as camphor which possessed several biological activities [6], in particular, a strong activity against several phytopathogens of the Fusarium species [21] and the major substance α-pinene noted to have activity against F. oxysporum [23]. Additionally, bornyl acetate and limonene have been recorded as having antimicrobial and insecticidal properties, and limonene is used as a fungicide [25,26]. In addition to that, the other main compounds, borneol, α-terpinyl acetate and chamazulene, have been reported to have the ability of growth inhibition of fungal pathogens [6,24,25,27], and not forgetting that in essential oil, even the minor compounds can exert biological effects due to synergistic effects between chemical classes [25]. Concerning the mechanism of action of essential oil, the antifungal activity of TaEO might be caused by the properties of terpenes/terpenoids that—due to their highly lipophilic nature and low molecular weight—are capable of disrupting the cell membrane, causing cell death or inhibiting the sporulation and germination of food spoilage fungi [43,44], which also can be caused due to synergistic effects between chemical classes [25]. Furthermore, several studies on antifungal activity reported that T. annuum essential oil has been able to inhibit the growth of numerous fungal species at different concentrations, including Alternaria solani, Botrytis cinerea, Helminthosporium oryzae, Pyricularia oryzae, Verticillium dahliae [9], Penicillium expansum [6], Aspergillus niger, Candida albicans and Cryptococcus neoformans [45].

Several research works investigated the antifungal activity of numerous plants’ crude extracts and essential oils against F. oxysporum f. sp. albedinis [1,2,3,46,47]. The study by Rahmouni et al. investigated the antifungal activity of essential oils of five medicinal and aromatic plants collected from northern Morocco against the same pathogen isolate F. oxysporum f. sp. Albedinis, and the results demonstrated that Origanum compactum (MIC = 2.5 and MFC = 5 µL/mL) showed better activity than the four plants Myrtus communis, Thymus satureioides, Lavandula dentata and Rosmarinus officinalis with MIC and MFC ranges between 10 and 40 µL/mL; in addition, these last four plants were also less active against Foa than the present essential oil [3]. On the other hand, Rahmouni et al. also evaluated the activity of the major components of its plant which included three molecules present in the current analysed essential oil as major compounds, namely, α-pinene, borneol and with a small percentage of α-terpineol, and these compounds possessed an anti-Fusarium effect [3]. The previous studies by Chibane et al. [47] and Chibane et al. [1] cited, respectively, that the essential oil of Cladanthus eriolepis and Asteriscus graveolens showed growth inhibition values of 86.20% and 100% against Foa at the concentration of 4 µL/mL, which is slightly higher than our concentration. Concerning the crude extracts, Bouhlali et al., previously determined the antifungal activity of five plant extracts, namely, Acacia cyanophylla, Cupressus atlantica, Eucalyptus torquata, Nerium oleander and Schinus molle, against Foa, and results showed that all extracts possessed antifungal activity, where the extracts of E. torquata and C. atlantica showed the strongest antifungal effect resulting in the inhibition of mycelial growth, sporulation and spore germination in a dose-dependent manner [2].

Overall, the crude extracts and essential oil of T. annuum showed a strong antifungal activity due to the ability of their major compounds to inhibit the growth of the pathogen. These compounds will be our future work to separately evaluate their anti-Fusarium activity and to understand the mechanisms and their connection with their antifungal action.

2.6. Antioxidant Activity

The antioxidant activity of the crude extracts TaME and TaHME of the T. annuum parts (stems, leaves and flowers) was evaluated using the DPPH (2,2-Diphenyl-1-picrylhydrazyl) assay, which is valid, accurate, quick, easy, economic and one of the most widely used methods for assessing the antioxidant properties of the extracts or purified compounds [48]. Figure 3 illustrates the increase of DPPH radical activities of the six extracts with the increase of the tested samples’ concentration (ranging from 0.25 to 4 mg/mL). Based on radical-scavenging activity inhibition (%), IC₅₀ values of the samples were graphically determined by their concentration providing 50% inhibition values (Figure 3 and . Each high IC₅₀ value indicates a lower antiradical potential [49].

Figure 3. Radical-scavenging activity (RSA) on DPPH radicals of T. annuum crude extracts. Values are expressed as the mean ± SD (n = 3).

As shown in , all the crude extracts exhibited a great antioxidant capacity as expected, considering the high amounts of total phenolic and total flavonoid contents in each part extract. The IC₅₀ values ranged between 0.22 and 0.65 mg/mL. The highest antioxidant effect was shown by TaHME (stems) with a value of 0.22 mg/mL, and the lowest one was observed in TaME (leaves) with a concentration of 0.65 mg/mL; in addition, there was no significant difference between the leaves’ and flowers’ antioxidant capacity in the two extract cases, TaME and TaHME. However, the antioxidant activity of the TaHME extracts was slightly better than TaME in the three parts . Ascorbic acid was used as a positive control and showed stronger antioxidant properties with the IC₅₀ value equal to 0.036 mg/mL. The phenolic compounds including phenolic acids and flavonoids are considered primary antioxidants, which have the ability to neutralize the free radicals or inhibit the production of free radicals from hydroperoxides by donating hydrogen atoms to lipid radicals [50]. The mechanism of antioxidant actions involved either hydrogen atom transfer, transfer of a single electron, sequential proton loss electron transfer, or chelation of transition metals [51].

