Erucastrum virgatum subsp. virgatum (Brassicaceae) Endemic to Sicily (Italy): Biological Properties and Phenolic Content of Leaf Extracts

TL;DR

This study compares different extraction methods for Sicilian Erucastrum virgatum leaves and evaluates their antioxidant and antimicrobial properties.

Contribution

The study provides a comparative analysis of green solvent extraction methods and their impact on the biological properties of Sicilian Erucastrum virgatum leaf extracts.

Findings

70% ethanol extracts showed better radical scavenging activity and higher polyphenol and flavonoid content than aqueous extracts.

Aqueous extracts exhibited stronger chelating activity compared to 70% ethanol extracts.

70% ethanol extracts showed weak antimicrobial activity against selected bacteria.

Abstract

The present work reports a comparative study between extracts obtained by different methods (ultrasonic bath‐assisted extraction, maceration, decoction, and Soxhlet extraction) from the leaves of Erucastrum virgatum subsp. virgatum collected in Sicily (Italy), using ethanol and water as “green” solvents. All the extracts were found to be nontoxic after preliminary toxicity evaluation by the Artemia salina lethality bioassay. The antioxidant properties of the extracts were investigated by in vitro methods based on diverse mechanisms. All the extracts showed moderate radical scavenging activity in the 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH) test and mild reducing power, with 70% ethanol extracts exhibiting better activity than aqueous ones. Conversely, all the aqueous extracts showed stronger chelating activity than that of the 70% ethanol ones. Weak antimicrobial properties against selected Gram‐positive and Gram‐negative bacteria were observed only for 70% ethanol extracts. The results of the spectrophotometric determinations highlighted higher total polyphenol and flavonoid contents for all the 70% ethanol extracts compared to the aqueous ones. Furthermore, the phenolic profile of the most promising extracts was characterized by HPLC‐PDA/ESI‐MS analysis.

Figures

Free radical scavenging activity (DPPH test) (A), reducing power (B), and ferrous ion chelating activity (C) of Erucastrum virgatum subsp. virgatum leaf extracts. UB: ultrasonic bath‐assisted extraction, Mac: maceration, Dec: decoction, Sox: Soxhlet extraction, Reference standard: BHT (A, B), EDTA (C). The results are expressed as the mean ± SD (n = 3).

Effect of UB‐70%EtOH, Mac‐70%EtOH, and Sox‐70%EtOH extracts on the metabolic activity of S. aureus strains (TTC assay). UB: ultrasonic bath‐assisted extraction, Mac: maceration, Sox: Soxhlet extraction. Columns: 1‐2, UB‐70%EtOH, 3‐4, Mac‐70%EtOH, 5‐6, Sox‐70%EtOH versus S. aureus ATCC 6538; 7‐8, UB‐70%EtOH, 9‐10, Mac‐70%EtOH, 11‐12, Sox‐70%EtOH versus S. aureus ATCC 43300. Rows: H, growth control.

HPLC‐PDA chromatograms of the phenolic compounds of UB‐H2O and Sox‐70%EtOH leaf extracts from Erucastrum virgatum subsp. virgatum, extracted at 280 nm wavelength. UB: ultrasonic bath‐assisted extraction, Sox: Soxhlet extraction. For peak identification, see Table 4.

Tables

TABLE 1. Free radical scavenging activity (DPPH test), reducing power, and ferrous ion chelating activity of Erucastrum virgatum subsp. virgatum leaf extracts.

Extract	DPPH IC50(mg/mL)	Reducing power mg ASE/g	Chelating activity IC50(mg/mL)
UB‐70%EtOH	2.05 ± 0.01 a	27.25 ± 1.11 a	0.42 ± 0.02 a
UB‐H2O	3.41 ± 0.29 b	23.75 ± 1.56 a	0.18 ± 0.02 b
Mac‐70%EtOH	2.32 ± 0.03a	41.58 ± 3.30 b	1.32 ± 0.01 c
Mac‐H2O	2.93 ± 0.22 c	11.27 ± 1.23 c	0.20 ± 0.02 b
Dec‐H2O	4.12 ± 0.04 d	19.84 ± 1.22 d	0.19 ± 0.03 b
Sox‐70%EtOH	1.88 ± 0.01 a	27.34 ± 1.61 a	1.47 ± 0.02 d
Standard	BHT 0.15 ± 0.01	—	EDTA 0.02 ± 6,84E‐05

TABLE 2. Antibacterial activity of Erucastrum virgatum subsp. virgatum leaf extracts.

	Inhibition zone diameters (mm)
Extract	S. aureus ATCC 6538	S. aureus ATCC 43300	E. coli ATCC 10536	P. aeruginosa ATCC 9027
UB‐70%EtOH	6	6	<6	<6
UB‐H2O	<6	<6	<6	<6
Mac‐70%EtOH	7	7	<6	<6
Mac‐H2O	<6	<6	<6	<6
Dec‐H2O	<6	<6	<6	<6
Sox‐70%EtOH	<6	8	7	6

TABLE 3. Quantitative determination of total polyphenols (calculated as gallic acid equivalents) and total flavonoids (calculated as quercetin equivalents) in the Erucastrum virgatum subsp. virgatum leaf extracts.

Extract	Total polyphenol mg GAE/g	Total flavonoids mg QE/g
UB‐70%EtOH	65.15 ± 1.78 a	52.07 ± 1.75 a
UB‐H2O	57.72 ± 1.32 b	24.90 ± 0.71 b
Mac‐70%EtOH	67.39 ± 0.78 a	65.95 ± 1,86 c
Mac‐H2O	54.32 ± 1.29 c	26.65 ± 0.45 b
Dec‐H2O	52.80 ± 1.17 c	26.01 ± 0.65 b
Sox‐70%EtOH	69.72 ± 1.16 d	58.33 ± 1.72 d

TABLE 4. Semi‐quantification of phenolic compounds in the UB‐H2O and Sox‐70%EtOH leaf extracts from Erucastrum virgatum subsp. virgatum through LC‐PDA/ESI‐MS analysis. Quantification of phenolic compounds was reported in mg/g of dried extract ± SD (n = 2).

