Phytochemical Compositions and Biological Activities of Essential Oil from Xanthium strumarium L.

The chemical composition of the essential oil (EO) from fresh cocklebur (Xanthium strumarium L.) leaves was investigated by GC-MS. The antimicrobial activity of the EO was tested against Gram-positive and Gram-negative bacteria and fungi. Scolicidal activity was assayed against Echinococcus granulosus protoscolices. In total, 34 compounds were identified, accounting for 98.96% of the EO. The main compounds in the EO were cis-β-guaiene (34.2%), limonene (20.3%), borneol (11.6%), bornyl acetate (4.5%), β-cubebene (3.8%), sabinene (3.6%), phytol (3.1%), β-selinene (2.8%), camphene (2.2%), α-cubebene (2.4%), β-caryophyllene (1.9%), α-pinene (1.8%) and xanthinin (1.04%). The antibacterial and antifungal screening of the EO showed that all assayed concentrations significantly inhibited the growth of Staphylococcus aureus, Bacillus subtilis, Klebsiella pneumoniae, Pseudomonas aeruginosa, Candida albicans and Aspergillus niger (MIC = 0.5 ± 0.1, 1.3 ± 0.0, 4.8 ± 0.0, 20.5 ± 0.3, 55.2 ± 0.0 and 34.3 ± 0.0 µg/mL, respectively). The scolicidal assay indicated that the EO exhibited a significant activity against E. granulosus protoscolices. To the best of our knowledge, this is the first report on the scolicidal activity of X. strumarium. Because of the emergence of antimicrobial drug resistance, the study of new effective natural chemotherapeutic agents, such as the X. strumarium EO, possibly with low side effects, represents a very promising approach in biomedical research.


Introduction
For a long time, aromatic and medicinal plants have played an important role as (phyto) therapeutic agents of both pharmacological and economic relevance [1][2][3][4]. In developing countries, due to economic constraints, nearly 80% of the population still depends on plant extracts as a source of natural remedies. Noteworthily, the excessive and repeated use of pharmaceuticals in modern medicine has caused the selection of antibiotic resistant microbial strains, thus reducing the number of antibiotics available to treat clinical infections [5][6][7][8][9][10], therefore, the use of medicinal and aromatic plants as a source of new therapeutic agents continues to be a pivotal element in traditional health care systems [10]. In addition, phytochemicals from these plants may also serve as precursors or lead compounds for the development of new pharmaceuticals [3,11,12].
Essential oils are complex mixtures of lipophilic, volatile and aromatic plant secondary metabolites. The principal constitutes of essential oils include mono-and sesquiterpenes, arising from the isoprenoid pathway, and their oxygenated derivatives such as ketones, alcohols, aldehydes, esters, oxides and phenols [23]. Several studies have reported the biocide activity of essential oils against many different agents, including clinically relevance pathogens [24][25][26].

Antibacterial, Antifungal and Scolicidal Activities
The antibacterial and antifungal activity results are summarized in Tables 2 and 3, respectively. X. strumarium essential oil significantly inhibited the growth of Gram-positive (S. aureus and B. subtilis) and Gram-negative (K. pneumoniae) bacteria (p < 0.05). MIC for S. aureus, B. subtilis and K. pneumoniae were 0.5 ± 0.1, 1.3 ± 0.0 and 4.8 ± 0.0 µg/mL of essential oil, respectively. S. aureus was the most sensitive microorganism, because of its very low MIC. P. aeruginosa was slightly inhibited in the disc diffusion assay, and its MIC was 20.5 ± 0.3 μg/mL of essential oil in the broth dilution assay. In addition, the essential oil significantly inhibited C. albicans and A. niger (p < 0.05), at all the assayed concentrations. MIC for C. albicans and A. niger were 55.2 ± 0.0 and 34.3 ± 0.0 µg/mL of essential oil, respectively.
The mortality rates of E. granulosus protoscolices after treatment with different concentrations of X. strumarium leaf essential oil are reported in Table 4. As exposure time and essential oil concentration increased, percentage mortality rised. Therefore, exposure to the essential oil for 60 min, at 2.5, 5, 10 and 20 mg/mL resulted in 58.7%, 64.48%, 68.48% and 79.22% inhibition, respectively. After 60 min, the mortality in the control was 43.56%. Table 2. Antibacterial activity of Xanthium strumarium L. leaf essential oil against gram-positive and gram-negative bacterial strains.

Bacillus subtilis
Klebsiella pneumoniae Pseudomonas aeruginosa 10 42  According to Scherer et al. [32], leaves of X. strumarium exhibited powerful antimicrobial activity against S. aureus, Escherichia coli, Salmonella thyphimurium, P. aeruginosa and Clostridium perfringens. In addition, they showed that S. aureus was the most susceptible microorganism followed by E. coli and P. aeruginosa, while S. typhimurium and C. perfringens were the most resistant to the X. strumarium essential oil. Rad et al. [13] investigated the antibacterial activity of X. strumarium on methicillin-susceptible (MSSA) and methicillin-resistant S. aureus (MRSA), showing that the plant extracts were effective on both strains, though their antibacterial activity was higher on the MSSA one. Similarly, Jawad et al. [34] reported that X. strumarium extract exhibited antimicrobial activity against S. aureus, B. subtilis, Proteus vulgaris, Candida pseudotropicalis and C. albicans. Gautam et al. [35] investigated X. strumarium extracts for in vitro antimycobacterium activity, and found that the ethylacetate and MeOH-petroleum ether extracts were effective against Mycobacterium smegmatis and M. tuberculosis. Amerjothy et al. [36] studied the hexane, alcoholic and ethylacetate extracts of Xanthium indicum Koen leaves for their antimicrobial activity. Hexane extract showed significant inhibition against P. aeruginosa, S. aureus, Aspergillus niger and C. albicans; ethylacetate extract inhibited S. aureus, A. niger and E. coli; alcoholic extract was active only against S. aureus. Antifungal activity of X. strumarium was also documented against both pathogenic and non-pathogenic fungi by Bisht and Singh [37], due to the presence of terpenes, limonene and carveol. Among the most representative constituents found in our essential oil, the sesquiterpene β-caryophyllene was extensively investigated because of its several biological activities, including antimicrobial [38,39], insecticidal [40,41], anti-inflammatory [42,43], anticarcinogenic [44][45][46][47][48] and local anaesthetic [49] activities.
Similarly, many studies showed the antimicrobial activity of α-pinene and eugenol on Gram-positive bacterial strains (S. aureus, Streptococcus pyogenes, S. epidermidis and Streptococcus pneumoniae) and fungi (Cryptococcus neoformans and C. albicans) [23,50,51]. In our study, both α-pinene (1.8%) and eugenol (trace amount) were detected in X. strumarium essential oil, as well as limonene (20.3%) and linalool (0.9%) ( Table 1) [52]. Aggarwal et al. [53] reported that limonene was particularly efficient in inhibiting the proliferation of a variety of microorganisms that cause food spoilage. Özek et al. [54] demonstrated that linalool enantiomers possessed the same antimicrobial activity against several microorganisms, specifically against the protozoan Plasmodium falciparum and the fungus Botrytis cinerea.
Xanthinin (1.04%) was found in X. strumarium essential oil (Table 1). This compound was previously isolated from the extracts of X. spinosum and was active against Colletotrichum gloesporoides, Trichothecium roseum, Bacillus cereus and Staphylococcus aureus [58]. Little et al. [59] reported that alcoholic extract of xanthinin in concentration of 0.01%-0.1% showed high antimicrobial activity against fungi and gram-negative bacteria.
Inoue et al. [60] examined the bactericidal activity of three diterpenes, i.e. phytol, terpenone and geranylgeraniol, showing that these compounds were effective against S. aureus. Similarly, Pejin et al. [61] investigated the antimicrobial activity of phytol against eight bacterial and eight fungal strains. It was proven phytol to be active against all tested bacteria and fungi. The amount of phytol in X. strumarium essential oils was 3.1% (Table 1).
Maggiore et al. [62] reported the efficacy of Thymus vulgaris and Origanum vulgare essential oils and thymol on E. granulosus protoscoleces and cysts [63]. Mahmoudvand et al. [64] studied scolicidal activity of black cumin seed (Nigella sativa) essential oil on hydatid cysts, and thymoquinone, p-cymene, carvacrol and longifolene were found to be the main components of the essential oil. To the best of our knowledge, this is the first report on the scolicidal activity of X. strumarium.

