Torilis arvensis ethanolic extract: Phytochemical analysis, antifungal efficacy, and cytotoxicity properties

Abdallah Khalil; Eman A. Abdelwahab; Omaima A. Sharaf; Abdulaziz A. Al-Askar; Przemysław Kowalczewski; Ahmed Abdelkhalek; Said Behiry

doi:10.1515/chem-2024-0113

Article Open Access

Torilis arvensis ethanolic extract: Phytochemical analysis, antifungal efficacy, and cytotoxicity properties

Abdallah Khalil , Eman A. Abdelwahab , Omaima A. Sharaf , Abdulaziz A. Al-Askar , Przemysław Kowalczewski , Ahmed Abdelkhalek and Said Behiry

Published/Copyright: November 29, 2024

Published by

Become an author with De Gruyter Brill

Submit Manuscript Author Information Explore this Subject

From the journal Open Chemistry Volume 22 Issue 1

Abstract

The aim of the current study is to assess the phytochemical contents, antifungal activity, and cytotoxicity characteristics of an ethanolic extract derived from the entire Torilis arvensis plant. High-performance liquid chromatography examination of the extract revealed that the primary phenolic components were benzoic, o-coumaric, and vanillic acids with concentrations of 259.1, 220.4, and 111.3 µg/g of extract, respectively. The highest flavonoids were catechol (117.9 µg/g) and kaempferol (108.7 µg/g). The extract is notable for its high concentration of long-chain saturated and unsaturated fatty acids, as well as its presence of 17 gas chromatography-mass spectrometry bioactive chemicals. Three soil-borne pathogenic fungi, Rhizoctonia solani, Fusarium solani, and Fusarium oxysporum, were molecularly identified and assessed for the antifungal activity of the extract. The extract showed the highest growth inhibition against R. solani, F. oxysporum, and F. solani at 300 µg/mL, with inhibition rates of 88.9, 71.5, and 67.8%, respectively. T. arvensis treatments were generally non-toxic after proceeding with cytotoxicity assay on the onion root tip cells, with no chromosomal abnormalities detected even at the highest concentration (300 µg/mL). These findings highlight the potential of T. arvensis extract as a safe and effective antifungal agent with a rich phytochemical profile.

Keywords: Torilis arvensis ; secondary metabolites; HPLC; GC-MS; soil-borne fungi; cytotoxicity

1 Introduction

The application of natural-origin bioactive components in combating microbial diseases is on the rise, reflecting the ongoing advancements in phytochemistry knowledge [1]. Plants are an excellent source of antioxidants due to their diverse composition and the abundance of secondary metabolites in their tissues [2]. Polyphenolic compounds are natural antioxidants found throughout various parts of plants and exhibit nonenzymatic antioxidant characteristics [3]. Furthermore, plant extracts containing phenolic compounds exhibit a variety of biological actions, including antibacterial, antiviral, antifungal, and anti-inflammatory properties [4,5,6,7]. The antifungal properties of polyphenols are well-documented, e.g., ellagic acid, gallic acid, and caffeic acid phenethyl ester have exhibited antifungal activity against Candida and Cryptococcus yeasts [8]. Additionally, vanillin and tannic acid have been shown to exhibit antifungal properties against several fungal species, including Fusarium graminearum, Aspergillus flavus, Aspergillus fumigatus, Fusarium solani, and Penicillium digitatum [9].

One of the common weeds imposed on many cereals and plants is hedge parsley, Torilis arvensis (Huds.) (Apiales: Apiaceae), which features a spiny and hairy structure. Some Torilis species possess an obvious disinfectant and antimicrobial role against several plants’ pathogens [10]. Several analytical studies have been conducted for the crude extract and essential oil of different parts of T. arvensis. The headspace solid-phase microextraction of the aerial parts of T. arvensis subsp. neglecta revealed 34 compounds, which were significant for essential oil screening [10]. These included spathulenol, β-farnesene, and β-caryophyllene. Gas chromatography profiling of the essential oil extracted from the leaves and stems of the common hedge parsley plant (T. arvensis) revealed the presence of several compounds, such as trans-β-farnesene, β-cubebene, cis-α-farnesene, β-caryophyllene, and nuciferyl acetate. However, the paramount sesquiterpene ester, (+)-(Z)-lanceol acetate, was only found in the fruit essential oil [11]. In crude extracts and phytochemical screenings from various provinces, the Torilis genus exhibits an imbalance between phenolic and terpene compounds. The isolated terpenes from the Torilis genus have demonstrated significant advancements in their antimicrobial and biological properties [12].

Phytopathogenic fungi pose significant challenges in agricultural management. Fusarium oxysporum is a major vascular wilt pathogen that causes root rot and damping-off in more than 150 types of crops [13]. Fusarium solani is a common soil-borne pathogen responsible for wilt and rot issues affecting nearly 111 plant species across 87 genera [14]. The Rhizoctonia genus encompasses numerous species, with R. solani being recognized as the most harmful soil-borne pathogen. Its form and capacity to affect a wide range of crops globally stand in stark contrast to those of other species [15]. Consequently, the objective of our investigation was to assess the impact of an ethanolic extract derived from the whole Torilis arvensis plant on the growth of three soil-borne fungal species: Fusarium oxysporum, Fusarium solani, and Rhizoctonia solani. Additionally, the cytotoxicity of various extract concentrations was evaluated. The extract was analyzed using high-performance liquid chromatography (HPLC) and gas chromatography-mass spectrometry (GC-MS) to identify the phytochemical composition of secondary metabolites.