To our knowledge, the antioxidant potential of different parts of T. annuum has not been evaluated by any previous studies. For this reason, comparisons will be made with other Tanacetum species. Herin, several studies investigated the antiradical activity of Tanacetum species using aerial parts mixed or separated and different solvents; therefore, their results varied from one sample to another. Baranauskienė et al. performed the antioxidant activity of T. vulgare, which gave the best result using water extract with EC₅₀ = 1.33 mg/mL (EC = effect concentration); this finding was lower than the one obtained in the current study [52]. T. vulgare was also the objective of a study by Devrnja et al., investigating the antioxidant activity of methanolic extracts of stems, leaves and flowers, with obtained IC₅₀ values of 0.080, 0.077 and 0.058 mg/mL, respectively [19]. Furthermore, Esmaeili et al. [18] evaluated the antioxidant properties of the ethanolic extracts of the aerial part of six Tanacetum species (T. budjnurdense, T. hololeucum, T. chiliophyllum, T. sonboli, T. tabrisianum and T. kotschyi), and the results showed IC₅₀ values ranged between 0.060 and 0.157 mg/mL, which were better than our findings. Overall, the Moroccan T. annuum could be considered a good source of antioxidant compounds.

3. Materials and Methods

3.1. Chemical Material

Alkane standards (C₈–C₂₀ and C₂₁–C₄₀), Folin–Ciocalteu phenol reagent, l-ascorbic acid, dimethyl sulfoxide (DMSO), PDA (Potato Dextrose Agar), RPMI-1640 and all individual phenolic compounds were obtained from Sigma-Aldrich, Co., (St. Louis, MO, USA). Acetonitrile, diethyl ether and ethyl acetate were purchased from Merck KGaA (Darmstadt, Germany). Acetone, hexane and n-hexane were acquired from CABLO ERBA Reagent, S.A.S (Val de Reuil Cedex, France). SAAF (Mancozeb 63% + Carbendazim 12%) was obtained from LAKORALE (Casablanca, Morocco) and GOLDAZIM 500 SC = 500 g/L (Carbendazim 50%) from PHILEA (Casablanca, Morocco). DPPH (2,2-diphenyl-l-picrylhydrazyl) was obtained from Alfa Aesar (Ward Hill, MA, USA). Methanol and all other solvents were purchased from Honeywell (St. Muskegon, MI, USA).

3.2. Plant Material

The aerial parts (stems, leaves and flowers) of the plant Tanacetum annuum were harvested during the flowering period of July–September 2020 at the Biological and Ecological Interest Site (SIBE) of Ben Karrich, a site of biological and ecological interest belonging to the Tetouan province in northwestern Morocco (35°30′50.6″ N. 5°26′13.9″ W, 220 m of altitude, thermo-Mediterranean vegetation level, a subhumid bioclimatic level at temperate winter, and siliceous substrate). The plant identification was based on the name given by specialists in mass cultivation of this plant and confirmed in our laboratory using the determination key “Practical Flora of Morocco: Manual of Determination of Vascular Plants” [53]. The different aerial parts of the plant were dried in the shade at room temperature for 7 days. They were then crushed, and the resulting fine powder (20 mesh) was stored in a dark glass bottle until the preparation of the plant’s EO and crude extracts.

3.3. Preparation of Essential Oil and Crude Extracts

The isolation of the TaEO was made from the aerial parts of the T. annuum by hydrodistillation for 3 h, using a Clevenger-type apparatus, where 100 g of fine powder was immersed in a 1000 mL flask of distilled water. The obtained oil was stored at −25 °C in a dark glass vial until use.

The crude extract preparation of the three parts, stems, leaves and flowers, was carried out by using two solvents, methanol and hydro-methanol (methanol/water, 80:20, v/v), and following a procedure used previously by Erbiai et al. [54] with some modifications. One gram of powder of each part was added to 10 mL of used solvents; then, the mixture was agitated using a magnetic stirrer at room temperature for 24 h and filtered through Whatman grade 4 qualitative filter paper. The unfiltered part was again extracted two times using the same procedures. The combined extracts were evaporated under reduced pressure at 40 °C by using a rotary vacuum evaporator to give dry extracts, which were then weighed and stored at −25 °C for further use.

The preparation of TaEO and T. annuum crude extracts was performed in triplicate and the extraction yield was calculated for each sample.

