Nizioł-Łukaszewska et al. Lipids in Health and Disease (2018) 17:280 https://doi.org/10.1186/s12944-018-0929-8 RESEARCH Open Access Antioxidant activity and cytotoxicity of Jerusalem artichoke tubers and leaves extract on HaCaT and BJ fibroblast cells Zofia Nizioł-Łukaszewska, Dominika Furman-Toczek and Martyna Zagórska-Dziok* Abstract Background: Extracts from plants, rich in antioxidants may be used as active ingredients of many preparations, mainly due to their antioxidant, regenerative and anti-aging properties. The work involved a comprehensive evaluation of the Jerusalem artichoke (Helianthus tuberosus L.) leaf and tuber extract as a multifunctional raw material. Methods: The plant extracts were prepared by using ultrasound-assisted extraction method (UAE).The content of total phenolic and flavonoid compounds of extracts were determined spectrophotometrically using the Folin-Ciocalteu method and aluminium nitrate nonahydrate, respectively. Antioxidant activity of extract was analyzed using DPPH free radical scavenging assay and the effect of the investigated extracts on the proliferation of keratinocytes (HaCaT) and fibroblasts (BJ) was measured. To detect of intracellular reactive oxygen species level in tested cells, the fluorogenic dye H2DCFDA was used. In the next step, the ability of obtained extracts to regulate the expression of genes (SOD-1, Nox-4) involved in oxidative stress in cells was evaluated. Results: As a result of the conducted research, it was shown that leaf extract exhibit a higher content of phenols and flavonoids comparing to tuber extracts (5.07 and 7.14 fold higher, respectively). The opposite trend was observed after proliferation assay with Neutral Red test. It was shown that tuber extract in all applied concentrations (25–500 μg·ml− 1) had a positive effect on fibroblast growth. The leaf extract showed proliferative activity only for the smallest tested concentrations (25–100 μg·ml− 1). Similar trends were observed for HaCaT cells. The distinct effect of leaves and tuber extract on the generation of ROS was observed in HaCaT cells. In the present study, it was shown that tuber and leaf extracts may increase the expression of the ROS SOD-1 inactivating enzyme gene in the fibroblast cell line. There were no significant differences in gene expression of the ROS Nox-4 producing enzyme. In the case of keratinocytes, the opposite effect was observed. Conclusions: The study suggest that Jerusalem artichoke leaves and tubers extracts affect the cell proliferation and can alter the expression of genes related to oxidative stress. Keywords: Jerusalem artichoke, Antioxidant activity, ROS, SOD-1, Nox-4 Background molecules that are generated in all cells during normal It is commonly known that oxidative stress caused by physiological and biochemical processes. Excessive pro- free radicals and their derivatives is responsible for dis- duction of free radicals can cause cellular and tissue in- turbing redox homeostasis [1]. It also is one of the pri- jury through nonspecific modification and disruption of mary factors involved in the development of chronic phospholipids, proteins and nucleic acid [4]. ROS have a disorders, aging, cancer, degenerative neuronal damage, negative effect primarily on organelle and cell mem- diabetes mellitus and coronary heart ailment [2, 3]. Re- branes. Their excess entail peroxidation of membrane active oxygen species (ROS) are a group of unstable lipid and modification of membrane protein. As a result, the structure of the membranes is changing and also * Correspondence: [email protected] their functions are disturbed. Reactive oxygen species University of Information Technology and Management in Rzeszow, causes alternations in cell membrane permeability and Kielnarowa 386a, 36-020 Tyczyn, Poland © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Nizioł-Łukaszewska et al. Lipids in Health and Disease (2018) 17:280 Page 2 of 12 fluidity, intracellular enzyme and ion channels activity fractures, swellings and to reduce pain. These applica- [5]. These radicals can also may cause DNA damage, tions are closely related to the activity of Helianthus leading to mutagenic changes and cell death [6]. Fortu- tuberosus L. leaves, which had shown anti-inflammatory, nately, well-functioning cells have the ability to defend antispasmodic, antimicrobial, antifungal, antipyretic and against the destructive effects of free radicals by the en- analgesic effects [19–21]. dogenous systems which consist of