2 Materials and methods

2.1 Isolation and characterization of fungal isolates

The selected fungal strains were obtained and purified from a tomato field in Alexandria governorate, Egypt. The morphology of the isolated strains was observed using a light microscope. The Campbell et al. [16] morphological identification handbook was utilized to precisely determine the genus classification. A fungal genomic extraction kit (Thermo Fisher Scientific PureLink Genomic DNA Mini Kit, Waltham, Massachusetts, USA) was used to get DNA from each fungal isolate that had been growing on potato dextrose agar (PDA) medium for 7 days. The amplification of the ribosomal internal transcribed spacer (ITS) was conducted using specific primers for ITS1 and ITS4, as described by White et al. [17]. The polymerase chain reaction (PCR) was controlled by the settings proposed by Soliman et al. [18]. After amplification, the PCR products were purified and sequenced using the Sanger method. The acquired sequences were annotated and compared to the GenBank database using NCBI alignments. The fungal sequences were then submitted to the GenBank database to obtain accession numbers. The phylogenetic tree was constructed to compare the sequenced isolates with the most likely in the GenBank database.

2.2 Cold-extraction technique of T. arvensis extract

The T. arvensis whole plant (TAWP) materials were collected from El-Hamam city in Marsa Matrouh Governorate, Egypt, near the geographical coordinates 30°50′25.8″N 29°23′56.0″E. We verified that the chosen plants were free from any observable morphological disease symptoms. The Department of Plant Production, a constituent unit of the Faculty of Agriculture at Saba Basha, Alexandria University, Egypt, identified the collected plants. The plant specimen voucher was stored in the herbarium and assigned the number 08-95-ALX. The plant materials were then transported to the laboratory to eliminate any debris or impurities by subjecting them to a 30 min washing process using running tap water. The TAWP was air-dried at a constant temperature of 25 ± 3°C for 2 weeks. Using a grinding mill, a pulverized form of the completely dried component was produced. A total of 500 mL of ethanol solvent with a purity of 96% was designated for the cold extraction process of 100 g of the dehydrated powder. After a week, the ethanolic extract solution was filtered using filter paper and concentrated using a rotary evaporator. Subsequently, the acquired raw extracts were placed in airtight glass containers and kept at a temperature below 0°C until they were required [19,20].

2.3 Antifungal assay

The extract’s efficacy in inhibiting the growth of plant-damaging fungus strains was assessed using the poisoned food technique described by Heflish et al. [21]. Varying quantities of the botanical extract were introduced to PDA plates, yielding concentrations of 0, 100, 200, and 300 μg/mL. Subsequently, a 5 mm disc of fungal specimens (7 days old). Subsequently, the disc was positioned at the midpoint of every treated medium plate. The plates were placed in an incubator and maintained at a consistent temperature of 25°C for 7 days. The experimental design incorporated three replicates. The antifungal activity was assessed by quantifying the percentage suppression of fungal mycelial growth after incubation. To compute, we used the following equation:

Mycelial growth inhibition ( % ) = [ ( A 0 − A t ) / A 0 ] × 100 ,

where A ₀ represents the average diameter of the untreated fungal growth, and (A _t) signifies the average diameter of the fungal growth following treatment.

2.4 Cytotoxicity assay

Onion (Allium cepa L.) root tips (3n = 18) were utilized for cytological analysis. Four concentrations of TAWP extract were prepared, along with a control (0, 100, 200, and 300 µg/mL). For each concentration, three healthy onions were grown in a glass jar (250 mL) at room temperature for 48 h. The root tips or meristems measured 1–1.5 cm in length, allowing for the observation of various stages of mitotic division. After collecting all the root tips, the material was fixed in a solution of 95% ethanol and 3:1 glacial acetic acid (v/v) for 24 h. Following that, the tips of the roots were removed from the fixation solution, submerged in a 70% ethanol solution, and stored in a refrigerator (4°C) while they were studied as previously explained [22].

The various stages of mitosis in onions were observed both before and after treatments with TAWP extract. Furthermore, the percentage of mitotic index M (%) for each treatment is determined using the following formula: M% = (number of dividing cells/total number of cells) × 100. The entire set of slides was examined, and a karyotyping system was used to capture micrographs of the aberrant cells at a consistent level of magnification. The frequency of chromosomal abnormalities was assessed using the method (A _b%) = (number of aberrant cells/total number of dividing cells) × 100 [23].

2.5 HPLC instrumentation and operating conditions

To find out how much polyphenolic material was in the ethanolic extract of TAWP, an Agilent 1260 Infinity HPLC Series system with a quaternary pump was used. The HPLC was conducted under specific conditions. A Zorbax Eclipse Plus C18 column, measuring 100 mm in length and 4.6 mm in inner diameter, was employed for the separation process. The separation was conducted at a temperature of 30°C. To achieve component separation, we employed a tertiary linear elution gradient. The gradient consisted of acetonitrile, methanol, and water, with a concentration of 0.2% H₃PO₄ (v/v) of HPLC-grade. The mixture was administered to the participants via injection at a volume of 20 µL. Individual molecules were successfully separated and isolated using variable wavelength detector (VWD) specifically calibrated for 284 nm detection. HPLC-standard compound index: The raw material contained 23 different phenolic and flavonoid compounds, including benzoic acid, o-coumaric acid, catechol, vanillic acid, pyrogallol, quinol, gallic acid, p-hydroxybenzoic acid, catechin, chlorogenic acid, caffeic acid, syringic acid, p-coumaric acid, ferulic acid, ellagic acid, resveratrol, rosmarinic acid, myricetin, kaempferol, rutin, cinnamic acid, quercetin, and naringenin [24].

2.6 GC-MS instrumentation and operating conditions

The bioactive components in the TAWP’s ethanolic extract were detected using Agilent 6890 GC-MS equipment. The GC-MS system comprised an Agilent mass spectrometer and a direct capillary interface. Additionally, a capillary column made of fused silica, specifically the HP-5MS, was used. The column’s dimensions were 30 m in length and 0.25 mm in diameter, with a film thickness of 0.25 µm. The temperature of the column was increased from 50 to 230°C at a rate of 5°C/min. After being consistently maintained at this level for 2 min, the column eventually achieved a temperature of 290°C. We successfully identified the bioactive metabolites we had collected by doing searches in the MS library, particularly in the NIST and Wiley databases. For identification purposes, the mass spectra and retention times were compared with data from the Wiley and NIST MS lab databases. This was done to ensure accurate data collection [25].