3.4. GC-MS Analysis of Essential Oil

The chemical components of TaEO were characterized by GC-MS, following the protocol described early by Erbiai et al. [55]. Briefly, a volume of 10 µL of TaEO was diluted in 90 µL of n-hexane and analysed using gas chromatography (GC) (Trace 1300 gas chromatograph; Thermo Fisher Scientific, Waltham, MA, USA) coupled with mass spectrometry (MS) (ISQ single quadrupole mass spectrometer; Thermo Fisher Scientific). The GC was equipped with the TG5-MS capillary column (60 m × 0.25 mm i.d.; 0.25 µm film thickness) with a non-polar stationary phase (5% phenyl and 95% dimethylpolysiloxane). The oven temperature was programmed to increase from 40 °C to 350 °C at a rate of 5 °C/min. The carrier gas used was helium with a flow rate of 1.2 mL/min. The components of TaEO were identified based on Kovats retention indices (RI) relative to those of a homologous series of known standards of alkanes mixture (C₈–C₂₀ and C₂₁–C₄₀) and with the spectral data obtained from the National Institute Standard and Technology (NIST) databases of the corresponding substance. Data acquisition and analysis were performed by Software Thermo XcaliburTM 2.2 SP1.48 and NIST MS Search 2.2 Library 2014, respectively.

3.5. Determination of Bioactive Compound Contents

The content determination of total phenolic (TPC), total flavonoid (TFC) and total carotenoids (β-carotene (Tβ-CC) and lycopene (TLC)) in T. annuum leaves and stems crude extracts was carried out using spectrophotometric methods [54].

TPC determination was performed by following the Folin–Ciocalteu method. A total of 1 mL of each crude extract solution (1 mg/mL) was mixed in a tube with 5 mL of Folin–Ciocalteu reagent (diluted at 1/10) and 4 mL of sodium carbonate (7.5%). The tube was mixed by vortex for 15 s and allowed to react by standing in the dark for 30 min at 40 °C. Afterwards, the mixture absorbance was measured at 760 nm against a blank. Gallic acid was used as a standard for obtaining the calibration curve and the results of different samples were expressed in mg of gallic acid equivalents (GAE) per gram of dry crude extract (DCE).

TFC was estimated by the aluminium chloride colourimetric method. Herein, a volume of 250 µL of each diluted plant extract (1 mg/mL) was mixed with 1100 µL of distilled water and 75 µL of NaNO₂ solution (5%). After 5 min, 75 µL of AlCl₃ solution (10%) was added and allowed to stand for a further 6 min; then, 1000 µL NaOH solution (4%) was added and mixed by vortex apparatus. The mixture was allowed to stand for another 15 min before measuring the absorbance against the blank at 510 nm. The TFC existing in the extracts was qualified using the standard calibration curve of flavonoid, (+)-catechin, and the results were expressed as mg of catechin equivalents (CE)/g of DCE.

The concentration of β-carotene and lycopene for each extract was determined following a procedure previously described by the authors Nagata and Yamashita [56]. Two hundred grams of fine powder was mixed with 5 mL of acetone–hexane mixture (4:6) for one minute. The solution was filtrated through Whatman N° 4 paper and the absorbance (A) was measured at 453, 505, 645, and 663 nm. The amounts of β-carotene and lycopene were determined using the following the equations: β-carotene (mg/100 mL) = [(0.216 × A₆₆₃) − (1.22 × A₆₄₅) − (0.304 × A₅₀₅) + (0.452 × A₄₅₃)] and Lycopene (mg/100 mL) = [(−0.0458 × A₆₆₃) + (0.204 × A₆₄₅) + (0.372 × A₅₀₅) − (0.0806 × A₄₅₃)].

3.6. HPLC-MS Analysis of Individual Phenolic Compounds

3.7. Evaluation of Antifungal Activity

3.8. Evaluation of Antioxidant Activity

The antioxidant activity of Blue Tansy crude extracts was evaluated using an assay of free DPPH^• (2,2-diphenyl-1-picrylhydrazyl) radical-scavenging activity (RSA) using a modified version of the methodology outlined in previous studies [54,62]. A volume of 300 µL of the plant samples or standard (ascorbic acid) at concentrations ranging from 4 to 0.25 and 1 to 0.06 mg/mL, respectively, was added to 2.7 mL of methanol solution of DPPH. The mixture was incubated in darkness for 30 min at room temperature, subsequently, the absorbance was measured at 517 nm using a UV-Vis spectrophotometer against blank. The percentage of DPPH scavenging activity was calculated as % inhibition of DPPH using the following equation: RSA (%) = [(A_DPPH − A_Sample)/A_DPPH] × 100. Samples or standard concentration providing 50% DPPH scavenging (IC₅₀) was graphically calculated. Ascorbic acid was used as a reference standard in this study.

3.9. Data Analysis

All the measurements were made in triplicate, and the results were expressed as mean ± standard deviation (SD). The statistical significance was calculated by the application of a one-way ANOVA test, followed by post hoc Tukey’s multiple comparison tests with p ≤ 0.05 level considered significant, by using GraphPad Prism 8.0.1 software.

