Accumulation of phenolic compounds in in vitro cultures and wild plants of Lavandula viridis L’Hér and their antioxidant and anti-cholinesterase potential Patrícia Costa a , Sandra Gonçalves a , Patrícia Valentão b , Paula B. Andrade b , Anabela Romano a,⇑ a IBB-CGB, Faculty of Sciences and Technology, University of Algarve, Campus de Gambelas, Ed. 8, 8005-139 Faro, Portugal b REQUIMTE/Laboratório de Farmacognosia, Departamento de Química, Faculdade de Farmácia, Universidade do Porto, Rua de Jorge Viterbo Ferreira, n.° 228, 4050-313 Porto, Portugal article info Article history: Received 9 January 2013 Accepted 9 March 2013 Available online 20 March 2013 Keywords: Wild plants In vitro cultures Phenolic compounds Rosmarinic acid Free radical scavenging Anti-cholinesterase activity abstract In this study, we evaluated the phenolic profile, antioxidant and anti-cholinesterase potential of different extracts from wild plants and in vitro cultures of Lavandula viridis L’Hér. The HPLC–DAD analysis allowed the identification and quantification of 3-O-caffeoylquinic, 4-O-caffeoylquinic, 5-O-caffeoylquinic and rosmarinic acids, and luteolin and pinocembrin. Water/ethanol extract from in vitro cultures contained the highest amount of the identified phenolic compounds (51652.92 mg/kg). To investigate the antioxi- dant activity we used Trolox equivalent antioxidant capacity, oxygen radical absorbance capacity, Fe 2+ chelation activity and the inhibition of Fe 2+ -induced lipid peroxidation in mouse brain homogenates (in vitro). Overall, all the extracts from both wild plants and in vitro cultures exhibited ability to scavenge free radicals, to chelate Fe 2+ and to protect against lipid peroxidation. In addition, the extracts from L. vir- idis were active in inhibiting both acetylcholinesterase and butyrylcholinesterase (Ellman’s method). Our findings suggest that L. viridis in vitro cultures represent a promising alternative for the production of active metabolites with antioxidant and anti-cholinesterase activity. Ó 2013 Published by Elsevier Ltd. 1. Introduction Alzheimer’s disease (AD) is a progressive and complex neurode- generative disorder characterized by the occurrence of senile pla- ques and neurofibrillary tangles. Amyloid beta (Ab) peptide is the main component of senile plaques and is highly involved in the pro- gression of AD. Accumulated evidences suggest that Ab peptide, in the presence of biometals and oxygen, generates reactive oxygen species and causes lipid peroxidation in neuronal cell membranes (Butterfield and Lauderback, 2002; Rauk, 2008). Nowadays, the most efficient therapeutic approach to AD is based on cholinesterases inhibitors in order to enhance the concentration of acetylcholine (ACh) in the synaptic cleft (Wilkinson et al., 2004). Natural products might slow the progression of AD because they can simultaneously protect neurons from oxidative stress (Ramassamy, 2006) and act as cholinesterases inhibitors (Hostettmann et al., 2006). The production of phytochemicals is affected by environmental and physiological conditions and their chemical synthesis is com- plex due to their structures and specific stereo-chemical character- istics (Smetanska, 2008). Biotechnological approaches, particularly plant tissue cultures, are found to have significant potential for the production of high-value phytochemicals. In vitro propagation methods allow the selective, rapid and effective production of sec- ondary metabolites with no seasonal constraints and independent of geographical and soil conditions. Lavandula species (Lamiaceae) produce valuable compounds for food, pharmaceutical and cosmetic industries (Boelens, 1995). Re- cently, Gonçalves and Romano (2013) reviewed the application of in vitro propagation methods to Lavandula spp. and demonstrated the usefulness of plant tissue culture technique for the production of valuable secondary metabolites overcoming the limitations of conventional propagation methods. Lavandula viridis L’Hér is an aromatic shrub endemic to the southwest Iberian Peninsula that is used in traditional medicine. The essential oil from this species has antioxidant, anti-cholinesterase (Costa et al., 2012a) and 0278-6915/$ - see front matter Ó 2013 Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.fct.2013.03.006 Abbreviations: Ab, amyloid beta; AAPH, 2,2 0 -azobis(2-methylpropionamidine) dihydrochloride; ABTS Å+ , 2,2 0 -azinobis(3-ethylbenzothiazoline-6-sulfonic acid) rad- ical cation; ACh, acetylcholine; AChE, acetylcholinesterase; AD, Alzheimer’s disease; ATCI, acetylthiocholine iodide; AUC, area under the curve; BChE, butyrylcholinest- erase; BHT, butylated hydroxytoluene; BTCI, butyrylthiocholine chloride; DTNB, 5,5 0 -dithiobis [2-nitrobenzoic acid]; EDTA, ethylenediaminetetraacetic acid; HPLC– DAD, high-performance liquid chromatography–diode array detection; MDA, malondialdehyde; MS, Murashige and Skoog medium; ORAC, oxygen radical absorbance capacity; SDS, sodium dodecylsulfate; TBA, thiobarbituric acid; TBARS, thiobarbituric acid-reactive substances; TE, Trolox equivalents; TEAC, Trolox equivalent antioxidant capacity; Trolox, 6-hydroxy-2,5,7,8-tetramethylchromane- 2-carboxylic acid. ⇑ Corresponding author. Tel.: +351 289800910; fax: +351 289818419. E-mail address: aromano@ualg.pt (A. Romano). Food and Chemical Toxicology 57 (2013) 69–74 Contents lists available at SciVerse ScienceDirect Food and Chemical Toxicology journal homepage: www.elsevier.com/locate/foodchemtox