# The Essential Oils ofRhaponticum carthamoides Hairy Roots and Roots of Soil-Grown Plants: Chemical Composition and Antimicrobial, Anti-Inflammatory, and Antioxidant Activities

Authors: Ewa Skała; Patrícia Rijo; Catarina Garcia; Przemysław Sitarek; Danuta Kalemba; Monika Toma; Janusz Szemraj; Dariusz Pytel; Halina Wysokińska; Tomasz Śliwiński
Journal: Oxidative Medicine and Cellular Longevity
 (2016)

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## Abstract

The essential oils were isolated by hydrodistillation from the hairy roots (HR) and roots of soil-grown plants (SGR) ofRhaponticum carthamoides and were analyzed by GC-MS method. In the both essential oils 62 compounds were identified. The root essential oils showed the differences in the qualitative and quantitative composition. The sesquiterpene hydrocarbons (55–62%) dominated in both essential oils. The major compounds of HR essential oil were cyperene, 13-norcypera-1(5),11(12)-diene, and cadalene while aplotaxene, nardosina-1(10),11-diene, and dauca-4(11),8-diene dominated in SGR essential oil. Both essential oils showed antibacterial activity especially againstEnterococcus faecalis (ATCC 29212) andPseudomonas aeruginosa (ATCC 27853) (MIC value = 125 µg/mL). HR and SGR essential oils also decreased the expression of IL-1β, IL-6, and TNF-α and the ROS level in LPS-treatment astrocytes. This is the first report to describe the chemical composition ofR. carthamoides essential oil from hairy roots, its protective effect against LPS-induced inflammation and ROS production in astrocytes, and its antimicrobial potential. The results show thatR. carthamoides hairy roots may be a valuable source of the essential oil and may be an alternative to the roots of soil-grown plants.


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## Body

## 1. Introduction

The last decade has seen increased interest in the use of essential oils from plants in the food, human health, and the pharmaceutical industry [1]. Different parts of plants can synthesize essential oils with diverse pharmacological activities, ranging from the cytotoxic, through the antioxidant, antimicrobial, and antiparasitic, to the anti-inflammatory [1, 2]. Their antibacterial activity makes essential oils suitable alternatives to antibiotics [3]. Many antibiotics can be not effective as bactericidal agents due to drug-resistancy of bacteria that have developed from the widespread use of antibiotics. Essential oils can be used as natural food preservatives against food spoilage and pathogenic bacteria [4]. They may be an alternative to currently used synthetic compounds, which may have toxic effects and may be carcinogenic [3, 4]. The antimicrobial activity of the essential oils is associated with different functional groups and the synergistic, antagonistic, or additive effects between them [4, 5]. Essential oils may also play an important role in scavenging free radicals which may lead to oxidative stress [1]. Oxidative stress and mitochondrial dysfunction caused by reactive oxygen species (ROS) may induce the inflammation [6]. The inflammation is connected with many neurological and neurodegenerative diseases, such as Parkinson’s disease, Alzheimer’s disease, multiple sclerosis, and other nervous pathogenesis [7].Rhaponticum carthamoides (Willd.) Iljin (Asteraceae) has been used for centuries in traditional Siberian medicine in the case of overstrain and weakness after illness [8]. The plant has demonstrated several biological activities, including adaptogenic, antioxidant, anticancer, and antimicrobial effects [8–10]. The raw material ofR. carthamoides comprises roots and rhizomes of three- to four-year-old plants which are used in various dietary supplements or nutraceutical preparations. The collection of raw material from natural habitat or cultivation is not effective because it requires the destruction of the whole plants [11]. An alternative may be the biotechnological approaches such as the hairy root cultures obtained byAgrobacterium rhizogenes transformation. Hairy root cultures are characterized by rapid increase of biomass in a short time (4–6 weeks) and genetic stability. The level of secondary metabolites in these cultures may be comparable to or higher than in roots of plants growing naturally [12]. In our previous study we obtained hairy roots ofR. carthamoides and evaluated their phytochemical profile in methanol extract [11].As no description currently exists of the chemical composition ofRhaponticum carthamoides essential oil from hairy roots (HR), the aim of the present study was to determine this. The anti-inflammatory and antioxidant activity of this essential oil in lipopolysaccharide- (LPS-) stimulated astrocytes and its antimicrobial potential were also examined. As the comparative material for phytochemical and biological studies essential oil from the roots of soil-grown plants (SGR) was used.


## 2. Material and Methods

### 2.1. Plant Materials

Hairy roots (HR) and the roots of soil-grown plants (SGR) ofR. carthamoideswere used as the materials. The transformation procedure, the establishment of hairy root cultures, and the obtainment of the roots of soil-grown plants have been described in our previous study [11]. The hairy roots were grown in liquid Woody Plant medium [13] on a rotary shaker at 80 rpm, under a 16/8 h light/dark photoperiod with a light intensity of 40 μmol m−2 s−1.


### 2.2. Isolation and GC-MS Analysis of Essential Oils

The essential oils of air-dried hairy roots and roots of soil-grown plants (about 50 g of each) were obtained by hydrodistillation, using a Clevenger-type apparatus for 5 h. Chemical analysis of essential oils composition was performed by GC-MS method using a Trace GC Ultra apparatus (Thermo Electron Corporation, Milan, Italy) with FID and MS DSQ II detectors and MS-FID splitter (SGE, Analytical Science). The analysis conditions (column, temperatures, etc.) were as previously reported [14]. The identification of the compounds was based on the comparison of their RIs and MS spectra with those stored in the NIST 98.1, Wiley 8th Ed., and MassFinder 4.1. computer libraries as well as literature data [15–17].


### 2.3. Antibacterial and Antifungal Activity

#### 2.3.1. Microorganisms Strains and Growth Conditions

Two Gram-positive bacteria species (Staphylococcus aureusATCC 25923 andEnterococcus faecalis ATCC 29212), two Gram-negative bacteria (Escherichia coli ATCC 25922 andPseudomonas aeruginosa ATCC 27853), and two yeasts (Saccharomyces cerevisiaeATCC 2601 andCandida albicans ATCC 10231) were used in this study. All bacteria were cultured at 37°C in Mueller-Hinton (Biokar Diagnostics, Beauvais, France) and the yeasts in Sabouraud dextrose agar (Difco).


#### 2.3.2. Determination of Minimum Inhibitory Concentration (MIC), Minimum Bactericidal Concentration (MBC), and Minimum Fungicidal Concentration (MFC)

The Minimum Inhibitory Concentration (MIC) of HR and SGR essential oils was evaluated by means of the twofold serial microdilution assay [18]. The essential oils dissolved in DMSO were diluted at concentrations ranging from 500 to 0.48 μg/mL, with Mueller-Hinton broth medium for bacteria and 5000 to 4.88 μg/mL with Sabouraud medium for yeasts. The antimicrobial activity of the solvent DMSO was also evaluated. Vancomycin (for Gram-positive bacteria), norfloxacin (for Gram-negative bacteria), and amphotericin B (for yeasts) were used as controls. The MIC values, presented in μg/mL, were considered as the lowest concentration of the essential oils that inhibited the growth of the microorganisms, after 24 h of incubation at 37°C. The microbial growth was measured with an Absorbance Microplate Reader set to 620 nm (Thermo Scientific Multiskan FC). Assays for each tested microorganism were carried out in triplicate.The Minimum Bactericidal Concentration (MBC) or Minimum Fungicidal Concentration (MFC) were evaluated for each set of wells. For the MIC evaluation, a loopful of broth was collected from those wells where any growth was observed and then was further inoculated on sterile Mueller-Hinton medium broth for bacteria and Sabouraud medium for yeasts by streaking. The plates were then inoculated with microorganism and were incubated at 37°C for 24 h. After incubation, the lowest concentration was considered as MBC (for bacteria) and MFC (for yeasts) and presented inμg/mL, at which no visible growth was observed.


### 2.4. Cell Culture of Human Normal Astrocytes and Cytotoxicity of HR and SGR Essential Oils and LPS

#### 2.4.1. Cell Culture of Human Normal Astrocytes

The normal human astrocytes were grown in AGM medium (Lonza, Basel, Switzerland) according to protocol of the manufacturer in atmosphere of 5% CO2, at 37°C.


