Introduction

Considering increasing global climate change and severe conditions that prevail throughout the world, cultivation of conventional crops is facing various limitations related with scarcity of good quality water, temperature increase, salinization, heavy metals contamination and degradation of soil properties, especially in arid and semi-arid regions of the Mediterranean basin [1, 2]. The common feature of the plant responses to different stresses is the production of reactive oxygen species (ROS) [3]. The overproduction of ROS in plants causes impairment of redox homeostasis, resulting in oxidative stress [4, 5]. The oxidative stress generated by ROS in plants has detrimental effects on crop production [6]. Therefore, it is of utmost importance to valorize other edible and medicinal species that can adapt under harsh conditions within the framework of saline agriculture, thus being good candidates as potential cash food and medicinal crops [7].

More than 100 halophyte species are promising candidates with important nutritional, medicinal and economic interests [2]. These salt-tolerant plants have developed adaptive responses, including the accumulation of highly bioactive molecules with powerful antioxidant capacities (e.g. phenolic compounds, antioxidant enzymes and vitamins), to cope with the production of ROS and to protect the cellular structures from these highly toxic entities [3, 8]. These natural antioxidants generally exhibit strong biological activities, such as radical scavenging, metal chelation and enzyme induction capabilities, leading to beneficial therapeutic properties. Thus, some halophytic species which accumulate them are used as ingredients in many traditional dishes, recipes and beverages [9,10,11,12]. Along, the recent advances in food science and technology dictate the evaluation of plant safety profile via metabolomic and toxicological studies to produce a safety and healthy food [8, 12].

The perennial halophyte sea fennel (Crithmum maritimum, Apiaceae) is a typical plant of the Mediterranean, Pacific and Atlantic coasts [4, 5, 13]. Interestingly, its growth is stimulated by low salinity, and it is tolerant to salt stress and climate variations [14], making it a promising crop in the context of biosaline agriculture. C. maritimum was recognized as a promising crop for human nutrition [15] since its leaves are a source of minerals and vitamins and have a convenient nutritional profile for human consumption and animal farming. Already, they are currently used as salads, pickles and to prepare soups [13, 14]. The areal parts of this perennial halophyte represent various economical interests, due to its high contents in flavonoids, carotenoids, vitamin C and substances with nutraceutical and antimicrobial properties [14]. In the Mediterranean regions, sea fennel has a good reputation as a traditional remedy. As an example, in Italy, the decoction of aerial parts has been used to treat whooping cough and pain, inflammations of urinary tracts and prostate, colics, as well as a liver-detoxifying remedy [16, 17].

Many plant extracts and plant-derived compounds have been tested using animal models for their potential protective activities against hepatotoxic agents such as carbon tetrachloride (CCl4) [18, 19]. Although the aerial parts of C. maritimum are widely studied in vitro [8, 12, 14, 20, 21], no in vivo studies using this plant have been reported hitherto. Therefore, the present study was undertaken to explore along with the antioxidant properties of C. maritimum leaf hydro-methanolic extract (CME), and the possible protective effects of C. maritimum (CM) leaves against CCl4-induced hepatotoxicity in rats.

Materials and methods

Plant material

Leaves of Crithmum maritimum L. were identified and sampled on rocks along the shoreline at “Sainte Anne du Portzic” by Professor Christian Magné (Brittany, France) in August 2016. The leaves were cleaned with deionized water, rapidly soaked, stored at − 20 °C and then freeze dried. The dry material was ground to a fine powder, and stored until analysis.

Methanolic extraction

One gram of sea fennel leaf powder was macerated in 10 mL of 80% methanol overnight under stirring at room temperature. The mixture was centrifuged for 10 min at 3500×g at 4 °C (Jouan CR3i centrifuge, Newport Pagnell, UK) and supernatant was collected, filtered (0.2 mm VWR International PBI, Milan, IT), and kept at 4 °C in the dark until use. The extraction was repeated twice on the pellet and the three filtered supernatants were gathered. This procedure resulted in a C. maritimum extract (CME) with a yield of 22%.

