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Oxidative stress protective effect of Dracocephalum multicaule essential oil against human cancer cell line a

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Mohammad Ali Esmaeili , Ali Sonboli & Mohammad Hossein Mirjalili

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Department of Biology, Medicinal Plants and Drugs Research Institute, Shahid Beheshti University, G.C., Evin 1983963113, Tehran, Iran b

Department of Agriculture, Medicinal Plants and Drugs Research Institute, Shahid Beheshti University, G.C., Evin 1983963113, Tehran, Iran Published online: 31 Jan 2014.

To cite this article: Mohammad Ali Esmaeili, Ali Sonboli & Mohammad Hossein Mirjalili (2014) Oxidative stress protective effect of Dracocephalum multicaule essential oil against human cancer cell line, Natural Product Research: Formerly Natural Product Letters, 28:11, 848-852, DOI: 10.1080/14786419.2014.881364 To link to this article: http://dx.doi.org/10.1080/14786419.2014.881364

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Natural Product Research, 2014 Vol. 28, No. 11, 848–852, http://dx.doi.org/10.1080/14786419.2014.881364

SHORT COMMUNICATION Oxidative stress protective effect of Dracocephalum multicaule essential oil against human cancer cell line Mohammad Ali Esmaeilia*, Ali Sonbolia* and Mohammad Hossein Mirjalilib

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Department of Biology, Medicinal Plants and Drugs Research Institute, Shahid Beheshti University, G.C., Evin 1983963113, Tehran, Iran; bDepartment of Agriculture, Medicinal Plants and Drugs Research Institute, Shahid Beheshti University, G.C., Evin 1983963113, Tehran, Iran (Received 27 November 2013; final version received 6 January 2014) In this study, we report the antioxidative and protective effect of essential oil of Dracocephalum multicaule on K562 cells. Our results demonstrated that monoterpenoids, including oxygenated and hydrocarbons, 71.5% and 28.3%, respectively, were the principal essential oils of D. multicaule. Perilla aldehyde (71.5%) and limonene (28.1%) were identified as the main components. Antioxidant studies based on the 2,2-diphenylpicrylhydrazyl assay indicated that the D. multicaule essential oil possesses a marked antioxidant and radical-scavenging activity with an IC50 value of 438.2 mg/mL. Pretreatment with essential oil and main constituents protected K562 cells 49.5% against H2O2-induced oxidative damage throughout increasing the activities of antioxidant enzymes and glutathione content in K562 cells. Collectively, D. multicaule essential oil and its main compounds especially in combinatory condition at a ratio of 7:3 with high antioxidant properties may be able to protect cells against oxidative stress induced by H2O2 through antioxidative mechanisms. Keywords: antioxidant activity; anticancer activity; Dracocephalum multicaule; essential oil; perilla aldehyde

1. Introduction Reactive oxygen species (ROS) including free radicals in the forms of superoxide anion, hydroxyl radicals, singlet oxygen and non-free radical species, such as hydrogen peroxide, are various forms of activated oxygen, resulting from oxidative biological reactions or exogenous factors. Overproduction of ROS can damage cellular biomolecules such as nucleic acids, lipids, carbohydrates, proteins and enzymes, resulting in several diseases (Halliwell 1995). Hydrogen peroxide plays an important role in the immune system and acts either directly or indirectly as a messenger molecule in the inflammation events and cell signalling pathways (Auroma et al. 1989). A body of data has indicated that the antioxidants directly terminate ROS, and radicalmediated oxidative reactions may be used as a method of prevention of ageing-associated diseases and health problems. This has led to an accelerated search for antioxidant principles, the identification of natural resources, and the isolation of active antioxidant molecules. Antioxidants have been detected in a number of agricultural and food products including cereals, fruits, vegetables and oil seeds (Kalt et al. 1996; Burits & Bucar 2000). Synthetic antioxidants such as butylated hydroxytoluene (BHT), butylated hydroxyanisole and propyl gallate have been widely used as antioxidants in the food industry (Nawar 1996), but safety concerns and reports on their involvement in chronic diseases have restricted their use in foods.