#### 2.4.2. Cytotoxicity of HR and SGR Essential Oils and LPS

The MTT assay was used to evaluate the cytotoxicity of HR and SGR essential oils and lipopolysaccharide (LPS) (Sigma-Aldrich, St. Louis, MO, USA). The normal human astrocytes were placed in 96-well culture plates (4 × 104 cells per well) and were treated with various concentrations of HR and SGR essential oils (0–3200 μg/mL) or LPS (1 μg/mL) for 24 h. As a control was used the cells incubated in the culture medium, for 24 h. The MTT assay was carried out according to the previous study [19]. All experiments were repeated three times.


### 2.5. Anti-Inflammatory Assay

#### 2.5.1. The Level Measurement of Pro- and Anti-Inflammatory Cytokines by Using ELISA Kit

The Multi-Analyte ELISArray Kits (Qiagen, Valencia, CA, USA) were used to estimate the level of IL-1β, IL-6, IL-8, IL-10, TNF-α, and GM-CSF in the supernatant of astrocytes incubated for 24 h with HR and SGR essential oils (25, 50, and 100 μg/mL) and LPS (1 μg/mL), only LPS (1 μg/mL), or only 25, 50, and 100 μg/mL of HR and SGR essential oils, according to protocol of the manufacturer. The cells incubated in the culture medium for 24 h were used as a control. The results were expressed as mean absorbance/OD (450 nm) value and were performed in triplicate.


#### 2.5.2. RT-PCR Analysis of Cytokines

The reverse transcription-polymerase chain reaction (RT-PCR) was used to determine cytokine expressions. The human astrocytes (4 × 105) were seeded in 6-well multidishes and were treated with 100 μg/mL of HR and SGR essential oils and LPS (1 μg/mL), only LPS (1 μg/mL), or only 100 μg/mL of HR and SGR essential oils for 24 h, at 37°C, 5% CO2. The control cells were incubated in the culture medium for 24 h. RNA was isolated using an RNA isolation kit (Blirt, Poland) according to the manufacturer’s protocol. The Reverse Transcription System (A&A Biotechnology, Poland) was used for cDNA synthesis according to the instruction of the manufacturer. PCR was carried out using the gene-specific primers for human (β-actin, IL-1β, IL-6, IL-8, IL-10, GM-CSF, and TNF-α). The products lengths were 509 bp, 93 bp, 296 bp, 425 bp, 476 bp, 292 bp, and 406 bp for β-actin, IL-1β, IL-6, IL-8, IL-10, GM-CSF, and TNF-α, respectively. The PCR conditions were as follows: initial denaturation at 95°C for 3 min, denaturation at 94°C for 1 min, annealing at 55–65°C for 1 min, and extension at 72°C for 2 min. PCR products were separated on a 1.5% agarose gel and stained with ethidium bromide. Further procedures are described in a previous study by Sitarek et al. [20]. The experiments were repeated three times.


### 2.6. ROS Formation Assay

The intracellular reactive oxygen species (ROS) levels were evaluated using 2′,7′-dichlorodihydrofluorescein diacetate (H2DCF-DA). The procedure was performed according to the previous study [21]. The astrocytes were seeded in 96-well plate, incubated only with LPS (1 μg/mL) or stimulated with LPS (1 μg/mL), and treated with HR and SGR essential oils (100 μg/mL) for 24 hours. Then, the cells were lysed, centrifuged at 10,000 ×g for 10 min, and incubated with 5 μM H2DCF-DA at 37°C for 30 min. ROS fluorescence (DCF) was measured in microplate reader with excitation at 485 nm and emission at 520 nm. The experiments were repeated three times.


### 2.7. Statistical Analysis

The values in this study are expressed as mean ± SD. The Shapiro-Wilk test was used for verification of the normality of the data. The Kruskal-Wallis test with multiple comparisons of average ranks was used to determine differences between samples with a nonnormal distribution. The one-way analysis of variance (ANOVA) and the subsequent Tukey post hoc test were used to evaluate the differences between samples with a normal distribution. The results were analyzed using STATISTICA 12.0 software (StatSoft, Poland). Differences ofp<0.05 were considered statistically significant.


## 2.1. Plant Materials

Hairy roots (HR) and the roots of soil-grown plants (SGR) ofR. carthamoideswere used as the materials. The transformation procedure, the establishment of hairy root cultures, and the obtainment of the roots of soil-grown plants have been described in our previous study [11]. The hairy roots were grown in liquid Woody Plant medium [13] on a rotary shaker at 80 rpm, under a 16/8 h light/dark photoperiod with a light intensity of 40 μmol m−2 s−1.


## 2.2. Isolation and GC-MS Analysis of Essential Oils

The essential oils of air-dried hairy roots and roots of soil-grown plants (about 50 g of each) were obtained by hydrodistillation, using a Clevenger-type apparatus for 5 h. Chemical analysis of essential oils composition was performed by GC-MS method using a Trace GC Ultra apparatus (Thermo Electron Corporation, Milan, Italy) with FID and MS DSQ II detectors and MS-FID splitter (SGE, Analytical Science). The analysis conditions (column, temperatures, etc.) were as previously reported [14]. The identification of the compounds was based on the comparison of their RIs and MS spectra with those stored in the NIST 98.1, Wiley 8th Ed., and MassFinder 4.1. computer libraries as well as literature data [15–17].


## 2.3. Antibacterial and Antifungal Activity

### 2.3.1. Microorganisms Strains and Growth Conditions

Two Gram-positive bacteria species (Staphylococcus aureusATCC 25923 andEnterococcus faecalis ATCC 29212), two Gram-negative bacteria (Escherichia coli ATCC 25922 andPseudomonas aeruginosa ATCC 27853), and two yeasts (Saccharomyces cerevisiaeATCC 2601 andCandida albicans ATCC 10231) were used in this study. All bacteria were cultured at 37°C in Mueller-Hinton (Biokar Diagnostics, Beauvais, France) and the yeasts in Sabouraud dextrose agar (Difco).


### 2.3.2. Determination of Minimum Inhibitory Concentration (MIC), Minimum Bactericidal Concentration (MBC), and Minimum Fungicidal Concentration (MFC)

The Minimum Inhibitory Concentration (MIC) of HR and SGR essential oils was evaluated by means of the twofold serial microdilution assay [18]. The essential oils dissolved in DMSO were diluted at concentrations ranging from 500 to 0.48 μg/mL, with Mueller-Hinton broth medium for bacteria and 5000 to 4.88 μg/mL with Sabouraud medium for yeasts. The antimicrobial activity of the solvent DMSO was also evaluated. Vancomycin (for Gram-positive bacteria), norfloxacin (for Gram-negative bacteria), and amphotericin B (for yeasts) were used as controls. The MIC values, presented in μg/mL, were considered as the lowest concentration of the essential oils that inhibited the growth of the microorganisms, after 24 h of incubation at 37°C. The microbial growth was measured with an Absorbance Microplate Reader set to 620 nm (Thermo Scientific Multiskan FC). Assays for each tested microorganism were carried out in triplicate.The Minimum Bactericidal Concentration (MBC) or Minimum Fungicidal Concentration (MFC) were evaluated for each set of wells. For the MIC evaluation, a loopful of broth was collected from those wells where any growth was observed and then was further inoculated on sterile Mueller-Hinton medium broth for bacteria and Sabouraud medium for yeasts by streaking. The plates were then inoculated with microorganism and were incubated at 37°C for 24 h. After incubation, the lowest concentration was considered as MBC (for bacteria) and MFC (for yeasts) and presented inμg/mL, at which no visible growth was observed.


## 2.3.1. Microorganisms Strains and Growth Conditions

Two Gram-positive bacteria species (Staphylococcus aureusATCC 25923 andEnterococcus faecalis ATCC 29212), two Gram-negative bacteria (Escherichia coli ATCC 25922 andPseudomonas aeruginosa ATCC 27853), and two yeasts (Saccharomyces cerevisiaeATCC 2601 andCandida albicans ATCC 10231) were used in this study. All bacteria were cultured at 37°C in Mueller-Hinton (Biokar Diagnostics, Beauvais, France) and the yeasts in Sabouraud dextrose agar (Difco).


