SUPPLEMENTARY MATERIAL
Chemical constituents, in vitro antioxidant and antimicrobial properties of ethyl acetate extract obtained from Cytisus triflorus L’Her Benabderrahmane W1,*, Amrani A4, Benaissa O1, Lores M 2, Lamas J P2, de Miguel T3, Benayache F 1, Benayache S 1 1
Unité de Recherche Valorisation des Ressources Naturelles, Molécules Bioactives, Analyses
Physicochimiques et Biologiques (VARENBIOMOL), Université Frères Mentouri Constantine 1, Route d’Aïn El Bey, 25000 Constantine, Algérie 2
Laboratory of Research and Development of Analytical Solutions Analiticas (LIDSA),
Analytical Chemistry Department. Faculty of Chemistry, Campus VIDA-USC, Santiago de compostela E-15782 3
Department of Microbiology and Parasitology, Faculty of Pharmacy at University of
Santiago de Compostela, Spain 4
Département de Biologie Animale, Faculté des Sciences de la Nature et de la Vie, Université
Frères Mentouri Constantine 1, Route de Aïn El Bey, 25000 Constantine, Algérie *Corresponding author:
[email protected] Abstract The present study investigates the phenolic composition, antioxidant and antimicrobial activities of an ethyl acetate extract from C. triflorus L’Her. The phytochemical study on the aerial parts of C. triflorus belonging to the Fabaceae family led to the isolation and structural elucidation of 5-Hydroxy-7-O-glucosylflavone (P1), 5-Hydroxy-7-O-galactosylflavone (P2), 5,7-Dihydroxy-flavone (P3), 5,7,3’-Trihydroxy,4’-methoxy-flavone (P4). These compounds were identified by 1D and 2D NMR combined analysis as well as by UV. Ethyl acetate extract of C. triflorus showed a significant and dose-dependent antioxidant activity in vitro, related to the presence of phenolics (180.33±12.22 µg GAE/mg) and flavonoids (16.78±1.54 µg QE/mg). The antimicrobial activity was evaluated in vitro against Staphylococcus aureus CECT 240
and Escherichia coli CECT 4099, by agar-diffusion
method. The most active antibacterial activity was expressed by ethyl acetate extract of C. triflorus against Gram-positive bacteria S. aureus. The gathered results suggest that C. triflorus polyphenols and flavonoids are closely associated to its antioxidant and antimicrobial properties. Keywords: Cytisus triflorus; Polyphenols; Flavonoids; Antimicrobial activity; RMN
Experimental Section Plant material The aerial parts of Cytisus triflorus L’Her., were collected locally Azazga (North of Algeria) in March 2011. The plant was authenticated by Professor Mohamed Kaabache (Botany department, Ferhat Abbas University, Setif, Algeria). Voucher specimen was deposited at the herbarium of the reseach unity VARENBIOMOL of the university of Constantine 1 (CTA 125/03/11). The samples were dried at room temperature, by hanging them for several days in a cool, dry place. The whole plants and the different parts (flowers, fruits, leaves) were evaluated separately aspotential sources of polyphenols. All data were finally expressed in dry weight (dw). Extraction and isolation procedures Total of 700 g air-dried and powdered aerial parts of Cytisus triflorus L’Her., were extracted with EtOH/H2O (80:20, v/v) for 24 h, three times. After filtration, the filtrate was concentrated and dissolved in H2O (400 mL). The resulting solution was successively extracted with petroleum ether (PE), chloroforme (CHCl3), Ethyl acetate (EtOAc) and nButanol (n-BuOH). In the EtOAc phase yellow needles appeared giving after filtration the compound 1 (1500 mg). The organic phases were filtered using common filter paper and concentrated in vacuum up to 35°C to obtain the following dry extracts: CHCl3 (2.08 g), EtOAc (4.03 g) and n-BuOH (15.21 g). The EtOAc was further fractioned by silica gel column chromatography, eluted with chloroforme containing increasing percentages of MeOH to obtain 20 fractions (F1-F20). F3 (22 mg) was purified on preparative plates of silica gel n-hexane/EtOAc 8:2 and nhexane/EtOAc 8:2, to obtain compound 3 (6 mg). Fraction 18 (122.2 mg) (CHCl 3/EtOAc, 80:20) was combined and chromatographed on a silica gel column (10 g) eluted with a mixture of hexane/EtOAc with increasing polarity to yield 9 subfractions. F6-1 (17.5 mg) eluted with (Hexane/EtOAc, 80:20) was submitted to preparative TLC (CHCl3/MeOH, 8:2) to give compound 2 (3 mg). Finally, F5 (20 mg) was purified on preparative plates of silica gel n-hexane/EtOAc 7:3 and n-hexane/EtOAc 8:2 compound 4 (4 mg). Chemicals and reagents Extraction solvents used were petroleum ether (PE), chloroforme (CHCl3), ethyl acetate (EtOAc), n-Butanol (n-BuOH), dimethyl sulfoxide (DMSO) and ethanol Ultrapure water was