Peak N	Compound	tR(min)	UV max(nm)	[M‐H]−	[M+H]+	Fragments	UB‐H2O	Sox‐70%EtOH	Ref.
1	Unknown	0.87	264	377	—	—	—	x	—
2	Galloylquinic acid isomer	1.06	273	343	345	191−[Quinic acid]	0.54 ± 0.033	—	[50]
3	Galloylglycerol isomer	2.19	262	243	245	—	0.34 ± 0.019	—	[50]
4	Gallic acid	2.74	271	169	—	—	0.31 ± 0.019	—	Standard
5	Unknown	9.88	279	275	277	—	X	x	—
6	Unknown	10.24	256, 287	—	295	—	X	x	—
7	Unknown	12.23	264	—	325	—	X	—	—
8	Unknown	21.22	263	579	581	—	—	x	—
9	Kaempferol deoxyhexosyl pentoside isomer	24.48	266, 347	563	565	433+[M+H‐pentose]; 287+[Kaempferol]	20.48 ± 0.886	32.25 ± 0.943	[50]
10	Kaempferol deoxyhexosyl hexoside isomer	25.32	265, 344	593	595	433+[M+H‐hexose; 287+[Kaempferol]	0.82 ± 0.029	1.22 ± 0.032	[51]
11	Kaempferol deoxyhexosyl pentoside isomer	26.21	266, 344	563	565	433+[M+H‐pentose; 287+[Kaempferol]	0.06 ± 0.005	0.23 ± 0.003	[50]
12	Kaempferol deoxyhexosyl deoxyhexoside isomer	30.51	266, 344	577	579	433+[M+H‐deoxyhexose; 287+[Kaempferol]	4.14 ± 0.183	6.39 ± 0.169	[51]
13	Kaempferol deoxyhexosyl deoxyhexosyl pentoside isomer	31.98	265, 342	709	711	433+[M+H‐pentose‐deoxyhexose; 287+[Kaempferol]	39.51 ± 1.983	61.25 ± 1.389	[51]
14	Kaempferol dicoumaroyl rhamnoside isomer	32.72	264, 343	723	725	433+[M+H‐2(Coumaric acid); 287+[Kaempferol]	0.37 ± 0.009	0.59 ± 0.056	[50]
15	Kaempferol deoxyhexosyl pentoside isomer	39.35	264, 343	563	565	433+[M+H‐pentose; 287+[Kaempferol]	0.31 ± 0.025	0.68 ± 0.031	[50]
16	Kaempferol coumaroyl dihexoside isomer	42.97	268, 334	755	757	433+[M+H‐2(hexose); 287+[Kaempferol]	0.39 ± 0.012	0.9 ± 0.051	[52]
17	Kaempferol deoxyhexosyl deoxyhexosyl caffeoyl pentoside isomer	52.06	265, 328	871	873	579+[M+H‐Caffeic acid‐pentose; 433+[M+H‐Caffeic acid‐pentose‐deoxyhexose]; 287+[Kaempferol]; 181+[Caffeic acid]	3.29 ± 0.145	6.49 ± 0.428	—
18	Kaempferol dicoumaroyl glucoside	52.58	266, 328, 361	739	741	433+[M+H‐Coumaric acid‐hexose]; 287+[Kaempferol]	0.35 ± 0.051	1.83 ± 0.021	[52]
19	Kaempferol deoxyhexosyl deoxyhexosyl caffeoyl pentoside isomer	55.73	265, 328	871	873	579+[M+H‐Caffeic acid‐pentose; 433+[M+H‐Caffeic acid‐pentose‐deoxyhexose]; 287+[Kaempferol]; 181+[Caffeic acid]	0.2 ± 0.032	5.09 ± 0.276	—
20	Kaempferol deoxyhexosyl deoxyhexosyl deoxyhexosyl pentoside isomer	58.09	266, 315	855	857	579+[M+H‐deoxyhexose‐pentose]; 433+[M+H‐deoxyhexose‐pentose‐deoxyhexose]; 287+[Kaempferol]	0.86 ± 0.051	1.82 ± 0.065	—
21	Kaempferol deoxyhexosyl deoxyhexosyl hexosyl hexuronide isomer	58.70	265, 329	915	917	579+[M+H‐hexose‐hexuronic acid]; 433+[M+H‐hexose‐hexuronic acid‐deoxyhexose]; 287+[Kaempferol]	1.1 ± 0.065	1.85 ± 0.174	—
22	Kaempferol derivative	59.63	265, 336	873	875	579+; 433+; 287+[Kaempferol]	1.98 ± 0.106	2.83 ± 0.043	—
23	Kaempferol deoxyhexosyl deoxyhexosyl deoxyhexosyl pentoside isomer	61.14	266, 315	855	857	579+[M+H‐deoxyhexose‐pentose]; 433+[M+H‐deoxyhexose‐pentose‐deoxyhexose]; 287+[Kaempferol]	—	0.96 ± 0.024	—
24	Kaempferol deoxyhexosyl deoxyhexosyl hexosyl hexuronide isomer	61.64	265, 329	915	917	579+[M+H‐hexose‐hexuronic acid]; 433+[M+H‐hexose‐hexuronic acid‐deoxyhexose]; 287+[Kaempferol]	—	1.44 ± 0.103	—
25	Kaempferol derivative	62.17	265, 336	873	875	579+; 433+; 287+[Kaempferol]	—	1.49 ± 0.032	—

TABLE 5. Yields of Erucastrum virgatum subsp. virgatum leaf extracts.

Erucastrum virgatum subsp. virgatum leaves		Extract	Yield %
Extraction procedure	Solvent
Ultrasonic bath‐assisted extraction	70% EtOH	UB‐70%EtOH	37.0%
	H2O	UB‐H2O	48.0%
Maceration	70% EtOH	Mac‐70%EtOH	32.0%
	H2O	Mac‐H2O	32.0%
Decoction	H2O	Dec‐H2O	32.0%
Soxhlet extraction	70% EtOH	Sox‐70%EtOH	38.7%

Full text

Introduction

1

Several species included in the Brassicaceae family have been recognized as valuable resources of natural compounds with nutritional and health benefits and a broad range of biological effects [1]. While the Brassica species are well‐studied, especially with regard to cultivated crops, a significant number of other Brassicaceae species remain quite unknown for their bioactive potential, including crop wild relatives and less common species. These unexplored species could represent novel resources of health‐promoting phytochemicals and unexploited traits for agriculture.

The genus Erucastrum C.Presl, belonging to the Brassicaceae family, includes about 25 species, annual, biennial, or perennial herbs, rarely suffruticose. [2] Morphological investigations on Brassicaceae have indicated that the genera Diplotaxis DC., Erucastrum C.Presl, and Brassica L., included in the tribe Brassiceae, subtribe Brassicinae, form a complex of species; their phylogenetic relationships have shown that the Diplotaxis genus presents almost all the primitive morphological characters, Brassica the more evolved ones, while Erucastrum is located in an intermediate position. [3]

Most of the Erucastrum species are mainly distributed in the western Mediterranean region and throughout much of Africa, with extensions into central and eastern Europe, the islands, and the Arabian Peninsula; many of them are steno‐endemic. [4, 5]

The genus Erucastrum has a history of use for food and medicinal purposes. In several African countries, species such as E. arabicum Fisch. & C.A.Mey. and E. abyssinicum (A.Rich.) O.E.Schulz are used as food and are part of traditional subsistence systems. They are also employed in traditional medicine to treat heartburn, diarrhea, and, for topical use, fungal infections. [6, 7, 8] In veterinary medicine, these species are used to treat diarrhea, arthritis, fractures, and neuropathic pain. [9] Despite their uses, to date, the Erucastrum species have no economic relevance and have been the subject of very few studies aimed at determining their bioactive potential.