## 2.3.2. Determination of Minimum Inhibitory Concentration (MIC), Minimum Bactericidal Concentration (MBC), and Minimum Fungicidal Concentration (MFC)

The Minimum Inhibitory Concentration (MIC) of HR and SGR essential oils was evaluated by means of the twofold serial microdilution assay [18]. The essential oils dissolved in DMSO were diluted at concentrations ranging from 500 to 0.48 μg/mL, with Mueller-Hinton broth medium for bacteria and 5000 to 4.88 μg/mL with Sabouraud medium for yeasts. The antimicrobial activity of the solvent DMSO was also evaluated. Vancomycin (for Gram-positive bacteria), norfloxacin (for Gram-negative bacteria), and amphotericin B (for yeasts) were used as controls. The MIC values, presented in μg/mL, were considered as the lowest concentration of the essential oils that inhibited the growth of the microorganisms, after 24 h of incubation at 37°C. The microbial growth was measured with an Absorbance Microplate Reader set to 620 nm (Thermo Scientific Multiskan FC). Assays for each tested microorganism were carried out in triplicate.The Minimum Bactericidal Concentration (MBC) or Minimum Fungicidal Concentration (MFC) were evaluated for each set of wells. For the MIC evaluation, a loopful of broth was collected from those wells where any growth was observed and then was further inoculated on sterile Mueller-Hinton medium broth for bacteria and Sabouraud medium for yeasts by streaking. The plates were then inoculated with microorganism and were incubated at 37°C for 24 h. After incubation, the lowest concentration was considered as MBC (for bacteria) and MFC (for yeasts) and presented inμg/mL, at which no visible growth was observed.


## 2.4. Cell Culture of Human Normal Astrocytes and Cytotoxicity of HR and SGR Essential Oils and LPS

### 2.4.1. Cell Culture of Human Normal Astrocytes

The normal human astrocytes were grown in AGM medium (Lonza, Basel, Switzerland) according to protocol of the manufacturer in atmosphere of 5% CO2, at 37°C.


### 2.4.2. Cytotoxicity of HR and SGR Essential Oils and LPS

The MTT assay was used to evaluate the cytotoxicity of HR and SGR essential oils and lipopolysaccharide (LPS) (Sigma-Aldrich, St. Louis, MO, USA). The normal human astrocytes were placed in 96-well culture plates (4 × 104 cells per well) and were treated with various concentrations of HR and SGR essential oils (0–3200 μg/mL) or LPS (1 μg/mL) for 24 h. As a control was used the cells incubated in the culture medium, for 24 h. The MTT assay was carried out according to the previous study [19]. All experiments were repeated three times.


## 2.4.1. Cell Culture of Human Normal Astrocytes

The normal human astrocytes were grown in AGM medium (Lonza, Basel, Switzerland) according to protocol of the manufacturer in atmosphere of 5% CO2, at 37°C.


## 2.4.2. Cytotoxicity of HR and SGR Essential Oils and LPS

The MTT assay was used to evaluate the cytotoxicity of HR and SGR essential oils and lipopolysaccharide (LPS) (Sigma-Aldrich, St. Louis, MO, USA). The normal human astrocytes were placed in 96-well culture plates (4 × 104 cells per well) and were treated with various concentrations of HR and SGR essential oils (0–3200 μg/mL) or LPS (1 μg/mL) for 24 h. As a control was used the cells incubated in the culture medium, for 24 h. The MTT assay was carried out according to the previous study [19]. All experiments were repeated three times.


## 2.5. Anti-Inflammatory Assay

### 2.5.1. The Level Measurement of Pro- and Anti-Inflammatory Cytokines by Using ELISA Kit

The Multi-Analyte ELISArray Kits (Qiagen, Valencia, CA, USA) were used to estimate the level of IL-1β, IL-6, IL-8, IL-10, TNF-α, and GM-CSF in the supernatant of astrocytes incubated for 24 h with HR and SGR essential oils (25, 50, and 100 μg/mL) and LPS (1 μg/mL), only LPS (1 μg/mL), or only 25, 50, and 100 μg/mL of HR and SGR essential oils, according to protocol of the manufacturer. The cells incubated in the culture medium for 24 h were used as a control. The results were expressed as mean absorbance/OD (450 nm) value and were performed in triplicate.


### 2.5.2. RT-PCR Analysis of Cytokines

The reverse transcription-polymerase chain reaction (RT-PCR) was used to determine cytokine expressions. The human astrocytes (4 × 105) were seeded in 6-well multidishes and were treated with 100 μg/mL of HR and SGR essential oils and LPS (1 μg/mL), only LPS (1 μg/mL), or only 100 μg/mL of HR and SGR essential oils for 24 h, at 37°C, 5% CO2. The control cells were incubated in the culture medium for 24 h. RNA was isolated using an RNA isolation kit (Blirt, Poland) according to the manufacturer’s protocol. The Reverse Transcription System (A&A Biotechnology, Poland) was used for cDNA synthesis according to the instruction of the manufacturer. PCR was carried out using the gene-specific primers for human (β-actin, IL-1β, IL-6, IL-8, IL-10, GM-CSF, and TNF-α). The products lengths were 509 bp, 93 bp, 296 bp, 425 bp, 476 bp, 292 bp, and 406 bp for β-actin, IL-1β, IL-6, IL-8, IL-10, GM-CSF, and TNF-α, respectively. The PCR conditions were as follows: initial denaturation at 95°C for 3 min, denaturation at 94°C for 1 min, annealing at 55–65°C for 1 min, and extension at 72°C for 2 min. PCR products were separated on a 1.5% agarose gel and stained with ethidium bromide. Further procedures are described in a previous study by Sitarek et al. [20]. The experiments were repeated three times.


## 2.5.1. The Level Measurement of Pro- and Anti-Inflammatory Cytokines by Using ELISA Kit

The Multi-Analyte ELISArray Kits (Qiagen, Valencia, CA, USA) were used to estimate the level of IL-1β, IL-6, IL-8, IL-10, TNF-α, and GM-CSF in the supernatant of astrocytes incubated for 24 h with HR and SGR essential oils (25, 50, and 100 μg/mL) and LPS (1 μg/mL), only LPS (1 μg/mL), or only 25, 50, and 100 μg/mL of HR and SGR essential oils, according to protocol of the manufacturer. The cells incubated in the culture medium for 24 h were used as a control. The results were expressed as mean absorbance/OD (450 nm) value and were performed in triplicate.


## 2.5.2. RT-PCR Analysis of Cytokines

The reverse transcription-polymerase chain reaction (RT-PCR) was used to determine cytokine expressions. The human astrocytes (4 × 105) were seeded in 6-well multidishes and were treated with 100 μg/mL of HR and SGR essential oils and LPS (1 μg/mL), only LPS (1 μg/mL), or only 100 μg/mL of HR and SGR essential oils for 24 h, at 37°C, 5% CO2. The control cells were incubated in the culture medium for 24 h. RNA was isolated using an RNA isolation kit (Blirt, Poland) according to the manufacturer’s protocol. The Reverse Transcription System (A&A Biotechnology, Poland) was used for cDNA synthesis according to the instruction of the manufacturer. PCR was carried out using the gene-specific primers for human (β-actin, IL-1β, IL-6, IL-8, IL-10, GM-CSF, and TNF-α). The products lengths were 509 bp, 93 bp, 296 bp, 425 bp, 476 bp, 292 bp, and 406 bp for β-actin, IL-1β, IL-6, IL-8, IL-10, GM-CSF, and TNF-α, respectively. The PCR conditions were as follows: initial denaturation at 95°C for 3 min, denaturation at 94°C for 1 min, annealing at 55–65°C for 1 min, and extension at 72°C for 2 min. PCR products were separated on a 1.5% agarose gel and stained with ethidium bromide. Further procedures are described in a previous study by Sitarek et al. [20]. The experiments were repeated three times.