2.7 Statistical analysis

Using the Statistical Analysis System (SAS), version 9.1.3 Service Pack 4 (SAS Institute, Cary, NC, USA, 2002), all laboratory experiment data were subjected to one-way ANOVA for variance analysis. The LSD test at the 0.05 significance level was employed to determine significant differences in the mean values.

3 Results

3.1 Fungal isolates identification

All fungal isolates were consistently recovered from tomato-infected root tissues that were cultured on PDA. The isolates were initially identified at the genus level based on their morphological characteristics. The colonies of Fusarium species displayed a white to pink coloration, with conidia that were observed to be microconidia and Fusarium-shaped under a light microscope. The Rhizoctonia-like isolate was characterized by septate hyphae that branched at approximately 90°. Sequencing of the ITS region of these isolates confirmed the morphological identifications. The NCBI-BLAST alignment and phylogenetic tree analysis of the annotated nucleotide sequence of the ITS region indicated that the three fungal isolates were identified as Fusarium oxysporum, Fusarium solani, and Rhizoctonia solani (Figure 1). The nucleotide sequence with annotations has been submitted to the GenBank database with the following accession numbers: OR116512 for the Fusarium oxysporum isolate FO-100, OQ891115 for the Fusarium solani isolate F69, and OR116532 for the Rhizoctonia solani isolate RHS-301. The nucleotide sequence of the three isolates demonstrated complete sequence coverage and similarity with other related species available in GenBank, particularly the Chinese Fusarium oxysporum isolate N-61-2 (Acc# MT560381), the Egyptian Fusarium solani isolate Wf12 (Acc# OL410542), and the Turkish Rhizoctonia solani isolate 35_693_AG4HGII_RHIZ (Acc# MT484259), as illustrated in Figure 1.

Figure 1

The phylogenetic relationships among the fungal isolates Fusarium solani F69 (OQ891115), Fusarium oxysporum FO-100 (OR116512), and Rhizoctonia solani RHS-301 (OR116532) were determined based on their ITS region sequences. This tree includes closely related fungal sequences from GenBank and is rooted with the outgroup isolate Pythium aphanidermatum KH1. The phylogenetic tree was constructed using the UPGMA method in MEGA program version 11. The reliability of the inferred phylogeny was assessed through 2,000 bootstrap replicates.

3.2 Antifungal activity of T. arvensis ethanolic extract

Following a 7-day incubation period, various concentrations of TAWP extract were applied to PDA media to evaluate their effectiveness in suppressing the growth of the fungal isolate, as shown in Table 1 and Figure 2. Meanwhile, the radial growth activities of the isolated strains were assessed by measuring their diameters. All doses of TAWP extract exhibited identical growth responses (diameter: 10 mm) against R. solani. The F. oxysporum showed the smallest increase in diameter (25.7 mm) when exposed to TAWP extract at a dose of 300 µg/mL. The growth of F. solani was significantly inhibited when PDA media was used at concentrations of 200 and 300 µg/mL, resulting in growth measurements of 32 and 29 mm, respectively. In general, the dosages of TAWP extract exhibited a significantly more potent inhibitory impact on the development of the selected isolates compared to the negative control (0.00 µg/mL) (Table 1). Based on the acquired results, all concentrations of TAWP extract demonstrated the highest level of growth inhibition (88.9%) against R. solani. Concentrations of 200 and 300 µg/mL PDA resulted in inhibitions of 64.4 and 67.8% against F. solani, respectively, in the second rank.

Table 1

Radial growth activity of the isolated fungi strains on contaminated PDA media with series concentrations of T. arvensis extract after 7 days of incubation

Concentrations (µg/mL)	Radial growth diameter (mm) ± SD*			Growth inhibition percentage (%)
Concentrations (µg/mL)	R. solani	F. oxysporum	F. solani	R. solani	F. oxysporum	F. solani
100	10 ± 0.0b	32.7 ± 1.15b	34.7 ± 0.58b	88.9	63.7	61.5
200	10 ± 0.0b	31.7 ± 0.58b	32 ± 1.73cb	88.9	64.8	64.4
300	10 ± 0.0b	25.7 ± 0.58c	29 ± 2.65c	88.9	71.5	67.8
Negative control**	90 ± 0.0a	90 ± 0.00a	90 ± 0.0a	0	0	0

*Standard deviation. **Untreated PDA media (0.00 µg/mL). The same letters adjoining the growth values per every column are insignificantly distinguished and computed by the LSD_0.05.

Figure 2

Antifungal activity of T. arvensis extract against Fusarium solani (a), Fusarium oxysporum (b), and Rhizoctonia solani (c) at concentrations 100, 200, and 300 µg/mL, compared to the control.

3.3 Genotoxicity evaluation of the extract

This study evaluated the effects of various concentrations of T. arvensis (TWAP) extract (100, 200, and 300 µg/mL) on mitotic division and chromosomal abnormalities in onion (Allium cepa L.), using water as a control. The root tips displayed typical stages of cell division, as detailed in Table 2. Following 48 h exposure to TWAP extract, cell counts of 1,446, 1,463, and 1,309 were recorded for the respective concentrations, with 784, 732, and 645 cells undergoing mitosis. This resulted in mitotic indices of 54.22, 50.03, and 49.27%, respectively, in comparison to the high mitotic index observed in the control. The T. arvensis treatments were generally non-toxic to the onion root tip cells, with no chromosomal abnormalities detected even at the highest concentration (300 µg/mL). A clear inverse relationship was noted between TWAP concentration and the mitotic index.