Phytochemical characterization and phenolic compound profiling by LC–ESI–MS analysis

The total phenolic content was determined by the Folin Ciocalteu colorimetric method [22] and expressed as mg of gallic acid equivalents/g dry weight (mg GAE/g DW). The total flavonoid contents were quantified using the aluminum chloride colorimetric method [23] and expressed as mg catechin equivalent (CE)/g DW. The total flavonols were measured according to the method described by [24] and expressed as mg quercetin equivalent (QE)/g DW. The phenolic profile of C. maritimum extract was characterized by LC–ESI–MS analysis.

Determination of antioxidant activities (DPPH test and ORAC assay)

The scavenging activity on DPPH radical of methanolic extracts of C. maritimum leaves extracts was determined following the method reported by Sokmen et al. [25]. The Oxygen Radical Absorbance Capacity (ORAC) of C. maritimum leaf extract was evaluated according to the method reported by Bacchiocca et al. [26].

In vivo hepatoprotective assay

Animal treatments

This study was performed using male Wistar rats with body weight of 180–200 g. The animals received food and drinking water ad libitum and were maintained in cages under a 12 h light/dark cycle at room temperature with 55% relative humidity. The rats were separated into four groups of five animals: control rats (CNT), rats supplemented daily during 5 days with a water suspension of C. maritimum leaves by gavage at 300 mg/kg bw (CM), rats injected intra-peritoneally (i.p.) with a single dose of 1.5 mL CCl4/kg bw dissolved in corn oil (CCl4) [18] and sacrificed after 24 h, and rats given daily a water suspension of C. maritimum leaves at 300 mg/kg bw during 5 days followed on day fifth by a single i.p. dose of 1.5 mL CCl4/kg bw (50% in corn oil) (CCl4 + CM). Rats from each group were sacrificed at the end of day 6. The dose of the suspension of C. maritimum leaves was selected on the basis of previous studies on other halophyte plants [27]. Rats from each group were sacrificed at day sixth 24 h after the last injection. At the sacrifice, the animals did not show any visual symptoms.

All animal procedures were performed with the approval of the Local Ethical Committee and in accordance with the European Communities Council Directive of 24 November 1986 (86/609/EEC).

Biological sample collection

The blood samples were drawn by cardiac puncture and centrifuged at 3000×g for 10 min. The serum was stored at − 20 °C until analyses. Liver was carefully removed, washed with ice-cold saline solution and then stored at − 80 °C for future analysis. A portion of liver was collected and fixed in 10% formalin solution and immediately processed for histological study.

Serum biochemical analysis

The levels of creatinine and the activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were determined using a semiautomatic clinical chemistry analyser (model ARCO Biotecnica Instruments, SPA, Italy).

Hepatic oxidative stress biomarkers

The malondialdehyde (MDA) concentration of liver homogenates was analyzed as marker of lipid peroxidation according to [28], with some modifications. A standard curve was made from a solution of the hydrolyzed 1,1,3,3-tetramethoxypropane (TEP) in water. MDA concentrations were calculated as µmol/g tissue. The protein oxidation level was determined by the carbonyl protein assay according to [29]. Concentration of carbonylated proteins was calculated as nmol/g tissue. The glutathione (GSH) content was determined following the method described by [30].

Liver enzyme assays

The microsomal and cytosolic fractions from liver were prepared according to Longo et al. [50]. Protein content was determined according to the method of [31], using bovine serum albumin (BSA) as the standard.

The heme oxygenase activity was spectrophotometrically determined by quantifying the bilirubin produced from the reduction of biliverdin [32]. To determine catalase (CAT) activity, hydrogen peroxide (H2O2) was used as substrate [33]. The degradation of H2O2 was detected by a decrease in absorbance at 240 nm for 1 min and the enzyme activity was expressed as µmol H2O2 consumed per minute per mg of protein.