*Corresponding authors. Email: [email protected]; [email protected] q 2014 Taylor & Francis

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Therefore, international attention has been directed towards natural antioxidants mainly from plant sources as an alternative to prevent deterioration of foods during processing and storage (Claudio & Hector 2000). The antioxidant activity of the essential oils has formed the basis of many applications, including fresh and processed food preservation, pharmaceutical and natural therapies. The genus Dracocephalum L. (Lamiaceae) is represented in the flora of Iran by eight annual and perennial species of which three are endemic to Iran (Rechinger 1982). Dracocephalum multicaule Montbar. & Auch. grows in the northwestern Iran (West Azarbaijan province). Infusion of the aerial flowering parts of the plant is used locally in the treatment of colds and gastrointestinal disorders. There are several reports on the biological and pharmacological properties of some isolated active compounds from different Dracocephalum species. Limonene and a-terpineol have been reported to be responsible for the antinociceptive activity of the oil of Dracocephalum kotschyi (Golshani et al. 2004). Citral and limonen-10-al isolated from Iranian Dracocephalum subcapitatum were found to be the most effective compounds against epimastigotes of Trypanosoma cruzi (Saeidnia et al. 2005). Xantomicrol as a potent anticancer agent has been identified from the hydro-alcoholic extract of D. kotschyi (Jahaniani et al. 2005) The antioxidant activity of the methanolic extract of Dracocephalum moldavica has been investigated (Povilaityte et al. 2001). Flavonoids (Oganesyan & Mnatsakanyan 1992), flavonoid glycosides (Oganesyan 1993) and minor flavonoids (Oganesyan 2009) of D. multicaule growing in Armenia have already been investigated. The goals of this study were to determine (1) the essential oil composition of the plant, (2) the antioxidant activity of the essential oil and its major compounds using the 2,2diphenylpicrylhydrazyl (DPPH) radical-scavenging assay and the b-carotene-linoleic acid blenching assays and (3) the protective effect of essential oil in oxidative stress damage of K562 cells induced by hydrogen peroxide. 2. Results and discussion 2.1. Chemical composition of the essential oil Hydrodistillation of the air-dried and ground aerial parts of D. multicaule gave a whitish yellow essential oil with the yield of 1.2% (w/w). In total, four components representing 99.8% of the essential oil were characterised (Table S1). The oil was rich in oxygenated (71.5%) and hydrocarbon (28.3%) monoterpenes, including perilla aldehyde (71.5%) and limonene (28.1%) as the major constituents, respectively. Recently, perilla aldehyde (54.3%) was also found to be the main constituent of the essential oil of Dracocephalum surmandinum from Iran (Sonboli et al. 2010). As far as our literature survey could ascertain, the essential oils of Dracocephalum species studied previously have shown qualitative and quantitative differences when compared with that of the species reported here, particularly in the levels of the principal components. Some of the Dracocephalum species exhibited different types of essential oil composition including, sabinene (55.2%) in Dracocephalum aucheri (Ahmadi & Mirza 2001), neral (32.1%) in D. moldavica (Sonboli et al. 2008) and citral (29.3%) in D. kotschyi (Yaghmai & Tafazzoli 1988) originating from Iran. 2.2. Antioxidant activity of D. multicaule essential oil and main constituents Radical-scavenging activity of the essential oil from aerial parts of plant and its major components (limonene and perilla aldehyde) were evaluated by means of the DPPH radical assay. The essential oil of D. multicaule demonstrated a concentration-dependent scavenging activity by quenching DPPH radicals (data not shown). The hydrogen-donating activity, measured using the DPPH test, revealed that the essential oil of D. multicaule with EC50 value