According to the data portal Euro+Med PlantBase, Erucastrum virgatum comprises four subspecies: E. virgatum C.Presl subsp. virgatum, subsp. baeticum (Boiss.) Gómez‐Campo, subsp. brachycarpum (Rouy) Gómez‐Campo, and subsp. pseudosinapis (Lange) Gómez‐Campo. [10] From a phytogeographic viewpoint, there is a substantial disjunction between the nominal subspecies, that is, E. virgatum subsp. virgatum, endemic to Southern Italy (Sicily, Calabria, and Basilicata), and the other E. virgatum subspecies, which are endemic to southern and eastern Spain, primarily in the Betic Mountains. [5, 11, 12, 13, 14, 15]

Among the Erucastrum species present in Italy, E. virgatum subsp. virgatum is the only one growing in Sicily. [12] It is a perennial herb 30‐60 cm tall, somewhat glaucous with a very branched stem. Basal leaves are lyrate with (0‐)1‐2 pairs of lateral segments and an irregularly triangular terminal segment, coarsely dentate; the cauline leaves are progressively reduced, all glabrous except for scattered setae on the upper surface. The flowers are gathered in elongated racemes, with erect‐patent sepals and light‐yellow petals. The fruit is a siliqua ending in a conical beak. [13, 16] Erucastrum virgatum subsp. virgatum blooms between May and June; it is found on ruins, fallow lands, and pastures at an altitude of 0–600 m above sea level. [17]

Ethnobotanical studies report on the use of leaves and flowering shoots of E. virgatum subsp. virgatum in traditional Sicilian cuisine. [18, 19, 20] Despite its food use, no studies regarding the phytochemical characterization or evaluation of the biological properties of this species are available in the literature. Therefore, in continuation of our investigations into species included in the Brassicaceae family growing wild in Sicily, it seemed interesting to undertake a study on E. virgatum subsp. virgatum as a new potential resource of health‐promoting phytochemicals.

The present work was designed to select a suitable method for obtaining bioactive‐rich extracts from the leaves of E. virgatum subsp. virgatum. For this purpose, conventional and unconventional extraction methods were utilized, namely ultrasonic bath‐assisted extraction (UB), maceration (Mac), decoction (Dec), and Soxhlet extraction (Sox), employing ethanol and water as “green” solvents in compliance with the principles of sustainability. The extracts obtained were investigated for their antioxidant and antimicrobial properties. Furthermore, their toxicity was preliminarily assessed by the Artemia salina Leach lethality bioassay. The phenolic content of the extracts was determined by spectrophotometric methods, and the phenolic profile of the most promising extracts was characterized by HPLC‐PDA/ESI‐MS analysis to highlight any differences in the chemical composition in relation to the biological activities examined.

Results and Discussion

2

Artemia salina Leach Lethality Bioassay

2.1

Nowadays, Artemia salina has been well accepted by the scientific community worldwide as a model organism for preliminary toxicity estimation. In the last decades, the use of the A. salina lethality bioassay as a reliable alternative for early toxicity screening of plant extracts or pure compounds has been reported in many literature data. [21] This experimental model is simple, rapid, cost‐effective, and can be used to detect the general toxicity for a broad spectrum of natural products. [22]

The results of the lethality bioassay carried out for the E. virgatum subsp. virgatum leaf extracts revealed no evidence of toxicity towards A. salina nauplii, indicating their potential safety for further exploration. This conclusion was based on Clarkson's toxicity criterion; indeed, the brine shrimp larvae were all alive after 24 h of exposure to extracts at all tested concentrations, resulting in the LC_50_ above 1000 µg/mL. [23]

Antioxidant Activity

2.2

Oxidative stress, an imbalance between free radicals and the body's antioxidant defense systems, can negatively affect several cellular structures, such as membranes, lipids, proteins, lipoproteins, and deoxyribonucleic acid (DNA), playing an essential role in the pathogenesis of numerous chronic and degenerative disorders including cancer, neurological disorders, diabetes mellitus, and cardiovascular disorders, as well as accelerating the aging process. [24, 25] During the last decades, much research has been focused on vegetables, fruits, and other plant sources as major resources of natural effective antioxidants able to prevent the harmful effects linked to oxidative stress, due to the associated health benefits. [26] Given the food use of E. virgatum subsp. virgatum, it seemed interesting to investigate the antioxidant potential of the leaves and to identify the most suitable extraction method for obtaining high concentrations of antioxidant compounds.

Antioxidant activity is a process that involves several mechanisms and is influenced by numerous factors; for this reason, it cannot be assessed thoroughly by using a single method. In the present work, different in vitro assays were used to achieve an overview of the antioxidant properties of E. virgatum subsp. virgatum leaf extracts, each selected to evaluate a specific antioxidant mechanism: hydrogen atom transfer (HAT), single electron transfer (SET), and chelation of transition metals reaction‐based assays. [27]

The DPPH test, based on mixed mode mechanism (HAT/SET), measures the scavenging potential of an antioxidant by using the synthetic free radical DPPH. [25, 28] The results of the DPPH assay carried out for E. virgatum subsp. virgatum leaf extracts are shown in Figure 1A and Table 1. In the range of concentrations tested, the different extracts exhibited weak to moderate radical scavenging activity compared to the standard BHT. All the 70% EtOH extracts were found to be more active than the aqueous ones; the highest activity was observed for the Sox‐70%EtOH extract, which achieved about 50% inhibition at the concentration of 2 mg/mL, followed by UB‐70%EtOH. This result was confirmed by the IC_50_ values, which were 1.88 ± 0.01 mg/mL and 2.05 ± 0.01 mg/mL for Sox‐70%EtOH and UB‐70%EtOH, respectively. Based on the IC_50_ values calculated for the extracts, the radical scavenging activity decreases in the following order: Sox‐70%EtOH > UB‐70%EtOH > Mac‐70%EtOH > Mac‐H_2_O > UB‐H_2_O > Dec‐H_2_O.

Free radical scavenging activity (DPPH test) (A), reducing power (B), and ferrous ion chelating activity (C) of Erucastrum virgatum subsp. virgatum leaf extracts. UB: ultrasonic bath‐assisted extraction, Mac: maceration, Dec: decoction, Sox: Soxhlet extraction, Reference standard: BHT (A, B), EDTA (C). The results are expressed as the mean ± SD (n = 3).