Ethoxycoumarin-O-deethylase (ECOD) and ethoxyresorufin O-deethylase (EROD) were determined fluorometrically by measuring the formation of 7-hydroxycoumarin and resorufin, respectively [34]. The pNPH activity was determined by measuring the formation of p-nitrocatechol [35]. The DT-diaphorase activity was assayed by following the reduction in dichlorophenolindophenol at 630 nm [36].

Histopathological analysis

Liver samples (n = 5) were collected from the rats of each experimental group for histological analysis and fixed in 10% neutral buffered formalin. After overnight fixation, livers were routinely dehydrated (throughout alcohol series 40%, 70%, 95%, 100% and xylene) and embedded in paraffin wax blocks, sectioned at a thickness of 5 μm and stained with haematoxylin and eosin [37]. All the slide sections were examined under a light microscope.

Statistical analyses

Statistical analyses were performed using XLStat 2016®. Results were expressed as mean ± standard deviation (SD) and experiments were conducted at least in triplicate. Significant differences (p < 0.05) were assessed by one-way analysis of variance (ANOVA). If significant, the pairwise multiple comparison tests Tukey or Dunn's were applied.

Results and discussion

Antioxidant properties

The content of total polyphenols, flavonoids, flavonols and the antioxidant capacity were measured in C. maritimum leaf extract and depicted in Table 1. The extract contained 26.25 mg GAE/g DW of polyphenols, 15.6 mg CE/g DW of flavonoids, and 12.2 mg QE/g DW of flavonols. Similar results were found in sea fennel plants harvested during summer from the Breton cliffs [14] or in Tighzert region (Bejaïa, Algeria) [21]. Overall, sea fennel was confirmed to be rich in phenolic compounds, compared to other Apiaceae such as Foeniculum vulgare [38] or Eryngium maritimum [14].

Table 1 Phenolic contents, DPPH scavenging activity and ORAC value in hydro-methanolic extract of C. maritimum leaves
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The in vitro antioxidant capacity and the radical scavenging activity of CME, as measured using the ORAC and the DPPH assays, are shown in Table 1. CME had 11,253 ORAC units per gram of DW, and a DPPH IC50 of 0.25 mg/mL. Other studies reported high antioxidant activities of C. maritimum extract [8, 14, 39]. The concomitant high phenolic contents and antioxidant activities confirm that phenolic compounds are likely major contributors to the antioxidant activities of C. maritimum extracts [12, 40], as commonly considered for halophytic species [41].

Phytochemical profile of CME extract

The phenolic profile of C. maritimum extract was characterized by LC–ESI–MS analysis and depicted in Fig. 1. Seventeen different compounds were detected and quantified, totalizing 18.19 mg/g DW (Fig. 1, Table 2). Other authors have reported a similar yield of these phytochemical compounds extracted from C. maritimum although with different extraction methods [12, 21]. Besides, [14] observed by NMR analysis that the most part of methanolic extract of this plant is represented by soluble sugars and organic acids (malate and quinate).

Fig. 1
figure 1

LC–ESI–MS analysis (280 nm) of soluble phenolic compounds in hydro-methanolic extract of C. maritimum. Peak numbers refer to the compounds shown in Table 2