Table 2

Number of cells observed, dividing cells, mitotic index (%), abnormal cells, and percentage of aberrations (%) in onion root tips after exposure to various concentrations of T. arvensis extract

Concentrations (µg/mL)	Treatments	OC	DC	M%
100	Control	1,290	967	74.96
100	Treated	1,446	784	54.22
200	Control	1,324	1,016	76.74
200	Treated	1,463	732	50.03
300	Control	1,336	980	73.35
300	Treated	1,309	645	49.27

Control: onion root tips treated with water, Treated: onion root tips treated with various concentrations of T. arvensis extract after 48 h of exposure, OC: observed cells number, DC: divided cells number, M%: mitotic index, AC: abnormal cells number, A _b%: aberrations percentage.

3.4 HPLC analysis of T. arvensis ethanolic extract

The TAWP extract contained a total of 17 phytochemical components, indicated by their peak areas (mAU, milli-Absorbance Unit), retention times (min), and concentrations (µg/g) as shown in Figure 3 and Table 3. The major phenolic constituents identified in the TAWP extract were benzoic acid (259.04 µg/g), o-coumaric acid (220.36 µg/g), and vanillic acid (111.31 µg/g). A lot of different types of minor phenolic compounds were also found. These include p-hydroxy benzoic acid, catechin, chlorogenic acid, caffeic acid, syringic acid, p-coumaric acid, ferulic acid, ellagic acid, and resveratrol. The primary flavonoid found in the TAWP extract was catechol (117.93 µg/g), then kaempferol, at a concentration of 108.71 µg/g. Furthermore, there are small flavonoids present, namely, rutin and quercetin.

Figure 3

HPLC chromatogram of phenolic and flavonoid compounds of T. arvensis ethanolic extract.

Table 3

Analysis of phenolics and flavonoids of the T. arvensis ethanolic extract using HPLC

Detected compounds	Retention time (min)	Concentration (µg/g)
Catechol	5.43	117.93
p-Hydroxy benzoic acid	7.66	61.63
Catechin	9.01	10.56
Chlorogenic	9.26	38.64
Vanillic acid	9.70	111.31
Caffeic acid	10.09	43.58
Syringic acid	10.37	27.61
p-Coumaric acid	12.92	2.37
Benzoic acid	14.03	259.04
Ferulic acid	15.39	22.20
Rutin	16.70	47.17
Ellagic acid	16.91	46.17
o-Coumaric acid	17.07	220.36
Resveratrol	19.58	26.36
Cinnamic acid	20.14	26.73
Quercetin	21.62	71.40
Kaempferol	24.25	108.71

3.5 GC-MS profile of T. arvensis ethanolic extract

The GC-MS data of TAWP extract throughout a retention time extended from 0.00 to 36.46 min contained 17 phyto-components (Figure 4). All these detected phyto-components were compared with the libraries of Wiley Registry 8E, Replib, Nist_msms, and Mainlib, besides relative abundance area (%) (Table 4). Major bioactive moieties (highest abundant area) in TAWP extract were displayed by nizatidine (43.75%), 2-propanamine, 2-methyl- (18.50%), 2-methylenebrexane (10.52%), and benzyl chloride (7.49%).

Figure 4

GC-MS chromatogram of T. arvensis ethanolic extract.

Table 4

Chemical composition of T. arvensis ethanolic extract using GC-MS analysis

RT (min)	Relative abundance%	Compounds	Chemical group
3.95	7.49	Benzyl chloride	—
10.07	0.71	Dodecanal	Long-chain fatty aldehyde
11.27	0.42	Decane, 1-chloro-	Non-aromatic halogenated hydrocarbons
11.37	1.17	1-Dodecanol	Fatty acid alcohol
11.95	43.75	Nizatidine	Triazole
14.00	0.49	Oxirane, tetradecyl-	Fatty acyls
15.22	0.67	1-Hexadecanol	Fatty acid alcohol
15.71	18.50	2-Propanamine, 2-methyl-	—
19.55	3.02	Hexadecanoic acid, methyl ester	Long-chain saturated fatty acids
20.24	1.93	n-Hexadecanoic acid	Long-chain saturated fatty acids
22.22	1.89	9,12-Octadecadienoic acid (Z,Z)-, methyl ester	Long-chain unsaturated fatty acids
22.32	2.47	9-Octadecenoic acid (Z)-, methyl ester	Long-chain unsaturated fatty acids
22.49	13.74	2-Methylenebrexane	—
22.73	0.53	Methyl stearate	Fatty acid methyl esters
22.99	2.32	9-Octadecenoic acid (Z)-	Long-chain unsaturated fatty acids
27.03	0.90	(9e,12e)-9,12-Octadecadienoyl chloride	—

4 Discussion

The study of phytochemistry has mainly focused on enhancing our comprehension of the roles played by bioactive compounds sourced from plants [26]. Recent recommendations emphasize the need for more research into the active chemicals discovered in the crude extract of T. arvensis, which may have previously unknown antifungal properties [10,12]. The findings of this investigation indicate that all doses of TAWP ethanolic extract exhibited the most potent inhibitory effects on R. solani in comparison to the other fungal isolates examined. At concentrations of 200 and 300 µg/mL, F. solani exhibited a notable inhibition, placing it in the second rank. At 300 µg/mL, the TAWP extract demonstrated reduced efficacy in inhibiting the growth of F. oxysporum. Multiple studies have revealed that the phenols, terpenes, and chemotypes found in the Torilis genus plant demonstrate significant antibacterial properties against various plant diseases.

In this research, o-coumaric acid and catechol, two HPLC-phenolic compounds found in the ethanolic extract of TAWP, may be thought of as bioactive agents that stop the growth of this particular fungus. Previous studies showed that coumaric acid posed a slight inhibitory effect against R. solani [27]. Furthermore, naturally generated catechol could confer a significant anti-growth effect against F. oxysporum [28] and R. solani [29]. Several studies have previously supported our claim that some HPLC-minor phenolic compounds in TAWP extract may be able to inhibit fungal growth. Free gallic acid could inhibit the growth of F. oxysporum f. sp. niveum [30]. Gallic acid, which is one of the many phenols found in Coccoloba uvifera L. leaf extract, was thought to inhibit R. solani from growing [31]. Catechin is one of the main HPLC-polyphenolic moieties found in Pinus wallichiana leaf extract. It suppresses the mycelial growth of F. oxysporum f. sp. cubense [32]. Nawrocka et al. [33] also found that synthesized catechin enhanced by Trichoderma atroviride in cucumber could inhibit the mycelia growth of R. solani. The naturally synthesized chlorogenic acid in the grafted root of watermelon provided anti-mycelial growth for F. oxysporum [34] and R. solani [35]. Caffeic acid exhibited a slight antigrowth activity on F. oxysporum f. sp. niveum [36]. However, low levels of syringic acid could make the Fusarium pathogens that live in the rhizosphere of C. sativus L. seedlings more active, while relatively high levels could have the opposite effect [37].

Ferulic acid, a phenolic compound found in leaf extracts of wild grapevine Vitis spp., has the potential to inhibit the growth of F. oxysporum [38]. Ellagic acid in Musa paradisiaca L. flakes extract may have antimicrobial activity against R. solani [31]. Pyrogallol, a natural source, is considered a safe antifungal agent against various fungi strains, such as Aspergillus flavus and Candida albicans. The role of pyrogallol may be considered a pro-oxidant that could generate sufficient quantities of reactive oxygen species [39]. The presence of the p-hydroxybenzoic acid moiety in the TAWP extract at a low concentration of 61.63 µg/g was also unexpected, as it demonstrated the ability to inhibit growth. This observation was noted by Wu et al. [40], who discovered that incorporating p-hydroxybenzoic acid at concentrations of 800 mg/L or lower into the culture medium of F. oxysporum f. sp. niveum resulted in a slight enhancement of its growth. At a concentration of 1,600 mg/L, this acid may significantly reduce the mycelial mass and conidial germination rate. However, the TAWP extract contains a considerable level of benzoic acid (259.04 µg/g), which is a key compound that could inhibit the growth of F. oxysporum. Wu et al. [41] previously established that incorporating benzoic acid in amounts greater than 200 mg/L into the medium of F. oxysporum f. sp. niveum and R. solani significantly suppressed their mycelial growth and biomass. No inhibition was observed in Fusarium solani when grown on an artificial medium containing benzoic acid [42]. Isolated resveratrol can prevent some fungal pathogens from growing [43]. Borrego-Muñoz et al. [44] found that some synthetic bioisoster resveratrol analogs had important biological effects on F. oxysporum. Rosmarinic acid extracted from the fine roots of Ocimum basilicum L. significantly inhibited the growth of F. oxysporum [45].

Kaempferol, on the other hand, was one of the most important flavonoids found in the HPLC analysis of TAWP extract. This flavonoid appears to exhibit properties that inhibit the growth of the isolated fungi that were tested. Free kaempferol was used to treat seeds against the soil pathogen F. oxysporum, and it worked well as a control in a greenhouse [46]. Some minor flavonoids found through HPLC, such as rutin, cinnamic acid, and quercetin, might stop the growth of the chosen fungi that were isolated for study. Rutin, a flavonoid found in the peel extracts of Musa paradisiaca L., is considered an antimicrobial agent against R. solani [31]. The presence of free cinnamic acid significantly impaired the morphology and development of hyphae in F. oxysporum [47]. Two distinct forms of isorhamnetin derived from quercetin might offer protection against F. oxysporum in infected onions, A. cepa L. [48], as well as R. solani and F. solani [49].

GC-MS tests showed that the TAWP ethanolic extract contains long-chain saturated fatty acids (LCSFAs). These include hexadecanoic acid (C16:0), n-hexadecanoic acid (C16:0), and methyl ester (C17:0). These LCSFAs have a lot of hydrophobic groups that could help them stick to the cell membrane of microbes. According to Guimarães and Venâncio [50], these LCSFAs were thought to effectively stop the growth of the present fungal isolates. On the other hand, they found that the cis-form LCUFAs in TAWP extract, which are represented by 9,12-octadecadienoic acid (Z,Z)-, methyl ester (C19:2), 9-octadecanoic acid (Z)-(C18:1), methyl ester (C19:2), and 9-octadecanoic acid (Z)-(C18:1), have the greatest impact on the cell membrane of microbes. However, the identified long-chain fatty aldehyde, dodecanal, had no proven role as an antimicrobial agent; it was thought to trigger signaling [50]. The cytotoxicity assessment of the TAWP extract revealed that all tested concentrations were non-toxic to the root tip cells of Allium cepa L., indicating that the extract is safe for use at these levels. This safety, combined with its potent antifungal properties, suggests that the TAWP extract could be effectively utilized in agricultural systems without harming plant cells. The work provides valuable insights into the antifungal properties of T. arvensis extract. However, a broader approach to extraction methods, a wider range of concentrations, and the inclusion of additional fungal isolates could strengthen the findings. Additionally, exploring specific antifungal mechanisms and conducting genotoxicity assessments on diverse organisms would enhance the study’s relevance.