produced in the laboratory with a Milli-Q gradient system (Millipore, Bedford, MA, USA). The Folin-Ciocalteu phenol reagent was obtained from Sigma–Aldrich (Steinheim, Germany). Other chemicals that are needed to determine the spectrophotometric indexeswere methanol (95%) (Panreac, Castellar del Vallès, Barcelona, Spain), ascorbic acid, FeSO4, trichloroacetic acid (TCA), Thiobarbituric acid (TBA), sodium carbonate (Na2CO3, Panreac) and aluminum trichloride (AlCl3, Merck). 2,2-Diphenil-1-picrylhydrazyl (DPPH, Sigma, St. Louis, USA) was used to determine the scavenging activity of the grape marc extracts. Pure polyphenols gallic acid 99% (CAS 149-91-7), and querectin 98% (CAS 117-39-5) were supplied by Sigma–Aldrich (St. Louis, USA). Culture media used for the antibacterial activity studies were: Tripticasein Soy Broth (TSB), Tripticasein Soy Agar (TSA) from Cultimed (Barcelona, Spain) and Mueller-Hinton Agar (MH) from Oxoid (Basingstoke, Hampshire, UK) Evaluation of the antioxidant activity Determination of Total Phenolic Content (TPC) The total phenolic content was determined using the Folin-Ciocalteu colorimetric method described by (Singleton et al; 1999). Briefly, 20 µL of sample were diluted with 1580 μL of distilled water and then mixed well with 100 μL of 2N Folin-Ciocalteu reagents. Wait for between 30 sec and 8 min, and then add 300 µL 20% (w/v) sodium carbonate solution, the mixture was then incubated at 20°C for 2 hours. The absorbance was measured at 765 nm. Gallic acid concentrations ranging from 50–500 µg/mL were prepared and the calibration curve was obtained giving a linear fit (R2 = 0.991). The samples were analyzed in triplicate. The concentration of total phenolic compounds was determined as µg of gallic acid equivalents (GAE) per 1 mg of extract (dry weight).
Determination of Total Flavonoids Content (TFC) The total flavonoids were estimated by the aluminium chloride method described by (Wang et al., 2008). Briefly, 500 µL of the sample, 500 µL of AlCl3 (2%) methanol solution was added. The absorbance was measured at 420 nm after 1 h incubation at room temperature. Quercetin concentrations ranging from 0 to 1200 µg/mL were prepared and the standard calibration curve gave a linear fit (R2 = 0.990). The samples were analyzed in triplicate. Flavonoid content was calculated as µg of quercetin equivalents (QE) per 1 mg of extract using the following equation (Absorbance = 0.34 × quercetin (μg) + 0.015).
Free Radical DPPH Scavenging Assay
The DPPH assay was carried out according to the procedure of (Braca et al., 2001), with minor modifications. In this study, different volumes (1, 2.5, 5, 10, 20, 30, 50 and 75 µg/mL) of extract were mixed with a methanolic solution of DPPH radical (0.004%, 3 mL). The mixture was kept in dark. 30 min later, the absorbance was measured at 517 nm. The free radical scavenging activity is expressed as the inhibition percentage of free radicals by the sample and calculated using following the formula:
Where A control is the absorbance of control and A sample is the absorbance of sample at 517 nm. The IC50 (the concentration of antioxidant which eliminate 50% of DPPH radicals) was defined for the extract and a standard antioxidant (vitamin C) used as control. The study was carried out in triplicate at each concentration (n = 3). Assays of lipid peroxidation using vitellose A modified thiobarbituric acid-reactive species (TBARS) assay described by (Cao and Ikeda., 2009) was used to measure the lipid peroxide formed, using egg vitellose homogenates. Briefly 10% homogenate were incubated with different dose of ethyl acetate extract or vitamin C in the presence of 50 µL FeSO4 (0.07 M) at 37°C for 1 h. Reaction was stopped by addition of 1ml trichloroacetic acid (TCA 20%), and 1.5 mL thiobarbituric acid (TBA 1%) in succession, and the solution was then heated at 100°C for 15 min. After centrifugation at 4000 rpm for 20 min to remove precipitated protein the absorbance was measured at 532 nm. The lipid peroxidation scavenging activity (I %) was calculated by the following equation:
Where A control is the absorbance of control and A sample is the absorbance of sample. Evaluation of the antimicrobial activity Bacterial strains and culture conditions Tests of antimicrobial activity have been carried out using two bacterial strains from the Spanish, Type Culture Collection (CECT): Gram positive strain Staphylococcus aureus CECT 240 and Gram negative strain Escherichia coli CECT 4099. Both cultures were maintained at -80°C in tryptic soy broth (TSB) containing 15% glycerol and reactivated in tryptic soy agar plates (TSA), made from TSB medium supplemented with 2% agar-agar. TSB and TSA
