Accumulation of hydroxybenzoic acids and other biologically active phenolic acids in shoot and callus cultures of Aronia melanocarpa (Michx.) Elliott (black chokeberry)

Agnieszka Szopa 0 Halina Ekiert 0 Boz_ ena Muszyn ska 0 0 A. Szopa (&) H. Ekiert B. Muszynska Department of Pharmaceutical Botany, Jagiellonian University , Collegium Medicum, ul. Medyczna 9, 30-688 Krakow, Poland Phenolic acids are plant metabolites important in phytotherapy and also in cosmetology. In this study, proliferating shoot and callus cultures of Aronia melanocarpa were established and maintained on Linsmaier and Skoog (L-S) medium containing different levels of a-naphthaleneacetic acid (NAA) and 6-benzyladenine (BA), ranging from 0.1 to 3.0 mg l-1. Methanolic extracts from the biomass of these cultures and from the fruits of soil-grown plants were used to determine the amounts of free phenolic acids and cinnamic acid using the high-performance liquid chromatography (HPLC) method. Out of a total of twelve analyzed compounds, all of the extracts contained four of them: caffeic acid, p-hydroxybenzoic acid, syringic acid, and vanillic acid. Moreover, shoot extracts also contained salicylic acid (o-hydroxybenzoic acid), while callus extracts contained p-coumaric acid. On the other hand, fruit extracts also contained both salicylic acid and p-coumaric acid. The total amount of the analyzed compounds in extracts from both shoot and callus cultures depended on the L-S medium used, and varied between 103.05 and 150.95 mg 100 g-1 dry weight (DW), and between 50.23 and 81.56 mg 100 g-1 DW, respectively. Both types of culture contained higher levels of phenolic acids than the fruit extracts (32.43 mg 100 g-1 DW). In shoot cultures, p-hydroxybenzoic acid and salicylic acid were the predominant metabolites (reaching 55.14 and 78.25 mg 100 g-1 DW, respectively), while in callus cultures, p-hydroxybenzoic acid (25.60 mg 100 g-1 DW) and syringic acid (41.20 mg 100 g-1 DW) were the main compounds. In fruit extracts, salicylic acid (15.60 mg 100 g-1 DW) and p-hydroxybenzoic acid (5.29 mg 100 g-1 DW) were predominant. - Phenolic acids, both benzoic and cinnamic acid derivatives and depsides, like chlorogenic acid and rosmarinic acid, are a pharmacologically attractive group of plant metabolites important in phytotherapy and also in cosmetology. Their anti-inflammatory, spasmolytic, cholagogic, hypolipemic, antiaggregatory and immunostimulating properties have long been known. Many recent studies have also confirmed strong antioxidant, antiradical and anticancer activities of some compounds of this group, e.g. caffeic acid, p-coumaric and protocatechuic acids (Laranjinha et al. 1994; Nardini et al. 1995; Rice-Evans et al. 1996; Sroka and Cisowski 2003; Wen et al. 2003; Ekiert and Czygan 2007; Sanchez-Maldonado et al. 2011). Hydroxybenzoic acids also possess many others very important biological properties. p-Hydroxybenzoic acid shows antimicrobial, antifungal, antisickling, and estrogenic activities (Pugazhendhi et al. 2005; Chong et al. 2009). o-Hydroxybenzoic acid (salicylic acid) has anti-inflammatory, antiseptic, antifungal, antipyretic, analgesic and keratolytic properties (Lin and Nakatsui 1998). Vanillic acid (monomethoxy-derivative of p-hydroxybenzoic acid) exhibits anti sickling and anthelmintic, as well as hepatoprotective activities (Itoh et al. 2009, 2010). Syringic acid (dimethoxyderivative of p-hydroxybenzoic acid), besides being an antioxidant, shows antibacterial and hepatoprotective activities, too (Kong et al. 2008, Itoh et al. 2009, 2010). Aronia melanocarpa (Rosaceae, Rosoideae) is a North American medicinal and culinary plant species that began to be cultivated in Europe from the turn of the 19th and 20th centuries. The fruits of this species owe their therapeutic potential to the presence of different groups of metabolites, of which flavonoids, anthocyanins, tannins, organic acids, pectins, vitamins and many bio elements are the most important. Of the phenolic acids, the presence of chlorogenic acid and its isomerneochlorogenic acid, has been documented so far (Kulling and Rawel 2008; Zdunczyka et al. 2002). The shikimic acid pathway is characteristic, partially or exclusively, of the biogenesis of the main groups of metabolites in this plant species (flavonoids, anthocyanins and tannins), and the same pathway is also characteristic of the biogenesis of all the groups of phenolic acids, i.e. benzoic acid and cinnamic acid derivatives, and that was the main argument for performing analysis of this group of compounds in A. melanocarpa biomass cultured in vitro (Dewick 1997). In addition, chemotaxonomic guidelines applicable to the subfamily Rosoideae were also taken into account. Salicylic acid and its derivatives are characteristic of this subfamily (Hegnauer 1973). Another argument was based on the biotechnological successes in the accumulation of some phenolic acids in plant in vitro cultures, e.g. rosmarinic acid (Makri and Kintzios 2004), protocatechuic acid (Ekiert et al. 2009; Szopa et al. 2012) and p-coumaric acid (Ekiert et al. 2008; Piekoszewska et al. 2008; Szopa et al. 2012). Fruit extracts from soil-grown plants were also analyzed for comparison. Aronia melanocarpa has not been a subject of biotechnological studies yet. Investigations of other species of the subfamily Rosoideae, e.g. Rubus chamaemorus or Rosa sp., have mostly focused on the development of micropropagation protocols (Rout et al. 1999; Thiem and S liwin ska 2003; Martinussen et al. 2004). In the present study, the accumulation of free phenolic acids and cinnamic acid the parent compound of one subgroup of these metabolites, was investigated for the first time in the biomass from established in vitro cultures differing in the degree of tissue differentiation (shoot and callus cultures), growing on five variants of Linsmaier and Skoog (L-S) (1965) medium supplemented with various concentrations of plant growth regulators (PGRs): auxina-naphthaleneacetic acid (NAA), and cytokininN6-benzyladenine (BA), in the concentration range from 0.1 to 3.0 mg l-1. Methanolic extracts from the biomass cultured in vitro and from the fruits of soil-grown plants were used to determine twelve compounds by the high-performance liquid chromatography (HPLC) method. The aim of the study was to find the best medium for free phenolic acids production in in vitro cultures of A. melanocarpa, to compare the biosynthetic capacity of the cells from in vitro cultures with that of the cells of soil-grown plants, and eventually to propose in vitro cultures as a potentially rich source of the investigated metabolites. Variants of L-S medium were tested to allow the possibility of comparing the results from A. melanocarpa in vitro cultures with the results from in vitro cultures of Ruta graveolens (Ekiert et al. 2009) and R.g. spp. divaricata (Ekiert et al. 2008; Piekoszewska et al. 2008) studied earlier in our laboratory for their capacity to produce free phenolic acids. Materials and methods Mature fruits of Aronia melanocarpa (Michx.) Elliott were collected in September 2010 from the plants growing in Rog (...truncated)