Phytochemical and pharmacological variability in Golden Thistle functional parts: comparative study of roots, stems, leaves and flowers

Abstract Scolymus hispanicus or the Golden Thistle, locally known as ‘Guernina’ or ‘Taghediwt’, is one of the most appreciated wild vegetables in Morocco. This study aims to characterise the functional chemical and pharmacological variability of Scolymus hispanicus parts (roots, stems, leaves and flowers). The chemical analysis revealed higher content of α-tocopherol in the flowers (2.79 ± 0.07 mg/100 g) and lead to the identification of 3 flavonoids and 13 phenolic acids, with high content of gallic acid in leaves (187.01 ± 10.19 mg/kg); chlorogenic (936.18 ± 92.66 mg/kg) and caffeic (4400.14 ± 191.43 mg/kg) acids in flowers, roots were much more higher in sinapic acid (0.25 ± 0.03 mg/kg) compared to the other parts. Moreover, Scolymus hispanicus ethanolic extracts exhibited interesting antioxidant and antimicrobial properties, promising anti-amylase and anti-glucosidase activities and relevant diuretic effect that confirms its traditional uses.


Introduction
Wild plants are often associated with times of famine or food scarcity. Apparently, because of the inadequate information on their nutritional contents and potential to serve as a secure food, non-cultivated vegetables have not received much attention. Recently, interest in wild leafy vegetables has significantly increased around the world, due to the recent investigations and proofs of high nutritional value associated with potential health benefits (Sánchez-Mata et al. 2012). Until recently, wild food use in Morocco has been significantly under-reported in the literature (Powell et al. 2014;Nassif & Tanji 2013). The Golden Thistle (Scolymus hispanicus) locally known as 'Guernina' or 'Taghediwt' , is one of the most appreciated wild vegetables in Morocco, Spain and other Mediterranean countries (Polo et al. 2009). The part used for consumption is stems after being peeled from spines raw or cooked with meat. In Morocco, the stems are consumed raw or cooked as a vegetable with meat after being peeled from spines (Nassif & Tanji 2013), while in Spain the central nerve of the prickly basal leaves are boiled and then usually lightly fried with a bit of garlic, cured ham and scrambled eggs, often also served as a garnish for the traditional Spanish dish cocido. In addition, the root has been employed as a coffee substitute and its flowers as a colouring alternative to saffron (Tardío & Pardo-de-Santayana 2016). Scolymus hispanicus have been linked to many medicinal properties such as diuretic, depurative, digestive, choleretic and lithiuretic (Polo et al. 2009). However, few papers investigated its pharmacological properties; for instance the antispasmodic, spasmogenic (Kirimer et al. 1997), anti-protozoal, cytotoxic (Camacho et al. 2003 and antioxidant activities (Pieroni et al. 2002;Morales et al. 2014) have been reported. In this focus, the present study aims to determine which part of the plant contains high levels of nutrient compounds, antioxidants molecules and exhibit diuretic and glucosidase inhibitory activities.
HPLC-MS analysis of ethanolic extracts from Scolymus hispanicus allowed identifying three flavonoids and thirteen phenolic acids, by means of comparison based on retention times and mass fragmentation pattern with data obtained from reference standards. The concentrations of each compounds calculated from calibration curves are reported in Table 1. Interesting differences were recorded in the extracts; Roots were much more higher in sinapic acid (0.25 ± 0.03 mg/kg); Leaves in gallic (187.01 ± 10.19 mg/kg), syringic (3.60 ± 0.16 mg/kg), p-coumaric (17.25 ± 1.33 mg/kg), salicylic (46.50 ± 3.22 mg/kg), and p-hydroxybenzoic (116.98 ± 7.21 mg/kg) acids, also in pyrogallol (4.67 ± 0.34 mg/kg) and resveratrol (0.1133 ± 0.0213 mg/kg). On the other hand, flowers were rich in chlorogenic

Antioxidant activities
The antioxidant effects of Scolymus hispanicus extracts using the DPPH, ABTS and FRAP methods are illustrated in Table S1. Flowers extract exhibited the most potent antioxidant activity in all assays. Radical scavenging activity and ferric reducing ability of the Golden thistle is linked to its tocopherol and phenolic contents. Antioxidant activity of Scolymus hispanicus was reported previously from leaf stalks (Pieroni et al. 2002) and basal leaves (Morales et al. 2014).

Antidiabetic properties
In order to explore the in vitro antidiabetic activity, Scolymus hispanicus extracts were screened for the α-amylase and α-glucosidase inhibitory properties and compared to the reference drug acarbose. Inhibitory activities of the extracts were evaluated at different concentrations and results were given in Figure S2. The IC 50 values for anti-amylase and anti-glucosidase activities were much lower than that of acarbose. IC 50 for amylase inhibition were 1299.75 μg/mL, 1538.46 μg/mL, 1640.35 μg/mL and 1632.65 μg/mL for roots, stems, leaves and flowers, respectively. While, acarbose inhibitory concentration was 396.42 μg/mL. In the same way, the α-glucosidase inhibitory effect of the extracts was lower than acarbose. In term of IC 50 , significant differences were registered between Scolymus hispanicus and the reference: roots (2088.11 μg/mL), stems (1267.90 μg/mL), leaves (2388.34 μg/mL) flowers (1271.45 μg/mL) and acarbose (199.53 μg/mL). Antidiabetic activity of Scolymus hispanicus was reported previously in a streptozotocin model of diabetes (Ozkol et al. 2013). However, this is the first report of anti-enzymatic activities against key enzymes in hyperglycaemia.

Diuretic effect and acute toxicity
Animals treated with 2000 mg/kg of the extracts did not show any change in their behavioural pattern, body weight and food consumption during the acute toxicity study. Oral administration of the plant extracts increased the urinary output. The urinary excretions at 24 h after treatment with Scolymus hispanicus and 2000 mg/kg were 3.05 ± 0.69, 4.69 ± 0.47, 4.44 ± 0.71 and 5.34 ± 0.83 mL/100 g for roots, stems, leaves and flowers, respectively. Flowers, stems and leaves values were significantly higher (p < 0.05) when compared to the control group (3.04 ± 0.84 mL/100 g). Therefore, Scolymus hispanicus led to a relative increase in diuresis of around 54.27% in leaves, 46.05% in stems and 75.65% in flowers compared to the control group. Moreover, the urinary volume found in the extract (except roots) treated animals after 24 h was not different to that from the diuresis obtained in animals treated with hydrochlorothiazide (HCT), as noted in the diuretic index (Table S3). Urinary electrolytes (sodium and potassium) excretion was measured and the results were summarised in Table  S3. Scolymus hispanicus and HCT treated group presented higher concentrations of Na + and K + when compared to the control rats. Hence, Scolymus hispanicus extracts at 2000 mg/kg increased sodium excretion by around 112.30% to 175.98% and potassium with 125.25% to 275.83% when compared to control values. Similarly, the reference drug HCT significantly increased sodium (74.26%) and potassium (90.02%) excretion compared with the control group.

Antimicrobial assay
The Golden Thistle extracts were tested against five species of bacteria (

Conclusion
In conclusion, we report here the relevant antioxidant effect of Scolymus hispanicus functional parts and their antidiabetic activities via α-glucosidase and α-amylase inhibition. Furthermore, our in vivo assay brings into evidence the traditional uses of Scolymus hispanicus as diuretic agent. Moreover, the antibacterial activity of the extract could be of interest. This diverse and interesting pharmacological variability between Scolymus hispanicus parts (roots, stems, leaves and flowers) is mainly linked to its chemical composition.