4. Conclusions

Bayoud, the biggest destructive disease of palm trees by the fungal pathogen Fusarium oxysporum f. sp. albedinis (Foa) urgently requires the discovery and development of new compounds that can control or aid in controlling this disease. The present study permitted evaluation for the first time of the crude extracts’ chemical composition of Tanacetum annuum aerial parts; proved the antioxidant activity of extracts; and demonstrated the antifungal activity of extracts and essential oil against Bayoud disease (Foa). The results proved that the plant extracts contained important bioactive compounds, mainly the phenolic compounds (total and individual (19 substances)), as well as the essential oil being composed of 39 volatile compounds classed mostly into the monoterpenoids class (85.22%). This richness and variety of compounds present in T. annuum has contributed strongly to antioxidant and anti-Foa activity. The crude extracts demonstrated a strong antioxidant property with small IC₅₀ values. Regarding anti-Foa, all extracts of T. annuum exhibited potent capacity at low concentrations. Therefore, it can be suggested that T. annuum essential oil, crude extracts and a combination of essential oil and commercial fungicides, which showed a strong antifungal capacity, could be a useful alternative to protect and combat fungal diseases, in particular Foa. However, further study is required to evaluate the anti-Foa activity of the major compounds identified in T. annuum, separately and/or in combination, and to understand the mechanisms and their connection with their antifungal action.