In a previous study evaluating the antioxidant activity of the most consumed wild edible plants in northeastern Ethiopia, methanol leaf extracts of two Erucastrum species, E. arabicum and E. abyssinicum, were investigated for their free radical scavenging activity in the DPPH assay. The results showed stronger activity for these extracts than those of E. virgatum subsp. virgatum, with IC_50_ values equal to 100.58 µg/mL and 139.69 µg/mL, respectively. [8]

The reducing power, a SET‐based assay, is determined by monitoring the direct reduction of potassium ferricyanide [Fe(CN)6] [3]^−^ to potassium ferrocyanide [Fe(CN)6]^4−^. The results of the reducing power assay performed for E. virgatum subsp. virgatum leaf extracts are shown in Figure 1B and Table 1. All the extracts exhibited only mild reducing power compared to the standard BHT. Also in this test, all 70% EtOH extracts displayed better activity than the aqueous ones; differently from the DPPH test, the highest activity was observed for the Mac‐70%EtOH extract, followed by Sox‐70%EtOH and UB‐70%EtOH, which exhibited superimposable reducing power. This result is also confirmed by the mg ASE/g values, which were 41.58 ± 3.30, 27.34 ± 1.61, and 27.25 ± 1.11 for Mac‐70%EtOH, Sox‐70%EtOH, and UB‐70%EtOH, respectively. Based on the mg ASE/g values, the activity of the extracts decreases in the following order: Mac‐70%EtOH > Sox‐70%EtOH = UB‐70%EtOH > UB‐H_2_O > Dec‐H_2_O > Mac‐H_2_O.

The ability of an extract or pure compound to chelate transition metals is considered a secondary mechanism for estimating antioxidant activity. Several studies reported in the recent literature have shown that transition metals, particularly Fe^2+^ and Cu^2+^, are involved in the pathogenesis of various diseases, such as cardiovascular and neurodegenerative diseases like Alzheimer's and Parkinson's. [29] The chelating activity of E. virgatum subsp. virgatum leaf extracts was assessed by evaluating the formation of the Fe^2+^‐ferrozine complex.

The results of the ferrous ion chelating activity assay performed for E. virgatum subsp. virgatum leaf extracts are shown in Figure 1C and Table 1.

Contrary to the findings of the other tests, all the aqueous extracts exhibited strong chelating properties, much higher than those of the 70% EtOH extracts, achieving more than 90% activity at the highest concentration tested (2 mg/mL), close to the reference standard EDTA. Among the extracts, the lowest IC_50_ value was observed for the UB‐H_2_O (0.18 ± 0.02 mg/mL), followed by Dec‐H_2_O (0.19 ± 0.03 mg/mL). Based on the IC_50_ values, the activity of the extracts decreased in the following order: UB‐H_2_O > Dec‐H_2_O > Mac‐H_2_O > UB‐70%EtOH > Mac‐70%EtOH > Sox‐70%EtOH.

It should be pointed out, however, that the biological relevance of antioxidant properties assessed by in vitro tests is only preliminary. Further validation using cellular and in vivo models is certainly needed to establish their relevance for nutraceutical or functional food applications with potential health benefits.

Antimicrobial Activity

2.3

Due to the serious threat of the rising trend of microbial resistance and the side effects associated with the overuse or misuse of conventional antibiotics, the search for biologically active compounds from plant sources for the treatment of prevalent infectious diseases has demanded widespread efforts in recent times, becoming the main goal of much research. [30] The antimicrobial properties of several species included in the Brassicaceae family have been previously demonstrated. [31] In this context, another objective of the present work was to explore the potential antimicrobial properties of E. virgatum subsp*. virgatum* leaf extracts.

The results obtained from the disc diffusion test demonstrated that only the 70% EtOH extracts exhibited weak antibacterial activity. The data presented in Table 2 show inhibition zones for the UB‐70%EtOH, Mac‐70%EtOH, and Sox‐70%EtOH extracts. Specifically, the UB‐70%EtOH and Mac‐70%EtOH extracts exhibited inhibitory activity against S. aureus ATCC 6538 and S. aureus ATCC 43300, while the Sox‐70%EtOH extract exhibited inhibitory activity against S. aureus ATCC 43300, E. coli ATCC 10536, and P. aeruginosa ATCC 9027.

Regarding the determination of minimum inhibitory concentration (MIC) of 70% EtOH extracts, Mac‐70%EtOH showed a MIC of 8 mg/mL, while UB‐70%EtOH exhibited only partial antibacterial activity at the same concentration (MIC ≥ 8 mg/mL) against S. aureus ATCC 43300. In contrast, no antibacterial activity was observed for the Sox‐70%EtOH extract at the highest concentration tested (8 mg/mL). These results were supported by the TTC assay, demonstrating inhibition of bacterial metabolic activity, as indicated by absent or decreased red formazan production for the Mac‐70%EtOH and UB‐70%EtOH extracts, respectively (Figure 2). A slight reduction in red formazan production was also observed in samples tested against S. aureus ATCC 6538 (Figure 2, row A, 8 mg/mL). However, the antimicrobial effects observed in this study should be considered preliminary and exploratory, and further studies are needed to confirm their biological relevance.

Effect of UB‐70%EtOH, Mac‐70%EtOH, and Sox‐70%EtOH extracts on the metabolic activity of S. aureus strains (TTC assay). UB: ultrasonic bath‐assisted extraction, Mac: maceration, Sox: Soxhlet extraction. Columns: 1‐2, UB‐70%EtOH, 3‐4, Mac‐70%EtOH, 5‐6, Sox‐70%EtOH versus S. aureus ATCC 6538; 7‐8, UB‐70%EtOH, 9‐10, Mac‐70%EtOH, 11‐12, Sox‐70%EtOH versus S. aureus ATCC 43300. Rows: H, growth control.

To the best of our knowledge, there are no data in the literature regarding the evaluation of the antimicrobial activity of Erucastrum spp., except for a single study conducted by Esazah et al., who investigated the antifungal properties of E. arabicum against Candida albicans, based on its use in traditional medicine in Uganda. [7] The results of both the disc diffusion test and MIC determination showed low activity for the ethanol extract obtained by the maceration process (MIC = 0.5 g/mL).

