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Winter wild fennel leaves as a source of anti-inflammatory and antioxidant polyphenols
⁎Corresponding author. Tel.: +39 0823 274572; fax: +39 0823 274571. severina.pacifico@unina2.it (Severina Pacifico)
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Received: ,
Accepted: ,
This article was originally published by Elsevier and was migrated to Scientific Scholar after the change of Publisher.
Peer review under responsibility of King Saud University.
Abstract
In the course of a screening program on the seasonal phenol composition of wild Mediterranean medicinal and aromatic plants, broadly used for culinary purposes, Foeniculum vulgare Mill. was the focus of the present study. Hydroalcoholic extracts from fennel freeze-dried leaves, collected in different seasons along 2012 and 2013 years, were quali-quantitatively analyzed through LC/MS/MS techniques. Winter extract contained, beyond several hydroxycinnamoyl quinic acids and flavonol glycosides, two chromone derivatives. Flavonol hexuronides were the main spring sample constituents. Phenol profile differences among the extracts influenced massively their bioactivity. When the antioxidant screening was performed, winter extract effectively scavenged DPPH• and ABTS•+ and reduced Fe3+. Although all the extracts did not show cytotoxicity, they were differently able to exert cytoprotection in H2O2-oxidized cell systems and to affect COX-2 gene expression in THP-1 cells. The most active one was winter extract, which inhibited COX-2 expression by 40%, whereas spring sample showed a weak pro-inflammatory capability.
Keywords
Foeniculum vulgare Mill
LC-ESI-MS/MS analysis
Polyphenols
Antioxidant activity
Cytoprotection
COX-2 inhibition
1 Introduction
Traditional medicine is an important part of human health care in many developing and developed countries, increasing their commercial value. Although the use of medicinal plants in therapy has been known for centuries in all parts of the world, the demand for herbal medicines has grown massively in recent years (Kartal, 2007). According to World Health Organization, a medicinal plant is a plant organism containing compounds that can be used for therapeutic purposes or which are precursors of semi-synthetic pharmaceutical species. The synergistic and/or additive action of plant chemicals finds its strength in the formulation of pharmacologically active phytocomplexes (Pacifico et al., 2013), whose preparation requires proper knowledge on the drug plant chemical constitution, as well as its variation over the plant life. This latter represents a key issue when wild medicinal plants are used. It is well known that the content of active ingredients in a medicinal plant is not constant; it undergoes significant variations, due to endogenous and/or exogenous factors (Galasso et al., 2014). In particular, environmental conditions heavily influence the development and the ability to synthesize secondary metabolites in spontaneous medicinal plants, which, even today, play a strategic role in the production of plant-based products (Ramakrishna and Ravishankar, 2011). Indeed, although the cultivation of medicinal species is in constant increase, it is still a marginal reality, and an amount between 75% and 90% of medicinal plants commercialized in the world still comes from wild harvest (Rapporto ISMEA – Osservatorio delle piante officinali, giugno, 2013).
In the Mediterranean area several medicinal and aromatic plants grow and develop, still today, spontaneously in rural areas. Among them, Foeniculum vulgare Mill., commonly called fennel, has been extensively used in traditional medicine since ancient times, not only in Europe but also in several parts of the world. In fact, it has been attested to have a number of therapeutic properties related to digestive, endocrine, reproductive and respiratory systems (Badgujar et al., 2014; Piras et al., 2014). The osteoprotective effects of a F. vulgare ethanol extract were also reported (Mahmoudi et al., 2013). Although the volatile chemical composition of wild fennel samples, harvested in different geographical areas, was broadly reported (Piccaglia and Marotti, 2001; Díaz-Maroto et al., 2006) different scientific evidences suggest that the various components of fennel (seeds, leaves and fruits) are rich in molecules with antioxidant activity (Oktaya et al., 2003; Parejo et al., 2004a,b; De Marino et al., 2007; Ghanem et al., 2012). The beneficial properties of fennel appeared to be due to its content of natural polyphenols, particularly abundant in the shoots (Barros et al., 2009). Plant polyphenols are molecules biosynthesized from the shikimate-derived phenylpropanoid and/or the polyketide pathway(s) and chemically feature more than one phenolic ring and devoid of any nitrogen-based functional group in their most basic structural expression (Quideau et al., 2011). As the interest in dietary polyphenols has been evaluated in several epidemiological studies, which indicated an inverse association between the intake of foods rich in these compounds and the incidence of diseases such as cardiovascular diseases, diabetes mellitus, and cancer (Quideau et al., 2011 and references therein; Rodrigo et al., 2014), the definition of the environmental conditions that favor the concentration of bioactive polyphenols could represent a strategy for the formulation of fennel products with healthy actions. To this aim in the present study four hydroalcoholic extracts from wild F. vulgare leaves, collected in the four different seasons along 2012 and 2013 years, were prepared by ultrasound-assisted maceration. The phenol profile of the obtained extracts was achieved through LC–MS/MS techniques. An assessment of the bioactivity of the extracts in terms of antioxidant, cytotoxic and anti-inflammatory effectiveness was also carried out.
2 Materials and methods
2.1 Plant collection and fractionation
Leaves from F. vulgare were collected in the wild in Durazzano (Italy) on tenth day of July and October 2012 and of January and April 2013, and identified by Dr. Assunta Esposito of the Second University of Naples. A voucher specimen has been deposited at the Herbarium of the Department of Environmental, Biological and Pharmaceutical Sciences and Technologies of the Second University of Naples. Three replicate samples (10.0 g each) of F. vulgare leaves for each collection time were ground in a porcelain mortar and pestle chilled with liquid N2, until particles of homogeneous size were obtained. Frozen powdered samples were lyophilized using a FTS-System Flex-DryTM instrument (SP Scientific, Stone Ridge, NY, USA). Aliquots of dried leaves (1.0 g) underwent ultrasound-assisted extraction (Dr. Hielscher UP 200S, Germany) using a hydro-alcoholic solution (H2O:MeOH; 1:1, v:v) as a extracting solvent. Four sonication cycles were performed (30 min each) in order to achieve the maximum recovery of the leaf metabolic content. At the end of each sonication cycle, samples were centrifuged at 2044g for 10 min in a Beckman GS-15R centrifuge (Beckman Coulter, Milano, Italy) fitted with rotor S4180. Obtained supernatants were dried under vacuum by a rotary evaporator (Heidolph Hei-VAP Advantage, Germany) to yield crude extracts: FvSu = summer extract; FvAu = autumnal extract; FvWi = winter extract; and FvSp = spring extract). All the extracts were stored at −20 °C until use.