## 2.6. ROS Formation Assay

The intracellular reactive oxygen species (ROS) levels were evaluated using 2′,7′-dichlorodihydrofluorescein diacetate (H2DCF-DA). The procedure was performed according to the previous study [21]. The astrocytes were seeded in 96-well plate, incubated only with LPS (1 μg/mL) or stimulated with LPS (1 μg/mL), and treated with HR and SGR essential oils (100 μg/mL) for 24 hours. Then, the cells were lysed, centrifuged at 10,000 ×g for 10 min, and incubated with 5 μM H2DCF-DA at 37°C for 30 min. ROS fluorescence (DCF) was measured in microplate reader with excitation at 485 nm and emission at 520 nm. The experiments were repeated three times.


## 2.7. Statistical Analysis

The values in this study are expressed as mean ± SD. The Shapiro-Wilk test was used for verification of the normality of the data. The Kruskal-Wallis test with multiple comparisons of average ranks was used to determine differences between samples with a nonnormal distribution. The one-way analysis of variance (ANOVA) and the subsequent Tukey post hoc test were used to evaluate the differences between samples with a normal distribution. The results were analyzed using STATISTICA 12.0 software (StatSoft, Poland). Differences ofp<0.05 were considered statistically significant.


## 3. Results

### 3.1. Qualitative and Quantitative Differences in Composition of Essential Oils fromR. carthamoides Hairy Roots and Roots of Soil-Grown Plants

The essential oil yields ofR. carthamoides hairy roots (HR) and root of soil-grown plants (SGR) were 0.09% and 0.06% (dry weight, v/w), respectively. The essential oils were dark yellow.A comparative analysis of essential oils from roots of soil-grown plants and hairy roots by gas chromatography mass spectrometry (GC-MS) demonstrated that the chemical profiles of both essential oils were different. Table1 presents the chemical constituents of essential oils with their relative percentage amounts and retention indices. In the SGR essential oil, 41 compounds were identified, representing 89.7% of the total oil while 44 compounds were detected in the HR essential oils, amounting to 81.9% of the total identified components (Table 1).Table 1
Percentage of composition of essential oils fromRhaponticum carthamoides roots of soil-grown plants (SGR) and hairy roots (HR).
Peak number Constituent RI lit RI exp SGR [%] HR [%] Class of compound (1) α-Pinene 936 931 0.1 0.1 MH (2) Oct-1-en-3-ol 962 961 — t O (3) 2-Pentylfuran 981 978 — 0.8 O (4) α-Phellandrene 1002 998 0.2 — MH (5) p-Cymene 1015 1010 0.1 t MH (6) β-Phellandrene 1023 1021 t — MH (7) Limonene 1025 1022 0.4 0.6 MH (8) (E)-Oct-2-enal 1034 1032 — t O (9) p-Cymenene 1075 1072 0.8 — MH (10) (E)-Non-2-enal 1139 1136 — 0.1 O (11) p-Cymen-9-ol 1157 1157 2.1 t MO (12) Thymol 1267 1268 — t MO (13) Carvacrol 1278 1277 — t MO (14) (E,E)-Deca-2,4-dienal 1291 1288 — 0.1 O (15) Cyprotene 1322 1318 t 0.1 SH (16) 13-Norcypera-1(5),11(12)-diene 1352 1349 1.4 15.3 SH (17) α-Longipinene 1360 1360 t — SH (18) Cyperadiene 1365 1361 t 0.3 SH (19) Cyclosativene 1370 1368 0.2 0.3 SH (20) α-Copaene 1379 1376 0.6 0.3 SH (21) α-Funebrene 1385 1382 t — SH (22) Petasitene 1398 1398 4.3 — SH (23) β-Elemene 1389 1389 — 0.2 SH (24) Thymol methyl ether 1403 1401 — t MO (25) Cyperene 1404 1404 2.6 18.2 SH (26) Dehydroisolongifolene 1410 1411 0.2 1.8 SH (27) α-Cedrene 1418 1415 0.4 — SH (28) β-Caryophyllene 1420 1418 2.7 0.2 SH (29) trans- α -Bergamotene 1434 1433 4.2 — SH (30) Sesquisabinene A 1435 1435 1.1 — SH (31) β-Helmiscapene 1446 1443 — 0.2 SH (32) α-Helmiscapene 1447 1447 — 0.2 SH (33) (Z)-β-Farnesene 1447 1447 1.3 — SH (34) α-Humulene 1455 1453 T — SH (35) β-Santalene 1457 1455 0.4 — SH (36) Selina-3,7-diene 1460 1458 — 0.3 (37) Rotundene 1461 1459 T 1.6 SH (38) α-Acoradiene 1464 1464 0.4 — SH (39) γ-Gurjunene 1472 1468 — 0.1 SH (40) Selina-4,11-diene 1475 1472 — 0.6 SH (41) Dauca-4(11),8-diene 1530a 1472 10.2 — SH (42) Nardosina-1(10),11-diene 1484 1479 20.7 — SH (43) β-Selinene 1486 1484 — 4.6 SH (44) Pentadec-1-ene 1486 1487 0.5 0.2 O (45) α-Muurolene 1496 1495 1.3 5.5 SH (46) Isorotundene 1503 1497 0.4 1.7 SH (47) β-Bisabolene 1503 1500 0.5 — SH (48) (Z)-γ-Bisabolene 1505 1506 0.7 — SH (49) Premnaspirodiene 1516 1510 — 0.2 SH (50) δ-Cadinene 1520 1514 0.3 0.4 SH (51) Cyperene oxide 1524 1521 — 1.0 SO (52) α-Calacorene 1527 1529 T 0.9 SH (53) (E)-Nerolidol 1553 1545 0.3 — SO (54) β-Caryophyllene oxide 1578 1571 — 1.4 SO (55) α-Corocalene 1602 1602 0.2 2.0 SH (56) Longifolene aldehyde 1631 1620 — 0.3 (57) 2,5,8-Trimethyl-1-naphthol — 1635 1.1 9.6 O (58) β-Himachalol 1638 1634 — 0.6 SO (59) Cadalene 1659 1658 0.6 7.2 SH (60) Aplotaxene 1659 1659 27.3 t O (61) Cyperotundone 1684 1676 0.3 3.6 SO (62) Palmitic acid 1942 1946 — 1.3 O Total identified   89.7 81.9 Monoterpene hydrocarbons MH   1.6 0.7 Oxygenated monoterpenes MO   2.1 t Sesquiterpene hydrocarbons SH   54.7 62.2 Oxygenated sesquiterpenes SO   0.6 6.9 Other O   28.9 12.1 The concentrations of the main compounds were signified in bold. RI: relative retention index on Rtx-1 column, RI lit: relative retention index of literature. t: traces (percentage value less than 0.05%).Sesquiterpenes, especially sesquiterpene hydrocarbons (54.7–62.2%), dominated in both essential oils (Table1). However, in SGR essential oil, aplotaxene (all-cis-heptadeca-1,8,11,14-tetraene) was the main constituent (27.3%). Among the sesquiterpenes, nardosina-1(10),11-diene was identified as the most dominant compound (20.7%) in this essential oil according to its mass spectrum and retention index [15]. The second significant sesquiterpene was dauca-4(11),8-diene (10.2%) whose MS was identical with literature data, and its RI on the Rtx-1 column differed from the RI available on DB-5 column [16]. In addition, petasitene (4.3%) andtrans-α-bergamotene (4.2%) were presented in appreciable amounts in SGR essential oil (Table 1).The HR essential oil was characterized by the high quantities of cyperene (18.2%), 13-norcypera-1(5,11(12)-diene (15.3%), and cadalene (7.2%) (Table1). The concentrations of these components were significantly higher (7-, 11-, and 12-fold, resp.) than those in the SGR essential oil (Table 1). Additionally, the other major constituent of HR essential oil was 2,5,8-trimethyl-1-naphthol (9.6%) which was identified on the basis of MS (Table 1); however, this compound has not yet been found in nature and was probably an artifact. The major compounds identified in SGR essential oil such as nardosina-1(10),11-diene and dauca-4(11),8-diene, petasitene, andtrans-α-bergamotene were not detected in the HR essential oil, and aplotaxene was presented only in traces (Table 1).The HR and SGR essential oils were poor in monoterpenes, both hydrocarbons (0.7–1.6%) and oxygenated (0–2.1%) compounds.