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(defined as the concentration of test compound required to produce 50% maximal inhibition) of 438.2 mg/mL (Table S2) scavenges the DPPH radicals. When compared with BHT, essential oil has been found to be less effective than this synthetic antioxidant agent. The potential of the plant to inhibit lipid peroxidation was evaluated using the b-carotene-/linoleic acid bleaching test. Considering the data presented in Table S2, the inhibition value of linoleic acid oxidation was estimated as 54.2 ^ 3.4 in the presence of the D. multicaule essential oil. Compared with perilla aldehyde and limonene in single and/or mixture dose (ratio of 1:1, 7:3 and 3:7 v/v of perilla aldehyde and limonene, respectively) the antioxidant activity of the essential oil was potent in both the DPPH and b-carotene/linoleic acid bleaching tests (Table S2). It may be concluded that the presence of other compounds except two main constituents exerts a synergistic effect on the total oil antioxidant potential. 2.3. Protective activity of essential oil and its main constituents on oxidative stress induced by H2O2 in K562 cells As a continuation of our studies, we checked the ability of the oil at a concentration of 10% v/v and main constituents (perilla aldehyde and limonene) in single or binary mixture at a ratio of 7:3 on hydrogen peroxide-induced cytotoxicity in terms of ROS content, LDH release, GSH content, GPx and GR activity among the oil/main constituents-pretreated human erythromyeloblastoid leukaemia cell line (K562). As it is evident from Figure S1, pretreatment of the cells with essential oil and main constituents at ratio of 7:3 in binary mixture restored cell viability and decreased LDH release by the H2O2-induced oxidative stress. To gain insights into modifications in the redox cell state, we investigated the accumulation of ROS within cells by monitoring the oxidation of DHR, which accumulates mainly within the mitochondria, as a result of its lipophilic cation nature, where it is oxidised in the presence of peroxidases. Figure S1 shows that the exposure of the cells to hydrogen peroxide caused high increase in the content of ROS relative to H2O2 untreated control cells. Pretreatment of the cells with essential oil at concentration of 10% v/v and perilla aldehyde and limonene at a ratio of 7:3 diminished the levels of ROS by 49.5% and 39.8%, respectively. The antioxidant defence system comprises non-enzymatic and enzymatic constituents and plays a crucial role in the defence against oxidative stress. Reduced glutathione (GSH) is the main non-enzymatic antioxidant defence as a substrate in GSH peroxidase-catalysed detoxification of organic peroxides, by reacting with free radicals and by repairing free radical-induced damage through electron-transfer reactions (Scharf et al. 2003). It is generally assumed that GSH depletion reflects intracellular oxidation, whereas a balanced GSH concentration could be expected to prepare the cell against a potential oxidative insult (Alja et al. 2006; Goya et al. 2007). The intracellular GSH content of K562 cells was reduced to 40% of that of the untreated control following exposure of the cells to H2O2 for 24 h (Figure S2). The GSH contents of K562 cells, pretreated with plant oil and/or its main compounds, were increased almost relative to H2O2-treated cells (Figure S2). The remarkable decrease in the concentration of GSH induced by a hydrogen peroxide condition was partly prevented by pretreatment with essential oil or its main compounds, a reaction in line with that reported for other phytochemicals such as quercetin and hydroxytyrosol (Alja et al. 2006; Goya et al. 2007). These observations are consistent with the reduction of ROS level in cells, which may be explained by an increased consumption of GSH in quenching of ROS generated in H2O2 oxidative stress to protect cell against oxidation conditions. Changes in the activity of antioxidant enzymes can be considered as biomarkers of the antioxidant response (Goya et al. 2007). GPx catalyses GSH oxidation to GSSG at the expense of H2O2 or other peroxides (Gunzler et al. 1974) and GR recycles oxidised GSH back to reduced GSH (Goldberg & Spooner 1987); therefore, their activities are essential for the intracellular quenching of cell-damaging

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peroxide species and the effective recovery of the steady-state concentration of reduced GSH. The significant increase in the GPx and GR activities after treatment in the presence of hydrogen peroxide indicated a cell defence system response to oxidative stress condition. Despite this fact, the remarkable decrease in the activities of these two enzymes induced by hydrogen peroxide condition was prevented by pretreatment of cells with D. multicaule oil (Figure S2).

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3. Conclusion Considering the great interest in antioxidative properties of the essential oils and various extracts from many plants in both academia and the food industry, studying the endemic species may be of great interest, since their bioactive properties and secrets could be lost forever without being tapped. D. multicaule essential oil and its main compounds especially in combinatory condition at a ratio of 7:3 with high antioxidant properties may be able to protect cells against oxidative stress induced by H2O2 through antioxidative mechanisms. Supplementary material Experimental details relating to this article are available online, alongside Figures S1 and S2 and Tables S1 and S2. Acknowledgement Financial support by the Research Council of Shahid Beheshti University is gratefully acknowledged (research project no. 600/170).

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Oxidative stress protective effect of Dracocephalum multicaule essential oil against human cancer cell line.

In this study, we report the antioxidative and protective effect of essential oil of Dracocephalum multicaule on K562 cells. Our results demonstrated ...
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