2.2 RP-HPLC-ESI-MS/MS analyses
Chromatographic analyses were carried out on a Dionex Ultimate 3000 HPLC system (ThermoScientific Vienna, Austria) equipped with Ultimate 3000 RS pump, Ultimate 3000 RS autosampler, Ultimate 3000 RS Column Compartment and Ultimate 3000 RS diode array detector (DAD). A Phenomenex® Synergy RP-80A column (4.0 μm particle size, 150 × 2 mm) was used for chromatographic separation. The mobile phase consisted of A: 0.1% formic acid in water and B: acetonitrile. Starting with 5% B, a linear gradient was followed to 15% B at 10 min, then increasing to 35% B at 30 min, to 80% B at 40 min, and to 100% B at 45 min, continuing for 5 min, before re-equilibration to starting conditions. The flow rate was 0.3 mL/min and the injection volume was 5.0 μL. The DAD acquisition range was 190–450 nm.
The LC system was coupled to a LTQ-XL mass spectrometer (Thermo Scientific Vienna, Austria) with an ESI ion source and controlled by Thermo Tune Plus 2.7.0 software. ESI ion source operated in negative mode with the following parameters: dry gas flow (N2) 8.0 L/min with a capillary temperature set at 330 °C; source heater temperature set at 250 °C; sheath gas flow set at 50 arb; auxiliary gas flow set at 10 arb; source voltage set at 3.00 kV; source current set at 100 μA; capillary voltage set at −16 V. Mass spectra were recorded between m/z 50–2000. To obtain further structural information, these ions were trapped and fragmented. Collision-induced fragmentation experiments were performed in the ion trap using helium as a collision gas, with voltage cycles from 0.3 up to 2 V and collision energy set at 35 eV. The constituents were identified by comparison of their specific chromatographic data and MS/MS fragmentation patterns to literature data and reference compounds. Metabolites content was expressed in relation to quercetin, used as an external standard. For this purpose quercetin calibration curve was prepared using ten different concentrations of the flavonol (3.06, 6.125, 12.5, 25.0, 50.0, 100.0, 400.0, 600.0, 800.0, and 1000.0 μg/mL) injected in the same conditions of the samples.
2.3 Total phenol determination
Total phenol amount of the investigated crude extracts was determined according to the Folin–Ciocalteau procedure as previously reported (Pacifico et al., 2014a). Analyzed samples (1.0 mg/mL in DMSO) were mixed with 0.50 mL of Folin–Ciocalteau reagent (FCR) and 4.0 mL of Na2CO3 (7.5% w/v). After stirring reaction mixture at room temperature for 3 h, the absorbance was read at 765 nm using a Shimadzu UV-1700 spectrophotometer (Shimadzu, Salerno, Italy). Total phenol content of the samples was expressed as milligram gallic acid equivalents (GAEs) per 100 g of fresh material.
2.4 Scavenging capacity assessment
In order to evaluate the antiradical properties, different antiradical assays were employed (Galasso et al., 2014; Pacifico et al., 2014b). Each extract, previously dissolved in DMSO as stock solutions of 125.0 mg/mL, was evaluated at different levels (DMSO final concentration was equal to 0.1% (v:v)). Tests were carried out performing three replicate measurements for three samples (n = 3) of each extract (in total, 3 × 3 measurements). Recorded activities were compared to a blank arranged in parallel with the samples. Results are the mean ± SD values. Student t-test was applied in order to determine statistical significance (significance level was set at P < 0.05).
2.4.1 Determination of DPPH radical scavenging capacity
In order to estimate the DPPH• (2,2-diphenyl-1-picrylhydrazyl) scavenging capability, investigated extracts (0.625, 1.25, 2.50, 5.00, 10.0, 25.0 and 50.0 μg/mL, final concentration levels) were dissolved in a DPPH• methanol solution (9.4 × 10−5 M; 1.0 mL) at room temperature. After 30 min, the absorption at 515 nm was measured by a Shimadzu UV-1700 spectrophotometer in reference to a blank. The results were expressed in terms of ID50 values on the basis of the percentage decrease of the initial DPPH• absorption by the test samples. TEAC (Trolox® Equivalent Antioxidant Capacity) values were also calculated.
2.4.2 Determination of ABTS radical cation scavenging capacity
The determination of ABTS•+ solution scavenging capacity was estimated as previously reported. ABTS radical cation was generated by reacting ABTS ([2,2′-azinobis-(3-ethylbenzothiazolin-6-sulfonic acid)]; 7.0 mM) and potassium persulfate (2.45 mM). The mixture was allowed to stand in the dark at room temperature for 12–16 h. Thus, the ABTS•+ solution was diluted with PBS (pH 7.4) in order to reach an absorbance of 0.70 at 734 nm. Fennel extracts (0.625, 1.25, 2.50, 5.00, 10.0, 25.0 and 50.0 μg/mL, final concentration levels) were dissolved in 1.0 mL of diluted ABTS•+ solution. After 6 min of incubation, the absorption at 734 nm was measured by a Shimadzu UV-1700 spectrophotometer in reference to a blank. The results were expressed in terms of ID50 values on the basis of the percentage decrease of the initial ABTS•+ absorption by the test samples. TEAC values were also calculated.
2.4.3 Determination of Fe(III) reducing power
F. vulgare extracts (0.625, 1.25, 2.50, 5.00, 10.0, 25.0 and 50.0 μg/mL, final concentration levels) were dissolved in potassium hexacyanoferrate (0.1 M, 2.5 mL) and NaH2PO4/Na2HPO4 buffer (0.2 M, pH 6.6, 2.5 mL). The mixture was incubated at 50 °C for 30 min. Then, an aqueous solution of trichloroacetic acid (100.0 mg/mL) was added. After 5 min, an aliquot of the reaction mixture (2.5 mL) was mixed with 2.5 mL of deionized water and 0.5 mL of ferric chloride (1.0 mg/mL). The absorbance was measured at 700 nm. The increase in absorbance with reference to the blank estimates the reducing power. The slopes of the inhibition percentage as a function of concentration for each sample and Trolox® were determined. The TEAC values were expressed as mg Trolox® equivalents per 1 g of extract.
