Original Research Paper Acta Chromatographica 27(2015)4, 743–754 DOI: 10.1556/AChrom.27.2015.4.11 HPLC, Two-Dimensional TLC Determination of Phenolic Content, and an In Vitro Perspective to Antioxidant Potential of Euonymus verrucosus Scop Extracts W KUKULA-KOCH1,*, J WIDELSKI1, W KOCH2, AND K GŁOWNIAK1 Department of Pharmacognosy with Medicinal Plant Unit, Medical University of Lublin, Chodzki St., 20-093 Lublin, Poland 2Chair and Department of Food and Nutrition, Medical University of Lublin, 4a Chodzki St., 20-093 Lublin, Poland *E-mail: virginia.kukula@gmail.com Summary The presence of phenolic content in overground extracts of Euonymus verrucosus Scop – commonly growing in Europe – has been reported recently The chromatographical and spectral data revealed the presence of several simple phenolic acids (gallic, protocatechuic, p-hydroxybenzoic, vanillic, syringic, caffeic, p-coumaric, ferulic, and m-coumaric acids), both as free and conjugated with other secondary metabolites The comparison of two-dimensional TLC systems on cellulose stationary phases with HPLC– DAD reversed-phase chromatography was performed to assess a cheap and rapid technique in the identification process of major phenolic constituents 2,2-Diphenyl-1picrylhydrazyl (DPPH) radical-scavenging tests, expressed as IC50, revealed the most beneficial results for the fraction after alkaline hydrolysis and yielded 205 ± μg mL−1 Key Words: phenolics, 2,2-diphenyl-1-picrylhydrazyl, radical-scavenging activity, HPLC, chemical composition, warted spindle Introduction The Celastraceae family consisting of ca 50 genera and 800 species encompasses trees or shrubs, which are characterized by opposite leaves, bisexual flowers, flat calyx, and 3–5-lobed fruits [1] Euonymus verrucosus Scop., known as the warted spindle, is native to East Asia, but it is also commonly spread around Australia, Europe, North America, and Madagascar [2, 3] The plants contain mainly sesquiterpene alkaloids (e.g euoverrine and eunonymol), polyamines, sesquiterpenes, and proteins (lectin) [4–6] A few scientific papers confirm the presence of flavonoids in different species of the Euonymus gender [4, 7] Fruits of the Euonymus species have been used in traditional medicine – mainly in the TCM, but also (in Poland) as emetic and purgative drugs, whereas the bark displayed tonic, laxative, diuretic, and expectorant activity 0231–2522 © 2014 Akadémiai Kiadó, Budapest W Kukula-Koch et al 744 [2] Currently, the plant is known for its antibacterial, insecticidal, and antihyperglycemic properties [8–10] Furthermore, the lectin fraction from spindle trees is an indicator of highly malignant tumor cells [11] In the course of the conducted study, a new application of E verrucosus Scop (warted spindle) extracts as antioxidant agents is proposed Twodimensional thin-layer chromatography (2D-TLC) as well as highperformance liquid chromatography (HPLC) results confirm the presence of different phenolic compounds in the investigated extracts – both as free phenolic acids and phenolics bound with other secondary metabolites, which is confirmed by the results of respective hydrolyses carried out by Bartnik et al [12] The paper shows a comparison of two modern techniques: 2D-TLC and HPLC with diode-array detection (DAD) in the fast profiling of unknown extracts towards their phenolic content and antioxidant potential Based on recent scientific papers on the flavonoid content of Euonymus spp., the proposed results draw attention to another group of compounds with radical-scavenging properties – phenolic acids [4, 7] Phenolic substances (simple phenols, phenolic acids, flavonoids, or hydroxycinnamic acid derivatives) belong to the group of nutrients characterized by strong antioxidant capacity [13, 14] They may reduce oxidizing damage, which remains a confirmed cause of cancer and cardiovascular problems [15, 16] According to the obtained TLC and HPLC results, the presence of phenolic acids in the extracts of E verrucosus Scop has a vital role in the antioxidant potential of this plant species The fractions obtained in the qualitative analysis of phenolic content were investigated for their 2,2-diphenyl-1-picrylhydrayzl (DPPH)–scavenging properties Their composition clearly influenced the scavenging properties of the warted spindle The obtained results may be perceived as an indicator of antioxidant potential found in the warted spindle Experimental