### 3.2. The Higher Antibacterial Activity ofR. carthamoides HR and SGR Essential Oils against Gram-Positive and Gram-Negative Bacteria Than against Yeasts

In this study, the Minimum Inhibitory Concentration (MIC), the Minimum Bactericidal Concentration (MBC), and the Minimum Fungicidal Concentration (MFC) of HR and SGR essential oils were investigated against the Gram-positive bacteria, Gram-negative bacteria, and yeasts. It was found that both essential oils showed similar properties. The essential oils showed stronger antibacterial (MIC = 125–250μg/mL) than antifungal activity (MIC = 625–1250 μg/mL) (Table 2). The highest antibacterial activity with a MIC value of 125 μg/mL was observed againstEnterococcus faecalis andPseudomonas aeruginosa. No significant difference in susceptibility was found between Gram-positive bacteria and Gram-negative bacteria (Table 2). In order to address the bacteriostatic and bactericidal properties of the samples, the MBC values were also evaluated. The MBC values are no more than four times the MIC values observed (Table 2).Table 2
Antibacterial and antifungal activity of essential oils fromRhaponticum carthamoides hairy roots (HR) and roots of soil-grown plants (SGR). The Minimum Inhibitory Concentration (MIC), the Minimum Bactericidal Concentration (MBC), and the Minimum Fungicidal Concentration (MFC) of essential oils were determined using the microdilution assay and are presented in µg/mL.
Essential oil Microorganism HR SGR Positive control MIC MBC/MFC MIC MBC/MFC MIC MBC/MFC Staphylococcus aureus (ATCC 25923) 250 >500 125 >500 7.82a >500a Enterococcus faecalis (ATCC 29212) 125 >500 125 >500 1.95a >500a Pseudomonas aeruginosa (ATCC 27853) 125 >500 125 >500 <0.48b >500b Escherichia coli (ATCC 25922) 250 >500 250 >500 0.98b >500b Saccharomyces cerevisiae (ATCC 2601) 625 2500 1250 >2500 <0.48c >5000c Candida albicans (ATCC 10231) 625 2500 625 >5000 <0.48c >5000c V a n c o m y c i n   a; norfloxacinb; amphotericinc B. Data represent the median values of at least three replicates.


### 3.3.R. carthamoides HR and SGR Essential Oils Did Not Induce Cytotoxic Effect in Astrocytes

The MTT assay was used to determine the cytotoxic effect ofR. carthamoides essential oil from hairy roots and the roots of soil-grown plants (range: 0–3200 μg/mL) after 24 h incubation with astrocytes (Figure 1). The viability of cells was decreased below 60% at the higher concentration of essential oils, above 1600 μg/mL (Figure 1).Figure 1
The cell viability of astrocytes after 24 h treatment with essential oils fromRhaponticum carthamoides hairy roots (HR) and roots of soil-grown plants (SGR). The viability was determined by MTT assay. The values are the mean ± SD of three independent experiments. p∗<0.05 versus control; p#<0.05 HR essential oil versus SGR essential oil.


### 3.4. Decrease of IL-1β, IL-6, and TNF-α Levels in LPS-Stimulated Astrocytes after Treatment ofR. carthamoides HR and SGR Essential Oils

In this study we also evaluated the anti-inflammatory activity of HR and SGR essential oils in LPS-treated astrocytes. The levels of pro- and anti-inflammatory mediators in astrocytes incubated for 24 h with HR and SGR essential oils (25, 50, and 100μg/mL) and LPS (1 μg/mL), only LPS (1 μg/mL), or only 25, 50, and 100 μg/mL of HR and SGR essential oils were measured using the ELISA Kits (Figure 2). LPS treatment of astrocytes led to increased levels of IL-1β, IL-6, IL-10, and TNF-α in comparison to the control cells. Treatment of LPS-stimulated astrocytes with HR and SGR essential oils dose-dependently reduced the levels of proinflammatory cytokines as IL-1β, IL-6, and TNF-α but not IL-10, IL-8, or GM-CSF (Figure 2). In addition, the expression levels of IL-1β, IL-6, IL-8, IL-10, GM-CSF, and TNF-α in LPS-stimulated astrocytes were analyzed by RT-PCR after 24 h incubation with 100 μg/mL of HR and SGR essential oils (Figure 3). A single concentration of essential oils known to be the most effective for reducing of the levels of pro- and anti-inflammatory cytokines, as determined by an ELISA, was used in this experiment. RT-PCR analyses showed also that treatment with HR and SGR essential oils resulted in the downregulation of IL-1β, IL-6, and TNF-α mRNA expression in LPS-induced astrocytes (Figure 3). No change was observed in the mRNA expression of IL-10, IL-8, and GM-CSF in comparison with astrocytes treated with only LPS.Figure 2
Effects ofRhaponticum carthamoides HR and SGR essential oils on IL-1β, IL-6, IL-8, IL-10, TNF-α, and GM-CSF levels in LPS-stimulated astrocytes. The levels of pro- and anti-inflammatory cytokines in LPS-stimulated astrocytes were measured by ELISA Kit. Astrocytes were incubated for 24 h with 25, 50, and 100 μg/mL of HR and SGR essential oils and LPS (1 μg/mL). The data is represented as means ± SD and was obtained from three independent experiments. p∗<0.05 versus LPS-treated cells; p#<0.05 SGR essential oil versus HR essential oil.Figure 3
The effect ofRhaponticum carthamoides HR and SGR essential oils on the expression of the inflammatory-related gene (IL-1β, IL-6, IL-8, IL-10, GM-CSF, and TNF-α) in LPS-stimulated astrocytes. mRNA expression levels were measured by RT-PCR analysis. Astrocytes were incubated for 24 h with 100 μg/mL of HR or SGR essential oils and LPS (1 μg/mL). (a) The bar graph shows the relative density. The data is represented as means ± SD of three independent experiments. p∗<0.05 versus LPS-treated cells. (b)Lanes: 1: control; 2: cell incubated with SGR essential oil; 3: cell incubated with HR essential oil; 4: cell incubated with LPS; 5: cell incubated with SGR essential oil + LPS; 6: cell incubated with HR essential oil + LPS.
(a) (b)


### 3.5. Decrease of ROS Levels in LPS-Stimulated Astrocytes after Treatment ofR. carthamoides HR and SGR Essential Oils

Reactive oxygen species (ROS) levels in LPS-stimulated astrocytes were assessed by 2′,7′-dichlorofluorescein (DCF) fluorescence measurements. Astrocytes were treated with LPS (1μg/mL) and then incubated with HR and SGR essential oils (100 μg/mL) for 24 h. The treatment of astrocytes with LPS induced a significant increase in ROS levels in comparison with the control cells. Our results showed that treatment with HR and SGR essential oils reduced intracellular levels of ROS about 1.5-fold in LPS-stimulated astrocytes in comparison with the cells treated with only LPS (Figure 4).Figure 4
ROS production in LPS-stimulated astrocytes. Astrocytes were induced with LPS (1μg/mL) and then incubated for 24 h with 100 μg/mL ofRhaponticum carthamoides HR and SGR essential oils. Values are given as means ± SD of three independent experiments. p∗<0.05 versus control; p#<0.05 HR essential oil versus SGR essential oil.


