ACTA BIOLOGICA CRACOVIENSIA Series Botanica 61/1: 75–86, 2019 DOI: 10.24425/118060 POLSKA AKADEMIA NAUK ODDZIAŁ W KRAKOWIE PHENYLETHANOID GLYCOSIDES IN PLANTAGO MEDIA L. ORGANS OBTAINED IN IN VITRO CULTURES ANNA BUDZIANOWSKA*, MAŁGORZATA KIKOWSKA, MAGDALENA MAŁKIEWICZ, INGA KAROLAK AND JAROMIR BUDZIANOWSKI Department of Pharmaceutical Botany and Plant Biotechnology, Poznan University of Medical Sciences, Św. Marii Magdaleny 14, 61-861 Poznań, Poland Received October 16, 2018; revision accepted December 3, 2018 Micropropagation of Plantago media L. and the presence of phenolic compounds in organs of multiplied plants were investigated for the first time. Multiplication of plant material was achieved in shoot-tip cultures and via direct organogenesis on Murashige and Skoog (MS) medium with four variants of plant growth regulators (M1–M4). The best multiplication coefficient – 9.2 was obtained in seedling shoot-tip cultures on MS medium M3 with BA 0.2 mg/L and IAA 1.0 mg/L. Methanol extracts prepared separately from shoots and roots of in vitro- derived plantlets were found to contain typical of the genus Plantago L. phenylethanoid glycosides as the only phenolics. Acteoside and plantamajoside were the major compounds – both known to possess a wide range of promising biological activities applicable for medicinal (therapeutic) and cosmetic uses. Martynoside, as a trace constituent, was also found for the first time in the studied species. The quantitative screening of the extracts by TLC video densitometric method showed a higher content of acteoside in shoots (range 62.43–93.03 mg/g, dry weight) and plantamajoside in roots (range 22.45–44.08 mg/g); the highest recorded values – 93.03 mg/g and 44.08 mg/g, respectively, were found in the organs obtained on MS medium M4 with BA 2.0 mg/L. Keywords: acteoside, direct and indirect organogenesis, hoary plantain, micropropagation, phenyle- thanoids, plantamajoside, TLC densitometry INTRODUCTION 10-O-acetylaucubin, melittoside, monomelittoside, 10-aetylmonomelittoside), polysaccharides, Plantago media L. (Plantaginaceae) is a cosmo- phenylpropanoids (caffeic acid, chlorogenic acid, politan wild perennial, which can be found in including phenylethanoid glycosides – acetoside, the Polish flora together with nine other species plantamajoside (Fig. 1)) (Rønsted et al., 2003; (Tacik, 1967). According to Flora Europea, it has Olennikov et al., 2011), flavonoids (apigenin, one or a few rosettes consisting of elliptical to apigenin 7-O-glucoside, rutin) (Beara et al., 2009), ovate-elliptical hairy leaves gradually narrowed phenolic acids (p-hydroxybenzoic acid, vanilic acid, into a petiole, usually less than half as long as gentyzinic acid, syringic acid, p-coumaric acid, the lamina (Chater and Cartier, 1967). P. media ferulic acid) (Świątek, 1977). is a species with medicinal properties related The major phenolics in the genus Plantago to P. lanceolata and P. major and like them are phenylethanoid glycosides represented having similar usage (Jędrzejko, 1997). Plants mainly by acteoside and plantamajoside (Rønsted from the genus Plantago exhibit many medicinal et al., 2000; Rønsted et al., 2003). Acteoside activities, which are assumed to be affected by (verbascoside), which occurs in over 20 plant the occurrence of polysaccharides, iridoids, families of Asteridae (Schlauer et al., 2004; phenylethanoid glycosides, triterpenes, flavonoids Alipieva et al., 2014), exhibits a wide range of and phenolic acids (Gonçalves and Romano, 2016). activities: antioxidant, antiproliferative, cytotoxic, In P. media the following chemical constituents anticancer, antimetastatic, anti-inflammatory, were found: iridoid glucosides (aucubin, and immunomodulatory (Alipieva et al., 2014). * Corresponding author, email: [email protected] PL ISSN 0001-5296 © Polish Academy of Sciences and Jagiellonian University, Cracow 2019 76 Budzianowska et al. Fig. 1. Chemical structures of acteoside, plantamajoside and martynoside. Moreover, acteoside, as an antioxidant compound, the presence of bioactive compounds in Plantago may be used in dermocosmetic and pharmaceutical inclined scientists to use the biotechnological topical formulations (Vertuani et al., 2011). methods to obtain a high and constant content of Acteoside and martynoside exhibit antiestrogenic desired metabolites in the biomass from shoots effects (Papoutsi et al., 2006). Plantamajoside, and roots of the micropropagated plantlets. In vitro which was found in three plant families (Ravn techniques provide a perfect opportunity to produce et al., 2015), has numerous biological and a large amount of Plantago media biomass, rich pharmacological activities: antioxidant, antibiotic, in bioactive compounds, without environmental antifungal, anti-inflammatory, cytotoxic, and restrictions. enzyme inhibitory (Ravn et al., 2015). The aim of the current study was to develop So far, in vitro propagation of Plantago species an efficient method for in vitro propagation was investigated for P. asiatica (Makowczyńska of Plantago media L. by comparison of the and Andrzejewska-Golec, 2003; Zeng, 2008), three techniques: development from shoot tips, P. camtschatica (Andrzejewska-Golec and regeneration via direct and indirect organogenesis. Makowczyńska, 2008), P. lanceolata (Fons Moreover, the biomass obtained from in vitro et al., 1998; Fons et al., 1999; Budzianowska cultures – shoots and roots of the micropropagated et al., 2004; Khawar et al., 2005), P. major plantlets from selected MS media was analyzed (Mederos et al., 1997/1998; Saker and Kawashity, for the presence of phenolic compounds to find 1998), P. maritima (Makowczyńska and phenylethanoids – acteoside and plantamajoside. Andrzejewska-Golec, 2009) and P. ovata (Barna To the best of our knowledge, this is the first report and Wakhlu, 1998; Pramanik et al., 1995). on P. media micropropagation and qualitative and The strong influence of environmental quantitative estimation of these phenylethanoids in conditions in the natural habitats of plants on in vitro cultures of this species. Phenyletanoid glycosides in Plantago media 77 MATERIAL AND METHODS Media composition. Culture media consisted of MS (Murashige and Skoog, 1962) media solidified with PLANT MATERIAL, MEDIA COMPOSITION, 0.8% (w/v) agar (Sigma-Aldrich) with 30 g/L (w/v) AND SURFACE STERILIZATION sucrose and appropriate plant growth regulators (PGRs) at various concentrations (Tables 1, 2, 3). Plant material. The voucher specimens of P. media The PGRs originated from Sigma-Aldrich. After (no. PM001/2012) is deposited in the Herbarium adjusting pH to 5.8, the media were autoclaved. of Department of Pharmaceutical Botany and The cultures were incubated in an air-conditioned Plant Biotechnology, Poznan University of Medical growth chamber (16/8 h photoperiod, 55 μmol/m2s1 Sciences, Poland. Mature seeds of P. media were light, temp. 21 ± 2°C). collected from the botanical garden of Department of Medicinal and Cosmetic Products, Poznan Surface sterilization of seeds. For aseptic University of Medical Sciences (52°25’30.7” N, culture initiation, the seeds were surface decon- 16°53’50.8” E), in July 2012. Botanical identity taminated. They were washed under running tap was confirmed by nuclear DNA content using water and then rinsed in distilled water followed flow cytometry with comparison to the reference by submerging in 70% (v/v) EtOH for 30 s to defat standard (Thiem et al., 2013). the seeds. The disinfection step was to treat the TABLE 1. Influence of PGRs on shoot ind uction (%) in Plantago media shoot-tip cultures in three subcultures. MS medium No. of subculture Abbrev. Cytokinin Auxin 1st(%) 2nd(%) 3rd(%) (mg/L) (mg/L) BA NAA M1 2.0 0.1 100 94.6 83.2 BA IAA M2 2.0 0.1 100 91.3 81.6 M3 2.0 1.0 100 75.0 73.3 M4 2.0 – 100 100 100 BA – 6-benzyladenine; IAA – indole-3-acetic acid; MS – Murashige and Skoog medium; NAA – α-naphthaleneacetic acid; 56, 174, 183 explants per 1st, 2nd and 3rd subculture, respectively. TABLE 2. Influence of PGRs on shoot formation (MC ± SE) originating from Plantago media seedling shoot tips. MS medium No. of passage Abbrev. Development from Cytokinin (mg/L) Auxin (mg/L) Shoot proliferation seedling tips 1st 2nd 3rd BA NAA M1 2.0 0.1 5.1 ± 0.1b 2.9 ± 0.1b 2.4 ± 01a BA IAA M2 2.0 0.1 4.9 ± 0.1b 1.3 ± 0.1c 3.0 ± 0.2a M3 2.0 1.0 9.2 ± 0.3a 3.6 ± 0.2a 2.8 ± 0.2a M4 2.0 – 2.1 ± 0.1c 4.0 ± 0.2a 2.6 ± 0.1a BA – 6-benzyladenine, IAA – indole-3-acetic acid, MS – Murashige and Skoog medium; NAA – α-naphthaleneacetic acid; Mean values within a column with the same letter are not significantly different at P = 0.05 (one-way analysis variance ANOVA followed by Duncan’s Multiple Range test /STATISTICA v.13; StatSoft, Inc. 2015). 78 Budzianowska et al. TABLE 3. Influ ence of PGRs and type of explant on shoot direct organogenesis and shoot proliferation (MC) in Plantago media. st nd rd MS Type of seedling expl. for 1 passage (1.) / rosettes for 2 (2.) – 3 (3.) passage PGRs medium Organogenesis Proliferation Hypocotyls Cotyledons Cotyledon- Hypocotyl- Leaf-derived Root-derived Leaves Cytokinin Auxin Roots derived derived Abbrev. shoots shoots (mg/L) (mg/L) shoots shoots 1. 1. 1. 1. 2. 3. 2. 3. 2. 3. 2. 3. BA NAA M1 2.0 0.1 1.5 – 4.2 2.3 2.4 2.5 – – 1.4 2.6 1.7 3.0 BA IAA M2 2.0 0.1 2.2 4.6 4.6 8.5 3.1 4.8 4.2 2.6 5.2 2.7 1.1 1.8 M3 2.0 1.0 0.8 2.8 3.4 6.8 2.6 2.9 0.7 1.9 1.8 2.9 0.9 2.3 M4 2.0 – 0.5 2.3 8.0 2.8 0.9 0.1 1.1 2.4 1.8 1.6 1.6 1.3 BA – 6-benzyladenine, IAA – indole-3-acetic acid, MS – Murashige and Skoog medium; NAA – α-naphthaleneacetic acid. seeds with commercial bleach at a concentration Direct organogenesis and shoot proliferation.