2.4.4 Determination of oxygen radical absorbance capacity (ORAC)
The ORAC assay was performed as follows: each extract (20 μL; 0.781, 1.56, 3.12, 6.25, 12.5 and 25.0 μg/mL, final concentration levels) and fluorescein (120.0 μL; 70 nM, final concentration) were preincubated for 15 min at 37 °C in 75 mM phosphate buffer (pH 7.4). Then 2,2′-azobis-(2-amidinopropane)-dihydrochloride (AAPH) solution (60.0 μL; 36 mM, final concentration) was rapidly added. In parallel with the test samples, a blank (FL + AAPH) and solutions of the reference antioxidant Trolox® (1–4 μM, final concentration levels) were properly prepared in PBS. The fluorescence decay (λex = 485 nm, λem = 525 nm) was recorded every minute for 120 min using a Tecan SpectraFluor fluorescence and absorbance reader. Antioxidant curves (fluorescence vs. time) were normalized to the curve of the blank. From the normalized curves, the area under the fluorescence decay curve (AUC) was calculated. Linear regression equations between net AUC (AUCantioxidant − AUCblank) and antioxidant concentration were calculated for all the samples. The antioxidant activity (ORAC value) was calculated by using the Trolox® calibration curve. The ORAC values were expressed as Trolox® equivalents (μM) per 100 g of extract.
2.4.5 Relative antioxidant capacity index determination (RACI)
RACI was calculated according to the method of Sun and Tanumihardjo (2007). The standard score was calculated as follows: (x − μ)/σ where x is the raw data, μ is the mean, and σ is the standard deviation. Standard scores have a mean of 0 and a standard deviation equal to 1.
2.5 Cytotoxicity assessment
2.5.1 Cell cultures
CCRF-CEM leukemia cells, and MDA-MB-231 breast cancer cells were grown in RPMI 1640 medium (Gibco, Invitrogen, Vienna, Austria) supplemented with 2.0 mM glutamine (Sigma, MO, USA), 10.0% heat-inactivated fetal bovine serum (FBS, PAA laboratories, Austria) and 1.0% Pen/Strep (PAA laboratories, Austria) at 37 °C in a humidified atmosphere containing 5% CO2. U251 glioblastoma cells, HCT-116 colon cancer cells were plated and grown under the same conditions, except that Dulbecco’s Modified Eagle Medium (DMEM, Gibco, Invitrogen, Vienna, Austria) was used instead of RPMI. Human MRC-5 lung fibroblasts were grown in Minimum Essential Medium (MEM, Gibco, Invitrogen, Vienna, Austria) equally supplemented with 2.0 mM glutamine, 10.0% FBS, and 1.0% Pen/Strep.
2.5.2 XTT cell viability test
Cytotoxicity was evaluated by the XTT test (Cell Proliferation Kit II, Roche Diagnostics, Mannheim, Germany), which allows the assessment of cell viability by determining the levels of activity of mitochondrial dehydrogenases toward 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT; Galasso et al., 2014). Samples of each extract were prepared as a stock solution of 20.0 mg/mL in DMSO and further diluted in culture medium (DMSO final concentration was equal to 0.5% (v:v)). Tests were carried out performing three replicate measurements (n = 3) for three samples of each extract (in total: 3 × 3 measurements). Recorded activities were compared to vehicle-treated (0.5% DMSO) control cells arranged in parallel with the samples.
MDA-MB-231, U251, HCT-116 and MRC-5 cells were diluted to a final concentration of 5 × 104 cells/mL. One hundred micro-liters of the cells were seeded into the wells of 96-well culture plates (flat bottom) and grown for 24 h at 37 °C in a 5% CO2 atmosphere before adding the extracts at final concentrations of 10.0, 25.0, 50.0, 100.0, and 250.0 μg/mL. Control cells were treated with 0.5% DMSO, which did not affect the growth of the cells.
In the case of CCRF-CEM cells, 100 μL of 1 × 104 cells/mL suspension was seeded in 96-well plates (flat bottom) and extracts were immediately added. After 24, 48 and 72 h exposure times, all the cells other than CCRF-CEM cells, were carefully washed with PBS to remove the maximum of potentially interfering phytochemicals before adding the freshly prepared XTT solution as specified by the manufacturer (Bunel et al., 2014). Briefly, XTT-labeling reagent and electron-coupling reagent were mixed in a ratio of 50:1 and 50 μL of this mixture was added to each well of the 96-well plate. XTT solution was added directly to CCRF-CEM cells at the above exposure times. The plates were incubated for 1.5 h (MDA-MB-231, U251, HCT-116 and MRC-5 cells) or 4.0 h (CCRF-CEM cells) at 37 °C in a 5% CO2 humidified atmosphere and read out after incubation using a Tecan Rainbow microplate reader (Tecan Group Ltd., Männedorf, Switzerland). Vinblastine (0.01 μM) served as a positive control. Results are the mean ± SD values. Student t-test was applied in order to determine statistical significance (significance level was set at P < 0.05).
2.5.3 Determination of cytoprotective effects in H2O2 induced oxidation cell systems
MRC-5, HCT-116 MDA-MB-231 and U251, cell lines were seeded in 96-multiwell plates at a density of 5 × 104 cells/mL, instead CCRF-CEM cell line was seeded in 96-multiwell plates at a density 1.0 × 104 cells/well. After 24 h of incubation, cells were treated with extracts (50.0 μg/mL, final concentration levels) for 24 h. Subsequently, all the cells (except to CCRF-CEM cells, to which hydrogen peroxide 0.5 mM was directly added) were gently washed with PBS to remove traces of samples and were challenged by H2O2 (0.75 mM vs U-251 and HCT-116; 0.4 mM vs MDA-MB 231 and MRC-5) for a further 1 h. H2O2 concentrations were chosen in order to obtain a viability reduction by approximately 40% toward the different cell lines. Cell viability (CV) was assessed by XTT assay.