Phytochemical Investigations

2.4

Determination of Total Phenolic and Flavonoid Contents

2.4.1

Plants produce a wide variety of naturally occurring antioxidants, differing in their structural features and mechanism of action. [32] Among plant‐derived antioxidant secondary metabolites, phenolic compounds constitute one of the most important groups, which have demonstrated remarkable antioxidant activity in both in vitro and in vivo investigations, playing a pivotal role in the prevention of the development of pathological conditions related to oxidative stress. [33] Within polyphenols, flavonoids are known as one of the main groups of naturally occurring compounds with antioxidant properties. [34, 35]

The antioxidant properties of polyphenols are associated with their ability to act as efficient free radical scavengers, as well as metal‐reducing and chelating agents. [36, 37] Indeed, in most cases, the antioxidant properties of plant extracts are associated with their total phenolic content, with a good correlation. [38]

Polyphenols are a complex group of heterogeneous compounds whose chemical structure varies from simple to highly polymerized. To achieve an efficient recovery of these compounds, and considering the complexity of plant matrices, the selection of the most suitable extraction procedure is necessary. Among conventional techniques, solid–liquid extraction methods are commonly applied for phenolic recovery from various plant matrices, such as maceration, performed at room temperature, decoction, and Soxhlet extraction, which employ high temperatures. The latter is used to exhaustively extract phenolic compounds, ensuring high extraction yields with much lower volumes of solvent and in a shorter time than maceration. As a valid alternative to conventional methods, novel unconventional extraction techniques have been developed in recent years, such as ultrasound‐assisted extraction at moderate temperatures (40–60°C), which can improve extraction efficiency more quickly than traditional methods. [39] It is well‐established that the composition and functionality of phenolic compounds are affected by several variables, including temperature, time, and solvent choice. Solvent selection is one of the most important steps in the extraction process that can influence the final phenolic composition and bioactivity of the extract. Polyphenols are generally more hydrophilic than lipophilic, and their relative lipophilicity depends on the number of hydroxyl groups they contain. In general, the highly hydroxylated aglycone forms of phenolic compounds are soluble in water, alcohols (ethanol, methanol), and their mixtures, while the less polar and highly methoxylated aglycone forms are soluble in less polar solvents. Since the hydroxyl groups of phenolic compounds contribute to antioxidant activity, more polar extracts usually possess higher antioxidant activity. [40] Water and ethanol are non‐toxic and sustainable green solvents, categorized under GRAS (generally recognized as safe), their use being favorable in the production of extracts for nutraceutical and functional food applications. [41] For this study, we selected water and a hydroethanolic mixture, that is, 70% EtOH, to recover phenolic compounds, including the more polar ones, such as some phenolic acids, which may not be completely solubilized in the pure organic solvent.

The total phenolic and flavonoid contents of E. virgatum subsp. virgatum leaf extracts were determined by using colorimetric assays. The results of the determinations are shown in Table 3.

The total phenolic content, evaluated by the Folin–Ciocâlteu assay, was found to be in the range from 52.80 ± 1.17 mg GAE/g (Dec‐H_2_O) to 69.72 ± 1.16 mg GAE/g (Sox‐70%EtOH), decreasing in the following order: Sox‐70%EtOH > Mac‐70%EtOH > UB‐70%EtOH > UB‐H_2_O > Mac‐H_2_O > Dec‐H_2_O. The results of the aluminum chloride assay showed that the total flavonoid content of the extracts ranged from 24.90 ± 0.71 mg QE/g (UB‐H_2_O) to 65.95 ± 1.86 mg QE/g (Mac‐70%EtOH), decreasing in the following order: Mac‐70%EtOH > Sox‐70%EtOH > UB‐70%EtOH > Mac‐H_2_O > Dec‐H_2_O > UB‐H_2_O.

The results of the determinations highlighted higher amounts of both phenolics and flavonoids for all the 70% EtOH extracts compared to the aqueous ones. Particularly relevant was the difference in the flavonoid content, being that of 70% EtOH extracts, approximately double that of the aqueous ones.

The results obtained from the different in vitro antioxidant assays highlighted better primary antioxidant properties for the 70% EtOH extracts, whereas the aqueous extracts exhibited strong chelating properties. With the aim of preliminarily establishing a potential relationship between the phenolic content and the antioxidant properties of the extracts, a correlation study was carried out with DPPH (IC_50_), reducing power (mg ASE/g), and ferrous ion chelating activity (IC_50_). A positive correlation was observed between the total polyphenol content and both the DPPH radical scavenging activity and the reducing power of the extracts, which was found to be strong in the first case (R ^2^ = 0.8044 and 0.5814, respectively). A positive correlation with the total flavonoid content was also evidenced for both tests (R ^2^ = 0.7021 and 0.6889). The correlation data seem to indicate an involvement of phenolic compounds, particularly of flavonoids, in the primary antioxidant properties of E. virgatum subsp. virgatum leaf extracts. On the other hand, no correlation was observed between the Fe^2+^chelating properties of the extracts and their total phenolic and flavonoid contents.

The higher phenolic and flavonoid content of the E. virgatum subsp. virgatum 70% EtOH extracts could also explain their antimicrobial effects, albeit weak. Indeed, the antibacterial properties of polyphenols derived from numerous plant sources have been documented by extensive research. [42] In particular, flavonoid compounds have been demonstrated to be antimicrobial agents against a wide range of pathogenic microorganisms, acting through different mechanisms. [43]

Characterization of the Phenolic Profile by HPLC‐PDA/ESI‐MS Analysis

2.4.2

Based on the results of the investigated activities, the two most promising leaf extracts from E. virgatum subsp. virgatum were selected among the 70% EtOH and the H_2_O ones, namely Sox‐70%EtOH, which showed the best radical scavenging properties, and UB‐H_2_O, exhibiting the lowest IC_50_ in the Fe^2+^ chelating activity assay. The phenolic composition of Sox‐70%EtOH and UB‐H_2_O was analyzed by HPLC‐PDA/ESI‐MS, in order to highlight the differences between the qualitative‐quantitative profile of the phenolic compounds that may underpin their observed biological effects. The chromatographic profiles and corresponding compound assignments, reported in Figure 3 and Table 4, respectively, revealed a complex mixture of phenolic constituents, with kaempferol derivatives being the most abundant and structurally diverse class in both extracts. LC‐MS profiles of both extracts are reported in Figure S1. Identification of phenolic compounds was carried out by combining retention times, UV spectra, and mass spectra of each peak with its standard, when available, and literature data. The Sox‐70%EtOH extract exhibited a richer and more diverse phenolic profile compared to the UB‐H_2_O extract, both in terms of the number of detected peaks and the total quantity of identified compounds. Notably, peak 13, tentatively identified as a kaempferol deoxyhexosyl deoxyhexosyl pentoside isomer, was the most abundant compound in both extracts, reaching a significantly higher concentration in Sox‐70%EtOH (61.25 ± 1.39 mg/g) than in UB‐H_2_O (39.51 ± 1.98 mg/g). Similarly, peak 9, another kaempferol deoxyhexosyl pentoside isomer, was present in both extracts with a higher abundance in Sox‐70%EtOH (32.25 ± 0.94 mg/g vs. 20.48 ± 0.89 mg/g in UB‐H_2_O), further indicating enhanced flavonoid solubility in ethanol‐based extraction.