Chemicals and Reagents Standards of phenolic acids (chlorogenic, ferulic, gallic, p-coumaric, p-hydroxybenzoic, protocatechuic, rosmarinic, quinic, sinapinic, syringic, and vanillic acid) as well as DPPH (95% purity) were purchased from Sigma-Aldrich (Steinheim, Germany) Acetic acid, acetonitrile, barium hydroxide, dichloromethane, diethyl ether reagent grade, DMSO, ethanol, formic acid, hydrochloric acid (37%), methanol reagent grade, sodium hydrocarbonate, sodium formate, sulphuric acid, and toluene reagent grade HPLC, 2D-TLC and an In Vitro Perspective 745 were produced by Avantor Performance Materials (Gliwice, Poland) Methanol gradient grade was produced by Merck (Darmstadt, Germany) Plant Material The herb of E verrucosus Scop (leaves, twigs and fruits) used in the course of the current survey was collected in the forest in Zwierzyniec, Poland, at the beginning of its fruiting time (September 2010) and was identified by Dr Michał Hajnos and Dr Stanisław Kwiatkowski A voucher specimen (No WK2010002) was deposited at the Department of Pharmacognosy with Medicinal Plant Unit, Medical University of Lublin, Poland Extraction Soxhlet Extraction Eighty-four grams of dried plant material were macerated twice in a Soxhlet apparatus with methanol – 24 h each time The obtained extracts were combined (3.51 g and 3.97 g, respectively) and evaporated to dryness under reduced pressure (ca 150 mbar) using the rotary evaporator The dried extracts underwent further separation of phenolic acids according to the method proposed by Bartnik et al which constituted the modification of Smolarz et al and Machalska et al [12, 17, 18] Separation The Separation of Phenolic Acids from the Extract According to the method of Machalska et al., the dried methanolic extract from overground parts of the warted spindle was partially dissolved in 25 mL of hot distilled water and subsequently filtered using cellulose filters (Avantor Performance Materials, Poland) [18] The procedure was repeated four times, and the filtrates were joined together The precipitate was thrown away together with the cellulose filter The water extract was then washed five times with 20 mL of diethyl ether each time Ether and aqueous extracts were analyzed separately Ether extract – free phenolic acids (EV-FPA, Fraction A) The ether extract was washed with five 10 mL portions of 5% solution of sodium hydrocarbonate in a separating funnel Next, the carbonate phase was collected W Kukula-Koch et al 746 separately, and a solution of 18% hydrochloric acid was added up to the pH of The carbonate extract of pH = was then placed in separating funnel and washed five times with 20 mL of diethyl ether The ether extracts contained free phenolic acids (FPA) from the plant material Water extracts – hydrolysis Acidic and alkaline hydrolyses were performed for water extracts to find the acidic or basic secondary metabolites with phenolic acids conjugated to their structures Alkaline hydrolysis Alkaline hydrolysis (EV-B, Fraction B) was performed on the half volume of water extract It was heated under cooler with barium hydroxide – Ba(OH)2 – in pH of 12, for h Next, the pH was slowly adjusted to 1.5 with the addition of 10% and later the 96% solution of sulphuric acid The extracts were moved to the separating funnel and were rinsed five times with 20 mL portions of diethyl ether Next, the ether extracts were collected separately from water extracts and rinsed five times with 5% solution of sodium hydrocarbonate (portions of 10 mL) Carbonate extracts were adjusted to the pH of with the addition of 18% hydrochloric acid and thoroughly shaken with five portions of 20 mL diethyl ether in a separating funnel Acidic hydrolysis (EV-C, Fraction C) Half of water extracts was transferred to a separating funnel and rinsed five times with 20 mL of diethyl ether Ether extracts were alkalized with the addition of five 10 mL portions of 5% solution of sodium hydrocarbonate Carbonate extracts were collected together and acidified with 18% solution of hydrochloric acid Next, carbonate extracts were transferred into a separating funnel and washed with five portions of diethyl ether Ether extracts contained phenolic acids unleashed after hydrolysis Identification Two-Dimensional Thin Layer Chromatography 2D-TLC Profiling Twenty milligrams of each extract were dissolved in mL of methanol (HPLC grade), filtered through a membrane filter (nylon filter