various enzymes The aim of our study was to determine the ability of ob- such as superoxidase dismutase (SOD), catalase (CAT) tained extracts to impact on cell metabolism, influence on and glutathione peroxide (GPX). An extremely import- the regulation of gene expression (SOD-1, Nox-4) and to ant role in the fight against damage caused by free radi- evaluate antioxidants activities of tubers and leaves of He- cals play antioxidants derived from diet. The rich source lianthus tuberosus L. extracts. The content of flavonoids of natural antioxidants are primarily plants, mainly and phenolic compounds was also determined. These ex- fruits, vegetables and herbs, which are now eagerly in- tracts were obtained by ultrasound-assisted extraction cluded in the daily diet. Plant compounds with antioxi- method (UAE) and pure ethanol was used as a solvent. It dant properties can be helpful in the therapy of diseases also has been investigated the ability of obtained extracts caused by the action of free radicals [7, 8]. They also can to impact on cell metabolism and regulate of gene expres- enhance the antioxidant system, reverse oxidative dam- sion (SOD-1, Nox-4) involved in oxidative stress in cells. age and protect against oxidative stress induced deterior- The experiments were performed on two human cell lines: ation. The antioxidant capacity of plant extracts is keratinocytes (HaCaT) and fibroblasts (BJ). mainly associated e.g. with phenolic, flavonoids, isoflavo- noids and anthocyanins content. Numerous studies had Methods indicated a correlation between the content of phenolic Plant material and extraction procedure compounds and flavonoids in plants and the antioxidant Helianthus tuberosus L. were planted in a sunny pos- activity of plant extracts [9, 10]. ition, in the first half of March, every 30 cm in a row. Helianthus tuberosus L. (Jerusalem artichoke) is a per- The distance between rows was about 80 cm. The tubers ennial herb from Asteraceae family, originating from the were planted in a depth of 10–15 cm. The leaves and tu- United States where it was cultivated by the indigenous bers of Helianthus tuberosus L. were collected from the inhabitants. It has a 1,5–3 m tall stem, large leaves, region of Subcarpathian Voivodeship in Poland during fleshy tubers and yellow sunflower-like flowers [11]. The the September 2017. The collected tubers and leaves content of compounds contained in Jerusalem artichoke were transported to the laboratory and prepared for fur- tubers depends strictly on the harvest conditions and ther analysis. To remove the soil and other impurities, topinambur clones. Helianthus tuberosus L. tubers are a the plant material was cleaned by washing with deion- rich source of carbohydrates [12]. It is one of the main ized water. Then, samples of leaves and tubers were used sources of inulin in higher plants, its content reaches up for solvent extraction. to 85% of the dry matter of tubers [13]. The content of The fresh plant extracts were prepared by using free sugars such as glucose, fructose and sucrose is ultrasound-assisted extraction method (UAE). UAE was much lower and rarely exceeds 6–8% of dry matter. Pro- performed according to the method described by Ying et teins present in topinambour tubers reaches up to 10% al. [22] in ultrasonic bath (Digital Ultrasonic Cleaner) of dry matter. It contains almost all essentials amino equipped with time controller. About 15 g of plant ma- acid such as tryptophan and threonine [11, 14] to the terial was packed to the glass tubes and extracted with a high content of inulin in topinambour tubers, it has 200 ml of ethanol in room temperature. The mixture been used in folk medicine, and found an application in was homogenized for 50 min (10 cycles for 5 min). Then, the treatment of many diseases such as diabetes and obtained extracts were collected and filtered through rheumatism. This polysaccharide also has a diuretic, Whatman filter paper No. 10 and evaporated at 50 °C aperients, cholagogue, spermatogenic, stomachic and using a rotary evaporator. Tuber and leaves extracts tonic effect [15]. Phytochemical studies demonstrated were stored in the dark in 4 °C for further analysis. that topinambour is a source of coumarins, polyacetyle- nic derivatives, unsaturated fatty acids and sesquiter- Total phenolic content determination penes. Aerial biomass of this plant contain cellulose, The total phenolic content of leaves and tubers Helianthus hemicelluloses, uronic acids, lignins, proteins