Infusions of Gamma Irradiated Aloysia Citrodora L. and Mentha X Piperita L
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Industrial Crops and Products 97 (2017) 582–590 Contents lists available at ScienceDirect Industrial Crops and Products jo urnal homepage: www.elsevier.com/locate/indcrop Infusions of gamma irradiated Aloysia citrodora L. and Mentha x piperita L.: Effects on phenolic composition, cytotoxicity, antibacterial and virucidal activities a,b c a a Eliana Pereira , Andreia I. Pimenta , Ricardo C. Calhelha , Amilcar L. Antonio , a b c a,∗ Lillian Barros , Celestino Santos-Buelga , Sandra Cabo Verde , Isabel C.F.R. Ferreira a Centro de Investigac¸ ão de Montanha (CIMO), ESA, Instituto Politécnico de Braganc¸ a, Campus de Santa Apolónia, 1172, 5300-253 Braganc¸ a, Portugal b GIP-USAL, Facultad de Farmacia, Universidad de Salamanca, Campus Miguel de Unamuno, 37007 Salamanca, Spain c Centro de Ciências e Tecnologias Nucleares, Instituto Superior Técnico, Universidade de Lisboa, Estrada Nacional 10 (km 139,7), 2695-066 Bobadela LRS, Portugal a r a t i c l e i n f o b s t r a c t Article history: The incorporation of plants in industry requires high standards of hygiene, being the raw materials obli- Received 28 September 2016 gatorily decontaminated. Irradiation is increasingly applied as a decontamination technique in several Received in revised form 5 January 2017 countries. This work aimed to evaluate the effects of gamma radiation on the phenolic composition, Accepted 5 January 2017 cytotoxicity, virucidal and antimicrobial properties of Aloysia citrodora L. (lemon verbena) and Mentha x Available online 13 January 2017 piperita L. (peppermint) infusions. Phenolic compounds profile was obtained by HPLC-DAD-ESI/MS and cytotoxicity was evaluated in human tumour and non-tumour cell lines; in virucidal efficacy, MNV-1 and Keywords: HAdV-5 viral titers were determined by plaque assay in Raw264.7 and A549 cell lines, respectively; and Gamma-radiation the antibacterial potential was evaluated against Gram-negative and Gram-positive bacteria. Radiation Aromatic herbs Cytotoxicity treatment caused a significant increase in total phenolic compounds of both plants, while the virucidal Virucidal activity efficacy was depend on the plant, the virus, and absorbed dose. S. aureus, with MIC and MBC values of Antibacterial activity 5 mg/mL, was the only bacteria sensible to the infusions of non-irradiated and 1 kGy irradiated lemon Phenolic compounds verbena and peppermint samples; gamma radiation appears to cause no alteration in the antibacterial potential of the studied plants. Peppermint infusion (irradiated at 10 kGy) showed the highest cytotoxic potential in all tumour cell lines. © 2017 Elsevier B.V. All rights reserved. 1. Introduction which provide health benefits due to their wide range of biological properties (Li et al., 2015; Gupta et al., 2016). Phenolic compounds The use of plants to improve the taste of foods, due to its sensory are potent antioxidants due to their capacity to scavenge reactive and preservative properties, has been a common practice through- oxygen species and chelate metal ions (Port’s et al., 2013). The most out generations (Güler et al., 2015). However, its employment also common compounds are flavonoids and phenolic acids, exhibit- covers the area of traditional medicine (Baydoun et al., 2015). The ing bioactive properties in terms of the cardiovascular, nervous increasing demand for healthier diets and the use of natural prod- and gastrointestinal systems, displaying anticarcinogenic, anti- ucts in prevention and/or treatment of health problems (Dhami hypertensive, antimutagenic, anti-nociceptive, anti-inflammatory, and Mishra, 2015) has led to an intensive search for plant bioac- immunomodulatory and hepatoprotective actions, as also angio- tive compounds (Gerson-Cwilich et al., 2006; Agyare et al., 2016; genesis inhibitory activity (Vaquero et al., 2010; Acosta-Estrada Giovannini et al., 2016; Jacobo-Herrera et al., 2016). et al., 2014; Bao et al., 2015). Plants are rich in phenolic compounds, terpenoids, alkaloids, However, plants easily suffer contaminations being absolutely quinones, carotenoids, sterols, glucosinolates and other sulphur- essential the application of techniques to eliminate microorgan- containing compounds (Embuscado, 2015; Gupta et al., 2016), isms such as fungi and bacteria, insects and others (Lung et al., 2015). The use of safe methodologies both for consumers and envi- ronment is mandatory (Machhour et al., 2011). Thus, irradiation is increasingly attracting attention, because it consists on a safe and ∗ Corresponding author. non-thermal decontamination and preservation technology, suit- E-mail address: [email protected] (I.C.F.R. Ferreira). http://dx.doi.org/10.1016/j.indcrop.2017.01.007 0926-6690/© 2017 Elsevier