2.6 COX-2 gene expression assay
2.6.1 Cell culture and reagents
Human leukemic monocytic cell line THP-1 (European Collection of Cell Culture; Item No. 88081201) was maintained in RPMI 1640 (Gibco®, NY, USA) supplemented with 2 mM l-glutamine, 10% heat-inactivated fetal bovine serum (FBS, Gibco®, NY, USA), 10 mM HEPES (N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid, Gibco®, NY, USA), 100 U/mL penicillin and 100 μg/mL streptomycin (Pen/strep, Gibco®, NY, USA) at 5% CO2 and 37 °C humidified atmosphere. For the initiation of monocyte-macrophage differentiation, 1 × 106 cells/24 wells were seeded in RPMI 1640 medium containing 12 nM PMA (Phorbol 12-myristate 13-acetate, Sigma, MO, USA) for 48 h. For the initiation of THP-1 monocyte-macrophage differentiation, 1 × 106 cells/24 wells were seeded in RPMI 1640 medium containing 12 nM PMA (Phorbol 12-myristate 13-acetate, Sigma, MO, USA) for 48 h. After differentiation, cells were treated with plant extracts (50 μg/mL) for 1 h and stimulated with 7.5 ng/mL final concentration LPS (lipopolysaccharide, Sigma, MO, USA) for additional 3 h. Cells treated with DMSO (⩽0.1%) were used as calibrator sample. Before RNA extraction, cells were washed 3 times with cold PBS (phosphate buffered saline, Gibco®, NY, USA) to remove non-attached cells.
2.6.2 RNA extraction and reverse transcription-polymerase chain reaction
Total RNA was extracted using GenElute™ Mammalian Total RNA Miniprep Kit (Sigma, MO, USA) and reverse transcribed to cDNA using High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, NY, USA) according to manufacturer’s manual. The thermal cycling conditions were set to 25 °C for 25 min, 37 °C for 120 min and 85 °C for 5 s.
2.6.3 Real-time PCR
COX-2 gene expression analysis was performed on ABI-7300 Real-Time PCR System (Applied Biosystems, NY, USA) using Pre-developed TaqMan® Assay (Applied Biosystems, NY, USA). Target cDNA was amplified using COX-2 primers: forward 5′-GAA-TCA-TTC-ACC-AGG-CAA-ATT-G-3′, reverse 5′-TCT-GTA-CTG-CGG-GTG-GAA-CA-3′ and COX-2 probe: FAM-5′-TCC-TAC-CAC-CAG-CAA-CCC-TGC-CA-3′-TAMRA (Applied Biosystems, NY, USA) under following cycling conditions: 50 °C for 2 min, 95 °C for 10 min, followed by 40 PCR cycles of 95 °C for 15 s and 60 °C for 1 min. Target genes were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and relative quantified using the 2−(ΔΔCT) method (Galasso et al., 2014).
3 Results and discussion
In order to identify changes in the metabolic profile in leaves of wild plants of F. vulgare Mill., LC-ESI-MS/MS analyses of hydroalcoholic extracts (FvSu, FvAu, FvWi, FvSp), prepared by ultrasound assisted maceration of leaf samples of herbaceous species collected in the different seasons along 2012 and 2013 years, were carried out. LC chromatograms, as well as TICs (Total Ion Current), of each extract are reported in Fig. 1. The identified metabolites are listed in Table 1.
| Peak number | RT (min) | [M−H]− | MSn | Tentative assignment | FvSu | FvAu | FvWi | FvSp |
|---|---|---|---|---|---|---|---|---|
| 1 | 1.5 | 341 | 281; 251; 221; 179 (→161; 143; 131; 119; 113; 89; 87; 71); 161; 143 | Gentiobiose (1) | + | + | + | + |
| 2 | 1.8 | 191 | 173 (→ 155;111;93); 127; 111 (→93;83) | Quinic acid (2) | + | + | + | |
| 3 | 9/10 | 353 | 191; 179 | 3-O-caffeoyl quinic acid (3) | + | + | + | + |
| 191; 179 | 5-O-caffeoyl quinic acid (4) | |||||||
| 191 | 1-O-caffeoyl quinic acid (5) | |||||||
| 173 | 4-O-caffeoyl quinic acid (6) | |||||||
| 4 | 14.5 | 367 | 191 (→179; 173; 161; 137; 127; 85) | 5-O-feruloyl quinic acid (7) | + | + | + | + |
| 5 | 17.0 | 609 | 301 (→283; 273; 257; 179; 151) | Quercetin-3-O-rutinoside (8) | + | + | ||
| 6 | 18.1 | 477 | 301 (→273; 257; 179; 151) | Quercetin-3-O-hexuronide (e.g. miquelianin; 9) | + | + | + | + |
| 7 | 19.7 | 505 | 463; 300/301 (→283; 273; 257; 179;151) | Quercetin-3-O-(6″-acetyl)hexoside (10) | + | |||
| 8 | 19.9 | 433 | 301 (→273; 257; 179; 151) | Quercetin-3-O-pentoside (11) | + | + | ||
| 9 | 20.1 | 447 | 285 (→267; 257; 243;241; 229; 213;199;151); 284 (→267; 256; 255) | Kaempferol-3-O-hexoside (12) | + | + | ||
| 10 | 20.2 | 549 | 505; 387 (→369; 225; 207; 179; 163); 225 (→163) | 12-O-(6′-caffeoylhexosyl)jasmonate (13) | + | |||
| 11 | 20.3 | 461 | 285 (→267; 257; 229; 213; 199 163) | Kaempferol-3-O-hexuronide (14) | + | + | + | + |
| 12 | 20.9 | 491 | 447; 315 (→300 →272; 271; 255; 254; 191; 179; 151; 135; 119; 107) | Isorhamnetin-3-O-hexuronide (15) | + | + | + | |
| 13 | 21.4 | 515 | 353; 191 (→179; 171; 135; 111; 85) | 3,5-Di-O-caffeoyl quinic acid (16) | + | + | + | + |
| 14 | 21.6 | 417 | 327; 285 (→229); 284 (→256; 255→227; 211) | Kaempferol-3-O-pentoside (17) | + | + | ||
| 15 | 22.6 | 233 | 191; 173 (→155; 111→93; 83; 81) | Acetyl quinic acid (18) | + | + | + | |
| 16 | 35.9 | 593 | 447; 315; 299; 285; 207 | Isorhamnetin-3-O-pentosyldeoxyhexoside (19) | + | + | ||
| 17 | 37.4 | 445 | 277 [→259; 233(→191; 177)] | 2″-Hydroxy-2″(3″,4″,5″-trihydroxylphenyl) ethyl-3-(3-(5′,7′-dihydroxychromone)-3-oxobutanoate (20) | + | |||
| 18 | 38.0 | 447 | 279 [→261(→259; 243; 233; 219; 205; 165; 109; 97; 83; 81)] | 2″-Hydroxy-2″-(3″,4″,5″-trihydroxylphenyl) ethyl- 3-(3-(5′,7′-dihydroxychromone)-3-hydroxybutanoate (21) | + |
Bold values highlight the [M−H]− ion and MS2 ions.
3.1 Metabolic profiling of extracts from F. vulgare Mill
The mass spectrum of metabolite 1 was in accordance with the presence of gentiobiose (glucosyl 1 → 6 glucose). The fragmentation of the [M−H]− molecular ion at m/z 341 gave product ions at m/z 179 and 161, associated with the glycosidic bonds cleavage. The presence of the ion at m/z 281, beside the ion a m/z 251, seemed to confirm this hypothesis (Garozzo et al., 1991).
On the basis of its fragmentation pattern, compound 2 was identified as quinic acid. In fact, the MS2 spectrum of the deprotonated molecular ion at m/z 191 showed the ion at m/z 173, resulting from dehydration of the deprotonated molecular ion, and fragment ions at m/z 127 and 111 (Galasso et al., 2014).
Between 9 and 10 min retention time four isomers of caffeoylquinic acid (3–6) co-eluted; they were previously found in fennel (Clifford et al., 2003). The analysis of MS/MS spectra, together with data reported in the literature, allowed the differentiation of the isomers. In fact, the deprotonated molecular ion at m/z 353 provided the fragment ion at m/z 173 ([quinic acid–H–H2O]−) as base peak only in the case of the 4-O isomer (6), while the fragmentation of the 1-O (5), 3-O (3) and 5-O (4) isomers leads to the formation of the ion at m/z 191, corresponding to deprotonated quinic acid, as the base peak. The last two isomers can be distinguished on the basis of the presence and different relative intensity of the ion at m/z 179, which is higher in the case of the 3-O isomer.
The study of the fragmentation pattern of metabolite 7 at m/z 367 allowed us to tentatively reveal the presence of 5-O-feruloylquinic acid. The MS2 mass spectrum showed, as base peak, ion at m/z 191. The 1-O, 3-O, 4-O and 5-O-feruloylquinic acid isomers show in their fragmentation patterns the ion at m/z 191, but only for 1-O and 5-O-isomers this is the base peak. Moreover, the presence and the relative intensity of the fragment ion at m/z 179, higher for the acid 5-O-feruloylquinic acid, allowed us to tentatively identify metabolite 7 (Clifford et al., 2003; Parejo et al., 2004a,b; Stalmach et al., 2009).
Metabolites 16 and 18 were found to be other quinic acid derivatives. In fact, in both cases the [M−H]− ion, once fragmented, provided as main fragment the ion at m/z 191, which in turn showed a fragmentation pattern superimposable to that of compound 2. In particular, metabolite 16 showed a deprotonated molecular ion at m/z 515, which underwent two neutral losses of 162 Da to give the ions at m/z 353 and 191, suggesting the presence of two caffeic acid moieties on the basic skeleton. The possible sites of esterification on quinic acid were represented by the 1,3, 3,4 and 4,5 positions. According to Clifford et al. (2003) the presence of the fragment ion at m/z 191 allowed us to exclude the 1,3 esterification. Furthermore, the 3,4 or 4,5 esterification was not supported by the presence of fragment ions at m/z 173 and 93, while the presence of product ions at m/z 171, 111 and 85 was characteristic of 3,5 esterification. From these experimental evidence, metabolite 16 was tentatively identified as 3,5-dicaffeoylquinic acid. The deprotonated molecular ion of compound 18 at m/z 233 underwent a loss of 42 Da, which could be indicative of an acetyl function. For this reason, this metabolite was identified as acetylquinic acid. Our MS data were not sufficient to determine the acetylation position.
The mass spectrum of metabolite 8 suggested the presence of quercetin-3-O-rutinoside (rutin). In fact, the collision induced decomposition of the deprotonated molecular ion at m/z 609 provided the fragment ion at m/z 301 as the base peak, likely losing a rutinose residue (−308 Da). To confirm the identity of compound 8, the LC/MS/MS data were compared with those of the rutin commercial standard.
Also metabolites 9–11 were tentatively identified as quercetin derivatives. In fact, the mass spectrum of compound 9 showed a deprotonated molecular ion at m/z 477, which provided in MS2 experiments the base peak at m/z 301, resulting from the loss of 176 Da. Comparing the fragmentation pattern of metabolite 9 with that reported by Parejo et al. (2004a,b), it was putatively identified as quercetin-3-O-glucuronide (miquelianin). Compound 10 showed a [M−H]− molecular ion at m/z 505, whose collision activated dissociation yielded product ions at m/z 463 and 301, indicating the loss of 42 and 204 Da ([M−H−42−162]−), respectively. These data were in agreement with the presence of quercetin-3-O-(6″-acetyl)hexoside. The MS/MS spectrum of metabolite 11 showed, beside the deprotonated molecular ion at m/z 433, fragment ions at m/z 301, due to the neutral loss of a pentose residue (−132 Da). Thus, this compound was tentatively identified as quercetin-3-O-pentoside.
Metabolites 12, 14 and 17 were identified as kaempferol derivatives. The MS/MS analyses allowed us to detect neutral losses of 162, 176 and 132 Da, which were characteristic of the presence of a residue of hexose, pentose and hexuronic acid, respectively. The ion at m/z 285 in all the cases was found to be the base peak. Moreover, the MS3 spectrum allowed us to discriminate between the flavonol kaempferol and the flavone luteolin, sharing the same molecular weight. In fact, our data were in accordance with Fabre et al. (2001), who reported the fragmentation pattern of both the isomers in negative ion mode, proving that only for the kaempferol the ion at m/z 229, corresponding to the fragment [M−H−2CO]−, was formed. Considering our experimental evidence, metabolites 12, 14 and 17 were tentatively identified as kaempferol-3-O-hexoside, kaempferol-3-O-hexuronide and kaempferol-3-O-pentoside, respectively.
Compound 13 was putatively identified as a dihydroxycinnamoyl hexoside of the 12-hydroxyjasmonic acid. The mass spectrum showed the [M−H]− molecular ion at m/z 549, which generated main fragment ions at m/z 505 ([M−H−CO2]−), m/z 387 (base peak), resulting from the loss of a 162 Da unit (probably corresponding to the neutral loss of a caffeic acid unit), and m/z 225 ([M−H−162−162]−). Based on tandem mass spectrometry experiments and on the relative retention time, metabolite 13 was hypothesized to be the esterification product between the carboxyl group of a caffeic acid residue and the hydroxymethyl function of the six-membered saccharide moiety. The proposed fragmentation pathway is reported in Fig. 2.
