Phenolic acids, antioxidant and antiproliferative activities of Naviglio® extracts from Schizogyne sericea (Asteraceae)

Abstract Schizogyne sericea, well-known as ‘salado’, is a halophytic shrub widespread on coastal rocks of Tenerife (Canary Islands). This plant is used traditionally as analgesic, astringent, anti-inflammatory and vulnerary agent. In the present work, we have analysed the aqueous and ethanolic extracts of S. sericea for the content of phenolic acids by HPLC-DAD. The dynamic solid–liquid Naviglio® extractor was used to extract the flowering aerial parts. Aqueous extracts showed higher levels of phenolics than ethanolic extracts. S. sericea extracts were rich in chlorogenic and isochlorogenic acids. The Naviglio® extracts obtained were assayed for in vitro biological activities, namely antioxidant, antimicrobial and cytotoxicity on tumour cells by DPPH, ABTS, FRAP, agar disc-diffusion and MTT methods, respectively. Results showed that aqueous extracts, being richer in phenolic acids, are endowed with relevant radical scavenging activity (TEAC values in the range 208–960 μmol TE/g) while ethanolic extracts exhibited noteworthy antiproliferative effects on tumour cells.


Introduction
Schizogyne sericea (L. fil.) DC. (syn. Chrysocoma sericea L. f., Inula schizogyne Masf.), wellknown as 'salado' , is a halophytic yellow-flowered shrub, up to 1 m tall, belonging to the Asteraceae family. This plant grows on coastal rocks and along roadsides of Canary and Selvagem Islands. Notably, it is very common in Tenerife and Gran Canaria (Anderberg 1991). In the traditional medicine of Canary Islands, the balm prepared with its aerial parts is used topically in the cure of wounds. Under decoction, S. sericea is also used as analgesic, astringent and anti-inflammatory agent. From the genus Schizogyne, a series of thymol derivatives was previously isolated and structurally elucidated, among which the 10-acetoxy-8-hydroxy-9-isobutyryloxy-6-methoxy-thymol was proven to exert anti-inflammatory activity on carrageenin-induced oedema in rats (González et al. 1988). on the contrary, diverse health-promoting compounds such as phenolic acids such as caffeoylquinic acids, that are abundant in the family Asteraceae, were not studied in deep in S. sericea. Epidemiological evidence has shown that an increased dietary intake of these natural polyphenols reduces the coronary heart disease, atherosclerosis effect and platelet aggregation (Holiman et al. 1996). Phenolic acids such as gallic, ferulic and caffeic acids are important dietary polyphenols endowed with antioxidative and vasodilatory activities (Sagratini et al. 2012). Chlorogenic acid was proven to inhibit carcinogenesis, to protect against oxidative stress, to improve the glucose metabolism, to reduce the risk of cardiovascular disease and to exhibit anti-obesity effects (Meng et al. 2013). Moreover, it can accelerate the process of excision wound healing by increasing the synthesis of collagen (Chen et al. 2013). Phenolic acids such as isochlorogenic and chlorogenic acids were also reported to exhibit antimicrobial activity (Zhu et al. 2004) and to be a potential treatment to prevent inflammatory diseases (Hwang et al. 2014).
The first aim of this work was to determine the level of phenolic acids in aqueous and ethanolic extracts of S. sericea harvested in different localities of Tenerife (Spain). For this purpose, we used a relative new extraction technique, called Naviglio® extraction, followed by HPLC-DAD analysis. The Naviglio® device is a rapid and dynamic solid-liquid extractor that, by generating a negative pressure gradient followed by a rapid equilibrium, forces the extraction of the analytes contained in the solid matrix (Naviglio's Principle). The Naviglio Extractor® can operate at room temperature or at subambient temperature, and it works applying a pressure increase on the surface of the liquid phase containing the solid material (matrix) to be extracted (Naviglio 2007;Naviglio et al. 2007Naviglio et al. , 2008. The device used consists of one extracting chamber equipped with a cylinder and a piston where, at the bottom, one porous set let the liquid phase and liquid soluble substances pass through, while the solid particles are blocked. Advantages of the Naviglio® extractor include low costs, rapid extraction, no production of by-products and the possibility to use aqueous solvents (Naviglio et al. 2007(Naviglio et al. , 2008.
Additionally, in the attempt to corroborate some ethnobotanical uses of the plant, we assessed the in vitro biological activity of polar extracts, namely the antioxidant, antimicrobial and cytotoxicity, on tumour cells by DPPH, ABTS, FRAP, agar disc-diffusion and MTT methods, respectively.

