Phytochemical composition of wormwood (Artemisia gmelinii) extracts in respect of their antimicrobial activity

Background Extracts from medicinal plants with phytochemicals with known antimicrobial properties can be an effective adjunct in the complex treatment of infectious diseases. This study aimed to evaluate the antimicrobial activity of wormwood extracts collected in Kazakhstan (Artemisia gmelinii Weber ex Stechm.), along with their phytochemical analysis. Methods The ethanolic and chloroform extracts were subjected to HPLC combined with quadrupole time-of-flight mass spectrometry method. For quantitative assessment of antimicrobial activity, minimal inhibitory concentration (MIC) of the tested extracts was determined by micro-dilution broth method for the panel of the reference microorganisms. Minimal bactericidal concentration (MBC) or minimal fungicidal concentration (MFC) were also determined. Results LC/MS analysis showed the presence of 13 compounds in the tested extracts, including flavonoids: apigenin, luteolin, rutin, two O-methylated flavonols (isorhamnetin, rhamnazine), coumarin compounds (umbelliferone, scopoletin and scopolin (scopoletin 7-glucoside), 3-hydroxycoumarin and 4-hydroxycoumarin), chlorogenic acid and two dicaffeoylquinic acid isomers. Quantitative HPLC analysis showed that umbelliferone was dominant in the chloroform extract while chlorogenic acid was identified as a main compound in the ethanolic extract. The antibacterial and antifungal activity of chloroform and ethanolic extracts was comparable. The most sensitive were the Gram-positive bacteria represented by staphylococci, Micrococcus luteus and Bacillus spp. (MIC = 1.25–5 mg/ml) and yeasts represented by Candida spp. (MIC = 2.5–5 mg/ml), irrespective of the assayed extract. Conclusions Extracts of wormwood Artemisia gmelinii have shown a wide spectrum of antibacterial and antifungal activity. Luteolin, rutin, isorhamnetin and scopolin were identified in A. gmelinii species for the first time. The determining of the most potential compounds of Artemisia gmelinii can be used to develop effective antibacterial and antifungal agents.


Background
Artemisia gmelinii Webb & Stechmann, also known as Gmelin's wormwood, is one from over 500 species in the genus Artemisia (Asteraceae). It is a perennial plant, reaching up to 50-150 cm of height, richly-branched, grayish-brown color, strongly woody at the bottom. Taproot with a thickness of 3-5 cm. Leaves are thick and mossy especially on the underside, however loosely on the upper side. Inflorescences are spherical (20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30) in head), small, slightly drooping, densely arranged, gathered in a short panicles. The fruit is achene (1,5 mm) finelystriped, brown. They bloom from August to October [1,2]. The plant occurs on the dry stony and rocky slopes, hills, semi-deserts, grasslands, wastelands, at the altitude of 1500-4900 m asl from Eastern Europe to Central Asia, China, Mongolia and Korea. Brought to the area of the United States, growing endemically in the state of Ohio. The plant's preference is a sandy and loamy, well drained soil [2].
The leaf and stem are used in Korea to treat hepatitis, hyperlipaemia and infected cholecystitis with flavonoids and sesquiterpenes being suggested as bioactive constituents. Dried parts of the plant have been applied in Tradition Chinese Medicine for treatment and prevention of chronic inflammations of the liver [3]. As the mechanism of action the inhibitive effects of lipid peroxidation and the downregulation of TNF-α mediated apoptosis were suggested [3].
As A. gmelinni is distributed widespread in the area of Kazakhstan and both composition and application are not well defined, the aim of the present studies was the evaluation of the antimicrobial activities of extracts of wormwood collected in Kazakhstan (Artemisia gmelinii Weber ex Stechm.) along with their preliminary phytochemical analysis.

Plant material and extraction
The aerial parts of wormwood were collected in July and August 2015 in the generative phase at the foothills of the Trans-Ili Alatau of the Republic of Kazakhstan. Raw material drying was carried out by air-shadow method, in the well-ventilated accommodations, out of direct sunlight. The harvesting was produced in accordance with the principles of the GACP standard. The collected raw materials were identified in accordance with the requirements of the State Pharmacopoeia of the Republic of Kazakhstan by Danylov Mikhail Petrovich, botanic specialist in Botanical Garden, Institute of Botany and Phytointroduction of Kazakhstan Academy of Sciences in Almaty, Kazakhstan. The vouchers have been deposited in herbarium of Institute of Botany and Phytointroduction of Kazakhstan Academy of Sciences, receiving identification numbers ASM-AG 2015/01.
A technology for obtaining dry extract from the raw material of wormwood with the use of 70% ethanol by percolation has been developed. Extraction of BAS was due to convective diffusion to complete depletion, then further purification and thickening of the extract to a moisture content of no more than 5% (dry extract) was carried out. The method of obtaining 100 g of dry extract from 4 kg of raw materials consisted of the following steps: soaking of raw materials, maceration pause (infusion), percolation (direct transition of the extractant through the raw material layer), purification and thickening of the extract as well as identification of the extract. The extract output was 2.5% ± 0.26.
The preparations of 30 g of thick extract from 1455 g of raw material by the method of circulation extraction with the use of a volatile chloroform solvent were carried out in a classical manner in a "Soxhlet" type installation. The resulting thick extract was a viscous mass of dark brown color, with a specific smell of a bitter taste. The extract output was 2.1% ± 0.71.
Exactly 20 mg of each extracts were weighed and then dissolved in 10 ml of a mixture of methanol-water 8:2 v/ v. Extracts were subjected to fingerprint analysis with application of HPLC after passing syringe filters (PTFE, 0.45 μm).

Reagents
Standard substances for HPLC analysis were purchased in the Sigma-Aldrich® Co. and ChromaDex Inc.

GC-MS analysis
GC-MS was performed with a Shimadzu GC-2010 Plus gas chromatography instrument coupled to a Shimadzu QP2010 Ultra mass spectrometer equipped with fused silica capillary column ZB-5 MS (30 m, 0.25 mm i.d.) with a film thickness of 0.25 μm (Phenomenex). Analysis were performed according to previously published method [14]. The oven temperature program was initiated at 50°C, held for 3 min, then increased at the rate of 8°C min-1 to 250°C, held for 2 min. The spectrometers were operated in electron-impact (EI) mode, the scan range was 40-500 amu, the ionization energy was 70 eV and the scan rate was 0.20 s per scan. Injector, interface and ion source were kept at 250, 250 and 220°C, respectively. Split injection (1 μL) was conducted with a split ratio of 1:20 and helium was used as carrier gas of 1.0 mL min-1 flow-rate. The retention indices were determined in relation to a homologous series of n-alkanes (C8-C24) under the same operating conditions. Thujone was identified using a computer-supported spectral library [15].

Antimicrobial activity assay in vitro
The ethanolic and chloroform extracts were screened for antibacterial and antifungal activities by micro-dilution broth method according to both the European Committee on Antimicrobial Susceptibility Testing (EUCAST) (www. eucast.org) using Mueller-Hinton broth and Mueller-Hinton broth with 5% lysed sheep blood for growth of non-fastidious and fastidious bacteria, respectively or RPMI with MOPS for growth of fungi as we described elsewhere [16].

Statistical analysis
All analysis were done in triplicates in order to prove their reproducibility. The results were expressed as mean ± SD. Mann-Whitney test using GraphPad InStat 3 was performed to determine the significance of MIC values between the extracts. Statistical significance was set at p < 0.05.

Phytochemical analysis
Prior to comprehensive sample analysis, extracts from A. gmelinii with different polarities (ethanol, chloroform) were subjected to HPLC-DAD and LC-MS analysis in order to systemise the knowledge about phytochemical composition. Preliminary HPLC analysis resulted in identification of: umbelliferone (1), chlorogenic acid (2), rutin (3), scopoletin (7), luteolin (10) and apigenin (12). The numbering in parentheses is assigned according to the results of the following LC-MS analysis. Other unknown compounds (coumarins, flavonoids and phenolic acid derivatives) were also detected. Obtained HPLC-DAD (λ = 325 nm) chromatograms are presented on Additional files 1 and 2: Figures S1, S2. Quantitative determination of the compounds identified by this method was performed on the basis of 3-n determinations and converted into the content in 1 g of investigated extracts [mg/g]. The content of identified compounds is presented in Table 1. In both investigated extracts umbelliferone was identified, with 3-times higher content in chloroform extract compared to ethanolic. A simple coumarin scopoletin as well as two flavones -luteolin, apigenin were detected in chloroform extract and absent in ethanolic one. Chlorogenic acida polar depside, was dominant in ethanolic extract and absent in chloroform one. Additionally rutin was detected.
To confirm this, as well as identified other active constituents, ethanolic and chloroform extracts were subjected to LC-MS. 13 compounds were identified, as described below and listed in Table 2  Quantitative determination of known toxic phytochemicals thujone was performed by GC-MS method for both extracts. The content was between 0,12-0,16%thus we assume as low and without influence on the activity.

