Antimicrobial activities, toxicity and phenolic composition of Asphodeline anatolica E. Tuzlaci leaf extracts from Turkey

Abstract The antimicrobial activity of acetone, methanol and aqueous extracts of Asphodeline anatolica E. Tuzlaci leaves was evaluated against American type culture collection, food and clinical isolates (Listeria monocytogenes and Staphylococcus aureus including methicillin-resistant strains-MRSA). Biofilm formation, toxicity and characterisation of the polyphenolic content were analysed. The acetone extract demonstrated a higher antibacterial activity against S. aureus including MRSA strains, L. monocytogenes and Pseudomonas aeruginosa than against other extracts. No effect was observed in biofilm formation. The extracts resulted non-toxic against Artemia salina Leach. The phytochemical screening of extracts indicated that they mainly contained six polyphenols identified as catechin 3-O-gallate, protocatechuic acid, diosmin, rutin, cirsimaritin and kaempferol glucoside. This study is the first report on antimicrobial activity and phenolic content of A. anatolica and contributes to enrich the literature data on the biological properties of this plant. A. anatolica leaves have a potential as source of natural antimicrobial compounds.


Introduction
The genus Asphodeline belongs to the Xanthorrhoeaceae family (until recently included in the family Liliaceae) and comprises of 14 species worldwide. It is present in South-west Asia, Middle Eastern countries and the Mediterranean region. In Turkey, this genus is represented by 20 taxa, 11 of which are endemic in this country (Tuzlaci 1987). The Asphodeline species are known as çiriş plants and are abundant especially in the mountains and steppes of the inner Anatolia region of Turkey. Several Asphodeline species are consumed in salads, others have significant applications in traditional medicine. For example, Asphodeline damascena and Asphodeline cilicica are used for earaches while Asphodeline globifera is used as a medicament for alleviating haemorrhoids symptoms. Previous studies demonstrated that the antioxidant activity of extracts from several Asphodeline species, including Asphodeline anatolica, correlated with their phenolic content (Zengin & Aktumsek 2014;Karadeniz et al. 2015). Polyphenols are secondary metabolites produced by higher plants, which play multiple essential roles in plant physiology and have potential health properties on the human organism, mainly as antioxidants, anti-allergic, anti-inflammatory, anticancer, antihypertensive and antimicrobial agents (Daglia 2012). Several studies reported the antimicrobial activity of polyphenols from tea (Yi et al. 2014), almond skins (Mandalari et al. 2010), Juniperus spp. (Marino et al. 2010;Miceli et al. 2011;Taviano et al. 2013) and propolis (Koru et al. 2007). The antimicrobial properties of certain classes of polyphenols have been proposed either to develop new food preservatives, due to the increasing consumer pressure on the food industry, to avoid synthetic preservatives, or, considering the increase in microbial resistance against conventional antibiotic therapy, to develop innovative therapies for the treatment of microbial infections (Saavedra et al. 2010).
Based on this information, we evaluated antimicrobial activity, toxicity and phenolic composition of methanol, acetone and aqueous leaf extracts of A. anatolica.

Results and discussion
The phytochemical analysis of polyphenols revealed the presence of cirsimaritin, kaempferol glucoside, rutin, protocatechuic acid and catechin 3-O-gallate in acetone extract, and rutin, diosmin, cirsimaritin, catechin 3-O-gallate, protocatechuic acid and kaempferol glucoside in methanol extract. The content of polyphenols in water extract was very low (Table S1).
The acetone and methanol extracts showed higher efficacy towards Gram-positive than against Gram-negative bacteria, but they had no activity against the yeast. Moreover, the acetone extract proved to have higher bacteriostatic activity against Staphylococcus aureus, Listeria monocytogenes and Pseudomonas aeruginosa than methanol extract. On the contrary, water extract showed no activity against all the micro-organisms at the concentrations tested (Table S2). The extracts had no significant inhibitory effect on biofilm formation (less than 20% respect to control).
All extracts did not display any toxicity against brine shrimps (LC 50 > 1000 μg ml −1 ). Polyphenols comprise a wide variety of molecules with polyphenol structure and are generally divided into flavonoids and non-flavonoids, potentially useful structures for the development of new chemotherapeutic agents (Daglia 2012). The antimicrobial and resistance modifying potentials of natural compounds have been reported (Cushnie & Lamb 2011). Gram-positive strains were the most sensitive bacteria against the acetone and methanol extracts. This has long been explained by the presence of an outer-membrane permeability barrier in Gram-negative bacteria limits access of the antimicrobial agents to their targets in the bacterial cells (Hendra et al. 2011). The acetone extract showed the higher bacteriostatic activity against S. aureus (including MRSA strains) and L. monocytogenes than methanol extract. This major activity could be explained by the interactions of different concentrations of polyphenols retained in the acetone extract. The combination of the main constituents of the acetone extract -cirsimaritin, diosmin and kaempferol glucoside -probably had a better activity than the main constituents of methanol extract -catechin-3-O-gallate, diosmin and rutin. Flavones, such as cirsimaritin, rutin and diosmin probably owe their activity to their ability to complex with extracellular and soluble proteins and to complex with bacterial cell walls (Cowan 1999). A hydroxyl group at position 5 in flavones is important for their activity against MRSA (Kumar & Pandey 2013). Diosmin showed no direct antibacterial activity on MRSA strains (Chan et al. 2013). Flavonols, such as kaempferol glucoside, due to their hydrophobicity, are capable of penetrating cell phospholipid membranes (Cushnie & Lamb 2011;Daglia 2012). Kaempferols isolated from Dodonaea viscosa Jacq. var. augustifolia leaf extracts showed antibacterial activity against S. aureus, Enterococcus faecalis, Escherichia coli and P. aeruginosa (Teffo et al. 2010). Flavan-3-ols, such as catechin-3-O-gallate, inhibit nucleic acid synthesis. In addition, compounds in the flavonol, flavan-3-ol and flavone classes have been shown to inhibit energy metabolism (Cushnie & Lamb 2011).
In conclusion, this study has demonstrated a potential good antibacterial activity of A. anatolica leaves against S. aureus (including MRSA) and moderate vs. L. monocytogenes. The higher antibacterial activity of the acetone extract could be due to different polyphenols concentrations in it with respect to those present in the methanol extract, even the involvement of other different secondary metabolites able to affect microbial growth cannot be excluded.
However, in order to prove this, it would be necessary to isolate the phenolic compounds from each extract and test them separately or in combination.

Conclusions
This study reports for the first time the antibacterial activity and phenolic content of A. anatolica leaves and contributes to enriching the literature data on the biological properties of this plant. A. anatolica leaves have a potential as a source of natural antimicrobial compounds.