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Table 2 Phenolics and allied compounds (mg/g DW) identified in the C. maritimum leaves hydro-methanolic extract using LC–ESI–MS analysis
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Our analyses showed that the most represented phenolic classes in CME are phenolic acids, flavonoids and flavonols. Indeed, the high content in phenolic acids (and allied compounds) in the leaves of C. maritimum extract was mainly composed of chlorogenic acid (28.36%), quinic acid (organic acid) (20.12%), neochlorogenic acid (9.40%) and trans ferulic acid (6.32%). Among the flavonoids, rutin was the major compound detected. Besides, kaempferol (7.75%) and quercetrin (6.54%) were the most abundant flavonols [14] reported that C. maritimum extract exhibited a significant concentration of chlorogenic acid and quinic acid. Our analyses showed the presence of six hydroxycinnamic acids (n° 2, 3, 4, 7, 8, 9 in Table 2), in accordance with the results described by [12] in the water infusion or decoction of sea fennel leaves. However, these authors did not detect free quinic and caffeic acids [21] reported the HPLC/DAD–ESI–MS analysis of the hydro-methanolic extract of aerial parts of C. maritimum. They identified the six hydroxycinnamic acids we also measured and, at a lower level, 3-coumaroylquininic acid, cis-5-coumaroylquininic acid, 3,4-dicaffeoylquinic acid and 3,5-dicaffeoylquinic acid. The presence in the boiling water extract of sea fennel of these six major phenolic acids was also reported by [20]. On the other hand, among the flavonoids and flavonols in C. maritimum extract, only rutin and hyperoside have been previously reported [42], though kaempferol was found here as the major flavonol. All these secondary metabolites have been associated with the strong antioxidant activity of several vegetables and their health-promoting effects, as for example the modulation of glucose and lipid metabolism and the protective effects against ROS damages [43].

In vivo effects of CCl4 treatment and CM administration

CCl4 induces toxicity by generating the reactive intermediates trichloromethyl (CCl3*) and trichloromethyl peroxyl radical [44]. The activation of CCl4 is performed by the hepatic cytochrome P450 system and in particular by the P450 2E1 isoform [45]. These radicals alkylate cellular proteins and other macromolecules including polyunsaturated fatty acids, giving rise to lipid peroxidation (MDA) and protein carbonylation. Moreover, they cause damages to hepatic cells and kidney, leading to a leakage of aminotransferases (ALT and AST) and creatinine in plasma [45,46,47]. The GSH level, which is the prominent antioxidant in liver, is reduced by the reaction with hydrogen peroxide and hydroperoxides formed by the CCl4 treatment [48].

To investigate a possible protective role of CM against hepatotoxicity, we treated rats by CCl4. We used a suspension of C. maritimum leaves and not the extract at the light of recent studies that show how the administration of an extract can lead to a significant different modulation pathways of xenobiotic metabolizing enzymes, compared to the whole vegetable [49]. As a consequence, AST and ALT levels in plasma increased markedly (p < 0.01), indicating CCl4-induced liver injury (Table 3). Moreover, CCl4 treatment caused a strong oxidative stress, as seen by the significant increase of MDA level and protein carbonylation, as well as the decrease of the hepatic glutathione content and plasmatic creatinine (Table 3). These metabolic markers were not affected by the CM treatment alone highlighting that the dose of CM administered for 5 days was not toxic for animals. Interestingly, the pre-treatment of rats with CM prior to administration of CCl4 partially maintained control levels of GSH, protein carbonylation and creatinine. Moreover, CM pre-treatment significantly reduced the activities of ALT and AST (p < 0.05), when compared to CCl4-treated group. All these observations strongly suggest a protective role of CM against CCl4 toxicity, though it failed to impede lipid peroxidation (TBARS levels being similar in the CCl4 and CM + CCl4 groups).

Table 3 Effect of Crithmum maritimum leaves water suspension (CM) on plasma ALT and AST activities, and creatinine, as well as on MDA, protein carbonyls and glutathione contents in the liver of CCl4-treated rats
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To see the effect of CCl4 on drug metabolizing system, we have measured some P450-dependent activities such as EROD, ECOD, pNPH and other activities of phase II enzymes. As shown in Fig. 2, the administration of CM at the dose of 300 mg/kg bw did not significantly affect the activities of hepatic ECOD, EROD, and pNPH, which are biomarkers of multiple P450 isoforms, P450 1A1/2, and of P450 2E1, respectively [50]. The CCl4 treatment markedly induced the pNPH activity compared to control group, whereas EROD and ECOD activities were strongly decreased. Some studies have reported the reduction of many P450 activities after application of CCl4, and conversely the induction of P450 2E1 to perform the CCl4 metabolism, since this compound is a selective substrate of this enzyme [43]. Noteworthy, EROD and particularly pNPH did not change much in the CCl4-intoxicated rats when pre-treated with CM.