media were always used for bacterial growth except for the case of diffusion tests, which were performed on plates of Mueller-Hinton Agar (MH). The bacterial cultures were always grown at 37°C. In the case of liquid cultures, erlenmayer flasks containing TSB medium and the bacterial strains were incubated in an orbital shaker at 37°C and 200 rpm. Preparation of the extracts for antimicrobial tests The extracts were first dried to eliminate the extraction solvent and dissolved in 50:50 DMSO : H2O (Because the extracts were not soluble in water) to a final concentration of 200 mg/mL. Agar diffusion tests 50 microliters of an overnight culture were spread onto MH agar plates. Then six wells, each 10 mm in diameter, were cut out of the agar and 20 µL of melted agarose were poured into each well to seal the bottom. Once the agarose solidified, 150 microliters of either extract or purified polyphenol were poured into each well and the plates were incubated for 24 hours. Antimicrobial activity of the samples was qualitatively assessed by the presence or absence of inhibition haloes. In the case of samples which were available only in a very small amount, a modification of this method was used. Thus, after spreading the bacterial strain onto MH agar plates and left to dry, a drop of each sample was poured on it. Inhibition was assessed by means of clear zones were the drops were poured. Samples exhibiting antimicrobial activity were selected to keep out quantitative inhibition tests. Determination of Minimum Inhibitory Concentration (MIC) The Minimum Inhibitory Concentration (MIC) generally is the lowest concentration of antimicrobial capable of inhibiting any visible growth after an incubation period of 18 to 24 hours. In order to assess the MIC, TSA plates containing different concentrations of each selected extract were prepared with the following procedure. Increasing volumes of this extract were added to the bottles containing sterile TSA medium at 55ºC to reach final concentrations ranging from 0.06 to 4 mg/mL and gently mixed to homogenization. The mixtures were then poured into sterile petri dishes and left to solidify. 50 µL of the 10-4 dilution of an overnight bacterial culture were spread onto each plate and incubated for 24 hours. Bacterial growth was assessed by means of observation of colonies. The minimal extract concentration at which no colonies were observed after 24 hours was the MIC.
Determination of Minimum Bactericidal Concentration (MBC) The Minimum Bactericidal Concentration (MBC) is the lowest concentration of an antibacterial agent required to kill a particular bacterium. MBCs were determined as follows : Wells of a 96-multiwell microplate were filled with a total amount of 100 mL of TSB medium and serial dilutions of extract, leading to decreasing concentrations from 100 mg/mL to 0.195 mg/mL. Then each well was inoculated with 30 µL of an overnight bacterial culture and incubated for 24 hours. Then, 50 µL of each well were spread onto TSA plates and incubated again for 24 hours. Bacterial growth on TSA plates was assessed by means of observation of colonies. Bactericidal Concentrations were those present in the wells, which lead to no bacterial growth after incubation on TSA plates Table S1. The phenolic and flavonoid contents of EtOAc extract of C. triflorus.
Specie
C. triflorus
Total phenolic
Flavonoid
content
concentration
µg GAE/mg extract
µg QE /mg extract
180.33 ± 12.22
16.78 ± 1.54
Extract
Ethyl acetate
Table S2. MICs (mg/mL): Growth of S. aureus in presence of different concentrations of EtOAc extract of C. triflorus. MBCs values (mg/mL) of EtOAc extract of C. triflorus against S. aureus in the micro dilution assay. Minimal Inhibitory Concentrations (MICs) Concentration
EtOAc
(H2O/DMSO)
mg/ml
Extract
(Blank)
0.25
++
++
0.5
+
++
0.75
-
++
Minimum Bactericidal Concentrations (MBCs) Concentration
EtOAc
Blank
mg/ml
Extract
(H2O/DMSO)
25
++
++
50
+
++
100
-
++
(++ represents normal colony size, + small colony size, - no growth) Ethyl acetate extract Vitamin C
Inhibition (%)
120 100 80 60 40 20 0 0
100
200
300
400
500
600
Concentration (µg/mL) Figure S1. Effect of ethyl acetate extract of C. triflorus and standard vitamin C on nonenzymatic lipid peroxidation in egg vitellose homogenate induced by the FeSO4. Values are expressed as mean ± SD ( = 3)
Figure S2. Determination of MIC values of ethyl acetate extract of C. triflorus (mg/mL) B: blank (H2O/DMSO); 1 : 0.5 mg/mL; 2 : 0.75 mg/mL
Figure S3. Determination of MBC values of E: ethyl acetate extract of C. triflorus (mg/mL), (E-1): 100 mg/mL, (E-2): 50 mg/mL, (G-1): blank (H2O/DMSO)
Figure S4. 1H NMR and 2D-NMR (COSY, HMQC and HMBC) spectra of compound P1. (DMSO, 500MHz).
Figure S5. 1H NMR and 2D-NMR (COSY, HSQC and HMBC) spectra of compound P3. (Acetone, 500MHz).
Figure S6. 1H NMR (MeOD, 400 and 500MHz) and 2D-NMR (HSQC and HMBC) spectra of compound P4. (MeOD, 500MHz).
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