## 3.1. Qualitative and Quantitative Differences in Composition of Essential Oils fromR. carthamoides Hairy Roots and Roots of Soil-Grown Plants

The essential oil yields ofR. carthamoides hairy roots (HR) and root of soil-grown plants (SGR) were 0.09% and 0.06% (dry weight, v/w), respectively. The essential oils were dark yellow.A comparative analysis of essential oils from roots of soil-grown plants and hairy roots by gas chromatography mass spectrometry (GC-MS) demonstrated that the chemical profiles of both essential oils were different. Table1 presents the chemical constituents of essential oils with their relative percentage amounts and retention indices. In the SGR essential oil, 41 compounds were identified, representing 89.7% of the total oil while 44 compounds were detected in the HR essential oils, amounting to 81.9% of the total identified components (Table 1).Table 1
Percentage of composition of essential oils fromRhaponticum carthamoides roots of soil-grown plants (SGR) and hairy roots (HR).
Peak number Constituent RI lit RI exp SGR [%] HR [%] Class of compound (1) α-Pinene 936 931 0.1 0.1 MH (2) Oct-1-en-3-ol 962 961 — t O (3) 2-Pentylfuran 981 978 — 0.8 O (4) α-Phellandrene 1002 998 0.2 — MH (5) p-Cymene 1015 1010 0.1 t MH (6) β-Phellandrene 1023 1021 t — MH (7) Limonene 1025 1022 0.4 0.6 MH (8) (E)-Oct-2-enal 1034 1032 — t O (9) p-Cymenene 1075 1072 0.8 — MH (10) (E)-Non-2-enal 1139 1136 — 0.1 O (11) p-Cymen-9-ol 1157 1157 2.1 t MO (12) Thymol 1267 1268 — t MO (13) Carvacrol 1278 1277 — t MO (14) (E,E)-Deca-2,4-dienal 1291 1288 — 0.1 O (15) Cyprotene 1322 1318 t 0.1 SH (16) 13-Norcypera-1(5),11(12)-diene 1352 1349 1.4 15.3 SH (17) α-Longipinene 1360 1360 t — SH (18) Cyperadiene 1365 1361 t 0.3 SH (19) Cyclosativene 1370 1368 0.2 0.3 SH (20) α-Copaene 1379 1376 0.6 0.3 SH (21) α-Funebrene 1385 1382 t — SH (22) Petasitene 1398 1398 4.3 — SH (23) β-Elemene 1389 1389 — 0.2 SH (24) Thymol methyl ether 1403 1401 — t MO (25) Cyperene 1404 1404 2.6 18.2 SH (26) Dehydroisolongifolene 1410 1411 0.2 1.8 SH (27) α-Cedrene 1418 1415 0.4 — SH (28) β-Caryophyllene 1420 1418 2.7 0.2 SH (29) trans- α -Bergamotene 1434 1433 4.2 — SH (30) Sesquisabinene A 1435 1435 1.1 — SH (31) β-Helmiscapene 1446 1443 — 0.2 SH (32) α-Helmiscapene 1447 1447 — 0.2 SH (33) (Z)-β-Farnesene 1447 1447 1.3 — SH (34) α-Humulene 1455 1453 T — SH (35) β-Santalene 1457 1455 0.4 — SH (36) Selina-3,7-diene 1460 1458 — 0.3 (37) Rotundene 1461 1459 T 1.6 SH (38) α-Acoradiene 1464 1464 0.4 — SH (39) γ-Gurjunene 1472 1468 — 0.1 SH (40) Selina-4,11-diene 1475 1472 — 0.6 SH (41) Dauca-4(11),8-diene 1530a 1472 10.2 — SH (42) Nardosina-1(10),11-diene 1484 1479 20.7 — SH (43) β-Selinene 1486 1484 — 4.6 SH (44) Pentadec-1-ene 1486 1487 0.5 0.2 O (45) α-Muurolene 1496 1495 1.3 5.5 SH (46) Isorotundene 1503 1497 0.4 1.7 SH (47) β-Bisabolene 1503 1500 0.5 — SH (48) (Z)-γ-Bisabolene 1505 1506 0.7 — SH (49) Premnaspirodiene 1516 1510 — 0.2 SH (50) δ-Cadinene 1520 1514 0.3 0.4 SH (51) Cyperene oxide 1524 1521 — 1.0 SO (52) α-Calacorene 1527 1529 T 0.9 SH (53) (E)-Nerolidol 1553 1545 0.3 — SO (54) β-Caryophyllene oxide 1578 1571 — 1.4 SO (55) α-Corocalene 1602 1602 0.2 2.0 SH (56) Longifolene aldehyde 1631 1620 — 0.3 (57) 2,5,8-Trimethyl-1-naphthol — 1635 1.1 9.6 O (58) β-Himachalol 1638 1634 — 0.6 SO (59) Cadalene 1659 1658 0.6 7.2 SH (60) Aplotaxene 1659 1659 27.3 t O (61) Cyperotundone 1684 1676 0.3 3.6 SO (62) Palmitic acid 1942 1946 — 1.3 O Total identified   89.7 81.9 Monoterpene hydrocarbons MH   1.6 0.7 Oxygenated monoterpenes MO   2.1 t Sesquiterpene hydrocarbons SH   54.7 62.2 Oxygenated sesquiterpenes SO   0.6 6.9 Other O   28.9 12.1 The concentrations of the main compounds were signified in bold. RI: relative retention index on Rtx-1 column, RI lit: relative retention index of literature. t: traces (percentage value less than 0.05%).Sesquiterpenes, especially sesquiterpene hydrocarbons (54.7–62.2%), dominated in both essential oils (Table1). However, in SGR essential oil, aplotaxene (all-cis-heptadeca-1,8,11,14-tetraene) was the main constituent (27.3%). Among the sesquiterpenes, nardosina-1(10),11-diene was identified as the most dominant compound (20.7%) in this essential oil according to its mass spectrum and retention index [15]. The second significant sesquiterpene was dauca-4(11),8-diene (10.2%) whose MS was identical with literature data, and its RI on the Rtx-1 column differed from the RI available on DB-5 column [16]. In addition, petasitene (4.3%) andtrans-α-bergamotene (4.2%) were presented in appreciable amounts in SGR essential oil (Table 1).The HR essential oil was characterized by the high quantities of cyperene (18.2%), 13-norcypera-1(5,11(12)-diene (15.3%), and cadalene (7.2%) (Table1). The concentrations of these components were significantly higher (7-, 11-, and 12-fold, resp.) than those in the SGR essential oil (Table 1). Additionally, the other major constituent of HR essential oil was 2,5,8-trimethyl-1-naphthol (9.6%) which was identified on the basis of MS (Table 1); however, this compound has not yet been found in nature and was probably an artifact. The major compounds identified in SGR essential oil such as nardosina-1(10),11-diene and dauca-4(11),8-diene, petasitene, andtrans-α-bergamotene were not detected in the HR essential oil, and aplotaxene was presented only in traces (Table 1).The HR and SGR essential oils were poor in monoterpenes, both hydrocarbons (0.7–1.6%) and oxygenated (0–2.1%) compounds.


## 3.2. The Higher Antibacterial Activity ofR. carthamoides HR and SGR Essential Oils against Gram-Positive and Gram-Negative Bacteria Than against Yeasts

In this study, the Minimum Inhibitory Concentration (MIC), the Minimum Bactericidal Concentration (MBC), and the Minimum Fungicidal Concentration (MFC) of HR and SGR essential oils were investigated against the Gram-positive bacteria, Gram-negative bacteria, and yeasts. It was found that both essential oils showed similar properties. The essential oils showed stronger antibacterial (MIC = 125–250μg/mL) than antifungal activity (MIC = 625–1250 μg/mL) (Table 2). The highest antibacterial activity with a MIC value of 125 μg/mL was observed againstEnterococcus faecalis andPseudomonas aeruginosa. No significant difference in susceptibility was found between Gram-positive bacteria and Gram-negative bacteria (Table 2). In order to address the bacteriostatic and bactericidal properties of the samples, the MBC values were also evaluated. The MBC values are no more than four times the MIC values observed (Table 2).Table 2
Antibacterial and antifungal activity of essential oils fromRhaponticum carthamoides hairy roots (HR) and roots of soil-grown plants (SGR). The Minimum Inhibitory Concentration (MIC), the Minimum Bactericidal Concentration (MBC), and the Minimum Fungicidal Concentration (MFC) of essential oils were determined using the microdilution assay and are presented in µg/mL.
Essential oil Microorganism HR SGR Positive control MIC MBC/MFC MIC MBC/MFC MIC MBC/MFC Staphylococcus aureus (ATCC 25923) 250 >500 125 >500 7.82a >500a Enterococcus faecalis (ATCC 29212) 125 >500 125 >500 1.95a >500a Pseudomonas aeruginosa (ATCC 27853) 125 >500 125 >500 <0.48b >500b Escherichia coli (ATCC 25922) 250 >500 250 >500 0.98b >500b Saccharomyces cerevisiae (ATCC 2601) 625 2500 1250 >2500 <0.48c >5000c Candida albicans (ATCC 10231) 625 2500 625 >5000 <0.48c >5000c V a n c o m y c i n   a; norfloxacinb; amphotericinc B. Data represent the median values of at least three replicates.