Phenolic profile of Naviglio® extracts
The amounts of phenolic acids in the Naviglio® extracts of S. sericea harvested in three different locations of Tenerife are reported in Table 1. Gallic acid, caffeic acid, trans-ferulic acid, chlorogenic acid and isochlorogenic acid were present in both ethanolic and aqueous extracts of all samples analysed; meanwhile, neochlorogenic acid was missing in the ethanolic extracts of S. sericea from Los Roques and La Barranquera. Chlorogenic acid was the most abundant compound, ranging from 125.4 to 2075.0 μg/g. It was found in higher concentration in the aqueous extracts (597.2-2075.0 μg/g) rather than in ethanolic ones (125.4-471.6 μg/g). In both extracts, the highest value was detected in the sample from Palm Mar and the lowest in that from La Barranquera. Also isochlorogenic acid was very abundant in all samples, ranging from 60.1 to 1333 μg/g. The lowest amount was detected in the ethanolic extract from La Barranquera and the highest amount in the aqueous one from Palm Mar. Neochlorogenic acid was quite abundant in the three aqueous extracts, ranging from 244.1 (La Barranquera) to 519.4 (Palm Mar) μg/g, but it was present only in one ethanolic extract (Palm Mar, 48.9 μg/g).
The high levels of chlorogenic acids found in this study are of great interest because of their beneficial effects on human health (Meng et al. 2013). Furthermore, the high levels of chlorogenic acid may support the traditional use of the plant as a vulnerary and anti-inflammatory agent. trans-Ferulic acid was quite abundant in the three aqueous extracts, ranging from 66.61 (La Barranquera) to 151.3 μg/g (Palm Mar), but it was detected in low level in the ethanolic extracts. Finally, low levels of gallic acid were detected in all S. sericea extracts.
Comparing the samples of different provenience, those from Los Roques were particularly rich in caffeic acid (112.8 and 483.0 μg/g in ethanolic and aqueous extracts, respectively). overall, the highest amount of all analysed compounds was detected in Naviglio® extracts from Palm Mar, with values of 1302.0 μg/g (ethanolic extract) and 4181.5 μg/g (aqueous extract) of total phenolic content. These plant extracts might enjoy the particular microclimate of Palm Mar that is warmer and more arid with respect to the other localities of the Isle. The total content of analysed compounds in ethanol extracts from this locality was two and five times higher than those from Los Roques and La Barranquera, respectively. As regards the aqueous extracts, the difference was not so high between the three localities.
Generally, water was more effective in extracting phenolic acids from the plant matrix using Naviglio® extractor, being the aqueous extracts (total content range 1333.8-4181.5 μg/g) richer than ethanolic ones (229.8-1302.0 μg/g). This result is consistent with  (Bastos et al. 2007).
The total phenolic content of S. sericea ethanolic extracts, as determined by the Folin-Ciocalteu method and expressed as GAE (gallic acid equivalent), is reported in Table 2; it was 69.9, 21.2 and 23.5 mg/g for samples from Palm Mar, Los Roques and La Barranquera, respectively. In comparison, the total phenolic content of the aqueous extracts was higher than ethanolic ones, with value of 61.0, 54.7 and 40.4 mg/g for the same locations, respectively. These data confirmed the results of HPLC analysis (Table 1). The ethanol extract as well as the aqueous extract of sample from Palm Mar contained the highest amount of phenolic compounds.