References

1. Chibane, E.; Essarioui, A.; Ouknin, M.; Boumezzourh, A.; Bouyanzer, A.; Majidi, L. Antifungal Activity of Asteriscus Graveolens (Forssk.) Less Essential Oil against Fusarium oxysporum f. sp. albedinis, the Causal Agent of “Bayoud” Disease on Date Palm. Moroc. J. Chem. 2020, 8, 456–465. [Google Scholar] [CrossRef]
2. Bouhlali, E.D.T.; Derouich, M.; Ben-Amar, H.; Meziani, R.; Essarioui, A. Exploring the Potential of Using Bioactive Plant Products in the Management of Fusarium oxysporum f.sp. albedinis: The Causal Agent of Bayoud Disease on Date Palm (Phoenix dactylifera L.). Beni-Suef Univ. J. Basic Appl. Sci. 2020, 9, 46. [Google Scholar] [CrossRef]
3. Rahmouni, A.; Saidi, R.; Khaddor, M.; Pinto, E.; Da Silva Joaquim Carlos Gomes, E.; Maouni, A. Chemical Composition and Antifungal Activity of Five Essential Oils and Their Major Components against Fusarium oxysporum f. sp. albedinis of Moroccan Palm Tree. Euro-Mediterr. J. Environ. Integr. 2019, 4, 27. [Google Scholar] [CrossRef]
4. Dihazi, A.; Jaiti, F.; WafaTaktak; kilani-Feki, O.; Jaoua, S.; Driouich, A.; Baaziz, M.; Daayf, F.; Serghini, M.A. Use of Two Bacteria for Biological Control of Bayoud Disease Caused by Fusarium oxysporum in Date Palm (Phoenix dactylifera L.) Seedlings. Plant Physiol. Biochem. 2012, 55, 7–15. [Google Scholar] [CrossRef]
5. Stappen, I.; Wanner, J.; Tabanca, N.; Bernier, U.R.; Kendra, P.E. Blue Tansy Essential Oil: Chemical Composition, Repellent Activity Against Aedes Aegypti and Attractant Activity for Ceratitis Capitata. Nat. Prod. Commun. 2021, 16, 1934578X21990194. [Google Scholar] [CrossRef]
6. Belcadi, H.; Aknouch, A.; El Amrani, S.; Chraka, A.; Lachkar, M.; Mouhib, M.; Zantar, S.; Ibnmansour, A. Gamma-Irradiation Effect on the Chemical Composition and Antibacterial Activity of the Moroccan Tanacetum annuum L. Essential Oil. Heliyon 2023, 9, e16625. [Google Scholar] [CrossRef]
7. Haddar, S.E.; Greche, H.; Bakri, Y.; Benjouad, A. Chemical Composition and Anti-Prolifertaive Properties of the Essential Oil of Tanacetum annuum L. Moroc. J. Biol. 2008, 4, 17–23. [Google Scholar]
8. Hmiri, S.; Amrani, N.; Rahouti, M. Détermination in Vitro de l’activité Antifongique Des Vapeurs d’eugénol et d’huiles Essentielles de Mentha pulegium L. et de Tanacetum annuum L. Vis-à-Vis de Trois Champignons Responsables de La Pourriture Des Pommes En Post-Récolte. Acta Bot. Gall. 2011, 158, 609–616. [Google Scholar] [CrossRef]
9. Greche, H.; Hajjaji, N.; Ismaïli-Alaoui, M.; Mrabet, N.; Benjilali, B. Chemical Composition and Antifungal Properties of the Essential Oil of Tanacetum annuum. J. Essent. Oil Res. 2000, 12, 122–124. [Google Scholar] [CrossRef]
10. Greche, H.; Ismaili-Alaoui, M.; Zrira, S.; Benjilali, B.; Belanger, A.; Hajjaji, N. Composition of Tanacetum annuum L. Oil from Morocco. J. Essent. Oil Res. 1999, 11, 343–348. [Google Scholar] [CrossRef]
11. Barrero, A.F.; Sánchez, J.F.; Altarejos, J.; Zafra, M.J. Homoditerpenes from the Essential Oil of Tanacetum annuum. Phytochemistry 1992, 31, 1727–1730. [Google Scholar] [CrossRef]
12. Greche, H.; Mrabet, N.; Ismaili-Alaou, M.; Hajjaji, N.; Benjilali, B. Détermination Des Paramètres de La Standardisation de l’huile Essentielle de Tanacetum annuum L. In Recherches sur les Plantes Aromatiques et Médicinales; Institut National des Plantes Médicinales et Aromatiques (INPMA): Mezraoua (Taounate) et Fès, Morocco, 2009; pp. 114–129. [Google Scholar]
13. Zaim, A.; Benjelloun, M.; EL Harchli, E.H.; Farah, A.; Meni Mahzoum, A.; Alaoui Mhamdi, M.; El Ghadraoui, L. Chemical Composition and Acridicid Properties of the Moroccan Tanacetum annuum L. Essential Oils. Int. J. Eng. Sci. 2015, 5, 13–19. [Google Scholar]
14. El Mannoubi, I. Impact of Different Solvents on Extraction Yield, Phenolic Composition, in Vitro Antioxidant and Antibacterial Activities of Deseeded Opuntia Stricta Fruit. J. Umm Al-Qura Univ. Appl. Sci. 2023, 9, 176–184. [Google Scholar] [CrossRef]
15. Liharaka Kidaha, M.; Alakonya, A.E.; Nyende, A.B. Bioactivity Determination of Methanol and Water Extracts for Roots and Leaves of Kenyan Psidium guajava L. Landraces against Pathogenic Bacteria. SpringerPlus 2013, 2, 670. [Google Scholar] [CrossRef] [PubMed]