Fig. 2
figure 2

Effect of Crithmum maritimum leaves water suspension (CM) on ECOD, EROD and pNPH activities in hepatic microsomes of CCl4-induced rats. Values are expressed as mean ± SD of five rats in each group. Values with different letters are significantly different (p < 0.05)

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As observed for the P450-dependent oxidative metabolism, the activity of DT-diaphorase, CAT and heme oxygenase enzymes was not significantly affected by the CM treatment alone, compared to control (Table 3). Conversely, CCl4 treatment significantly increased the DT-diaphorase and heme oxygenase activities and reduced that of CAT (Table 4). These results are in agreement with the literature showing that CCl4 significantly decreases CAT activity [51], whereas it induces the activity of DT-diaphorase and heme oxygenase as an adaptive response to oxidation injury [52, 53]. Interestingly, pre-treatment of rats with CM partially mitigated the above-mentioned changes. Overall, these findings indicate the partial preventive protection against hepatic damages by CM administration at 300 mg/kg bw for 5 days. The disease prevention by natural products is at the heart of the scientific debate and the dose of the substance taken can be crucial. In the last decade, the scientific community has experienced how several compounds, specifically hypothesized to protect or reduce risk, have actually increased the incidence of the disease they hoped to prevent [54, 55].

Table 4 Effect of C. maritimum leaves water suspension (CM) on liver parameters in CCl4-treated rats
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Histopathological analysis

Histopathological examination of the rat liver tissues from the different groups is shown in Fig. 3. Control group (Fig. 3a, b) showed normal liver morphology with well-preserved cytoplasm and prominent nucleus in hepatocytes; same tissue architecture was observed in the liver from CM group (Fig. 3c, d), indicating that the CM alone had no negative effects on the liver anatomy and functionality. Conversely, treatment with CCl4 induced severe injuries in liver tissues such as cytoplasmic vacuolization of hepatocytes, cellular swelling, fatty degeneration, obvious tissue necrosis, congested central vein, and infiltration by inflammatory cells (Fig. 3e, f, arrows). Such observations confirm those previously reported by [53] and [56].

Fig. 3
figure 3

Photomicrographs of hepatic tissue in control and experimental treated rats. liver sections stained with hematoxylin and eosin (magnification ×50 and ×100). a, b Control group; c, d CM-treated rats; e, f CCl4-treated rats; and g, h CCl4 + CM-treated rats, arrows indicates lipids and erythrocyte infiltration in central vein

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Noteworthy, CM pre-treatment mitigated histological features of CCl4-induced liver injuries and clearly ameliorated the pathological changes in liver tissues: steatosis and hepatocyte vacuolization were alleviated and the liver anatomy looked similar to those of the control or CM groups, indicating an hepatoprotective effect of the plant (Fig. 3g, h). The remarkable anti-steatosis effect of CM pre-treatment is likely due to the antioxidant compounds present in the plant leaves, since recent studies have shown that the use of natural antioxidants can positively act against steatosis and fibrosis [19, 56].

Conclusion

The present study confirmed that the hydro-alcoholic extract of C. maritimum leaves contains abundant soluble polyphenols with important antioxidant properties as demonstrated by their high ORAC and DPPH-scavenging activities. The phytochemical analysis using LC–ESI–MS allowed us to identify 17 compounds among hydroxycinnamic acids, flavonoids and flavonols. Moreover, our study used the highly toxic CCl4 as a model to induce acute liver damage. The administration of a suspension of C. maritimum leaves significantly prevented rats from the toxic effects of CCl4, as evidenced by its ability to partially restore the hepatic activities of some P450 enzymes, antioxidant enzymes such as catalase, DT-diaphorase and heme oxygenase, and the contents of GSH and protein carbonyls. Moreover, the histopathological results also suggested that C. maritimum has no adverse effects. Thus, the medicinal properties of C. maritimum are broaden by this work, and its richness in bioactive compounds with a great antioxidant potential makes CM a good candidate to be used as natural food and/or as a source of natural bio-antioxidants.