HPLC‐PDA chromatograms of the phenolic compounds of UB‐H2O and Sox‐70%EtOH leaf extracts from Erucastrum virgatum subsp. virgatum, extracted at 280 nm wavelength. UB: ultrasonic bath‐assisted extraction, Sox: Soxhlet extraction. For peak identification, see Table 4.

The exclusive detection of several complex glycosylated kaempferol derivatives in Sox‐70%EtOH, such as peaks 23‐25, suggests that ethanol may facilitate the extraction of higher molecular weight or more hydrophobic phenolic constituents.

Additionally, the Sox‐70%EtOH extract contained a broader array of acylated flavonoids, such as kaempferol dicoumaroyl glucoside (peak 18) and caffeoyl‐conjugated species (peaks 17 and 19), which are often associated with enhanced antioxidant capacity due to extended conjugation and increased radical‐stabilizing potential. [44, 45]

Several studies have demonstrated that the antioxidant ability of flavonoid compounds is generally positively correlated with the number of hydroxyl groups; actually, the presence of many of these, especially in the B ring, leads to an increase in antioxidant activity. Kaempferol contains four hydroxyl groups, two of them situated on ring A and one on each of the rings B and C, which is an essential feature for good radical scavenging activity. [46] In a previous work, Gao et al. demonstrated moderate DPPH radical scavenging activity for several isolated kaempferol derivatives, suggesting that the active center of flavonol glycosides is located in the aglycone moiety and that the more glycosidic residues present, the less active they are for the molecule. [47] This could explain the moderate radical scavenging properties also found for Sox‐70%EtOH extract, despite the high number and content of glycosylated kaempferol derivatives.

On the other hand, the aqueous UB‐H_2_O extract was characterized by a simpler profile, predominantly composed of early‐eluting low molecular weight phenolics, including gallic acid (peak 4) and its derivatives such as galloylquinic acid and galloylglycerol isomers (peaks 2 and 3), not detected in the Sox‐70%EtOH extract. These small phenolic acids are known for their strong metal‐chelating properties, which may partly explain the superior Fe^2^ ^+^‐chelating activity of UB‐H_2_O observed in this assay. [48]

In a previous study, the occurrence of kaempferol and quercetin mono‐, di‐, and tri‐glycosides was highlighted in the leaf extracts of some cultivated Erucastrum spp. using paper chromatography and thin‐layer chromatography. [49] Notably, the phenolic profile of the leaves of E. virgatum subsp. virgatum growing wild in Sicily did not reveal the presence of quercetin derivatives.

Conclusions

3

This work reports the first investigations on the phenolic composition, the antioxidant and antimicrobial potential, and the toxicity of different leaf extracts from E. virgatum subsp. virgatum, an edible species growing wild in Sicily (Italy). Overall, the results obtained evidenced the significant impact of extraction techniques and solvents on the recovery of bioactive compounds and their corresponding activities. The results of the antioxidant assays indicate that the extracts are a source of different antioxidant natural compounds acting by multiple mechanisms, their extraction being influenced by the selected conditions, especially the solvent utilized. The Sox‐70%EtOH extract, richer in highly glycosylated and acylated kaempferol derivatives, showed greater radical scavenging properties, while the aqueous UB‐H_2_O extract, enriched in simple galloyl compounds, exhibited more pronounced metal‐chelating activity.

Data obtained from antimicrobial tests showed a weak activity against selected Gram‐positive and Gram‐negative bacteria only for the 70% EtOH extracts. Notably, all the extracts were found to be nontoxic after preliminary toxicity evaluation by the A. salina lethality bioassay.

This study provides new insights into the phytochemical composition and the biological activities of E. virgatum subsp. virgatum, an edible species that could be regarded as a promising source of bioactive metabolites along with other well‐known Brassicaceae species. These early findings pave the way for further investigations into the antioxidant properties and toxicity of the leaf extracts involving cellular systems and in vivo studies, as well as in‐depth characterization of the phytochemical profile, to establish their relevance for nutraceutical or functional food applications with potential health outcomes.

Experimental Section

4

Chemicals and Reagents

4.1

Ethanol 96% (EtOH) was purchased from VWR (Milan, Italy). 2,2‐diphenyl‐1‐picrylhydrazyl, butylated hydroxytoluene (BHT), potassium hexacyanoferrate (III), iron (III) chloride hexahydrate, L‐ascorbic acid, sodium phosphate monobasic monohydrate, potassium phosphate dibasic, trichloroacetic acid, iron (II) chloride, 3‐(2‐Pyridyl)‐5,6‐diphenyl‐1,2,4‐triazine‐4′,4′′‐disulfonic acid sodium salt (ferrozine), ethylenediaminetetraacetic acid disodium salt dihydrate (EDTA), dimethyl sulfoxide (DMSO), Mueller Hinton agar, Mueller Hinton broth, 2,3,5‐triphenyltetrazolium chloride (TTC), Folin‐Ciocâlteu reagent, sodium carbonate, gallic acid, LC‐MS grade water (H_2_O), acetonitrile (ACN), formic acid, and quercetin 3‐O‐rhamnoside, were obtained from Merck Life Science (Merck KGaA, Darmstadt, Germany). Methanol (MeOH) was purchased from Honeywell—Riedel‐de Haën (Seelze, Germany).

Plant Material

4.2

The leaves of Erucastrum virgatum subsp. virgatum were collected in the locality of Scaletta Superiore, municipality of Scaletta Zanclea (Messina, Sicily, Italy) in January 2024, from limestone cliffs. Taxonomic identification was confirmed by Dr. Fabio Mondello, Department of Chemical, Biological, Pharmaceutical, and Environmental Sciences, University of Messina. Voucher specimens of the plant have been deposited in the herbarium of the “Pietro Castelli” Botanical Garden of the University of Messina (accession numbers: 9119 and 9120).

Extraction Procedures

4.3

After collecting, the leaves were cleaned, ground, frozen, and lyophilized to preserve bioactive compounds. The lyophilized material was subjected to different extraction procedures, using ethanol (EtOH) and water (H_2_O), environmentally friendly:

Ultrasonic bath‐assisted extraction: The plant material was powdered and then subjected to preliminary maceration with 70% EtOH (1:10 w/v) and distilled H_2_O (1:10 w/v) at 25°C for 1 h. The extractions were carried out with the same solvents (1:10 w/v) using an ultrasonic bath at 50°C for 15 min; each extract was then separated by filtration. This procedure was repeated two more times.