Compounds 15 and 19 were putatively identified as isorhamnetin-3-O-hexuronide and isorhamnetin-3-O-pentosyldeoxyhexoside. Deprotonated compound 15 at m/z 491, once fragmented, gave product ions at m/z 447 (−44 Da) and 315 (−176 Da), allowing us to hypothesize the presence of a hexuronic acid linked to flavonol isorhamnetin at the C-3 position. The subsequent fragmentation of the ion at m/z 315 ([isorhamnetin-H]−) confirmed the presence of a methylquercetin. In fact, it provided the ion at m/z 300, resulting from the loss of a methyl radical, beside lower intensity fragments, useful for the identification of flavonoids and arising from the breaking of two C–C bonds following retro Diels–Alder reactions involving the C ring (Cuyckens and Claeys, 2004).
Compound 19 showed the [M−H]− molecular ion at m/z 593, which in turn yielded product ions at m/z 447 (−146 Da) and 315 ([M−H−146−132]−), according to the presence of a pentosyldeoxyhexosyl moiety linked to the molecular skeleton of isorhamnetin.
Metabolites 20 and 21 were assumed to have a chemical structure containing a chromone-like skeleton. Chromanone derivatives were previously isolated and characterized in F. vulgare by Kitajima et al., 1999). In particular, compound 20 was tentatively identified as 2″-hydroxy-2″-(3″,4″,5″-trihydroxyphenyl)ethyl-3-(3-(5′,7′-dihydroxychromone)-3-oxobutanoate. The MS2 spectrum showed, beside the deprotonated molecular ion at m/z 445, fragment ions at m/z 277, which, undergoing collision-induced dissociation, gave the ions at m/z 259 and 233. Finally, the MS4 spectrum of the ion at m/z 233 showed fragment ions at m/z 191 and 177. The hypothesized fragmentation pathway is reported in Fig. 3. Briefly, the formation of the product ion at m/z 277 could be due to the loss of a 5-(oxyran-2-yl)benzene-1,2,3-triol moiety (−168 Da). Subsequently, the ion at m/z 277 could lose a CO2 (−44 Da) unit to give the peak at m/z 233, or it may undergo a rearrangement in which the shift of the acidic proton from C-2 position to the carboxyl group is followed by the dehydration with a consequent formation of the ion at m/z 259. Finally, the ion at m/z 233 fragmented giving the ions at m/z 191 (−42 Da) and 177 (−56 Da), which could correspond to the partial or complete breaking of the acylic side chain, leaving the deprotonated chromonic unit.
Compound 21 showed a [M−H]− molecular ion at m/z 447 and the fragment ion at m/z 279, both being two mass unit higher than those observed for metabolite 20. The MS3 fragmentation pattern of the ion at m/z 279 resulted perfectly superimposable with that of compound 20. For this reason it has been hypothesized that metabolite 21 differed only by the presence of a hydroxyl substituent at the C-3 position. Thus, it was putatively identified as 2″-hydroxy-2″-(3″,4″,5″-trihydroxyphenyl)ethyl-3-(3-(5′,7′-dihydroxychromone)-3-hydroxybutanoate.
3.2 Quantitative analysis of F. vulgare extracts
Winter sample (FvWi) was the richest in phenolic compounds, while in summer sample (FvSu) the lowest metabolic content was present. In particular, among the identified metabolites, compounds 1, 3–7, 9, 14 and 16 were found in all the hydroalcoholic extracts from F. vulgare (Table 2). Gentiobiose (1) was particularly abundant in FvWi sample, which showed a content 2.71, 3.57 and 3.78-fold higher than that of summer, autumnal and spring samples, respectively. Thus, its relative abundance seemed strongly influenced by harvest time. Compounds 20 and 21 were identified only in winter sample, and their amounts were estimated equal to 20.49 and 37.10 μg of quercetin equivalents per g of extract, respectively. The same sample, together with the autumnal one, contained appreciable amounts of quercetin-3-O-pentoside (11), kaempferol-3-O-pentoside (17) and isorhamnetin-3-O-pentosyldeoxyhexoside (19).
| Compounds | FvSu | FvAu | FvWi | FvSp |
|---|---|---|---|---|
| Gentiobiose (1) | 4.48 | 3.38 | 12.08 | 3.19 |
| Quinic acid (2) | 4.40 | n.d. | 0.75 | 6.87 |
| 3-O-caffeoyl quinic acid (3) | 5.89 | 7.52 | 8.60 | 10.69 |
| 5-O-caffeoyl quinic acid (4) | ||||
| 1-O-caffeoyl quinic acid (5) | ||||
| 4-O-caffeoyl quinic acid (6) | ||||
| 5-O-feruloyl quinic acid (7) | 1.17 | 1.29 | 0.04 | 0.49 |
| Quercetin-3-O-rutinoside (8) | 0.01 | 0.01 | n.d. | n.d. |
| Quercetin-3-O-hexuronide (e.g. miquelianin; 9) | 19.93 | 25.29 | 27.13 | 44.25 |
| Quercetin-3-O-(6″-acetyl)hexoside (10) | 14.50 | n.d. | n.d. | n.d. |
| Quercetin-3-O-pentoside (11) | n.d. | 5.50 | 4.89 | n.d. |
| Kaempferol-3-O-hexoside (12) | n.d. | 1.12 | n.d. | 3.35 |
| 12-O-(6′-caffeoylhexosyl)jasmonate (13) | 0.43 | n.d. | n.d. | n.d. |
| Kaempferol-3-O-hexuronide (14) | 2.05 | 5.19 | 8.60 | 12.85 |
| Isorhamnetin-3-O-hexuronide (15) | 2.28 | n.d. | 2.26 | 10.56 |
| 3,5-Di-O-caffeoyl quinic acid (16) | 0.26 | 4.13 | 1.27 | 0.36 |
| Kaempferol-3-O-pentoside (17) | n.d. | 3.06 | 3.98 | n.d. |
| Acetyl quinic acid (18) | 1.11 | 4.92 | 2.92 | n.d. |
| Isorhamnetin-3-O-pentosyldeoxyhexoside (19) | n.d. | 2.13 | 3.29 | n.d. |
| 2″-Hydroxy-2″-(3″,4″,5″-trihydroxylphenyl) ethyl-3-(3-(5′,7′-dihydroxychromone)-3-oxobutanoate (20) | n.d. | n.d. | 20.49 | n.d. |
| 2″-Hydroxy-2″-(3″,4″,5″-trihydroxylphenyl) ethyl-3-(3-(5′,7′-dihydroxychromone)-3-hydroxybutanoate (21) | n.d. | n.d. | 37.10 | n.d. |
3.3 Antioxidant activity of F. vulgare leaf hydroalcoholic extracts
Hydroalcoholic extracts from fennel leaves were evaluated for their antioxidant capacity through the application of different tests (Table 3). Folin–Ciocalteu method (based on Folin Ciocalteu Reagent, FCR) highlighted the seasonal variation of total polyphenol content (TPC, Total Polyphenol Content). In fact, FvWi extract showed the highest TPC value (100.0 mg GAE per g). TP content strongly decreased in spring extract (59.3 mg GAE/g) and autumnal one (40.5 GAE mg/g), reaching its lowest level in FvSu extract (29.4 GAE mg/g).
