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Research Journal of Pharmacognosy (RJP) 1(4), 2014: 35-39 Received: July 2014 Accepted: Aug 2014 Original article Antibacterial activity of ethanol extract and fractions obtained from
Taraxacum mongolicum flower
H. Qiao, T.J. Sun* School of Basic Medicine, Shanxi Medical University, Taiyuan 030001, PR China Abstract
Background and objectives: Resistance towards reveling antibiotics has captured great interest in
evaluating the antimicrobial properties of the natural plants. Taraxacum mongolicum is widely used as
a folklore medicinal plant for its diuretic, antirheumatic and anti-inflammatory properties. Though
there are some reports on antimicrobial properties of Taraxacum mongolicum, studies on antibacterial
abilities of its flower are limited and it was decided to evaluate the antibacterial properties of the
flowers in the present study. Methods: The antibacterial properties of ethanol extract of Taraxacum
mongolicum flower, and its fractions (petroleum ether, ethyl acetate (ET), and aqueous fractions) were
examined through agar disc diffusion method, and the minimum inhibitory concentration (MIC) was
determined. Four Gram-negative and two Gram-positive bacteria were used in the study. Results: The
antibacterial test results showed that the ET fraction strongly inhibited the growth of all of the
microorganisms, especially Pseudomonas aeruginosa and Bacillus subtilis (with MIC values of 125
μg/mL and 62.5 μg/mL, respectively), whereas the ethanol extract and the other two fractions
demonstrated moderate and weak activities, respectively. Conclusion: The ET fraction obtained from
Taraxacum mongolicum flowers presented high antibacterial activity and might be suggested for use
as a natural preservative ingredient in pharmaceutical industries.
Keywords: antibacterial activity, minimum inhibitory concentration, Taraxacum mongolicum
Introduction
Nowadays, multiple drug resistance has been
roadsides and rural sites. It is a well-known developed due to the indiscriminate use of traditional medicine with a long history, having commercial antimicrobial drugs [1]. Therefore, diuretic, anti-inflammatory, anti-oxidative, anti- there is a growing tendency for replacing carcinogenic activity, etc. [6]. In China, T. synthetic additives with natural ones and this has mongolicum is used as medicine (treatment of brought about great interest in evaluating the mastitis, tonsillitis), food (nutritious plant) and antimicrobial properties of natural products [2-5]. livestock feed (for cows and goats). T. Taraxacum mongolicum Hand.-Mazz. mongolicum produces antibacterial compounds (Asteraceae) is a perennial weed and is widely that may act to reduce or control the bacterial distributed in the warmer temperate zones of the growth and there are some reports describing its antibacterial activity [7,8]. For example, Gao has Available at: http://rjpharmacognosy.ir Copy right 2014 by the Iranian Society of Pharmacognosy *Corresponding author: [email protected]; [email protected], Tel/Fax: +86351-4135645 Qiao H. and Sun T.J. reported that ethanol extracts of the dried aerial Preparation of stock and sample solutions parts of T. mongolicum had demonstrated Stock solutions of the ethanol extract and antibacterial activity which may be due to the fractions (10 mg/mL) were prepared using presence of phenylpropanoids and sesquiterpene dimethyl sulfoxide (DMSO). For assessing the lactones [7]; meanwhile Qian has revealed that minimum inhibitory concentration (MIC), the oligosaccharides derived from T. mongolicum stock solutions were serially diluted with Mueller showed high antibacterial activity against Hinton broth containing 1% Tween 20 to obtain Escheriachia concentrations between 62.5 and 8000 μg/mL. Staphylococcus aureus [8]. However, studies on The extract and fractions were filtered with 0.45 the antimicrobial properties of Taraxacum μm syringe filters (Minisart, Sartorius, Gottingen) mongolicum flower were found to be limited. Thus, this work was aimed to assess the antibacterial potential of the ethanol extracts and Antibacterial activity assay its fractions of T. mongolicum flowers against Preparation of bacterial culture The following bacterial strains were obtained from the Department of Microbiology & Collection of samples (Escherichia Vulgaris, Fresh flowers of Taraxacum mongolicum with no Pseudomonas aeruginosa, Klebsiella apparent physical, insect and microbial damage pneumoniae), were collected from the University Garden of (Staphylococcus aureus, Bacillus subtilis). Shanxi Medical University, Taiyuan. The flower Bacterial strains were grown in Muller Hinton petals were carefully removed and were freeze- broth for 24 h at 37 °C. Before using the culture dried (FD-1A-50, Bilon, Shanghai) for 48 h at for antibacterial assay, culture broth was serially -50 °C. Samples were powdered (mesh size 20), diluted using above sterile fresh broth medium to covered with aluminum foil and stored at 4 °C get a cell number of 1.0×106 CFU/mL. Disc diffusion assay Extraction and fractionation The standard disc diffusion method described by About 10 g of powdered T. mongolicum flower Bauer et al. [9] was followed. Whatman filter was soaked in 50 mL of 75% ethanol for 2 days paper (No. 1) discs of 6 mm diameter were and then filtered off using sterile Whatman No. 1 impregnated with 10 μL of the solution containing ethanol extract and fractions (at a concentrated to dryness under reduced pressure concentration of 10 mg/mL). And then these using a rotary flash evaporator (N-1000, Eyela, discs were evaporated at 37 °C for 24 h. The Tokyo) to yield 6.43% ethanol extract. The solvents used for dissolving the extracts served as ethanol extract was sequentially partitioned with the negative controls, while reference antibiotics petroleum ether (PT), ethyl acetate (ET), and gentamicin (10 μg per disc) and tetracycline (10 water (WT) to yield the following fractions: PT, μg per disc) were used as the positive controls for ET and WT fractions which were evaporated to dryness. The extract and fractions were stored