Maceration: The plant material was powdered and then macerated with 70% EtOH (1:10 w/v) and distilled H_2_O (1:10 w/v) under stirring for 24 h; each extract was then separated by filtration. The procedure was repeated two more times.

Decoction: The plant material was extracted with distilled H_2_O (1:20 w/v) at a temperature of 100°C for 30 min. The extract was then separated by filtration.

Soxhlet Extraction: The plant material, previously powdered, underwent a preliminary maceration with 70% EtOH for 1 h. Soxhlet extraction was then performed with 70% EtOH (1:20 w/v) for 12 h.

The obtained extracts were concentrated to dryness using a rotary evaporator; the yields of the extracts, calculated referring to 100 g of lyophilized plant material, are reported in Table 5.

Artemia salina Leach Lethality Bioassay

4.4

The potential toxicity of E. virgatum subsp. virgatum leaf extracts were assessed using an in vivo bioassay, the Artemia salina Leach lethality test, by determining the median lethal concentration (LC_50_) according to the method described by Meyer and colleagues, with some modifications. [53] Brine shrimp eggs were placed in a hatchery dish containing artificial seawater (32 g of sea salt/L) and incubated under continuous light using a 60 W lamp at a temperature ranging from 24–26°C for hatching. After 24 h from hatching, nauplii were collected and used for the assay. Specifically, 10 brine shrimp larvae were incubated for 24 h at 24°C–26°C in artificial seawater containing different amounts of the extracts dissolved in DMSO, achieving final concentrations ranging from 10 to 1000 µg/mL in a total volume of 5 mL. At the time point, the surviving larvae were counted, and the median lethal concentration (LC_50_) values were determined. Control groups included seawater and seawater with 2% DMSO, that is, the highest solvent concentration used in the assay, which was found to be nontoxic to brine shrimp larvae in preliminary experiments. The test was performed in triplicate. To assess the toxicity level of the extracts, Clarkson's toxicity criterion was used. [23]

Antioxidant Activity

4.5

Radical Scavenging Activity

4.5.1

The radical scavenging activity of E. virgatum subsp. virgatum leaf extracts were assessed using the 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH) assay, according to the method of Ohnishi and colleagues. [54] The concentrations tested ranged from 0.0625 to 2 mg/mL, with butylated hydroxytoluene (BHT) used as a reference standard. Briefly, 0.5 mL of each sample solution was added to 3 mL of a 0.1 mM DPPH methanol solution. The mixture was incubated in the dark for 20 min at room temperature, after which the absorbance was measured at 517 nm using a model UV‐1601 spectrophotometer (Shimadzu, Milan, Italy). The scavenging activity was measured as the decrease in absorbance of the samples versus the control, prepared by replacing the sample solution with an equal volume of solvent. The results are expressed as mean radical scavenging activity (%) ± standard deviation (SD) and mean 50% inhibitory concentration (IC_50_) ± SD.

Reducing Power

4.5.2

The reducing power of E. virgatum subsp. virgatum extracts were determined using the potassium ferricyanide method, evaluating spectrophotometric detection of Fe^3+−^Fe^2+^ transformation, according to the method described by Oyaizu. [55] The extracts were tested in the concentration range 0.0625–2 mg/mL, and BHT was used as a standard. A 1 mL aliquot of each sample solution was mixed with 2.5 mL of phosphate buffer (0.2 M, pH 6.6) and 2.5 mL of 1% potassium ferricyanide. The mixture was incubated at 50°C for 20 min and then rapidly cooled. Afterward, 2.5 mL of 10% trichloroacetic acid was added, and the mixture was centrifuged at 3000 rpm for 10 min at 4°C. Finally, a volume of 2.5 mL of the supernatant was combined with 2.5 mL of distilled water and 0.5 mL of 0.1% w/v ferric chloride. A blank was prepared similarly, replacing the sample solution with an equal volume of solvent. The reaction was incubated in the dark for 10 min at room temperature, and the absorbances were measured at 700 nm by a spectrophotometer. The results are expressed as mean absorbance ± SD and mg of ascorbic acid equivalent (ASE)/g extract ± SD, calculated from an ascorbic acid calibration curve.

Ferrous Ion (Fe2+) Chelating Activity

4.5.3

The chelating activity of E. virgatum subsp. virgatum extracts were assessed by evaluating the formation of the Fe^2+^‐ferrozine complex, according to the method described by Kumar and colleagues. [56] Ethylenediaminetetraacetic acid (EDTA) was used as a reference standard, and the extracts were tested in the concentration range of 0.0625 to 2 mg/mL. For the assay, 1 mL of the sample solution was mixed with 0.5 mL of methanol and 0.05 mL of 2 mM ferrous chloride. Then, a 0.1 mL volume of 5 mM ferrozine solution was added, followed by vigorous shaking and incubation in the dark at room temperature for 10 min. At the timepoint, the absorbances were measured spectrophotometrically at 562 nm. The chelating activity was measured as the decrease in absorbance of the samples versus the control, prepared by replacing the sample solution with an equal volume of solvent. The results are expressed as mean inhibition of the ferrozine‐Fe^2+^ complex formation (%) ± SD and IC_50_ ± SD.

Antimicrobial Activity

4.6

The study of the antimicrobial activity of E. virgatum subsp. virgatum leaf extracts were tested in vitro against reference strains from the American Type Culture Collection (ATCC) by the disc diffusion test and the determination of minimum inhibitory concentration (MIC). Specifically, representative Gram‐positive and Gram‐negative bacteria were used, including Staphylococcus aureus ATCC 6538, methicillin‐resistant S. aureus ATCC 43300, Escherichia coli ATCC 10536, and Pseudomonas aeruginosa ATCC 9027. The strains were stored at −70°C in Micro‐banks (ProlabDiagnostics, Neston, UK).

Disc Diffusion Test

4.6.1

The initial screening of the antimicrobial activity of E. virgatum subsp. virgatum extracts were performed using the disc diffusion test, according to the method previously reported. [57] Petri plates containing Mueller–Hinton agar were inoculated with 5 µL of overnight culture standardized at a concentration of 1 × 10^8^ CFU/mL. Sterile filter paper discs (6 mm diameter) were placed on the inoculated agar surface and impregnated with 15 µL of each extract, dissolved in dimethyl sulfoxide (DMSO) at a concentration of 200 mg/mL. After 24 h of incubation at 37°C, the antibacterial activity was expressed as the inhibition zone (mm) produced by the extracts. The test was performed in duplicate, with negative (DMSO) and positive controls (ciprofloxacin 5 µg) included.