Plant Material
The Xanthium strumarium L. leaves were collected between August-September 2013 from area of Hamun Lake of Zabol (31°1'43'' N, 61°30'4'' E), Sistan and Baluchestan Province, Iran. The plant was taxonomically identified at the Department of Botany of Shahid Beheshti University of Medical Sciences, Tehran, Iran, where a voucher specimen was conserved.

Essential Oils Extraction
Fresh leaves (1 kg) were detached from the stem and dried in the shade for 96 h. Then, they were chopped and hydro-distilled for 3 h utilizing an all-glass Clevenger-type apparatus. The distillate was saturated with sodium chloride (NaCl) (Merck, Darmstadt, Germany) and the oil was extracted with n-hexane (Merck) and dichloromethane (Merck). The essential oil obtained was dried over anhydrous sodium sulphate (Sigma-Aldrich, St. Louis, MO, USA) and stored at 4 °C before gas chromatography coupled to mass spectrometry (GC-MS) analysis and bioassays.

Identification of Essential Oil Constituents
The leaf essential oil was analyzed by GC-MS. A Shimadzu 17A gas chromatograph coupled with a Shimadzu QP-5000 quadrupole mass spectrometer and Varian 3800 gas chromatograph coupled with FID detector was used. The extracted compounds were separated on DB-5 fused silica capillary column (30 m × 0.25 mm × 0.25 µm film thickness). Helium was used as carrier gas with a 1.0 mL/min flow rate. The analyses were carried out by a splitless injection (1 µL), with the injector set at 230 °C. The oven temperature program used was 60-240 °C at 3 °C /min and the final temperature was held for 8 min. The GC/MS interface and FID detector were sustained at 240 °C and 250 °C, respectively. Retention indices for all constituents were determined based on the method using n-alkanes as standard. Retention indices were determined using retention times of n-alkanes that were injected after the essential oil under the same chromatographic conditions. All data were acquired by collecting the full-scan mass spectra within the scan range 50-550 amu. Compounds were recognized using comparison of their mass spectra with the Wiley GC-MS Library and Adams Library [65,66].