in bacteria, respectively. Discs of T. mongolicum tightly sealed collection bottles at -20 °C until extract/fractions, the time of the experiments. tetracycline were placed on Muller Hinton agar plates where the bacterial culture was swabbed on the surface of the agar and incubated for 24 h RJP 1(4), 2014: 35-39 Antibacterial activity of Taraxacum mongolicum flower extracts at 37 °C. The antibacterial activity was evaluated 1. The ethanol extract and the ET fraction by measuring the zones of inhibition, and the exhibited inhibitory activitiy against both Gram- diameters of these zones were measured in negative and Gram-positive bacteria, while the millimeters against the test organisms. All PT and WT fractions were inactive against all six experiments were carried out in triplicate and the tested bacteria. mean values were accounted for results. The results showed that P. aeruginosa and B. subtilis were highly susceptible to the ET fraction, Determination inhibitory though this fraction was also active against E. concentration (MIC) coli, P. vulgaris, K. Pneumoniae and S. aureus. The MIC values of the ethanol extract, fractions On the other hand, the ethanol extract was found and reference antibiotics were determined for to be active against P. aeruginosa and B. subtilis, bacterial cultures. About 95 μL of Muller Hinton and partially against E. coli, P. vulgaris, K. broth and 5 μL of inoculum containing 1.0×106 pneumoniae, and S. aureus. This agreement may CFU/mL was pipetted into designated wells of be explained by the presence of similar the 96-well microtiter plate, except for the compounds in the ethanol extract and the ET negative control wells which consisted of 100 μL fraction, whereas the higher antibacterial activitiy of Muller Hinton broth. Then 100 μL different might be attributed to the presence of high concentrations of the extract/fractions (ranging flavonoids and phenolic acids in the ET fraction from 62.5 to 8000 μg/mL) were added to the [10]. The activity against both types of bacteria designated wells. The final volume in each well may be indicative of the presence of broad was 200 μL. Growth inhibitions or microbial spectrum of antibiotic compounds. Both positive growth was determined by measuring the optical density of the culture in the micro wells with demonstrated the greatest inhibitory activities different concentrations of T. mongolicum flower against all tested bacteria, while the negative extracts at 590 nm using a microplate reader. control DMSO did not show any inhibition zone Two controls were considered: Muller Hinton broth + bacterial suspensions to verify microbial Minimum inhibitory concentration tetracycline solutions (at concentrations 0.625, The ethanol extract and the ET fraction that 1.25, 2.5, 5.0, 10.0, 20.0 and 40.0 μg/mL) as showed antibacterial activities against Gram- positive control. Values obtained for positive and Gram-negative bacteria were mongolicum flower extracts were compared with evaluated for their MIC. The results have been the values from normal and the difference was listed in table 2. The data indicated that the considered as growth inhibition activity. MIC ethanol extract and the ET fraction exhibited was defined as the lowest concentration of the variable levels of antimicrobial activity against the investigated pathogens. The MIC values of microorganisms. All experiments were conducted the ET fraction against the tested Gram-negative in triplicate and the mean values were accounted bacteria ranged from 125 to 250 μg/mL and for Gram-positive bacteria from 62.5 to 250 μg/mL. Though these MIC values were higher than that Results and discussion
of the reference antibiotics gentamicin (against Antibacterial activity Gram-negative bacteria) and tetracycline (against The in vitro antibacterial activity of the ethanol Gram- positive bacteria), it paves way for the extract and the three fractions were assessed by potential use of T. mongolicum flower as a new the zone diameters and results were listed in table source of effective antibacterial compounds. Qiao H. and Sun T.J. Table 1. Antibacterial activity (mm) of Taraxacum mongolicum flower extract/fractions against human pathogens
Gram-negative bacteria P. vulgaris P. aeruginosa K. pneumoniae Gram-positive bacteria S. aureus B. subtilis Zone of inhibition was determined by agar disc diffusion assay. Results were presented as mean±SD. nd: not determined; PT: petroleum ether fraction; ET: ethyl acetate fraction; WT: aqueous fraction. Zone of inhibition <10 mm: inactive; 10-13 mm: partially active; 14-19 mm: active; >19 mm very active.
Table 2. Minimum inhibitory concentration of T. mongolicum flower extracts against microorganisms (μg/mL)
P. vulgaris P. aeruginosa K. pneumoniae S. aureus B. subtilis Results were presented as mean±SD. nd: not determined; ET: the ethyl acetate fraction. Antibacterial potency of the ET fraction against mongolicum these bacteria expressed in MIC indicated that antibacterial activity against the six mentioned this fraction was more effective against Gram- microorganisms. The results indicated that the ET μg/mL) at lower fraction of the ethanol extract could be suggested as a natural alternative to synthetic antimicrobial bacteria (lowest 125 μg/mL). Saha et al. have reported similar results for essential oils and methanol extracts of Ocimum species [11]. A possible explanation may reside in the possession This work was supported by the Research Fund of an outer membrane of Gram-negative bacteria, for the Doctoral Program of Shanxi Medical which restricts the diffusion of hydrophobic University (03201303). compounds through its lipopolysaccharide and protects the bacteria cell wall from leakage Declaration of interest
The authors declare that there is no conflict of Phytochemical analysis have revealed the interest. The authors alone are responsible for the presence of flavonoids, phenolic acids, and content of the paper. terpenoids in the T. mongolicum [14,15]. The antibacterial nature of the ethanol extract and the References
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