5 Conclusion

The TAWP ethanolic extract proved to be safe for plant cells while exhibiting the strongest growth inhibitory effect on Rhizoctonia solani. In terms of effectiveness, the extract also demonstrated notable antigrowth effects on Fusarium solani at concentrations of 200 and 300 µg/mL, while its impact on Fusarium oxysporum was less pronounced, observed only at the 300 µg/mL concentration. These findings can be attributed to the presence of certain phenolic compounds and flavonoids identified through HPLC analysis, as well as the profiles of long-chain saturated fatty acids (LCSFAs) and cis-long-chain unsaturated fatty acids (cis-LCUFAs) revealed by GC-MS. Consequently, it is recommended that TAWP extract be considered a promising option for controlling the growth of R. solani, with effective management of F. solani and F. oxysporum at relatively higher concentrations.

tel: +20-1007556883

aabdelkhalek@srtacity.sci.eg

Acknowledgements

The authors would like to extend their appreciation to the Researchers Supporting Project number (RSP2024R505), King Saud University, Riyadh, Saudi Arabia.

Funding information: The research is financially supported by Researchers Supporting Project Number (RSP2024R505), King Saud University, Riyadh, Saudi Arabia.
Author contributions: Conceptualization and methodology: A.K., S.B., and A.A. Software and validation: S.B., E.A., O.A., P.K., and A.A. Formal analysis and writing-original draft: A.K., O.A., S.B., E.A., A.A.Al., P.K., and A.A. Supervision: A.A. All co-authors reviewed the final version and approved the manuscript before submission.
Conflicts of interest: The authors declare no conflicts of interest.
Data availability statement: The datasets used and/or analyzed through this study are accessible from the corresponding author upon reasonable request.

References

[1] Bashir S, Behiry S, Al-Askar AA, Kowalczewski PŁ, Emaish HH, Abdelkhalek A. Antibacterial, antifungal, and phytochemical properties of Salsola kali ethanolic extract. Open Life Sci. 2024;19:20220962.10.1515/biol-2022-0962Search in Google Scholar PubMed PubMed Central

[2] Bédoui I, Nasr HBen, Ksouda K, Ayadi W, Louati N, Chamkha M, et al. Phytochemical composition, bioavailability and pharmacokinetics of scorzonera undulata methanolic extracts: antioxidant, anticancer, and apoptotic effects on MCF7 cells. Pharmacogn Mag. 2024;20:218–29.10.1177/09731296231207231Search in Google Scholar

[3] Schneider C, González-Reyes M, Vergara C, Fuica-Carrasco C, Zapata P. Antioxidant and antifungal activities and characterization of phenolic compounds using high-performance liquid chromatography and mass spectrometry (HPLC-MS) in empetrum rubrum vahl ex willd. Plants. 2024;13:497.10.3390/plants13040497Search in Google Scholar PubMed PubMed Central

[4] Ben-Nasr H, Badraoui R. Approach of utilizing Artemisia herbs to treat covid-19. Brazilian. J Pharm Sci. 2022;58:e20345.10.1590/s2175-97902022e20345Search in Google Scholar

[5] Badraoui R, Siddiqui AJ, Bardakci F, Ben-Nasr H. Ethnopharmacology and ethnopharmacognosy: current perspectives and future prospects. In: Adnan M, Patel M, Snoussi M, editors. Ethnobotany and ethnopharmacology of medicinal and aromatic plants. Boca Raton: Taylor & Francis Group, CRC Press; 2023. p. 115–28.10.1201/b22842-7Search in Google Scholar

[6] Abdelkhalek A. Expression of tomato pathogenesis related genes in response to tobacco mosaic virus. J Anim Plant Sci. 2019;29:1596–602.Search in Google Scholar

[7] Abdelkhalek A, Király L, Al-Mansori ANA, Younes HA, Zeid A, Elsharkawy MM, et al. Defense responses and metabolic changes involving phenylpropanoid pathway and PR genes in squash (Cucurbita pepo L.) following cucumber mosaic virus infection. Plants. 2022;11(15):1908. 10.3390/plants11151908.Search in Google Scholar PubMed PubMed Central

[8] Possamai Rossatto FC, Tharmalingam N, Escobar IE, d’Azevedo PA, Zimmer KR, Mylonakis E. Antifungal activity of the phenolic compounds ellagic acid (EA) and caffeic acid phenethyl ester (CAPE) against drug-resistant Candida auris. J Fungi. 2021;7:763.10.3390/jof7090763Search in Google Scholar PubMed PubMed Central

[9] Ríos-López AL, Dávila-Aviña J, González GM, Flores-Maldonado O. Antifungal and antivirulence activity of vanillin and tannic acid against Aspergillus fumigatus and Fusarium solani. Curr Microbiol. 2024;81:156.10.1007/s00284-024-03678-wSearch in Google Scholar PubMed

[10] Kiliç Ö. Essential oil composition of Torilis arvensis subsp. neglecta from Bingöl (Turkey). MOJ Food Process Technol. 2018;6:419–21.10.15406/mojfpt.2018.06.00198Search in Google Scholar

[11] Saad HEA, El-Sharkawy SH, Halim AF. Composition of the essential oils of the leaves and stems of Torilis arvensis. Pharm Acta Helv. 1995;70:85–7.10.1016/0031-6865(94)00053-XSearch in Google Scholar

[12] Nasreen N, Semmar N, Farman M, Lacaille-Dubois M-A, Ahmed NS. Phytochemical, geographical, and pharmacological retrospect of genus Torilis. Curr Top Med Chem. 2023;23:2300–31.10.2174/1568026623666230727163658Search in Google Scholar PubMed

[13] Salem MZM, Mohamed AA, Ali HM, Al Farraj DA. Characterization of phytoconstituents from alcoholic extracts of four woody species and their potential uses for management of six Fusarium oxysporum isolates identified from some plant hosts. Plants. 2021;10:1325.10.3390/plants10071325Search in Google Scholar PubMed PubMed Central

[14] Bogale M, Steenkamp ET, Wingfield MJ, Wingfield BD. Diverse Fusarium solani isolates colonise agricultural environments in Ethiopia. Eur J Plant Pathol. 2009;124:369–78.10.1007/s10658-008-9422-ySearch in Google Scholar