## 3.3.R. carthamoides HR and SGR Essential Oils Did Not Induce Cytotoxic Effect in Astrocytes

The MTT assay was used to determine the cytotoxic effect ofR. carthamoides essential oil from hairy roots and the roots of soil-grown plants (range: 0–3200 μg/mL) after 24 h incubation with astrocytes (Figure 1). The viability of cells was decreased below 60% at the higher concentration of essential oils, above 1600 μg/mL (Figure 1).Figure 1
The cell viability of astrocytes after 24 h treatment with essential oils fromRhaponticum carthamoides hairy roots (HR) and roots of soil-grown plants (SGR). The viability was determined by MTT assay. The values are the mean ± SD of three independent experiments. p∗<0.05 versus control; p#<0.05 HR essential oil versus SGR essential oil.


## 3.4. Decrease of IL-1β, IL-6, and TNF-α Levels in LPS-Stimulated Astrocytes after Treatment ofR. carthamoides HR and SGR Essential Oils

In this study we also evaluated the anti-inflammatory activity of HR and SGR essential oils in LPS-treated astrocytes. The levels of pro- and anti-inflammatory mediators in astrocytes incubated for 24 h with HR and SGR essential oils (25, 50, and 100μg/mL) and LPS (1 μg/mL), only LPS (1 μg/mL), or only 25, 50, and 100 μg/mL of HR and SGR essential oils were measured using the ELISA Kits (Figure 2). LPS treatment of astrocytes led to increased levels of IL-1β, IL-6, IL-10, and TNF-α in comparison to the control cells. Treatment of LPS-stimulated astrocytes with HR and SGR essential oils dose-dependently reduced the levels of proinflammatory cytokines as IL-1β, IL-6, and TNF-α but not IL-10, IL-8, or GM-CSF (Figure 2). In addition, the expression levels of IL-1β, IL-6, IL-8, IL-10, GM-CSF, and TNF-α in LPS-stimulated astrocytes were analyzed by RT-PCR after 24 h incubation with 100 μg/mL of HR and SGR essential oils (Figure 3). A single concentration of essential oils known to be the most effective for reducing of the levels of pro- and anti-inflammatory cytokines, as determined by an ELISA, was used in this experiment. RT-PCR analyses showed also that treatment with HR and SGR essential oils resulted in the downregulation of IL-1β, IL-6, and TNF-α mRNA expression in LPS-induced astrocytes (Figure 3). No change was observed in the mRNA expression of IL-10, IL-8, and GM-CSF in comparison with astrocytes treated with only LPS.Figure 2
Effects ofRhaponticum carthamoides HR and SGR essential oils on IL-1β, IL-6, IL-8, IL-10, TNF-α, and GM-CSF levels in LPS-stimulated astrocytes. The levels of pro- and anti-inflammatory cytokines in LPS-stimulated astrocytes were measured by ELISA Kit. Astrocytes were incubated for 24 h with 25, 50, and 100 μg/mL of HR and SGR essential oils and LPS (1 μg/mL). The data is represented as means ± SD and was obtained from three independent experiments. p∗<0.05 versus LPS-treated cells; p#<0.05 SGR essential oil versus HR essential oil.Figure 3
The effect ofRhaponticum carthamoides HR and SGR essential oils on the expression of the inflammatory-related gene (IL-1β, IL-6, IL-8, IL-10, GM-CSF, and TNF-α) in LPS-stimulated astrocytes. mRNA expression levels were measured by RT-PCR analysis. Astrocytes were incubated for 24 h with 100 μg/mL of HR or SGR essential oils and LPS (1 μg/mL). (a) The bar graph shows the relative density. The data is represented as means ± SD of three independent experiments. p∗<0.05 versus LPS-treated cells. (b)Lanes: 1: control; 2: cell incubated with SGR essential oil; 3: cell incubated with HR essential oil; 4: cell incubated with LPS; 5: cell incubated with SGR essential oil + LPS; 6: cell incubated with HR essential oil + LPS.
(a) (b)


## 3.5. Decrease of ROS Levels in LPS-Stimulated Astrocytes after Treatment ofR. carthamoides HR and SGR Essential Oils

Reactive oxygen species (ROS) levels in LPS-stimulated astrocytes were assessed by 2′,7′-dichlorofluorescein (DCF) fluorescence measurements. Astrocytes were treated with LPS (1μg/mL) and then incubated with HR and SGR essential oils (100 μg/mL) for 24 h. The treatment of astrocytes with LPS induced a significant increase in ROS levels in comparison with the control cells. Our results showed that treatment with HR and SGR essential oils reduced intracellular levels of ROS about 1.5-fold in LPS-stimulated astrocytes in comparison with the cells treated with only LPS (Figure 4).Figure 4
ROS production in LPS-stimulated astrocytes. Astrocytes were induced with LPS (1μg/mL) and then incubated for 24 h with 100 μg/mL ofRhaponticum carthamoides HR and SGR essential oils. Values are given as means ± SD of three independent experiments. p∗<0.05 versus control; p#<0.05 HR essential oil versus SGR essential oil.


