Isolation and Identification of Compounds from Bioactive Extracts of Taraxacum officinale Weber ex F. H. Wigg. (Dandelion) as a Potential Source of Antibacterial Agents

Currently, the most effective treatment for recurrent urinary tract infections in women is antibiotics. However, the limitation for this treatment is the duration and dosage of antibiotics and the resistance that bacteria develop after a long period of administration. With the aim of identifying mainly novel natural agents with antibacterial activity, the present study was undertaken to investigate the biological and phytochemical properties of extracts from the leaves Taraxacum officinale. The structural identification of compounds present in hexane (Hex) and ethyl acetate (AcOEt) extracts was performed by mass spectrometry (GC-MS) spectroscopic techniques and nuclear magnetic resonance (NMR) with the major compounds corresponding to different sesquiterpene lactones (α-santonin, glabellin, arborescin, and estafiatin), monoterpene (9,10-dimethyltricycle [4.2.1.1 (2,5)]decane-9,10-diol), phytosterol (Stigmasta-5,22-dien-3β-ol acetate), terpenes (lupeol acetate, pregn-5-en-20-one-3β-acetyloxy-17-hydroxy, 2-hydroxy-4-methoxy benzaldehyde), and coumarin (benzofuranone 5,6,7,7-a-tetraaldehyde-4,4,7a-trimethyl). The results obtained show that the Hex extract was highly active against Staphylococcus aureus showing a MIC of 200 μg/mL and moderately active against Escherichia coli and Klebsiella pneumoniae with MIC values of 400 μg/mL and 800 μg/mL for the other Gram-negative strains tested with Proteus mirabilis as uropathogens in vitro. Therefore, the effective dandelion extracts could be used in the development of future products with industrial application.


Introduction
The increasing resistance of uropathogens to antibiotics and the recognition of the generally self-limiting nature of uncomplicated urinary tract infection (UTI) suggest that it is time to reconsider the empirical treatment of UTI by using antibiotics. Limitation for this treatment is the duration and dosage of antibiotics and the resistance that bacteria develop after a long period of administration [1]. Therefore, alternatives to the pharmaceutical industry approaches need to be considered. During our continuous exploratory search for new antibacterial extracts, we have selected the Chilean medicinal plant Taraxacum officinale Weber ex F. H. Wigg. (Asteraceae), commonly known as dandelion, for a systematic study on the chemical composition and potential antibacterial properties.
T. officinale is an herbaceous perennial plant of the family Asteraceae, native to Asia; it can be found growing in temperate regions of the world, including Chile in the III-IX region. Dandelion is considered a weedy species [2]; it 2 Evidence-Based Complementary and Alternative Medicine has numerous antioxidant, anti-inflammatory, antidiabetic, antimicrobial, and anticancer properties [3,4]. There are several references to treating bacterial infections and the use of traditional botanical remedies [5][6][7]. These properties have been attributed to the large number of bioactive compounds in their tissues, and several studies have reported a wide range of compounds, including terpenes, flavonoids, and phenolic compounds, which are mentioned as responsible for the medicinal activity of different plants [8,9]. For the genus Taraxacum, only few studies concerning its antimicrobial properties consider chemical identification of the extracts obtained and, most of the time, this identification is qualitative (e.g., using colorimetric methods indicating presence or absence). Authors report the presence of terpenoids, triterpenoids, steroids, coumarins, phenols, saponins, flavonoids, flavones, flavonols, chalcones, phlobatannins, and cardiac glycosides in antimicrobial extracts [10][11][12][13][14][15][16][17][18], but neither compound isolation nor further identification was performed. Therefore, based on the above reasoning and observations, the aim of the study was to explore the phytochemical of n-hexane and ethyl acetate extracts of the Taraxacum officinale plant and its antibacterial activity against selected uropathogenic bacterial strains.
Most of the metabolites isolated belonging to the group of terpenes (lupeol, lupeol acetate, -Amyrin, -sitosterol, and betulin) from both extracts were characterized and identified by different NMR experiments ( 1 H-NMR, 13 C NMR 1D, and 2D). In addition, all the spectra of these products were compared with literature data and with authentic samples acquired from Sigma-Aldrich. The structures of these terpenes are shown in Figure 1.    HO OH H Betulin    Recently, other authors have determined from ethanol extract three novel lupane-, bauerane-, and euphane-type triterpenoids and other known triterpenes present in dandelion roots, using spectroscopic analysis, which have potential anti-inflammatory activity [32,33]. Dandelion leaves in this study were found to be rich in triterpenes such as betulin, lupeol acetate, lupeol, -Amyrin, and -sitosterol from both extracts with potential antibacterial activity against pathogens that infect the urinary tract. In several investigations, the toxicity of dandelion was found to be low, due to absence of any significant toxins or alkaloids [4]. Therefore, it would be interesting to improve the biological activity by enhancing the extract with other additives to develop a pharmaceutical product for medicinal purposes.
Manning 2001 [34] mentioned that pollen lipids, dominated by long-chain unsaturated (linoleic and linolenic) and saturated (myristic and lauric) fatty acids, have bactericidal and antifungal properties, playing an important role in inhibiting the growth of the spore forming bacteria Evidence-Based Complementary and Alternative Medicine 5 Table 3: Fatty acids content in the T. officinale leaves collected from Temuco region, Chile (mg/g dry weight).