[15] Ajayi‐Oyetunde OO, Bradley CA, Ajayi-Oyetunde OO, Bradley CA, Ajayi‐Oyetunde OO, Bradley CA. Rhizoctonia solani: taxonomy, population biology and management of rhizoctonia seedling disease of soybean. Plant Pathol. 2018;67:3–17. 10.1111/ppa.12733.Search in Google Scholar

[16] Campbell CK, Johnson EM, Warnock DW. Identification of Pathogenic Fungi. Oxford, UK: John Wiley & Sons; 2013. 10.1002/9781118520055.Search in Google Scholar

[17] White TJ, Bruns T, Lee S, Taylor J. Amplification and Direct Sequencing of Fungal Ribosomal RNA Genes for Phylogenetics. PCR Protoc. 1990;18:315–22. 10.1016/b978-0-12-372180-8.50042-1.Search in Google Scholar

[18] Soliman SA, Al-Askar AA, Sobhy S, Samy MA, Hamdy E, Sharaf OA, et al. Differences in pathogenesis-related protein expression and polyphenolic compound accumulation reveal insights into tomato–Pythium aphanidermatum interaction. Sustainability. 2023;15:6551.10.3390/su15086551Search in Google Scholar

[19] Sobhy S, Al-Askar AA, Bakhiet EK, Elsharkawy MM, Arishi AA, Behiry SI, et al. Phytochemical characterization and antifungal efficacy of camphor (Cinnamomum camphora L.) extract against phytopathogenic fungi. Separations. 2023;10:189. 10.3390/separations10030189.Search in Google Scholar

[20] Behiry SI, Soliman SA, Al-Askar AA, Alotibi FO, Basile A, Abdelkhalek A, et al. Plantago lagopus extract as a green fungicide induces systemic resistance against Rhizoctonia root rot disease in tomato plants. Front Plant Sci. 2022;13:966929. 10.3389/fpls.2022.966929.Search in Google Scholar PubMed PubMed Central

[21] Heflish AA, Behiry SI, Al-Askar AA, Su Y, Abdelkhalek A, Gaber MK. Rhaphiolepis indica fruit extracts for control Fusarium solani and Rhizoctonia solani, the causal agents of bean root rot. Separations. 2023;10:369. 10.3390/separations10070369.Search in Google Scholar

[22] Raskar SV, Laware SL. Effect of zinc oxide nanoparticles on cytology and seed germination in onion. Int J Curr Microbiol Appl Sci. 2014;3:467–73.Search in Google Scholar

[23] Abdelsalam NR, Fouda MMG, Abdel-Megeed A, Ajarem J, Allam AA, El-Naggar ME. Assessment of silver nanoparticles decorated starch and commercial zinc nanoparticles with respect to their genotoxicity on onion. Int J Biol Macromol. 2019;133:1008–18. 10.1016/j.ijbiomac.2019.04.134.Search in Google Scholar PubMed

[24] Abdelkhalek A, El-Gendi H, Al-Askar AA, Maresca V, Moawad H, Elsharkawy MM, et al. Enhancing systemic resistance in faba bean (Vicia faba L.) to Bean yellow mosaic virus via soil application and foliar spray of nitrogen-fixing Rhizobium leguminosarum bv. viciae strain 33504-Alex1. Front Plant Sci. 2022;13:933498. 10.3389/fpls.2022.933498.Search in Google Scholar PubMed PubMed Central

[25] Youssef NH, Qari SH, Behiry SI, Dessoky ES, El-Hallous EI, Elshaer MM, et al. Antimycotoxigenic activity of beetroot extracts against Altenaria alternata mycotoxins on potato crop. Appl Sci. 2021;11:4239. 10.3390/app11094239.Search in Google Scholar

[26] Dubale S, Kebebe D, Zeynudin A, Abdissa N, Suleman S. Phytochemical screening and antimicrobial activity evaluation of selected medicinal plants in Ethiopia. J Exp Pharmacol. 2023;15:51–62. 10.2147/JEP.S379805.Search in Google Scholar PubMed PubMed Central

[27] Al-Luhaiby AAK, Hassan AK. Evaluation the ability of some organic compounds is protecting bean seedling against infection with Rhizoctonia solani. Plant Arch. 2020;20:86–90.Search in Google Scholar

[28] Yu S, Teng C, Liang J, Song T, Dong L, Bai X, et al. Characterization of siderophore produced by Pseudomonas syringae BAF.1 and its inhibitory effects on spore germination and mycelium morphology of Fusarium oxysporum. J Microbiol. 2017;55:877–84. 10.1007/s12275-017-7191-z.Search in Google Scholar PubMed

[29] Jiang S, Wang C, Shu C, Huang Y, Yang M, Zhou E. Effects of catechol on growth, antioxidant enzyme activities and melanin biosynthesis gene expression of Rhizoctonia solani AG-1 IA. Can J Plant Pathol. 2018;40:220–8. 10.1080/07060661.2018.1437775.Search in Google Scholar

[30] Wu HS, Wang Y, Zhang CY, Bao W, Ling N, Liu DY, et al. Growth of in vitro Fusarium oxysporum f. sp. niveum in chemically defined media amended with gallic acid. Biol Res. 2009;42:297–304. 10.4067/S0716-97602009000300004.Search in Google Scholar

[31] Ashmawy NA, Behiry SI, Al-Huqail AA, Ali HM, Salem MZM. Bioactivity of selected phenolic acids and hexane extracts from Bougainvilla spectabilis and Citharexylum spinosum on the growth of Pectobacterium carotovorum and Dickeya solani bacteria: An opportunity to save the environment. Processes. 2020;8:482. 10.3390/PR8040482.Search in Google Scholar

[32] Ain QU, Asad S, Ahad K, Safdar MN, Jamal A. Antimicrobial activity of Pinus wallachiana leaf extracts against Fusarium oxysporum f. sp. cubense and analysis of its fractions by HPLC. Pathogens. 2022;11:347. 10.3390/pathogens11030347.Search in Google Scholar PubMed PubMed Central