with pore size of 0.45 μm), transferred to a glass vial, and applied on cellulose-covered glass NP, TLC plates 10 × 10 cm (Merck, Germany) by TLC autosampler (CAMAG, Muttenz, Switzerland) The plate was dried in the air and conditioned for approximately 10 in the vapors of a solution: toluene–methanol–acetic acid (94:1:5 v/v/v) to prevent solvent-demixing effects Then, it was developed vertically over cm in a flat bottom chamber (CAMAG) at 21 °C and with relative humidity of 57% in the following solvent system: toluene–acetic acid– HPLC, 2D-TLC and an In Vitro Perspective 747 acetonitrile (75.5:10:7.5 v/v/v) After drying at room temperature for h, another development procedure was performed in the second direction on each TLC plate in the solution of sodium formate–formic acid–water (10:1:200 v/v/v) The TLC plates were observed under ultraviolet (UV) light both at 365 nm and 254 nm wavelengths A similar TLC plate was prepared for the mixture of reference solutions of caffeic, ferulic, p-coumaric, protocatechuic, syringic, vanillic, and phydroxybenzoic acids HPLC Profiling Ten milligrams of each extract (EV-FPA, EV-B, EV-C) were redissolved in mL of methanol (HPLC grade), filtered through a 0.45-μm nylon membrane filter (Waters,) and subjected to RP-HPLC–DAD analysis The Agilent 1100 system (Santa Clara, USA) coupled with an autosampler and a diodearray detector was employed for the profiling of the extract ChemStation software was used for data management For the HPLC– DAD phytochemical profiling, an RP 18 (250.0 mm × 4.6 mm, 5.0 μm) Discovery-Sigma Aldrich column was used, and the injection volume was set at 10 μL The mobile phase consisted of 2% aqueous acetic acid (solvent A) and acetonitrile-containing 2% acetic acid (solvent B) The following elution conditions were used: initial A–B (99.5:0.5); in 40 A–B (80:20); in 60 A–B (60:40); in 70 A–B (95.5:0.5); running time: 75 min; and in 15 conditioning of the column The flow rate was set at mL min−1, and the chromatograms were recorded at 260, 280, and 365 nm by monitoring spectra within a wavelength range of 200–500 nm, at room temperature Antioxidant Assay — DPPH Test All extracts (EV-FPA, EV-B, EV-C) were subjected to the DPPH test which was to measure their potential to quench free radicals The procedure applied in this study was a modification of the methods of Lee et al and Kukula-Koch et al [19, 20] In detail, ethanolic solution of 0.3 mM DPPH was prepared immediately before the analysis and 10 mg of each sample was dissolved in mL of dimethyl sulfoxide (DMSO) 0.1 mL of the prepared sample was transferred to a test tube containing 1.9 mL of DPPH radical solution The final concentration of each sample at the measurement step was 500 μg mL−1 at this stage Further dilutions were prepared from the stock solution: 250, 150, 100, 50, and 25 μg mL−1 and added to 1.9 mL of DPPH solution each time The reaction mixtures were subsequently incubated in a W Kukula-Koch et al 748 37 °C water bath for 30 min, and their absorbance was measured at 515 nm using UNICAM spectrophotometer, Helios, three times Ethanol was used as the negative control sample Percent inhibition of radicals was determined on the basis of comparison of the samples with a DMSO-treated control group The experiment was performed three times The values of UV absorbance were plotted on graphs to calculate the IC50 values of each extract The calculated IC50 values displayed the concentration of sample required to scavenge 50% DPPH free radicals DPPHscavenging activity was expressed as IC50 in μg mL−1 (see Table I) The lower the IC50 values, the greater the antioxidant activity To compare the scavenging properties of the investigated extracts, an IC50 value was calculated for ferulic acid as well Table I IC50 values of obtained fractions together with percentages of scavenged radicals depending on the sample’s concentration EV-FPA EV-B %b Concentrationa IC50c Concentrationa EV-C %b IC50c Concentrationa %b 500 68.2 500 90.7 500 53.2 250 46.1 250 72.1 250 44.6 150 32.5 150 10.9 150 27.12 100 29.9 100 5.1 100 26.1 50 22.9 50 4.8 50 25.5 25 18.1 25 2.7 25 21.8 Standard deviation IC50 of ferulic acid 290 ±5 205 ±8 IC50c 400 ±14 30 ± aExtracts’ dilution (μg mL−1) of scavenged radicals in comparison with the DMSO-treated blind probe cConcentration of extract scavenging half of radicals (μg mL−1) bPercent Results and Discussion Development of the Extraction