16. Nawaz, H.; Shad, M.A.; Rehman, N.; Andaleeb, H.; Ullah, N. Effect of Solvent Polarity on Extraction Yield and Antioxidant Properties of Phytochemicals from Bean (Phaseolus vulgaris) Seeds. Braz. J. Pharm. Sci. 2020, 56, e17129. [Google Scholar] [CrossRef]
17. Yapıcı, İ.; Altay, A.; Öztürk Sarıkaya, B.; Korkmaz, M.; Atila, A.; Gülçin, İ.; Köksal, E. In Vitro Antioxidant and Cytotoxic Activities of Extracts of Endemic Tanacetum erzincanense Together with Phenolic Content by LC-ESI-QTOF-MS. Chem. Biodivers. 2021, 18, e2000812. [Google Scholar] [CrossRef]
18. Esmaeili, M.A.; Sonboli, A.; Ayyari Noushabadi, M. Antioxidant and Protective Properties of Six Tanacetum Species against Hydrogen Peroxide-Induced Oxidative Stress in K562 Cell Line: A Comparative Study. Food Chem. 2010, 121, 148–155. [Google Scholar] [CrossRef]
19. Devrnja, N.; Anđelković, B.; Aranđelović, S.; Radulović, S.; Soković, M.; Krstić-Milošević, D.; Ristić, M.; Ćalić, D. Comparative Studies on the Antimicrobial and Cytotoxic Activities of Tanacetum vulgare L. Essential Oil and Methanol Extracts. S. Afr. J. Bot. 2017, 111, 212–221. [Google Scholar] [CrossRef]
20. Zengin, G.; Cvetanović, A.; Gašić, U.; Stupar, A.; Bulut, G.; Şenkardes, I.; Dogan, A.; Ibrahime Sinan, K.; Uysal, S.; Aumeeruddy-Elalfi, Z.; et al. Modern and Traditional Extraction Techniques Affect Chemical Composition and Bioactivity of Tanacetum parthenium (L.) Sch.Bip. Ind. Crop. Prod. 2020, 146, 112202. [Google Scholar] [CrossRef]
21. Huo, H.; Gu, Y.; Cao, Y.; Liu, N.; Jia, P.; Kong, W. Antifungal Activity of Camphor against Four Phytopathogens of Fusarium. Res. Sq. 2021, 1, 18. [Google Scholar] [CrossRef]
22. Dai, J.; Zhu, L.; Yang, L.; Qiu, J. Chemical Composition, Antioxidant and Antimicrobial Activities of Essential Oil from Wedelia Prostrata. EXCLI J. 2013, 12, 479–490. [Google Scholar] [PubMed]
23. Marei, G.I.K.; Abdelgaleil, S.A.M. Antifungal Potential and Biochemical Effects of Monoterpenes and Phenylpropenes on Plant. Plant Prot. Sci. 2018, 54, 9–16. [Google Scholar] [CrossRef]
24. Fanouriou, E.; Kalivas, D.; Daferera, D.; Tarantilis, P.; Trigas, P.; Vahamidis, P.; Economou, G. Hippocratic Medicinal Flora on the Greek Island of Kos: Spatial Distribution, Assessment of Soil Conditions, Essential Oil Content and Chemotype Analysis. J. Appl. Res. Med. Aromat. Plants 2018, 9, 97–109. [Google Scholar] [CrossRef]
25. Mssillou, I.; Agour, A.; Allali, A.; Saghrouchni, H.; Bourhia, M.; El Moussaoui, A.; Salamatullah, A.M.; Alzahrani, A.; Aboul-Soud, M.A.M.; Giesy, J.P.; et al. Antioxidant, Antimicrobial, and Insecticidal Properties of a Chemically Characterized Essential Oil from the Leaves of Dittrichia viscosa L. Molecules 2022, 27, 2282. [Google Scholar] [CrossRef]
26. Luo, W.; Wang, K.; Luo, J.; Liu, Y.; Tong, J.; Qi, M.; Jiang, Y.; Wang, Y.; Ma, Z.; Feng, J.; et al. Limonene Anti-TMV Activity and Its Mode of Action. Pestic. Biochem. Physiol. 2023, 194, 105512. [Google Scholar] [CrossRef]
27. Vaičiulytė, V.; Ložienė, K.; Švedienė, J.; Raudonienė, V.; Paškevičius, A. α-Terpinyl Acetate: Occurrence in Essential Oils Bearing Thymus Pulegioides, Phytotoxicity, and Antimicrobial Effects. Molecules 2021, 26, 1065. [Google Scholar] [CrossRef]
28. Greche, H.; Ismaili-Alaoui, M.M.; Hajjaji, N.; Zrira, S.; Bellanger, A.; Benjilani, B. Rendement et Composition Chimique de l’huile Essentielle de Tanacetum annuum L. in Plantes Aromatiques et Médicinales et Leurs Huiles Essentielles. In Proceedings of the Actes (Eds.), Plantes Aromatiques et Médicinales et Leurs Huiles Essentielles; I.A.V. Hassan II: Rabat, Morocco, 1997; pp. 169–176. [Google Scholar]
29. Albuquerque, B.R.; Heleno, S.A.; Oliveira, M.B.P.P.; Barros, L.; Ferreira, I.C.F.R. Phenolic Compounds: Current Industrial Applications, Limitations and Future Challenges. Food Funct. 2021, 12, 14–29. [Google Scholar] [CrossRef]
30. El Mihyaoui, A.; Charfi, S.; Erbiai, E.H.; Pereira, M.; Duarte, D.; Vale, N.; Candela Castillo, M.E.; Badoc, A.; Lamarti, A.; Esteves da Silva, J.C.G.; et al. Phytochemical Compounds and Anticancer Activity of Cladanthus Mixtus Extracts from Northern Morocco. Cancers 2023, 15, 152. [Google Scholar] [CrossRef]