Antimicrobial activity
Reference strains of bacteria and fungi enlisted in Table 3 were taken for preliminary screening of antimicrobial activity using micro-broth dilution method. It was shown that the assayed extracts inhibited growth of the refer-

Discussion
Different Artemisia species are described as producers of metabolites with an antimicrobial activity [10,[17][18][19]. However, to our best knowledge, this is so far the first report of antimicrobial activity of A. gmelinii extracts. The phytochemical characteristic of tested extracts was different, but in general only slightly higher activity against Gram-negative bacteria was noticed for ethanolic extract in comparison to the chloroform one. In ethanolic A. gmelinii extract the high level of chlorogenic acid was observed. Recent studies showed that chlorogenic acid bound to the outer membrane, disrupted the membrane, exhausted the intracellular potential, and released cytoplasm macromolecules, which led to the death of cell [20]. That may be also an explanation of a more frequent bactericidal activity of ethanolic extract for most of the tested strains. Determining the MBC value allowed to establish the activity to kill bacteria or inhibit its growth. Antimicrobial agents are usually regarded as bactericidal if MBC value is higher no more than four times the MIC value [21]. The low values of MBC/MIC ratio (1-4) for ethanolic extract suggested its bactericidal power for majority of bacterial and fungal strains except for bacteriostatic activity against spore-forming B. cereus.
The data shown by Poiata et al. [22] indicated that among ethanol, methanol and hexane extracts from Artemisia absinthium, Artemisia annua and Artemisia vulgaris alcoholic extracts were more effective against   tested microorganisms. However, all plants' extracts have moderate or no activity against Gram-negative bacteria. The higher microbial activity of n-hexane, ethyl acetate and menthol extracts observed in other studies, could be related to the higher concentration of active antimicrobial agents like terpenoids, phenolics and volatile oils in Artermisia spp. [17,23]. It is widely thought that some coumarins, including umbelliferone, scopoletin and furanocoumarins play important fungicidal roles in plants. In general, umbelliferone shows no antifungal activities and very weak antibacterial activities irrespective of the test method and sample purity [24][25][26]. In our study, the chloroform extract of A. gmelinii showed higher amount of umbelliferone that may be the cause of lower value of MFCs for tested fungi in comparison to the ethanolic extract.
Standardized MIC testing methods were used by Ahameethunisa and Hopper [10,17] to determine the antimicrobial activity of A. nilagirica and A. parviflora extracts from India. Higher activity of both etanolic and chloroform A. nilagrica extracts was revealed in their studies in comparison to the one presented in our study what may be caused by different geographical origin of the plants and species and their phytochemical composition. Chloroform extracts of A. nilagrica were high in alkaloids, flavonoids, phenols, and terpenoids, similarly to the ethanolic extract which was richer with tannins, saponins and quinines, though [10].
In other studies agar well diffusion method was the only one used to detect antimicrobial activities of A. annua leaf extract [19], A. absinthium extract [26] and Artemisia sp. methanolic extracts recovered by different extraction techniques [18]. The well or disk diffusion methods provide qualitative results, appropriate for screening selection only. This method is not appropriate to give quantitative results allowing for MIC dtermination, because it is not possible to define precisely the amount of the extract diffused into the agar medium. Sometimes it is impossible to distinguish whether the lack of antimicrobial activity is a result of the extract's action or a lack of its diffusion to agar. Moreover, since the bacterial growth does not mean the bacterial death, this method cannot distinguish bactericidal or bacteriostatic effect of the extracts. We would like to point out that due to the use of standardized methods in antimicrobial activity testing of plant extracts or essential oils, data can be trustworthy and allow researchers to compare the results.
In general, 13 compounds were identified in the analyzed extracts. To the best of our knowledge, this is the first ever work that shows the identification of rhamnazine, umbelliferone, 3-hydroxycoumarin and 4-hydroxycoumarin in the genus Artemisia. Moreover, for the first time in A. gmelinii species, luteolin, rutin, isorhamnetin and scopolin were identified, even though these substances were previously determined in other species of Artemisia [27][28][29]. The presence of apigenin, scopoletin, chlorogenic acids and two dicaffeoylquinic acid isomers have also previously been reported [12,13,30]. Additionally it is worth to highlight that low level of thujone was determined in the tested extract so there should be no potential influence of the activity as well as no toxicity towards the human. Thujone is a natural monoterpene ketone present in variable amounts in a large number of plants, such as Salvia, Tanacetum and Artemisia species. Thujone has neurotoxic action, causing tonicclonic convulsions and also exhibits hepatotoxicity and porphyrogenic activity [31]. However at the same time it should be noted that the toxic activity of the extracts should not be associated exclusively with thujones as, in addition to the thujone content, the amount and toxicity of other constituents should be taken into consideration when making risk assessment and determining the regulatory status of plants in food and medicines [32].

Conclusion
There is an urgent need, to work towards the development of safer antimicrobial agents, that are expected to be renewable, non-petrochemical, naturally ecofriendly and easily obtainable. The extracts of wormwood Artemisia gmelinii have shown a wide spectrum of antibacterial and antifungal activity. For the first time in A. gmelinii species, luteolin, rutin, isorhamnetin and scopolin were identified. Determining of the most potential compounds of Artemisia gmelinii can be used to develop effective antibacterial and antifungal agents.