## 4. Discussion

The aim of this study was to identify and quantitatively determine the constituents in the essential oils ofRhaponticum carthamoides hairy roots (HR) and roots of soil-grown plants (SGR), to examine the anti-inflammatory and antioxidant activity of these essential oils in LPS-stimulated astrocytes, and to determine the antimicrobial potential of HR and SGR essential oils. This is the first report that describes the composition of HR essential oil and its biological potential. Some essential oil possesses many biological properties with antioxidant, antimicrobial, and anti-inflammatory activities [1, 2]. Because the essential oils are lipophilic they may easily enter to cells, disrupt the membrane, and/or make it permeable [22].The essential oil yield fromR. carthamoides HR was higher than those from SGR, 0.09% and 0.06%, respectively. The oil yields were higher than those obtained previously from roots and rhizomes ofR. carthamoides from the Czech Republic (0.04%) [17] and similar to those from the roots and rhizomes of plants growing in Poland (0.07–0.11%) [23]. In the present study, it was found that the essential oils from hairy roots and roots of soil-grown plants ofR. carthamoides differed significantly in their compositions. Qualitative and quantitative differences have also been observed between essential oils compositions from hairy roots and parent plant roots for other plant species [24, 25].In theR. carthamoides SGR and HR essential oils studied in this report, sesquiterpene hydrocarbons dominated, 54.7 and 62.2%, respectively. The sesquiterpene hydrocarbons were also the major group of the components inR. carthamoides essential oil obtained by Havlik et al. [17]. In contrast, Geszprych and Weglarz [23] found that oils from the roots and rhizomes of this species growing in Poland were abundant in monoterpenes. The SGR and HR essential oils in the present study were poor in both hydrocarbons (0.7–1.6%) and oxygenated (0–2.1%) compounds.The major compounds of sesquiterpene hydrocarbons in HR essential oil were cyperene (18%), 13-norcypera-1(5),11(12)-diene (15%), and cadalene (7%). The levels of these components were significantly higher than those evaluated in the SGR essential oil. Interestingly, Havlik et al. [17] found similar results for essential oil isolated fromR. carthamoides roots and rhizomes of commercial material and also showed that 13-norcypera-1(5),11(12)-diene (22.6%) and cyperene (17.9%) were the major compounds in this essential oil. Cadalene was identified for the first time inR. carthamoides root essential oil in this work.InR. carthamoides essential oil from roots of soil-grown plants aplotaxene (27%), nardosina-1(10),11-diene (21%), dauca-4(11),8-diene (10%), petasitene (4.3%), andtrans-α-bergamotene (4.2%) were the major components which were not detected in the hairy roots essential oil. Aplotaxene has also previously been found to dominate in essential oil fromR. carthamoides roots and rhizomes by Havlik et al. [17]. In addition, nardosina-1(10),11-diene and dauca-4(11),8-diene, petasitene, andtrans-α-bergamotene were found for the first time in theRhaponticum carthamoides root essential oil in the present study.In contrast, Geszprych and Weglarz [23] found geraniol (17–18.3%), β-caryophyllene (14.12–16.53%), and linalool (8.88–12.07%) to be main compounds in the essential oil of roots and rhizomes ofR. carthamoides. However, geraniol and linalool were not identified in the HR and SGR essential oils examined in the present work. The differences between the chemical compositions of the essential oils obtained in this study and those of others may be attributable to environmental factors (geographical, climatic, and seasonal), development stage, genetic variability, and the conditions of in vitro cultures [24–27]. Additionally, individual variability and chemotype exist within the same plant species, resulting in the differences in chemical compositions of the raw materials [28].Nevertheless, although qualitative and quantitative differences were found between theR. carthamoides SGR and HR essential oils, the biological activities, that is, the antimicrobial and anti-inflammatory properties, of these essential oils were similar. We suspect that this may be due to similar activities of the major components of the SGR and HR essential oils. While it is known that the biological activity of essential oils may be related with the major compounds, their action might be changed by different interaction with the minor components [22]. Perhaps, the activity of the essential oils is also dependent on the constituents ratio.The essential oils from hairy roots and roots of soil-grown plants showed antimicrobial properties with higher antibacterial (MIC = 125–250μg/mL) than antifungal (MIC = 625–1250 μg/mL) activities. The most susceptible bacteria strains wereEnterococcus faecalis(ATCC 29212) andPseudomonas aeruginosa(ATCC 27853) with a MIC value of 125 μg/mL. However, no significant difference in susceptibility between Gram-positive bacteria and Gram-negative bacteria was found, although it is known that the latter are less sensitive to essential oils because their outer membrane contains hydrophilic lipopolysaccharides (LPS). LPS inhibits the penetration of membrane by the different hydrophobic compounds of the essential oils [5, 22]. Essential oils and their hydrophobic components may disrupt the bacterial cell membranes by separating the lipids leading to inhibition of the functional properties of the bacterial cell [3, 22]. The strong antimicrobial activity (MIC = 32–256 μg/mL) also showedR. carthamoidesroot and rhizomes essential oil from commercial available material [17] but with lower activity againstE. faecalis(ATCC 29212) (MIC = 265 μg/mL) andP. aeruginosa(ATCC 27853) (MIC>512 μg/mL) than the root essential oils in the present study (MIC = 125 μg/mL). A pure compound is usually used as a bactericide if the MBC is no more than four times the MIC value [29] which was observed forR. carthamoides HR and SGR essential oils. Therefore, these essential oils may be effective as bactericidal agents.The antibacterial activity ofR. carthamoides root essential oils may be associated with the presence of the major components of essential oils such as cyperene, 13-norcypera-1(5),11(12)-diene, and cadalene in the HR essential oil and aplotaxene, nardosina-1(10),11-diene, and dauca-4(11),8-diene in the SGR essential oil. To the best of our knowledge, the microbiological activities of these compounds have not been investigated. In literature, we only found that essential oils fromCyperus rotundus andC. articulatuscontaining cyperene as the main component showed antimicrobial effects against different strains of bacteria and fungi [30, 31].The study also evaluates the anti-inflammatory activity ofR. carthamoides HR and SGR essential oils in LPS-induced astrocytes. Astrocytes are multifunctional glial cells in the central nervous system and can regulate the extracellular ion and neurotransmitter concentrations, control the release of antioxidant defences, and are associated with the immune response by the production of inflammatory mediators such as pro- and anti-inflammatory cytokines and chemokines, prostaglandin E, and nitric oxide [32–34]. The inflammation causes neuronal death and brain injury. The activation of astrocytes leads to the release of neutrophic factors which regulate the survival and function of nervous tissue [32]. Hence, the astrocytes are good cellular models in in vitro study to determine the potential anti-inflammatory effects of various agents, such as essential oils. TheR. carthamoides HR and SGR essential oils did not inhibit the viability of astrocytes at concentrations between 0 μg/mL and 800 μg/mL.Lipopolysaccharide (LPS) is the main component of the outer membrane of Gram-negative bacteria and is known as an inductor of inflammation in various cells, including astrocytes [33]. LPS is recognized by Toll-like receptor 4 (TLR4) of astrocytes and the LPS treatment of astrocytes resulting in activation of transcription factor (NF-κB) and mitogen-activated protein kinase (MAPK) which lead to the release of such inflammatory mediators as pro- and anti-inflammatory cytokines or the tumor necrosis factor (TNF-α) or the overproduction of reactive oxygen species (ROS) [33]. Oxidative stress and inflammation are connected with many neurodegenerative diseases, such as Parkinson’s disease, Alzheimer’s disease, multiple sclerosis, and other nervous pathogeneses [7]. Many studies have previously reported LPS induction of various inflammatory mediators in astrocytes [32, 33, 35].Many studies have also indicated that natural plant secondary metabolites may be used for the prevention of inflammation and oxidative damage-related neurodegenerative diseases [33, 35, 36]. In order to determine the anti-inflammatory effects of essential oils fromR. carthamoides hairy roots and roots of soil-grown plants, astrocytes were treated with HR and SGR essential oils and stimulated by LPS for 24 h. It was found that HR and SGR essential oils possessed anti-neuroinflammatory activity and were able to suppress the levels of IL-1β, IL-6, and TNF-α in LPS-stimulated astrocytes. In response to inflammation molecular signaling cascades are activated in astrocytes. IL-1β and TNF-α are secreted during acute inflammation and are important for the production of IL-6 [33]. However, the stimulation of IL-1β causes astrocytes to generate and release vascular endothelial growth factor, which increases blood-brain barrier permeability and promotes leukocyte extravasation [34]. IL-10, the anti-inflammatory cytokine, reduces of TNF-α level and shows neuroprotective effect by changes of intracellular calcium and mediates delay effectsvia NF-κB [33]. In addition, in response to IL-10, astrocytes release transforming growth factor-β (TGFβ) that suppresses the inflammation [34].Although the chemical profile of bothR. carthamoides root essential oils differed, these oils showed similar anti-inflammatory activity. The anti-inflammatory effects of major compounds of HR and SGR essential oils have not yet been reported. Only, aplotaxene which was the dominant compound in SGR essential oil demonstrated an immunosuppressive effect by the inhibition of T cell activationvia the reduction of IL-2 and CD69 expression. However, aplotaxene inhibited MAPK activation and the transcriptional activities of nuclear factor of activated T cells (NF-AT), NF-κB, and activator protein 1 (AP-1) [37].R. carthamoides HR and SGR essential oils were able to protect astrocytes against oxidative stress by the reduction of intracellular ROS levels in LPS-stimulated cells. ROS production is activated by the NADPH oxidase (NOX) [38] and by cyclooxygenase-2 (COX-2) as a side-product in the synthesis of prostanoids [35]. It is known from literature that ROS can activate various signaling molecules such as protein kinase C, MAPK, and NF-κB which play an important role in regulating the gene expression of various proinflammatory factors [39]. The overproduction of ROS may lead to an increase of TNF-α level [33]. This highlights the relationship between inflammation and oxidative stress.


## 5. Conclusions

This is the first report that describes the chemical composition ofRhaponticum carthamoides essential oil from hairy roots, its antimicrobial potential, and the protective effect of this essential oil against LPS-induced inflammation and ROS production in astrocytes. The results show thatR. carthamoides hairy roots may be used as a valuable source of essential oil and may represent an alternative to the roots of soil-grown plants. Our study suggests thatR. carthamoides essential oil from hairy roots may be used as an effective natural antibacterial and antioxidant agent as food preservative and for the treatment of some infectious diseases. Moreover, the antineuroinflammatory properties of this essential oil give it potential as a complementary therapy in the prevention of some central nervous system diseases especially neurodegenerative ones. However, further research is needed to isolate the main compounds from hairy roots essential oil and to determine their biological activity.



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