Antioxidant and antibacterial activities and polyphenolic constituents of Helianthemum sessiliflorum Pers.

Abstract In this study, the various extracts of aerial parts of Helianthemum sessiliflorum Pers. were examined in vitro for possible source of antioxidants and for antibacterial activity. The antioxidant activity was performed by DPPH radical scavenging method which showed that ethyl acetate extract possessed the best antioxidant potential (IC50 = 32.75 ± 2.07 μg/mL). The significant linear correlation was realised between the values of the total phenolic/flavonoid content and antioxidant activity of plant extracts. The ethyl acetate and n-butanol extracts showed moderate antibacterial activity. In addition, the phytochemical study of n-butanol extract afforded nine known phenolic compounds (1–9). This is the first report of six of them (1, 3, 5–8) in Cistaceae family. The structural identification of the isolated compounds was achieved using several spectroscopic methods.


Introduction
Large numbers of species of genus Helianthemum (Cistaceae family) are widely used in traditional medicine due to their notable pharmacological effects as analgesic (Alzetrini et al. 2009), phytoinhibitrice (Gonnet 1968), antioxidant, antimicrobial (Rivera et al. 2005), antiprotozal, antigiradial and antibacterial (Meckes et al. 1999;Barbosa et al. 2006). Helianthemum sessiliflorum Pers. is one of species of this genus that showed previously anti-inflammatory and analgesic activities (ermeli et al. 2012). In addition, the aerial parts of this plant are recommended in folk medicine in case of cutaneous lesion (Ustün et al. 2006).

Total phenolic and flavonoid contents
The total phenolic content (TPC) in the examined extracts of aerial parts of H. sessiliflorum was determined using Folin-Ciocalteu method, and gallic acid was used as a standard to plot the calibration curve (Supplementary material, Table S1). The concentration of flavonoids (TFC) in various H. sessiliflorum extracts was measured using spectrophotometric method with aluminium chloride. The results revealed that phenolic content ranged from 42.51 ± 1.01 to 1.31 ± 0.84 (μg GAe/mg) while the flavonoid content varied from 46.70 ± 0.22 to 0.23 ± 0.62 (μg Qe/mg). The highest concentration of TPC and TFC was found in ethyl acetate extract (42.51 ± 1.01 μg GAe/mg and 46.70 ± 0.22 μg of Qe/mg) followed by n-butanol extract (40.02 ± 2.81 μg GAe/ mg and 35.48 ± 0.36 μg of Qe/mg). This finding could be explained by our previous study carried out on the chemical composition of ethyl acetate extract of aerial parts of H. sessiliflorum (Benabdelaziz et al. 2015) indicating that this extract is rich in polyphenols including simple phenolics, flavonoids and lignans. Moreover, the present phytochemical investigation of n-butanol extract revealed the presence of many polyphenolic compounds.

Evaluation of antioxidant activity
The antioxidant activity of H. sessiliflorum extracts was determined in vitro using a methanol solution of DPPH reagent (Supplementary material, Table S2). ethyl acetate extract showed the highest DPPH free radical scavenging activity (23.75 ± 2.07 μg/mL) followed by n-butanol extract (94.03 ± 1.52 μg/mL). Quercetin was used as a reference compound (IC 50 = 8.5 ± 1.45 μg/mL) (Monika et al. 2011). It is interesting to consider the correlation between the content of total phenolics and flavonoids, and antioxidant activity of various extracts. To find the relationship between the antioxidant activity, TPC and TFC, we performed linear regression and correlation analysis of IC 50 values with TPC and TFC, the correlation coefficient values were depicted in (Supplementary material, Table S3). It is very important to indicate that ethyl acetate extract showed very strong correlation (r > 0.90) between TPC (r = 0.9627), TFC (r = 0.9598) and DPPH, but the coefficient of correlation was very weak in case of n-butanol extract (TPC: r = 0.3172 and TFC: r = 0.3834). These results suggested that phenolic and flavonoid compounds of ethyl acetate extract might contribute directly to their antioxidant action (Liu et al. 2009). However, the low correlation between TPC, TFC and antioxidant activity in case of n-butanol extract could be related to other antioxidant compounds contained in this extract (Kolar et al. 2011). This relation between the phenolic and flavonoid compounds and antioxidant activity has been observed also in the case of the desert truffle Terfezia boudieri (Pezizaceae) that forms a mycorrhizal association with its host plant H. sessiliflorum (Turgeman et al. 2011), where the extracts of this truffle contained the highest amounts of polyphenolics and displayed a powerful antioxidant activity (Dogan & Aydin 2013).

Evaluation of antibacterial activity
In the in vitro study of antibacterial activity of aerial parts of H. sessiliflorum, using the disc diffusion method, we tested eight bacterial species belonging to four different groups (Supplementary material). The obtained results showed that ethyl acetate extract was the most active against both Gram-positive and Gram-negative organisms tested with better activity against Streptococcus faecalis, Salmonella typhimurium and Aeromonas hydrophyla indicating zone of inhibition values of 33, 31.5 and 30.4 mm at 500 mg/mL, respectively (Supplementary material, Figure S2). Among bacterial organisms, S. faecalis, Escherichia coli and A. hydrophyla were the most sensitive to the n-butanol extract. Moreover, the two other water and cyclohexane extracts presented poor antibacterial activity against all tested bacterial species.
It might be assumed that antibacterial and antioxidant activities of H. sessiliflorum are due to the phenolic compounds. These secondary metabolites are characteristics of Helianthemum genus (Rubio-Moraga et al. 2013). Previous biological studies performed on some Helianthemum species such as Helianthemum alypoides, Helianthemum cinereum subsp. rotundifolium, Helianthemum hirtum, Helianthemum asperum, Helianthemum marifolium subsp. marifolium, Helianthemum syriacum, Helianthemum polygonoides and Helianthemum glomeratum (Rivera et al. 2005;Barbosa et al. 2006;Rubio-Moraga et al. 2013) showed a strong correlation between the antibacterial and antioxidant activities of this taxa and their polyphenolic composition.

Conclusion
The n-butanol extract of aerial parts of H. sessiliflorum afforded nine known phenolic compounds (1-9), where majority of them (1, 3, 5-8) were isolated for the first time from the family Cistaceae. The results obtained from the present study, together with our previous investigation of the ethyl acetate extract from the same parts of this plant will be helpful for the chemotaxonomic profile of the Helianthemum genus. On the other hand, ethyl acetate extract showed the best antioxidant activity, and significant correlation was found between the antioxidant activity and total phenolic and flavonoid contents which indicated that these phytocompounds are the major contributors of antioxidant capacities of this plant. Furthermore, both ethyl acetate and n-butanol extracts of aerial parts of H. sessiliflorum have great potential as antibacterial compounds against micro-organisms. This could explain the traditional use of this plant against skin infections.

Supplementary material
General experimental methods, supporting NMR information are available online.

Disclosure statement
No potential conflict of interest was reported by the authors.

Funding
This work was supported by the DGRSDT of the Algerian Minister of Higher education and Scientific Research [grant number PNR Project 8/u05/853]; School of Pharmaceutical Science, ePGL, University of Geneva, Geneva, Switzerland.