In Vitro Antimicrobial Assay.
The antibacterial effects of the extracts of T. officinale were evaluated against four uropathogenic bacteria ( Table 4). Evaluation of the dandelion extracts revealed that the extract Hex inhibited the growth of S. aureus by 89% at 200 g/mL and was a more effective inhibitor for the Gram-positive than for the Gram-negative strains (Table 4). K. pneumoniae was the lowest inhibited by the Hex extracts.
EtOAc extract showed low activity against E. coli. with 1600 g/mL and growth inhibition with a 97% value. However, this extract did not exert an antibiotic effect on Grampositive bacteria S. aureus in the concentrations tested (Table 4). Similar results have been reported where the ethanol extract of leaves of T. officinale had low antimicrobial activity against S. aureus, E. coli, and Salmonella abony [38].
In addition, it was observed that as the extract concentration increased, so does the bacteria growth inhibition. This inhibitory effect may be due to the presence of phenolic compounds, terpenes, tannins, flavonoids, alkaloids, and/or proteins in the plant extracts. Such compounds had been reported to have an active effect on the bacterial cells membrane, which may destroy these microorganisms [39,40].
In studying the antipathogenic properties of genus Taraxacum to combat infectious diseases, T. officinale is the most studied species, but it has shown various results depending on the extraction characteristics or on the bioassay performed. For example, a methanolic extract of T. officinale at 0.2 mg/mL was effective as antibacterial against Micrococcus luteus and Vibrio cholera with MIC values of 1.0 mg/mL and 12.5 mg/mL, respectively, but did not show any activity against S. aureus, Enterobacter faecalis, Enterococcus bacteria, V. cholera, Bacillus subtilis, Pseudomonas aeruginosa, K. pneumonia, or E. coli [12]. In another work, methanolic extracts of T. officinale show an activity between 0.003 and 0.5 mg/mL on S. aureus, P. aeruginosa, B. cereus, Shigella sonnei, S. enterica serovar Typhimurium, E. coli, K. pneumonia, Candida albicans, and C. neoformans with MIC values ranging from 0.04 to 5.0 mg/mL [11]. So, these extracts could be active or improve their antibacterial activity against other Gramnegative and Gram-positive strains, and this work provides preliminary information for the development and use of natural medicine with extracts of T. officinale in the control of disease against uropathogenic bacteria, as antimicrobial agents.

Collection and Plant
Material. The leaves of T. officinale were collected in Temuco, Cautín province (IX region), Chile, in May 2014 and authenticated by agronomist Lorena Jorquera, Ph.D., from "Pontificia Universidad Católica de Valparaíso", V region, Chile. The leaves were transported to the laboratory, washed 3 times with water and once with sterile distilled water (WDS), then air-dried on kraft paper, and placed in hot air oven at a temperature of 45.0 ∘ C for a period of 4 days until the weight became constant. The dried plant material was converted to a powdered form with the help of clean grinder.

Preparation of the Extracts. The extracts of T. officinale
(1,5 g, 1,5%) were obtained from dried plant material (100 g) suspended first in 400 mL of n-hexane and then the residues were further extracted with ethyl acetate (EtOAc) separately; that is, an extraction was performed sequentially and serially. The extraction was carried out by using an orbital shaker (150 rpm) at 25 ∘ C for 48 h at room temperature; then the extract was filtered through Whatman N ∘ 1 filter paper (Sigma-Aldrich, Darmstadt, Germany), concentrated under reduced pressure with a rotatory evaporator, and finally kept in the refrigerator (−4 ∘ C) until the analyses were made.

Isolation of Natural Compounds from
Extracts. The nhexane and EtOAc extracts were subjected to chromatography over a silica gel column (400 g) and eluted with mixtures of increasing polarity n-hexane and EtOAc. Fractions were combined based on TLC monitoring and purified by repeated CC on silica gel columns. Compounds were identified by chromatographic analysis and spectroscopic data, including 1 H-and 13 C-NMR, respectively, on a Bruker Avance 400 Digital NMR spectrometer operating at 400.1 MHz for 1 H and 100.6 MHz for 13 C, and comparisons with data reported in the literature were made.