Antioxidant activity
In this work, we evaluated the free radical scavenging capacity of S. sericea Naviglio® extracts using multiple assays such as DPPH, ABTS + and FRAP (Zorzetto et al. 2015a(Zorzetto et al. , 2015b and results are presented in Table 2. In the DPPH assay, the IC 50 value of the reference compound Trolox amounted to 4.9 μg/mL ( Table 3). The sample from Los Roques exhibited the highest antioxidant activity, with a TEAC value of 224 μmol TE/g for ethanolic extract (IC 50 value of 87.2 μg/mL) and 283 μmol TE/g for aqueous extract (IC 50 of 69.2 μg/mL).
Regarding the ABTS assay, the IC 50 value of Trolox was 3.5 μg/mL. Ethanol extract of sample from Los Roques showed the highest activity (IC 50 of 10.7 μg/mL), while, among the aqueous extracts, that from Palm Mar was the most active, with a TEAC value of 960 μmol TE/g (IC 50 of 14.6 μg/mL). The same was observed in the FRAP assay, where aqueous extracts exhibited the highest TEAC values (358-549 μmol TE/g vs. 141-365 μmol TE/g of ethanolic extracts). The three antioxidant assays did not show noteworthy differences between samples of different geographic origin. Generally, we observed that the aqueous extracts exhibited a bit stronger antioxidant capacity compared to the ethanolic extracts. This is in agreement with the higher total phenolic content of the aqueous extracts determined by HPLC-DAD analysis and Folin-Ciocalteu method (Tables 1 and 2). The strong antioxidant activity displayed by S. sericea extracts may be related to the high concentrations of phenolic acids, mainly chlorogenic acids that are reported as potent antioxidants with beneficial effects on health (Sato et al. 2011). These compounds inhibit lipid peroxidation and showed neuroprotective activities (Nakajima et al. 2007). The relatively strong antioxidant capacity of S. sericea may also justify the traditional uses in the treatment of wounds where oxidative stress and inflammation impair the wound healing process (Shetty et al. 2008). Table S1 reports the inhibition zone diameters (IZDs) of S. sericea Naviglio® extracts on a panel of pathogens. Activity of the extracts was in the range of 6.0 (no activity) to 10 mm (low activity) IZDs. For the different extracts of S. sericea, no activity was observed against Pseudomonas aeruginosa. The ethanolic extracts of S. sericea from Los Roques and La Barranquera showed little activity against the bacterial species Staphylococcus aureus and Enterococcus faecalis. The former extract was also responsible for the most growth inhibition of the fungal strain, while the latter showed no activity. Neither of the bacterial species were inhibited by the ethanolic extract of S. sericea from Palm Mar; however, low antifungal activity was detected. Aqueous extracts did not inhibit growth of E. faecalis, P. aeruginosa and Candida albicans. An extremely low activity of the aqueous extracts against S. aureus and Escherichia coli was observed. Hence, no association of inhibitory activity with the subdivision in gram-positive and gram-negative strains of bacteria could be made. The comparison of the three S. sericea extracts from samples collected in different localities showed no big differences. The activity of the ethanolic extracts on the yeast C. albicans might be ascribed to its content of thymol derivatives present (1986a;1986b, González et al. 1988). These compounds are soluble in ethanol and not in water, which explains why no activity is observed for the aqueous extracts at the assayed concentrations. Since the antimicrobial activity determined by the disc diffusion method was poor, the extracts were not further investigated by other susceptibility testing methods.

Cytotoxicity on tumour cells
Results of MTT assay showed a potential tumour cell growth inhibitory effect of S. sericea ethanolic extracts from Palm Mar on all tumour cell lines tested (Table 3). IC 50 values were 0.74, 0.32 and 0.52 μg/mL on A375, MDA-MB 231 and HCT116 tumour cell lines, respectively. The IC 50 values of cisplatin after 72 h were 0.43, 2.9 and 2.4 μg/mL, respectively. The cytotoxic activity of ethanolic extracts is probably due to the presence of thymol derivatives that are soluble in ethanol and not in water (González et al. 1988). These kinds of molecules are endowed with strong antiproliferative effects on different tumour cell lines (Zhu et al. 2004). Moreover, some of them can be used as active ingredients in patented anticancer formulations due to their excellent anti-tumour activity (US Patent 2001). According to the NCI plant screening programme, a crude extract is considered to possess important in vitro cytotoxic effect if the IC 50 value on tumour cells, after 48-72 h of incubation, is below 20 μg/mL (Boik 2001). Thus, the ethanolic extract of S. sericea is considered promising for the important IC 50 values displayed. on the above, it will be interesting to conduct further studies in order to purify and isolate the most active constituents and to determine their cytotoxicity on non-tumour cells, as well as its mode of action, and in vivo efficacy.

Experimental
Please see the online Supplementary Material for the 'Experimental' section.

Conclusions
In conclusion, HPLC-DAD analysis showed that S. sericea extracts are a rich source of dicaffeoylquinic acids such as chlorogenic acid and isochlorogenic acid. In some cases, their concentrations were close to those found in artichoke (Wang et al. 2003;Schütz et al. 2004). The plant material from Palm Mar contained the highest level of these compounds, and this may be related to the more favourable climatic conditions (warmer and more arid) of this locality that influenced their production. overall, the high antioxidant potential of S. sericea was demonstrated as the TEAC values in the DPPH, ABTS and FRAP assays were noteworthy. The high level of chlorogenic acids together with the strong antioxidant capacity may support the traditional use of S. sericea as a wound healing and anti-inflammatory agent. Finally, the ethanolic extract of S. sericea was able to exert strong cytotoxic effects on all tumour cell lines tested. These relevant inhibitory effects seem to be related to the occurrence of thymol derivatives previously reported, and surely deserve further studies in order to exploit this plant as a source of chemotherapeutic drugs.

Aknowledgements
Authors thank Dr. Evy Blomme from Gent University for her precious support in the chemical analyses and biological assays.

Disclosure statement
No potential conflict of interest was reported by the authors Funding This work was supported by the FAR 2014-2015 (Fondo di Ateneo per la Ricerca, FPI000044) of the University of Camerino.