Microbial Isolates, Antibacterial and Antifungal Activities
All microorganisms were obtained from the Persian Type Culture Collection (PTCC), Tehran, Iran. The essential oil was tested against three gram-negative bacteria: Different concentrations of essential oil were evaluated against bacteria and fungi by disc diffusion method [67]. In brief, microorganisms were cultured at 37 °C for 14-24 h and the densities were adjusted to 0.5 McFarland standards at A530 nm (10 8 CFU/mL). Then, 100 µL of the microbial suspensions (10 8 CFU/mL) were spread on nutrient agar (Merck) plates (100 mm × 15 mm). The discs (6 mm diameter) were separately impregnated with 10 µL of different concentrations of essential oil (10,20,40,60,80 and 100 µg/mL) and placed on the inoculated agar. All the inoculated plates were incubated at 37 °C for 24 h. Ketoconazole (10 mg/disc), ampicillin (10 mg/disc) and gentamicin (10 mg/disc) were used as positive controls for fungi, gram-positive and gram-negative bacteria, respectively. Dimethyl sulfoxide (DMSO) was used as negative control. Antibacterial and antifungal activities were determined by measuring the zone of inhibition (mm). Minimal inhibitory concentration (MIC) values of the of essential oil versus each investigated microbial strain were determined by the microdilution assay in 96 multi-well microtiter plates, according to the standard procedure of the Clinical and Laboratory Standards Institute [68]. The bacterial and fungal strains were suspended in Luria-Bertani media and the densities were adjusted to 0.5 McFarland standard at 570 nm (10 8 CFU/mL). Essential oil was dissolved in 50% DMSO to a final concentration of 10 mL. Each strain was assayed with samples that were serially diluted in broth to obtain concentrations ranging from 512.0 to 0.06 µg/mL. Overnight broth cultures of each strain were prepared and the final microorganism concentration in each well was adapted to 10 6 CFU/mL. The optimal incubation conditions were 37 °C for 24 h. Medium without bacteria and fungi was the sterility control, whereas medium with bacteria and fungi, but without essential oil, was the growth control. The growth of bacteria and fungi was compared with that of the controls. The MIC values were visually detected and defined as the lowest essential oil concentrations with >95% growth inhibitory activity to the assessed microorganisms.

Scolicidal Activity
The Echinococcus granulosus protoscolices were obtained from the infected livers of calves killed in an abattoir used to study scolicidal activity. Animals were ethically treated according to the Helsinki Declaration. In this assay, hydatid fluid was collected together with protoscolices using the Smyth and Barrett method [69]. Briefly, hydatid fluid was conveyed to a glass cylinder. Protoscolices, settled at the bottom of the cylinder after 40 min, were washed 3 times with normal saline and their viability was confirmed by motility under a light microscope (Nikon Eclipse E200, Tokyo, Japan). Protoscolices were transferred into a dark receptacle containing normal saline and stored at 4 °C. Four concentrations of essential oil (2.5, 5, 10 and 20 mg/mL) were tested for 10, 20, 30 and 60 min. To prepare these concentrations, 25, 50, 100 and 200 µL of essential oil, added to test tubes, were dissolved in 9.7 mL of normal saline supplemented with 0.5 mL of Tween-80 (Merck) under continuous stirring. For each test, one drop of protoscolices-rich solution was added to 3 mL of essential oil solution, mixed slowly, and incubated at 37 °C. After each incubation period (10, 20, 30 and 60 min), the upper phase was gently removed so as not to disturb the protoscolices; then, 1 mL of 0.1% eosin stain was added to the remaining colonized protoscolices and mixed slowly. The supernatant was discarded after incubating for 20 min at 25 °C. The remaining pellet of protoscolices (no centrifugation performed) was smeared on a manually scaled glass slide, covered with a cover glass, and evaluated under a light microscope. The percentage of dead protoscolices was determined after counting a minimum of 600 protoscolices. In the control, protoscolices were treated only with normal saline + Tween-80.

Statistical Analysis
Essential oil was extracted and tested in triplicate for chemical analysis and bioassays. Data were subjected to analysis of variance (ANOVA) following an entirely randomized design to determine the least significant difference (LSD) at p < 0.05, using statistical software package (SPSS v. 11.5, IBM Corporation, Armonk, NY, USA). All results are expressed as mean ± SD.

Conclusions
Our results indicated X. strumarium as a promising source on antimicrobial agents, with potential in biomedical applications. However, in vivo studies on this medicinal plant are needed to determine pharmacokinetics and toxicity of the active components and their side effects. In addition, the antimicrobial, antifungal and scolicidal activities may be increased by purifying active constitutes and determining proper dosages for effective therapies. This would avoid the prescription of inappropriate treatments, a usual practice among many traditional herbal practitioners. Finally, a particular application of X. strumarium plant may involve the field of food hygiene, to reduce the risk of food contamination and to control the food-borne diseases.