Chromatographic
Analysis. The n-hexane and ethyl acetate extract was diluted with acetone, and analysis by gas chromatography (Hewlett Packard, Palo Alto, CA, USA) was carried out according to the method detailed elsewhere [41]. The operating conditions were as follows: on-column injection; injector temperature, 250 ∘ C; detector temperature, 280 ∘ C; carrier gas, He at 1.0 mL/min; oven temperature program, 40 ∘ C increased to 260 ∘ C at 4 ∘ C/min and then 260 ∘ C for 5 min, to achieve the best separation through a capillary Rtx-5MS column. The mass detector ionization employed an electron impact of 70 eV. Compounds in the chromatograms were identified by comparison of their mass spectra with those in the NIST11 library database [42]. Chromatographic peaks were considered "unknown" when their similarity index (MATCH) and reverse similarity index (RMATCH) were less than 850 and discarded in this identification process [43]. These parameters referred to the degree in which the target spectrum matches the standard spectrum in the NIST Library (the value 1000 indicates a perfect fit), by comparison of their retention index with those reported in the literature [44], for the same type of column or those of commercial standards, when available. The retention indices were determined under the same operating conditions in relation to a homologous n-alkanes series (C8-C36) by where is the number of carbon atoms in the smaller nalkane, is the number of carbon atoms in the larger nalkane, and Tr is the retention time. Components relative concentrations were obtained by peak area normalization.

Fatty Acids Determination.
The gas chromatography analysis was performed on an Agilent Technologies 7890B gas chromatograph using a 30 m capillary column, Supelco Omega-Wax, 0.25 mm (Agilent Corp. CA). Hydrogen was used as a carrier gas at a flow rate of 1 mL/min. Detection was with flame-ionization detection and areal quantitation was made with an auxiliary automatic integrator. A temperature of 170 ∘ C was used at the beginning, which was maintained for two minutes, rising from 2,5 ∘ C/min to a final temperature of 240 ∘ C, which was maintained for 3 min. Injector and detector remained at 250 ∘ C and 270 ∘ C, respectively. Methyl esters were prepared according to our previously described procedure [45] by treating the extracted oil with 0.5 M NaOH in methanol, followed by 14% BF3 in methanol treatment. The acid identification was made by comparing the relative retention times of FAME peaks from samples with standards. The results were recorded, processed, and expressed in relative percentage of each fatty acid.

3.7.
In Vitro Antimicrobial Assay. The minimum inhibitory concentration (MIC) of the plant extract was determined using the broth serial dilution method with modifications, following Andrews 2001 [46]. Briefly, the extracts were first dissolved in ethanol (ET) and the final concentration of ET in each microwell was less than 1%, which did not affect the growth of the test strain. A dilution range of the extracts from 12.5 g/mL to 1600 g/mL was tested. Later, 1.5 L of microbial suspension (1 × 10 6 CFU/mL) of each strain was inoculated into sterile 96-well microplates with equal volume of MHB broth and plant extracts. Then, the 96-well microplates were incubated aerobically at 37 ∘ C for 24 h in a shaker at 120 rpm. MIC was determined according to the OD 600 obtained in an Accu Reader M965 spectrophotometer. After the cultures were incubated at 37 ∘ C for 24 h, the minimum inhibitory concentration (MIC) was confirmed as the lowest concentration of the test extract that demonstrated no visible growth. The plates were incubated for 24 h at 37 ∘ C. Chloramphenicol and streptomycin were used as positive control tested at the same concentrations for bacterial strains and 1% ET was used as negative control with inoculum. Also, a negative control was used with 1% ET without inoculum to subtract the OD 600 obtained. Each concentration of the extracts was tested in triplicate. Each value represents the mean ± SD of three experiments performed in triplicate, for which the mean and standard deviation for each value were calculated.
Finally, the percentage of bacterial growth inhibition (GI) was calculated using the following formula: where Abs is the absorbance of the control treatment and Abs is the absorbance of samples treated with different extracts.

Conclusions
Different secondary metabolites of the n-hexane and ethyl acetate extracts were isolated from T. officinale (Compositae) leaves, mainly known as triterpenoids, and other unknown compounds to a lesser extent. Their presence and structures were elucidated by spectroscopic analyses. In addition, the fatty acids content was determined showing a higher content of palmitic and linolenic acids. Both extracts present antibacterial activity against uropathogenic clinical bacteria, being more active against Gram-positive bacteria (MIC 200 g/mL, 89% of bacterial inhibition). These results suggest that both extracts have potential as antibacterial uropathogenic disease agents.

Conflicts of Interest
The authors declare no conflicts of interest.

Authors' Contributions
Katy Díaz Peralta designed the research, performed extractions, separation, and purification of the compounds, performed the bioassays, and wrote the biological component of this document. Rolando Chamy Maggi contributed to research design, copywriting, and corrections. Luis Espinoza Catalán contributed to the structures determination by spectroscopic methods (1D, 2D NMR) and document writing. Alejandro Madrid Villegas contributed to the interpretation of the results (GC-M) and document writing. Leonardo Pizarro Dasso contributed to the determination of fatty acids by chromatographic methods. All authors read and approved the final document.