31. Di Carlo, G.; Mascolo, N.; Izzo, A.A.; Capasso, F. Flavonoids: Old and New Aspects of a Class of Natural Therapeutic Drugs. Life Sci. 1999, 65, 337–353. [Google Scholar] [CrossRef]
32. Miguel, F.G.; Cavalheiro, A.H.; Spinola, N.F.; Ribeiro, D.L.; Barcelos, G.R.M.; Antunes, L.M.G.; Hori, J.I.; Marquele-Oliveira, F.; Rocha, B.A.; Berretta, A.A. Validation of a RP-HPLC-DAD Method for Chamomile (Matricaria recutita) Preparations and Assessment of the Marker, Apigenin-7-Glucoside, Safety and Anti-Inflammatory Effect. Evid.-Based Complement. Altern. Med. ECAM 2015, 2015, 828437. [Google Scholar] [CrossRef]
33. Chen, S.; Yang, B.; Xu, Y.; Rong, Y.; Qiu, Y. Protection of Luteolin-7-O-Glucoside against Apoptosis Induced by Hypoxia/Reoxygenation through the MAPK Pathways in H9c2 Cells. Mol. Med. Rep. 2018, 17, 7156–7162. [Google Scholar] [CrossRef] [PubMed]
34. Babich, O.; Larina, V.; Krol, O.; Ulrikh, E.; Sukhikh, S.; Gureev, M.A.; Prosekov, A.; Ivanova, S. In Vitro Study of Biological Activity of Tanacetum Vulgare Extracts. Pharmaceutics 2023, 15, 616. [Google Scholar] [CrossRef] [PubMed]
35. Arif, T. Salicylic Acid as a Peeling Agent: A Comprehensive Review. Clin. Cosmet. Investig. Dermatol. 2015, 8, 455–461. [Google Scholar] [CrossRef] [PubMed]
36. Calvisi, L. Efficacy of a Combined Chemical Peel and Topical Salicylic Acid-Based Gel Combination in the Treatment of Active Acne. J. Cosmet. Dermatol. 2021, 20 (Suppl. S2), 2–6. [Google Scholar] [CrossRef]
37. Bączek, K.B.; Kosakowska, O.; Przybył, J.L.; Pióro-Jabrucka, E.; Costa, R.; Mondello, L.; Gniewosz, M.; Synowiec, A.; Węglarz, Z. Antibacterial and Antioxidant Activity of Essential Oils and Extracts from Costmary (Tanacetum balsamita L.) and Tansy (Tanacetum vulgare L.). Ind. Crop. Prod. 2017, 102, 154–163. [Google Scholar] [CrossRef]
38. Ivănescu, B.; Tuchilu, C.; Corciov, A.; Lungu, C.; Mihai, C.T.; Gheldiu, A.-M.; Vlase, L. Antioxidant, Antimicrobial and Cytotoxic Activity of Tanacetum vulgare, Tanacetum corymbosum and Tanacetum macrophyllum Extracts. FARMACIA 2018, 66, 282–288. [Google Scholar]
39. Khoulassa, S.; Elmoualij, B.; Benlyas, M.; Meziani, R.; Bouhlali, E.D.T.; Houria, B.; Alaoui, Y.E.H.; Haridas, S.; Guo, J.; Lipzen, A.; et al. High-Quality Draft Nuclear and Mitochondrial Genome Sequence of Fusarium oxysporum f. sp. albedinis Strain 9, the Causal Agent of Bayoud Disease on Date Palm. Plant Dis. 2022, 106, 1974–1976. [Google Scholar] [CrossRef]
40. Khalil, A.M.A.; Saleh, A.M.; Abo El-Souad, S.M.S.; Mohamed, M.S.M. Plants from a Semi-Arid Environment as a Source of Phytochemicals against Fusarium Crown and Foot Rot in Zucchini. AMB Express 2023, 13, 6. [Google Scholar] [CrossRef]
41. Ansari, M.; Anurag, A.; Fatima, Z.; Hameed, S. Natural Phenolic Compounds: A Potential Antifungal Agent. Microb. Pathog. Strateg. Combat. Sci. Technol. Educ. 2013, 1, 1189–1195. [Google Scholar]
42. BeşiRiK, N.Ş.; Göger, G. Antimicrobial Evaluation of the Patchouli (Pogostemon cablin Benth) Leaf Essential Oil Combination with Standard Antimicrobial Compounds. Int. J. Second. Metab. 2023, 10, 385–393. [Google Scholar] [CrossRef]
43. Nazzaro, F.; Fratianni, F.; Coppola, R.; De Feo, V. Essential Oils and Antifungal Activity. Pharmaceuticals 2017, 10, 86. [Google Scholar] [CrossRef] [PubMed]
44. Al-Zereini, W.A.; Al-Trawneh, I.N.; Al-Qudah, M.A.; TumAllah, H.M.; Al Rawashdeh, H.A.; Abudayeh, Z.H. Essential Oils from Elettaria cardamomum (L.) Maton Grains and Cinnamomum verum J. Presl Barks: Chemical Examination and Bioactivity Studies. J. Pharm. Pharmacogn. Res. 2022, 10, 173–185. [Google Scholar] [CrossRef]
45. Powers, C.N.; Satyal, P.; Mayo, J.A.; McFeeters, H.; McFeeters, R.L. Bigger Data Approach to Analysis of Essential Oils and Their Antifungal Activity against Aspergillus niger, Candida albicans, and Cryptococcus neoformans. Molecules 2019, 24, 2868. [Google Scholar] [CrossRef] [PubMed]
46. Mezouari, A.; Makhloufi, A.; Bendjima, K.; Benlarbi, L.; Boulanouar, A.; Makhloufi, K.; Jes&ugrave, M.D.; Gonzalez, S. Antifungal Activity of Acacia Tortilis Subsp. Raddiana Tar on Fusarium oxysporum f.sp. albedinis, the Cause of Bayoud Disease of the Date Palm in Southwest Algeria. Indian J. Agric. Res. 2019, 53, 713–717. [Google Scholar] [CrossRef]