[33] Nawrocka J, Szczech M, Małolepsza U. Trichoderma atroviride enhances phenolic synthesis and cucumber protection against Rhizoctonia solani. Plant Prot Sci. 2018;54:17–23. 10.17221/126/2016-PPS.Search in Google Scholar

[34] Ling N, Zhang W, Wang D, Mao J, Huang Q, Guo S, et al. Root exudates from grafted-root watermelon showed a certain contribution in inhibiting Fusarium oxysporum f. sp. niveum. PLoS One. 2013;8:e63383. 10.1371/journal.pone.0063383.Search in Google Scholar PubMed PubMed Central

[35] Martínez G, Regente M, Jacobi S, Del Rio M, Pinedo M, de la Canal L. Chlorogenic acid is a fungicide active against phytopathogenic fungi. Pestic Biochem Physiol. 2017;140:30–5. 10.1016/j.pestbp.2017.05.012.Search in Google Scholar PubMed

[36] Ahmad H, Matsubara Y. Antifungal effect of Lamiaceae herb water extracts against Fusarium root rot in Asparagus. J Plant Dis Prot. 2020;127:229–36. 10.1007/s41348-019-00293-x.Search in Google Scholar

[37] Zhang C, Liu H, Wang X, Long X, Huang A, Zhang J, et al. Inhibitory effects and mechanisms of cinnamaldehyde against Fusarium oxysporum, a serious pathogen in potatoes. Pest Manag Sci. 2024;80:3540–52.10.1002/ps.8058Search in Google Scholar PubMed

[38] Roy S, Nuckles E, Archbold DD. Effects of phenolic compounds on growth of colletotrichum spp. in vitro. Curr Microbiol. 2018;75:550–6. 10.1007/s00284-017-1415-7.Search in Google Scholar PubMed

[39] Gupta A, Jeyakumar E, Lawrence R. Pyrogallol: a competent therapeutic agent of the future. Biotech Env Sc. 2021;23:213–7.Search in Google Scholar

[40] Wu H-S, Shen S, Han J-M, Liu Y-D, Liu S. The effect in vitro of exogenously applied p-hydroxybenzoic acid on Fusarium oxysporum f. sp. niveum. Phytopathol Mediterr. 2009;48:439–46.Search in Google Scholar

[41] Wu H-S, Wang Y, Zhang C-Y, Gu M, Liu Y-X, Chen G, et al. Physiological and biochemical responses of in vitro Fusarium oxysporum f. sp. niveum to benzoic acid. Folia Microbiol (Praha). 2009;54:115–22.10.1007/s12223-009-0017-6Search in Google Scholar PubMed

[42] Mohamed S, Kordali Ş, Korkmaz M. Evaluation of the effect of benzoic acid on some plant pathogenic fungi. Int J Agric Nat Sci. 2018;1:3–5.Search in Google Scholar

[43] Alqahtani M, Almukainzi M, Alghoribi MF, El-Mahdy AM. Antivirulence effects of trans-resveratrol and curcumin on methicillin-resistant Staphylococcus aureus (MRSA) from Saudi Arabia. Life. 2024;14:491.10.3390/life14040491Search in Google Scholar PubMed PubMed Central

[44] Borrego-Muñoz P, Coy-Barrera E, Quiroga D. New strategies in the chemical control of Fusarium oxysporum using synthetic bioisosteres of secondary metabolites: a review of the synthetic methods for novel compounds with potential antifungal activity. Mini Rev Org Chem. 2024;21:794–810.10.2174/1570193X20666230516144539Search in Google Scholar

[45] Bais HP, Walker TS, Schweizer HP, Vivanco JM. Root specific elicitation and antimicrobial activity of rosmarinic acid in hairy root cultures of Ocimum basilicum. Plant Physiol Biochem. 2002;40:983–95.10.1016/S0981-9428(02)01460-2Search in Google Scholar

[46] Ouf SA, Abdel Hady FK, Elgamal MH, Shaker KH. Isolation of antifungal compounds from some Zygophyllum species and their bioassay against two soil-borne plant pathogens. Folia Microbiol (Praha). 1994;39:215–21.10.1007/BF02814651Search in Google Scholar

[47] Shalapy NM, Kang W. Fusarium oxysporum & Fusarium solani: identification, characterization, and differentiation the fungal phenolic profiles by HPLC and the fungal lipid profiles by GC-MS. J Food Qual. 2022;2022(1):4141480. 10.1155/2022/4141480.Search in Google Scholar

[48] Lee JH, Lee SJ, Park S, Jeong SW, Kim CY, Jin JS, et al. Determination of flavonoid level variation in onion (Allium cepa L.) infected by Fusarium oxysporum using liquid chromatography–tandem mass spectrometry. Food Chem. 2012;133:1653–7.10.1016/j.foodchem.2012.02.063Search in Google Scholar

[49] Xi K-Y, Xiong S-J, Li G, Guo C-Q, Zhou J, Ma J-W, et al. Antifungal activity of ginger rhizome extract against Fusarium solani. Horticulturae. 2022;8:983.10.3390/horticulturae8110983Search in Google Scholar

[50] Guimarães A, Venâncio A. The potential of fatty acids and their derivatives as antifungal agents: a review. Toxins (Basel). 2022;14:188. 10.3390/toxins14030188.Search in Google Scholar PubMed PubMed Central

Received: 2024-09-05

Revised: 2024-10-25

Accepted: 2024-11-11

Published Online: 2024-11-29

This work is licensed under the Creative Commons Attribution 4.0 International License.

Articles in the same Issue

https://doi.org/10.1515/chem-2024-0113

Keywords for this article

Torilis arvensis; secondary metabolites; HPLC; GC-MS; soil-borne fungi; cytotoxicity

Creative Commons

BY 4.0