| F. vulgare | ID50 ABTS•+ | ID50 DPPH• | TEAC ABTS•+ | TEAC DPPH• | TEAC Fe(III)RP | ORAC values | TPC |
|---|---|---|---|---|---|---|---|
| FvSu | 88.37 | 57.72 | 1.93 | 2.11 | 10.53 | 437.86 | 29.4 |
| FvAu | 64.41 | 55.18 | 2.65 | 2.21 | 11.91 | 141.55 | 40.5 |
| FvWi | 46.01 | 40.12 | 3.95 | 3.04 | 21.57 | 119.78 | 100.0 |
| FvSp | 245.79 | 321.51 | 0.69 | 0.38 | 2.84 | 168.33 | 59.3 |
| Trolox® | 1.71 | 1.22 |
The high TP content of FvWi extract seemed to be correlated with its antiradical properties. In particular, FvWi sample was able to scavenge ABTS•+ 5.33, 1.40 and 1.92-fold higher than FvSp, FvAu and FvSu, respectively. Unexpectedly, FvAu sample provided a scavenging power greater than FvSp, although this latter was quantitatively richer in polyphenolic constituents. Indeed, autumnal sample showed a percentage content of molecules as dicaffeoylquinic acid higher than that of sample spring-collected. In fact, when the percentage content of the individual constituents in each extract was calculated, it was observed that FvAu extract had a dicaffeoylquinic acid content about 17-fold higher than that of spring extract, whereas the content of other compounds, such as the miquelianin (9) and caffeoylquinic acids (3–6), was similar. The presence of two free catechol functions in the compound 16 could contribute massively to the observed radical scavenging activity (Goupy et al., 2003; Thavasi et al., 2006; Fiorentino et al., 2007). A similar behavior was observed when Fe3+ reducing power data were evaluated. FvSp sample exerted a reducing activity about 4-fold lower than FvAu sample. Also in this case, winter extract promoted the strongest reducing effectiveness (21.6 g of Trolox® equivalents per g of extract; Table 3).
Peculiar differences were found between the results obtained from the tests described above and those obtained by the ORAC method. In fact, the ORAC test data promoted the summer extract (FvSu) as the sample with highest radical scavenging capacity. On the basis of data obtained from individual tests and taking into account the relevant indices of correlation the RACI (Relative Antioxidant Capacity Index) value was calculated for each extract (Table 4a). Data obtained by ABTS method (Table 4b) were positively correlated with those obtained by the DPPH (R = 0.95) and Fe3+ RP (R = 0.99). The RACI index allowed us to compare data by the different adopted chemical methods providing a more comprehensive and dimensional determination of the antioxidant capacity of the investigated plant complexes. FvWi sample had the highest RACI value (0.844). In particular, RACI value allowed us to observe that the accumulation of metabolites able to confer antioxidant efficacy to fennel extracts was mainly exhibited during winter and summer seasons. Our findings are in accordance with Xu et al. (2011), who evaluated the influence of growing season (winter vs. summer) on the synthesis and accumulation of phenolic compounds and antioxidant properties in five grape cultivars, and found that both phenolic compounds and antioxidant properties of winter berries were significantly higher than those of summer berries for all the investigated cultivars.
| ABTS•+ RSC | DPPH• RSC | Fe3+ RP | ORAC value | TPC value | RACI | |
|---|---|---|---|---|---|---|
| (a) | ||||||
| FvSu | −0.275 | 0.157 | −0.154 | 1.487 | −0.869 | 0.069 |
| FvAu | 0.253 | 0.246 | 0.026 | −0.507 | −0.542 | −0.105 |
| FvWi | 1.207 | 0.989 | 1.282 | −0.653 | 1.397 | 0.844 |
| FvSp | −1.185 | −1.392 | −1.154 | −0.327 | 0.015 | −0.809 |
| ABTS | DPPH | Fe3+ RP | ORAC | TPC | |
| (b) | |||||
| ABTS | 1 | 0.953881 | 0.987824 | −0.31286 | 0.590033 |
| DPPH | 1 | 0.952099 | −0.02779 | 0.363763 | |
| Fe3+RP | 1 | −0.23409 | 0.631177 | ||
| ORAC | 1 | −0.64488 | |||
| TPC | 1 | ||||
Bold value underlines the RACI value of the most antioxidant extract.