Determination of Minimum Inhibitory Concentration (MIC)

4.6.2

Based on their activity against S. aureus strains, the 70% EtOH extracts were selected for MIC determination using the Clinical and Laboratory Standards Institute microdilution method, with minor modifications. [58] Briefly, extracts solubilized in DMSO at a concentration of 200 mg/mL were twofold serially diluted in Mueller–Hinton broth. Overnight cultures of S. aureus ATCC 6538 and S. aureus ATCC 43300 were standardized in fresh medium and inoculated into the wells to obtain a final concentration of 5 × 10^5^ CFU/mL. Microplates were incubated aerobically at 37°C for 24 h. The MIC was defined as the lowest extract concentration that inhibited visible bacterial growth compared with the control. To evaluate bacterial metabolic activity, 2,3,5‐triphenyltetrazolium chloride (TTC) was added to each well, followed by incubation at 37°C for 1 h. This tetrazolium salt is colorless in solution but turns red in the presence of metabolically active bacteria; thus, color development correlates with the number of viable cells. All experiments were performed in triplicate, including growth controls, and modal MIC values were reported.

Phytochemical Investigations

4.7

Determination of Total Phenolic and Flavonoid Content

4.7.1

The total phenolics and flavonoids contained in E. virgatum subsp. virgatum leaf extracts were quantitatively determined using colorimetric assays. The total phenolic content was estimated using the Folin–Ciocâlteu method. [59] For the assay, an aliquot of 0.1 mL of each sample solution was added to 0.2 mL of Folin–Ciocâlteu reagent, 2 mL of distilled water, and 1 mL of a 15% (w/v) sodium carbonate solution. The mixture was incubated in the dark at room temperature for 2 h, and the absorbance was measured at 765 nm using a spectrophotometer model UV‐1601 (Shimadzu, Milan, Italy). A linear calibration curve of gallic acid, used as the standard phenolic compound, was constructed, and the results were estimated as gallic acid equivalents (GAE) and expressed as mg GAE/g extract (dw) ± SD.

The total flavonoid content of the extracts was measured using the aluminum chloride colorimetric method. [60] Briefly, an aliquot of 0.5 mL of each sample solution was added to 1.5 mL of methanol (MeOH), 0.1 mL of 10% aluminum chloride, 0.1 mL of 1 M potassium acetate, and 2.8 mL of distilled water. The reaction mixtures were incubated at room temperature for 30 min, and the absorbance was spectrophotometrically measured at 415 nm. A linear calibration curve of quercetin, used as the standard phenolic compound, was constructed, and the results were estimated as quercetin equivalents (QE) and expressed as mg QE/g extract (dw) ± SD.

Characterization of the Phenolic Profile by HPLC‐PDA/ESI‐MS Analysis

4.7.2

The phenolic profile characterization of the two most promising leaf extracts from E. virgatum subsp. virgatum among the 70% EtOH and the H_2_O ones, selected based on the results of the investigated activities, namely UB‐H_2_O and Sox‐70%EtOH, was performed by high‐performance liquid chromatography coupled to a photodiode array and electrospray ionization mass spectrometry HPLC‐PDA/ESI‐MS.

Sample preparation: Each dried extract was redissolved in the same extraction solvent and filtered through a 0.45 µm Acrodisc nylon membrane (Merck Life Science, Merck KGaA, Darmstadt, Germany) prior to HPLC‐PDA‐ESI/MS.

HPLC‐MS analysis conditions: Chromatographic analysis was accomplished by means of a Shimadzu HPLC system (Kyoto, Japan) equipped with a CBM‐20A controller, two LC‐30AD dual‐plunger parallel‐flow pumps, a DGU20A5R degasser, a CTO‐20AC column oven, a SIL‐30AC autosampler, an SPD‐M20A photodiode array detector, and an LCMS‐2020 single quadrupole mass spectrometer, with the employment of ESI source operated in negative and positive ionization modes.

Chromatographic separations were carried out on an Ascentis Express RP C18 column (150 × 2.1 mm; 2.7 µm) (Merck Life Science, Merck KGaA, Darmstadt, Germany). The employed mobile phase was composed of two solvents: water (solvent A) and ACN (solvent B), both acidified with 0.10 % of formic acid (v/v). The flow rate was set at 0.5 mL/min, under gradient elution: 0 min–0% B, 10 min–10% B, 20 min–11% B, 30 min–15% B, 50 min–18% B, 65 min–23% B, 70 min–100% B, 75 min–100% B. The injection volume was 2 µL. PDA detection was applied in the range of 200–400 nm and monitored at a wavelength of 280 nm (sampling frequency: 12.5 Hz, time constant: 0.160 s). MS conditions were as follows: scan range and the scan speed were set at a mass‐to‐charge ratio (m/z) 100–1200 and 7500 amu/s, respectively; Event time: 0.3 s, nebulizing gas (N_2_) flow rate: 1.5 L/min, drying gas (N_2_) flow rate: 15 L/min, interface temperature: 350°C, heat block temperature: 300°C, desolvation line temperature: 300°C, desolvation line voltage: 1 V, interface voltage: −4.5 kV.

Calibration curves of Gallic acid (y = 12999x + 54347, R ^2^ = 0.9996, LOD = 0.041, LOQ = 0.125) and Quercetin 3‐O‐rhamnoside (y = 8054x + 27465, R ^2^ = 0.9997, LOD = 0.034, LOQ = 0.103) were employed for the quantification of the polyphenolic content in the sample extracts. Each analysis was performed in six repetitions. Data acquisition was performed using Shimadzu LabSolution software version. 5.97.

Statistical Analysis

4.8

The results of total phenolic and flavonoid content determinations and antioxidant assays were obtained from the average of three independent experiments. Statistical comparison of the data was performed using the one‐way analysis of variance (ANOVA), followed by Tukey's multiple comparisons test (GraphPAD Prism Software for Science, San Diego, CA, USA). P‐values < 0.05 were considered statistically significant.

Author Contributions

Conceptualization: Maria Fernanda Taviano and Fabio Mondello Investigation: Benedetta Galletta, F.M, Federica Davì, Francesco Cacciola, Roberto Laganà Vinci, and Maria Fernanda Taviano Data curation: Francesco Cacciola, Luigi Mondello, Antonia Nostro, Natalizia Miceli, and Maria Fernanda Taviano Supervision: Maria Fernanda Taviano Writing – original draft: Benedetta Galletta, Francesco Cacciola, Roberto Laganà Vinci, and Maria Fernanda Taviano writing – review and editing: Francesco Cacciola, Fabio Mondello, Luigi Mondello, Antonia Nostro, Natalizia Miceli, and Maria Fernanda Taviano All authors have read and agreed to the published version of the manuscript.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Supporting File 1: cbdv71110‐sup‐0001‐SuppMat.docx