The Influence of Different Oregano Species on the Antioxidant Activity Determined Using HPLC Postcolumn DPPH Method and Anticancer Activity of Carvacrol and Rosmarinic Acid

The aim of this study was to evaluate concentration-dependent antioxidant and anticancer activities of CA and RA in ethanol extracts of three different Oregano species (Origanum onites L., Origanum vulgare L., and Origanum vulgare ssp. hirtum). The study revealed the highest RA antioxidant activity in O. vulgare ssp. hirtum (9550 ± 95 mmol/g) and the lowest in O. vulgare L. (2605 ± 52 mmol/g) (p < 0.05). The highest CA amount was present in O. onites L., which was 1.8 and 4.7 times higher (p < 0.05) than in O. vulgare ssp. hirtum and O. vulgare L., respectively. The anticancer activity was evaluated on human glioblastoma (U87) and triple-negative breast cancer (MDA-MB231) cell lines in vitro. RA anticancer activity was negligible. CA and the extracts were about 1.5–2 times more active against MDA-MB231 cell line (p < 0.05) compared to U87 cell line. The anticancer activities of three tested extracts were similar against U87 cell line (p > 0.05) but they had different activities against MDA-MB231 cell line.


Introduction
There is a wide variety in nature of naturally occurring antioxidants that differ in their composition, physical and chemical properties, and action mechanisms [1]. Since the early work of Chipault et al., who have examined more than 70 spices and herbs, the interest in the antioxidant activity of spices has increased recently [2]. Since many of widely used synthetic antioxidants may cause negative health effects, herbs and spices could be the most important safe targets searching for the natural antioxidants [3]. Free radicals may disrupt normal or lead to the pathological cell metabolism causing diseases such as cancer, cirrhosis, and arteriosclerosis [4]. Natural antioxidants might play an important role in the free radical metabolism.
Many studies have demonstrated that spices and herbs that have high content of essential oils and phenolic compounds, such as Oregano, rosemary, and sage, serve as strong antioxidants [5]. In recent years, the efficiency of essential oils from Oregano species has been reported in many studies. Antioxidant activity of Oregano [6,7] is associated with its constituents rosmarinic acid (RA) and carvacrol (CA). Oregano essential oils and phenolic compounds have been shown to possess antioxidant, anticancer, antibacterial, antifungal, diaphoretic, carminative, antispasmodic, and analgesic activities [8,9]. Essential oils containing CA are the subject of increasing scientific interest because of their multiple biological effects, including antioxidant activity that is associated with the prevention of various degenerative diseases [9]. HPLC postcolumn assay is a highly promising 2 BioMed Research International method to investigate natural antioxidants with various derivatization techniques [10].
Some studies have used spectrophotometric method to determine a total antioxidant potency of Oregano [5,11]. It is important that the antioxidant activity of the herb can be defined not only by the essential oil, but also by phenolic components or their interaction with other components [11]. Many studies have determined antioxidant activity of essential oils (EOs), but only few studies have been published on the antioxidant activity of individual components, for example, carvacrol and thymol [11][12][13]. A highly effective technique, developed and modified by different researchers, is based on a model oxidation system that combines HPLC separation methods with the online postcolumn detection of radical-scavenging compounds [14][15][16]. Online postcolumn methods are employed in the rapid identification of antioxidants and determination of their activity [10]. Model oxidation systems of DPPH radicals possess important advantages when compared with other colorimetric methods and thus have been extensively used in screening of plant extracts for antioxidants [17].
The objective of this study was to evaluate concentrationdependent antioxidant and anticancer activities of CA and RA in ethanol extracts of three different Oregano species (Origanum onites L., Origanum vulgare L., and Origanum vulgare ssp. hirtum).

Plant Material.
Dried Origanum vulgare L. herb was purchased from "Svencioniu zoles," Lithuania; dried Origanum vulgare ssp. hirtum herb from "Ç engelköy spice and herbs center," Turkey; dried Origanum onites L. herb from "İnanTarım ECO DAB," Turkey. Dried herbs were identified by Dr. Professor Jurga Bernatoniene, Medical Academy, Lithuanian University of Health Sciences. Identification of the plant material was performed according to the requirements of the European Pharmacopoeia, Monograph number 01/2008:1833 corr.6.0. The tests used include a pharmacognostical investigation of the anatomical and microscopical characteristics and TLC fingerprinting. Voucher specimens (number L170709, L170710, and L170711) have been deposited at the Herbarium of the Department of drug technology and social pharmacy, Lithuanian University of Health Sciences, Lithuania. The dried herb particle size of O. vulgare L., O. vulgare ssp. hirtum, and O. onites was 125 m.

Preparation of Oregano Ethanol Extract.
Prior to preparation of extract Oregano herb was ground in a cross beater mill IKA A11 Basic Grinder (IKA Works, Guangzhou, China) and sieved using vibratory sieve shaker AS 200 basic (Retch, UK) equipped with a 125 m sieve. Powdered material (100 g) was then extracted with 1000 mL of 90% (v/v) ethanol in a round bottom flask by heat-reflux extraction performed in the water bath Memmert WNB7 (Memmert GmbH & Co. KG, Schwabach, Germany) at 95 ∘ C for 4 hours. Prepared extract was filtered using vacuum filter. These conditions were determined in our previous study as the best for the extraction of main active compounds of Turkish Oregano [18].

HPLC Analyses.
HPLC analysis has been carried out using Waters 2695 chromatography system (Waters, Milford, USA) equipped with Waters 996 PDA detector. Data was collected and analysed using a PC and the Empower2 chromatographic manager system (Waters Corporation, Milford, USA). For determination of RA ACE 5 C18 250 × 4.6 mm column (Advanced Chromatography Technologies, Aberdeen, Scotland) was used. Quantification of compounds was carried out by the external standard method. Standard stock solutions at a concentration of 1.0 mg/mL were prepared freshly in methanol and diluted in appropriate quantities to obtain a set of corresponding concentration ranges for the study of linearity. The regression coefficient ( 2 ) was 0.999 for all the calibration curves. A calibration curve for each of the compounds was constructed by plotting peak areas versus the respective compound concentration and calculated by linear regression analysis. The precision of the method was demonstrated for all analytes, since all the obtained relative standard deviation (RSD) values were lower than 5.0%. The concentration of compounds was expressed as g/g dry mass (DM). HPLC conditions for determination of RA: the two elution solvents were exchanged: the solvent A (methanol) and the solvent B (0.5% (v/v) acetic acid in water). The following linear gradient elution profile was used: 95% A/5% B-0 min, 40% A/60% B-40 min, 10% A/90% B-41-55 min, and 95% A/5% B-56 min. The flow rate was 1 mL/min and injection volume was 10 L. The effluent was determined at a wavelength of 329 nm. The linear calibration curve was made and expressed by the following quadratic equation: 2 (RA) = 0.999918, ( = 2.01⋅10 7 +5.52⋅10 3 ), the linearity range: 10-100 g/ml. (Figure 1(a)) [18]. For determination of CA: the mobile phase was composed of methanol and water (60/40, v/v). The flow rate was 0.6 mL/min and injection volume was 10 L. The absorbance was measured at 275 nm. The quantification has been carried out by the external standard method. The calibration curve was made and expressed by the following quadratic equation: 2 (CA) = 0.999751, ( = 4.99 * 10 9 + 1.08 * 10 3 ), the linearity range: 0.18-3 g/ml (Figure 1(b)) [18].

HPLC Postcolumn Antioxidant Detection Method.
According to Raudonis et al. [15], after applying the HPLC-PDA detection system, the mobile phase containing the analytes was entered into a reaction coil through a mixing tee where the reagents (DPPH solution) were supplied (split ratio,

Determination of Anticancer Activity by MTT Assay.
Cell viability was studied using the method of 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma). 100 L of cells was seeded in 96-well plates in triplicate (5000 cells/well) and incubated at 37 ∘ C for 24 hours. Then serial dilutions of the pure compounds (CA and RA) or extracts were made in microplates. Medium without cells was used as a positive control. Cells treated with medium containing 0.25% dimethylsulfoxide (DMSO) (in the case of compounds) or ethanol (in the case of extracts) served as a negative control. After 72-hour incubation at 37 ∘ C, 10 L of MTT was added in each well. After 3 hours the liquid was aspirated from the wells and discarded. Formazan crystals were dissolved in 100 L of DMSO, and absorbance was measured at a test wavelength of 490 nm and a reference wavelength of 630 nm using a multidetection microplate reader. The experiments were repeated three times independently and the results were given as means ± SD.

Statistical
Analysis. Statistical analysis was performed by one-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test with the software package Prism v. 5.04 (GraphPad Software Inc., La Jolla, CA, USA). The value of < 0.05 was taken as the level of significance.

Results and Discussion
In our previous study, we have used HPLC method for quantitative analysis of Oregano extract [16]. According to scientific literature the main active compounds in Oregano species herbs are phenolic compound-derived RA and CA from essential oil [4,5]. Our results confirmed that the main  compounds in all Oregano species were RA and CA (the chromatograms are shown in Figures 1(a) and 1(b)). The main differences between the species according to the amounts of RA and CA were shown in Figures 2 and 3. The highest yield of RA was revealed in . ssp. hirtum L. and the highest yield of CA in O. onites L.