3.4 Cytotoxic effects of F. vulgare leaf hydroalcoholic extracts
In order to obtain a cytotoxic profile of the investigated extracts and to fit out non-toxic and safe preparations, five different human cell lines (HCT-116, MDA-MB-231, U-251, CCRF-CEM and MRC-5) were treated with the investigated fennel extracts (dose level range 10–250 μg/mL; Table 5). After the considered exposure times, cell viability was tested through application of the XTT test. As metabolically active cells reduce XTT, the resulting formazan amount is proportional to their number. The results showed that the U-251 metabolic activity was weakly inhibited by the treatment with different doses of the four extracts, after 24, 48 and 72 h exposure times. Similarly, the viability of the MDA-MB-231 cells, after 48 h exposure time was reduced by almost 25%. The other cell lines tested did not appear to be affected by the treatment with the extracts, with some exceptions.
| FvSu | FvAu | FvWi | FvSp | VNB | |||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| L | M | H | L | M | H | L | M | H | L | M | H | 0.01 μM | |
| 24 h | |||||||||||||
| MRC-5 | ○ | ○ | ○ | ○ | ○ | ○ | ○ | ○ | ○ | ○ | ○ | ○ | ○ |
| CCRF-CEM | ○○ | ○○ | ● | ○○ | ○○ | ○ | ○○ | ○○ | ○ | ● | ○ | ● | ●● |
| HCT-116 | ○ | ○ | ○ | ○ | ○ | ○ | ○ | ○ | ○ | ○ | ○ | ○ | ●● |
| MDA-MB-231 | ○ | ○ | ○ | ○ | ○ | ○○ | ○○ | ○○ | ○○ | ○ | ○○ | ○○ | ●● |
| U-251 | ● | ● | ● | ● | ● | ○ | ● | ● | ● | ● | ● | ○ | ●●● |
| 48 h | |||||||||||||
| MRC-5 | ○ | ○ | ○ | ○ | ○○ | ○○ | ○ | ○ | ○○ | ○ | ○○ | ○○ | ○ |
| CCRF-CEM | ○ | ○○ | ○○ | ○ | ○○ | ○○ | ○○ | ○○ | ○○ | ○○ | ○○ | ○○ | ●●● |
| HCT-116 | ○ | ○ | ○ | ○ | ○ | ○ | ○ | ○ | ○ | ○ | ○ | ○ | ●●● |
| MDA-MB-231 | ● | ● | ● | ● | ● | ○ | ● | ● | ● | ● | ● | ● | ●●● |
| U-251 | ● | ● | ● | ● | ● | ● | ● | ● | ● | ● | ● | ● | ●●● |
| 72 h | |||||||||||||
| MRC-5 | ○ | ○ | ○○ | ○ | ○○ | ○○ | ○○ | ○○ | ○○ | ○○ | ○○ | ○○ | ○ |
| CCRF-CEM | ○○ | ○○ | ○○ | ○○ | ○○ | ○○ | ○○ | ○○ | ○○ | ○○ | ○○ | ○○ | ●●● |
| HCT-116 | ○ | ○ | ○ | ○ | ○ | ○ | ● | ○ | ○ | ○ | ○ | ○ | ●●● |
| MDA-MB-231 | ● | ○ | ● | ○ | ○ | ○ | ● | ○ | ○ | ○ | ○ | ○ | ●●● |
| U-251 | ● | ● | ● | ● | ● | ● | ● | ● | ● | ● | ● | ● | ●●● |
○ = cell viability ∼ untreated control; ○○ = cell viability increase > 25%; ● = cell viability decrease < 25%; ●● = cell viability decrease > 25% and <50%; ●●● cell viability decrease > 50%.
3.5 Cellular protection against H2O2 induced cell death
Hydrogen peroxide (H2O2) is known to be generated within human tissues as a by-product of metabolic processes which, if under conditions of oxidative stress, may accelerate the rate of cellular injuries (Sakulnarmrat et al., 2013). In order to evaluate the potential cellular protection of fennel extracts from H2O2 induced cell death, MRC-5 cells, as well as CCRF-CEM, HCT-116, MDA-MB-231 and U-251 cells, were pre-treated for 24 h with 50 μg/mL (dose at which the cells metabolic activity was not or weakly affected) and then challenged with H2O2 for 1 h. The adverse effect of H2O2 was diversely counteracted by the investigated fennel extracts (Fig. 4). In particular, FvWi sample induced a marked cytoprotective effect (up to 50%) toward MRC-5 cells and HCT-116, favored an increase in metabolic activity by 33% in MDA-MB-231 cells and protected weakly leukemia cells. Spring extract protected the HCT-116 (28%) and MRC-5 (38%) cells from oxidative insult. FvSu and FvAu extracts exerted cytoprotective effects toward colon cancer cells (38% and 13%, respectively).
3.6 Anti-inflammatory activity of fennel leaf extracts
All the extracts from F. vulgare leaves seemed to influence the expression of COX-2 gene in THP-1 macrophages. FvWi extract was the most active; it was capable of inhibiting the synthesis of COX-2 by 40%. The activity of FvAu and FvSu extracts was not entirely negligible (Fig. 5). The strong anti-inflammatory capability of FvWi extract could be due to the presence of the two chromononic derivatives (20 and 21) and to an optimum quantitative ratio of the metabolites constituting the extract. LC-ESI-MS/MS phenolic profile data seemed to support this assertion. In both the extracts miquelianin (9) content was almost equal to 40%. FvSp sample, which contained a massive amount of metabolite 9 and of other glucuronidated flavonols, showed a pro-inflammatory potential. Recently, Ishisaka et al. (2013) evaluating the inflammatory responses in LPS-stimulated RAW264 cells, observed that quercetin and miquelianin exerted distinct activities. In fact, when they evaluated the early phase of the COX-2 expression in the macrophages at 4 h after treatment with LPS, because no accumulation of the quercetin aglycone was observed within the 4 h-culturing of the cells in the presence of miquelianin, it was clearly demonstrated that the quercetin aglycone inhibited the LPS-induced COX-2 expression (of the protein and of mRNA), whereas miquelianin was not able to affect the COX-2 expression even at higher concentrations. However, the LPS-induced COX-2 expression was significantly inhibited in the miquelianin-treated cells in the presence of the β-glucuronidase enzyme. Moreover, it was reported that glucuronidation and/or sulfation of hydroxyl group(s) on polyphenols attenuates the anti-oxidative and anti-inflammatory activities and promotes their rapid excretion (Williamson et al., 2005). Thus, quercetin-3-glucuronide itself failed in inhibiting the anti-inflammatory gene expressions in macrophages and the β-glucuronidase-mediated conversion into the aglycone is essential for the inhibitory effects (Kawai, 2014).
4 Conclusions
The phytochemical analysis of hydroalcoholic herbal preparation based on fennel leaves aimed at establishing, preliminarily, their phenol composition variation during the year. LC–MSn based metabolic profiling analyses emphasized that fennel is a rich and perennial source of polyphenol ingredients. The opportune knowledge of phenols abiotic stress-induced expression in wild fennel plants could address the maintenance of their biodiversity safeguard and optimize their domestication in order to realize a production chain that exploits environmental conditions favorable to the production of active and safe polyphenol complexes, which could be used in different fields as plant-based products.
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