Method According to Cha et al [21], methanolic extracts from Euonymus spp contain the highest concentration of phenolics Therefore, the authors of this paper chose this extrahent to obtain the best possible results of the presented antioxidant assay HPLC, 2D-TLC and an In Vitro Perspective 400 749 400 PD A-280n m v bW A Fraction A 350 350 300 300 250 250 mA U 200 mA U 200 150 150 56 100 100 50 50 0 -50 -50 10 15 20 25 30 35 40 45 50 55 60 65 70 75 Minut e s PD A-28 0n m vbW B 700 700 Fraction B 600 600 500 500 m AU 400 m AU 400 300 300 200 100 200 100 0 10 15 20 25 30 35 40 45 50 55 60 65 70 75 Minut es PD A-28 0n m vbW C 500 2500 2250 000 2000 750 1750 500 1500 250 1250 000 1000 750 750 500 250 500 250 0 -250 -250 10 15 20 25 30 35 40 45 50 55 60 65 70 Minut es Fig HPLC chromatograms of fraction A (VE-FPA), fraction B (VE-B), and fraction C (VE-C) recorded at 280 nm (1 – gallic acid, – protocatechuic acid, – p-hydroxybenzoic acid, – vanillic acid, – caffeic acid, – syringic acid, – p-coumaric acid, – ferulic acid, and – m-coumaric acid) 75 m AU m AU Fraction C 250 W Kukula-Koch et al 750 The method of Machalska et al constituted a standard procedure in the qualitative analysis of phenolic acids’ content in extracts [18] Due to the conducted hydrolyses, it was possible to trace phenolic acids present as bound with other secondary metabolites in the extract The differences in qualitative compositions of three investigated fractions were significant The qualitative compositions of all obtained fractions are presented in Fig The retention times of all traced phenolic acids together with their UV maxima obtained for methanol are listed in Table II Table II Retention times and UV maxima of traced phenolic acids No Compound Rt (min) UV maxima Caffeic acid 25.8 212, 244, 297, 327 Ferulic acid 37.4 210, 322 Gallic acid 6.5 234, 269 m-Coumaric acid 47.0 227, 278, 330 Protocatechuic acid 13.7 233, 258, 292 p-Hydroxybenzoic acid 20.8 218, 255 p-Coumaric acid 34.6 232, 310 Syringic acid 27.3 231, 275 Vanillic acid 24.9 232, 259, 290 Identification Phenolic Composition and Identification of Phenolic Compounds by 2D-TLC The phenolic contents of the obtained extracts of E verrucosus Scop – EVFPA, EV-B, and EV-C – were analyzed by 2D-TLC, and their identification was accomplished by the comparison of retention times with those of respective standards The quantitative compositions of phenolic compounds on TLC plates are shown in Fig 2D-TLC technique enabled rapid identification of ferulic, p-coumaric, p-hydroxybenzoic, protocatechuic, and syringic acids, which constitute the major compounds of the extracts according to HPLC tests described below The TLC systems applied on a cellulose covered TLC plate enabled rapid two-dimensional separation of phenolic acids present in the extracts The RF values of separated phenolics were as follows: ferulic acid – 8.7, p-coumaric acid – 7.5, p-hydroxybenzoic acid – 4.8, protocatechuic acid – 2.5, and syringic acid – 7.6 HPLC, 2D-TLC and an In Vitro Perspective 751 Fraction A Fraction B 8 7 3 2 Fraction C Fig Two-dimensional separation of phenolic acids from fractions A, B, and C of Euonymus verrucosus methanolic extract in UV light at 365 nm (2 – protocatechuic acid, – p-hydroxybenzoic acid, – syringic acid, – p-coumaric acid, – ferulic acid) Thanks to the two-dimensional separation, ferulic, p-coumaric, and syringic acids could be well separated from one another, as their RF values are close to those of 1D development Even though this technique is perceived as less sensitive, major phenolic constituents of the extracts could be identified in a quick and cheap way 752 W Kukula-Koch et al Phenolic Composition and Identification of Phenolic Compounds by HPLC–DAD In the current study, phenolic compounds in the extracts from the aerial parts of E verrucosus Scop were identified by HPLC–DAD Identification was based on the chromatographic behavior relative to authentic standards and UV spectral data as well as on the comparison with literature data The identification of constituents of the warted spindle extracts is shown in Fig HPLC method applied to assess the extracts confirmed the presence of nine phenolic acids To the authors' knowledge, this is the first time these compounds have been recognized in the investigated plant species Detailed information on UV-data as well as retention times of traced phenolic acids are available in Table II