Antioxidant Activity.
Phenolic compounds and the essential oil are the main sources of antioxidant activity [11]. The spectrophotometric method has been often used for the determination of antioxidant activity as total antioxidant activities [4,9]. But still it is very difficult to obtain information on the contribution of individual compounds to the BioMed Research International 5 overall antioxidant effects due to complexity and diversity [19]. DPPH (2,2-diphenyl-1-picrylhydrazyl), a paramagnetic compound with an odd electron, is one of the most popular radicals used to evaluate antioxidant activities of pure substances and complex samples. HPLC postcolumn (bio) chemical assay determines activities of individual compounds present in the mixtures [20]. In the course of experimental investigations, HPLC-DPPH assays have been developed and applied for rapid screening and identification of antioxidants from the extracts of herbal medicines [21]. The HPLC-DPPH postcolumn methodology was used in further investigations of ethanol Oregano herb extracts of three different species and antioxidant activity of CA and RA has been determined. The antioxidant activity of the measured RA compound has been expressed as TEAC values (Trolox mmol/g) ( Figure 2). The results of DPPH postcolumn assay in terms of the TEAC values (mmol/g) for the measured RA compound showed statistically significant differences in antiradical response between three Oregano species. The highest antiradical response was obtained in O. vulgare ssp. hirtum (9550 ± 95 mmol/g) and the lowest in O. vulgare L. (2605 ± 52 mmol/g). Numerous studies have been published on total phenolic antioxidant activity of Oregano [11,22]. Madsen et al. [23] reported that the antioxidant activity of Oregano is due to a variety of components, and a significant correlation between antioxidant activity and total phenolic content of the spice extract has been found [24].
Numerous studies have been published on antioxidant activity of essential oil of Oregano [4,25,26]. The present study proved the antioxidant activity of the identified CA compound from three different Oregano species and it was expressed as TEAC values (Trolox mmol/g). Results (Figure 3) showed statistically significant differences in the antioxidant activities of the identified CA compounds between three different Oregano species. The highest CA amount was obtained from O. onites L. While the concentration of CA was 1.8 times higher ( < 0.05) in O. onites L. compared with O. vulgare ssp. hirtum, the concentration of CA was 4.7 times higher ( < 0.05) in O. onites L. compared with O. vulgare L. (Figure 1(b)). O. vulgare accumulated the lowest amounts of CA and it led to the smallest values of TEAC.
The antioxidant activity of RA and CA as separate compounds has not been extensively studied yet to our knowledge. In our study, RA and CA have been tested for the first time in order to determine active components of the extract or essential oil of Oregano. Thus, our study puts importance on raising awareness about antioxidant properties of the specific components of Oregano. The results showed that the potency of antioxidant activity of Oregano depends on the species due to the different quantities of active substances (RA and CA) which is in good agreement with Lindberg Madsen report demonstrating that antioxidant activities are different for the different species [23].

Anticancer
Activity. The anticancer activity of RA against tested cancer cell lines was negligible, even at the concentration of 50 M. Some other researchers [27,28] also found that RA alone showed little effect on cancer cell viability. CA possessed 1.2 times higher activity ( < 0.05) against MDA-MB231 cells compared to U87 cells (Figure 4(a)). It inhibited the growth of 50% of both tested cancer cells at the concentrations of 199 M (MD-MB231) to 322 M (U87). The determined activity of CA is comparable with the results from other studies [29,30].
All tested Oregano extracts showed antiproliferative activity in vitro. The extracts were about 2.1-2.9 times more active against MDA-MB231 cell line ( < 0.05) compared to U87 cell line (Figure 4(b)). This different activity could be due to the fact that glioblastoma cells are usually more resistant to chemo-and radiotherapy; they may contain accumulated gene mutations and epigenetic alterations [31].
The anticancer activities of all three tested extracts were very similar against U87 cell line ( > 0.05) (Figure 5), except the different activity against MDA-MB231 cell line ( Figure 6). The extracts prepared from O. vulgare ssp. hirtum Anticancer activity of extracts on U87 cells and O. vulgare L. were more active ( < 0.05; EC 50 values were 2.0 ± 0.3 mg/mL and 1.8 ± 0.4 mg/mL, resp.). Interestingly, the extracts possessed different activities on MDA-MB231 cell line depending on their concentration ( Figure 6). Extract prepared from O. onites L. had lower activity at the concentration of 0.25 mg/mL but was similarly active as the extract prepared from O. vulgare ssp. hirtum at the highest tested concentration of 1 mg/mL. Those different species contain different quantities of active substances and thus could possess different antiproliferative activities against the same cancer cell line [32].

Conclusion
The As a final conclusion of this study, the potency of antioxidant activity of Oregano varies in the species due to different quantities of RA and CA. The anticancer activity of the tested extracts was higher against human triple-negative breast cancer cell line compared to glioblastoma cell line. The anticancer activity of Oregano extracts depends on both the tested cell line and the species.

Conflicts of Interest
The authors declare that there are no conflicts of interest regarding the publication of this paper.