47. Chibane, E.M.; Ouknin, M.; Renucci, F.; Costa, J.; Majidi, L. Chemical Profile, Antioxidant and Antifungal Activity of Essential Oil from Cladanthus eriolepis. J. Essent. Oil Bear. Plants 2020, 23, 1296–1305. [Google Scholar] [CrossRef]
48. Kedare, S.B.; Singh, R.P. Genesis and Development of DPPH Method of Antioxidant Assay. J. Food Sci. Technol. 2011, 48, 412–422. [Google Scholar] [CrossRef]
49. Pokorný, J.; Yanishlieva, N.; Gordon, M. Antioxidants in Food: Practical Applications, 1st ed.; Woodhead Publishing: Cambridge, UK, 2001; ISBN 978-1-85573-463-0. [Google Scholar]
50. Shafi, A.; Jubair, A.; Hossain, M.A.; Hossain, M.M.; Azam, M.S.; Biswas, M. Free Radical-Scavenging Capacity and HPLC-DAD Screening of Phenolic Compounds from Pulp and Seed of Syzygium claviflorum Fruit. J. Agric. Food Res. 2021, 6, 100203. [Google Scholar] [CrossRef]
51. Zeb, A. Concept, Mechanism, and Applications of Phenolic Antioxidants in Foods. J. Food Biochem. 2020, 44, e13394. [Google Scholar] [CrossRef]
52. Baranauskienė, R.; Kazernavičiūtė, R.; Pukalskienė, M.; Maždžierienė, R.; Venskutonis, P.R. Agrorefinery of Tanacetum vulgare L. into Valuable Products and Evaluation of Their Antioxidant Properties and Phytochemical Composition. Ind. Crop. Prod. 2014, 60, 113–122. [Google Scholar] [CrossRef]
53. Fennane, M.; Ibn Tattou, M. Observations sur la flore vasculaire endémique, rare ou menacée du Maroc. Flora Mediterr. 1999, 9, 113–124. [Google Scholar]
54. Erbiai, E.H.; da Silva, L.P.; Saidi, R.; Lamrani, Z.; Esteves da Silva, J.C.G.; Maouni, A. Chemical Composition, Bioactive Compounds, and Antioxidant Activity of Two Wild Edible Mushrooms Armillaria mellea and Macrolepiota procera from Two Countries (Morocco and Portugal). Biomolecules 2021, 11, 575. [Google Scholar] [CrossRef] [PubMed]
55. Erbiai, E.H.; Maouni, A.; Pinto da Silva, L.; Saidi, R.; Legssyer, M.; Lamrani, Z.; Esteves da Silva, J.C.G. Antioxidant Properties, Bioactive Compounds Contents, and Chemical Characterization of Two Wild Edible Mushroom Species from Morocco: Paralepista flaccida (Sowerby) Vizzini and Lepista nuda (Bull.) Cooke. Molecules 2023, 28, 1123. [Google Scholar] [CrossRef]
56. Nagata, M.; Yamashita, I. Simple Method for Simultaneous Determination of Chlorophyll and Carotenoids in Tomato Fruit. Nippon Shokuhin Kogyo Gakkaishi 1992, 39, 925–928. [Google Scholar] [CrossRef]
57. Erbiai, E.H.; Bouchra, B.; da Silva, L.P.; Lamrani, Z.; Pinto, E.; da Silva, J.C.G.E.; Maouni, A. Chemical Composition and Antioxidant and Antimicrobial Activities of Lactarius sanguifluus, a Wild Edible Mushroom from Northern Morocco. Euro-Mediterr. J. Environ. Integr. 2021, 6, 43. [Google Scholar] [CrossRef]
58. Sghir, F.; Touati, J.; Mouria, B.; Touhami, A.O.; Filali-Maltouf, A.; Modafar, C.E.; Moukhli, A.; Douira, R.B.A. Variation in Pathogenicity of Fusarium oxysporum f. sp. albedinis on Two Cultures Associated with Date Palm of Moroccan Oasis. World J. Pharm. Life Sci. 2016, 2, 56–68. [Google Scholar]
59. Clinical and Laboratory Standards Institute. Reference Method for Broth Dilution Antifungal Susceptibility Testing of Filamentous Fungi, 2nd ed.; CLSI Document M38–A2; Clinical and Laboratory Standards Institute: Wayne, PA, USA, 2008. [Google Scholar]
60. Parker, R.A.; Gabriel, K.T.; Graham, K.D.; Butts, B.K.; Cornelison, C.T. Antifungal Activity of Select Essential Oils against Candida Auris and Their Interactions with Antifungal Drugs. Pathogens 2022, 11, 821. [Google Scholar] [CrossRef]
61. Afeltra, J.; Dannaoui, E.; Meis, J.F.G.M.; Rodriguez-Tudela, J.L.; Verweij, P.E. In Vitro Synergistic Interaction between Amphotericin B and Pentamidine against Scedosporium prolificans. Antimicrob. Agents Chemother. 2002, 46, 3323–3326. [Google Scholar] [CrossRef]
62. Heleno, S.A.; Barros, L.; Sousa, M.J.; Martins, A.; Santos-Buelga, C.; Ferreira, I.C.F.R. Targeted Metabolites Analysis in Wild Boletus Species. LWT—Food Sci. Technol. 2011, 44, 1343–1348. [Google Scholar] [CrossRef]