Fraction EV-B after alkaline hydrolysis was found to be the richest in phenolic acids Nine of them were identified on HPLC chromatogram, including gallic acid, protocatechuic acid, p-hydroxybenzoic acid, vanillic acid, syringic acid, caffeic acid, p-coumaric acid, ferulic acid, and m-coumaric acid Fraction EV-FPA (fraction A) rich in phenolics did not contain gallic acid This compound was not present as a free acid in the studied sample Fraction EV-C differed from the above samples The concentration of these compounds was significantly smaller (p < 0.05) Gallic acid was not present in this fraction, which took place in the case of sample EV-FPA Antioxidant Assay — DPPH Test The extraction conditions applied in this study were of significant influence on the phenolic content of samples The IC50 values calculated for the measured fractions are presented in Table I The table shows marked differences between tested samples and DPPH-scavenging properties Sample EV-B obtained with alkaline hydrolysis was characterized by the lowest IC50 value, which is due to its strongest antioxidant properties The elevated temperature did not lead to the decomposition of phenolic acids The IC50 value of EV-PTB (205 ± µg mL−1) remains higher than that of EV-FPA (290 ± μg mL−1) The weakest antioxidant activity was observed in the fraction after acidic hydrolysis Its IC50 value was calculated as 400 ± 14 μg mL−1 (see Table I) The compositions of HPLC chromatograms presented in Fig are relevant to the results of the performed DPPH assay HPLC, 2D-TLC and an In Vitro Perspective 753 To assess the antioxidant potential of the extracts, a sample of ferulic acid was included in the antioxidant activity assay as a reference compound with known radical-scavenging properties IC50 for ferulic acid in this method was calculated as 30 ± μg mL−1 On the basis of the above information, it can be concluded that overground parts of E verrucosus Scop display moderate antioxidant activity Conclusion The performed qualitative analysis of the phenolic content of the methanolic extracts of overground parts of E verrucosus Scop confirmed the presence of nine phenolic acids: gallic, protocatechuic, p-hydroxybenzoic, vanillic, syringic, caffeic, p-coumaric, ferulic, and m-coumaric acids This has been done for the first time for this plant species Phenolic compounds present both as free and conjugated to other metabolites were analyzed by HPLC–DAD and two-dimensional TLC HPLC with DAD detection traced nine phenolic acids in the extracts, also those of lower concentration The latter technique – the cheaper one – proved to be a rapid technique in the analysis of all major phenolic components of the extracts Furthermore, two-dimensional TLC enabled the identification of three phenolic acids with a similar retention factor: ferulic, p-coumaric, and syringic acids This technique may enable quick fingerprinting of extracts, as, within the course of the presented experiment, it visualized the major phenolic acids present in the samples The antioxidant capacity of all obtained extracts was measured in comparison to a standard ferulic acid The warted spindle tree extracts were found moderately active to scavenge free radicals The lowest IC50 value and the strongest antioxidant potential were calculated for fraction B containing phenolic acids after alkaline hydrolysis — 205 ± μg mL−1 The study on this species proved the antioxidant capacity of E verrucosus Scop for the first time As a result, the species extracts may be successfully applied as antioxidants, especially because E verrucosus Scop still remains a commonly spread species around Europe Acknowledgemnts The authors express their gratitude to Marta Infantes and Maria Ines Fernandes for their help and support in the realization of this project W Kukula-Koch et al 754 Declaration of Interest The authors report no conflict of interest The authors alone are responsible for the content and writing of the paper References [1] A.N Alvarenga and E.A Ferro, in: A Rahman (Eds) Studies in Natural Products Chemistry, Bioactive Natural Products (Part K), London, 2005, pp 635–702 [2] J.X Zhu, J Ren, J.J Qin, X.R Cheng, 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Composition and Identification of Phenolic Compounds by 2D -TLC The phenolic contents of the obtained extracts of E verrucosus Scop – EVFPA, EV-B, and EV-C – were analyzed by 2D -TLC, and their identification