B.V. All rights reserved. E. Pereira et al. / Industrial Crops and Products 97 (2017) 582–590 583 able for various food matrices, including medicinal herbs and spices 2.2.4. For virucidity determination assays l (EU, 1999; EFSA, 2011). DMEM media, FBS, -glutamine, penicillin and streptomycin, The application of gamma radiation to different plant species sodium pyruvate, non-essential amino acids, HEPES buffer were has proven its capacity to preserve chemical and nutritional prop- from Gibco, Life Technologies (Carlsbad, CA, USA). Agarose was erties (Pereira et al., 2014; Pinela et al., 2016). The present study from SeaKem ME; Lonza (Rockland, ME, USA) and neutral red was extended to evaluate gamma radiation (1 and 10 kGy) effects solution was from Sigma (St. Louis, MO, USA). Deionized filtered on the phenolic composition, cytotoxicity, virucidal and antimi- water (DI) was produced by the Milli-Q system Millipore (Millipore, crobial activity of the most consumed forms of Menta x piperita model A10, Millipore Iberica, Spain). L. (peppermint) and Aloysia citrodora L. (lemon verbena): infusions. 2.3. Infusion’s preparation 2. Materials and methods The infusions were obtained from the dried plant material. Each 2.1. Dried samples and gamma irradiation sample (2 g) was added to 200 ml of boiling distilled water and left to stand at room temperature for 5 min, and subsequently filtered Aloysia citrodora Paláu (lemon verbena) and Mentha piperita under reduced pressure through Whatman No. 4 paper (Pereira L. (peppermint) were provided by Pragmático Aroma Lda, Alfân- et al., 2015a). dega da Fé, Braganc¸ a, Portugal – a local producer. Dried samples were divided into three groups: control (non-irradiated, 0 kGy), 2.4. Evaluation of cytotoxic potential and samples irradiated with different gamma radiation doses (1 and 10 kGy). 2.4.1. General The irradiation was performed using a Co-60 experimental The cytotoxicity evaluation was accomplished according to a chamber (Precisa 22, Graviner Manufacturing Company Ltd., UK) procedure previously described by Pereira et al. (2016a). The results with an activity of 165 TBq (4.45 kCi), in September 2015, and were expressed in GI50 values, i.e., sample concentration that inhib- Fricke dosimeter was used to estimate dose rate for the applied ited 50% of the net cell growth. For positive control, ellipticine was irradiation geometry. During irradiation process, Amber Perspex used. routine dosimeters (batch X, from Harwell Company, U.K.) was used for estimate the dose, following a procedure previously described by Pereira et al. (2015a). The estimated dose rate was 2.4.2. In tumour cell lines 1.5 kGy/h, and the average absorbed doses by the plants samples Four human tumour cell lines, (MCF-7), non-small cell lung were 0.9 ± 0.1 kGy and 10.2 ± 0.6 kGy. The dose uniformity ratio cancer (NCI-H460), cervical carcinoma (HeLa) and hepatocellu- (Dmax/Dmin) was 1.1 In the text and tables, for simplicity, we con- lar carcinoma (HepG2) were used to determine the cytotoxicity sidered the values 0, 1 and 10 kGy, for the doses of non-irradiated (Pereira et al., 2016a). The cell growth inhibition was measured and irradiated groups, respectively. using sulforhodamine B assay (SRB), were the amount of pigmented cells is directly proportional to the total protein mass and therefore to the number of bounded cells. The bounded sulforhodamine B was 2.2. Standards and reagents measured at 540 nm using an ELX800 microplate reader (Bio-Tek Instruments, Inc; Winooski, VT, USA). 2.2.1. For gamma radiation Fricke dosimeter (a chemical solution sensitive to ionizing radia- tion) to estimate the dose and dose rate of irradiation was used. The 2.4.3. In non-tumour cell line preparation of this chemical solution was performed in the lab fol- This methodology was performed following a procedure pre- lowing the standards (ASTM, 1992) and Amber Perspex dosimeters viously performed by Pereira et al. (2016a). A cell culture PLP2 (batch V, from Harwell Company, Oxfordfordshire, UK). For pre- was obtained from porcine liver in the laboratory. SRB assay was pare the acid aqueous Fricke dosimeter solution was used ferrous followed to evaluate the growth inhibition. ammonium sulfate(II) hexahydrate, sodium chloride and sulfuric acid; all purchased from Panreac S.A. (Barcelona, Spain) with purity 2.5. Virucidal activity evaluation PA (proanalysis), and water treated in a Milli-Q water purification system (Millipore, model A10, Billerica, NA, USA). 2.5.1. Viruses and cell cultures ® TM Human Adenovirus type 5 (HAdV-5, ATCC VR-1516 ) was 2.2.2. For chemical analysis propagated in confluent monolayers of human lung carcinoma cells ® Acetonitrile 99.9% was of HPLC grade from Fisher Scientific TM A549 (ATCC CCL-185 ). Murine Norovirus type 1 (MNV-1) strain (Lisbon, Portugal). Phenolic compound standards: