hplc two dimensional tlc determination of phenolic content and an in vitro perspective to antioxidant potential of euonymus verrucosus scop extracts

The chroma-tographical and spectral data revealed the presence of several simple phenolic acids gal-lic, protocatechuic, p-hydroxybenzoic, vanilgal-lic, syringic, caffeic, p-coumaric,

0231–2522 © 2014 Akadémiai Kiadó, Budapest

HPLC, Two-Dimensional TLC Determination of

Phenolic Content, and an In Vitro Perspective to Antioxidant Potential of Euonymus verrucosus

Scop Extracts

W K UKULA -K OCH 1,* , J W IDELSKI 1 , W K OCH 2 , AND K G ŁOWNIAK 1

1 Chodzki St., 20-093 Lublin, Poland

4a Chodzki St., 20-093 Lublin, Poland

*E-mail: virginia.kukula@gmail.com

Summary The presence of phenolic content in overground extracts of Euonymus

verruco-sus Scop – commonly growing in Europe – has been reported recently The

chroma-tographical and spectral data revealed the presence of several simple phenolic acids

(gal-lic, protocatechuic, p-hydroxybenzoic, vanil(gal-lic, syringic, caffeic, p-coumaric, feru(gal-lic, 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 tech-nique in the identification process of major phenolic constituents 2,2-Diphenyl-1-picrylhydrazyl (DPPH) radical-scavenging tests, expressed as IC 50 , revealed the most beneficial results for the fraction after alkaline hydrolysis and yielded 205 ± 8 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 encom-passes 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

[2] Currently, the plant is known for its antibacterial, insecticidal, and anti-hyperglycemic 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 Two-dimensional thin-layer chromatography (2D-TLC) as well as high-performance 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 sub-stances (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

phe-nolic acids in the extracts of E verrucosus Scop has a vital role in the

anti-oxidant 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)–scaven-ging properties Their composition clearly influenced the scaven(DPPH)–scaven-ging prop-erties 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 hy-droxide, dichloromethane, diethyl ether reagent grade, DMSO, ethanol, formic acid, hydrochloric acid (37%), methanol reagent grade, sodium hy-drocarbonate, sodium formate, sulphuric acid, and toluene reagent grade

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 com-bined (3.51 g and 3.97 g, respectively) and evaporated to dryness under re-duced pressure (ca 150 mbar) using the rotary evaporator The dried ex-tracts 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 ex-tract was washed with five 10 mL portions of 5% solution of sodium hydro-carbonate in a separating funnel Next, the carbonate phase was collected

separately, and a solution of 18% hydrochloric acid was added up to the

pH of 3

The carbonate extract of pH = 3 was then placed in separating funnel and washed five times with 20 mL of diethyl ether The ether extracts con-tained free phenolic acids (FPA) from the plant material

Water extracts – hydrolysis Acidic and alkaline hydrolyses were per-formed 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 per-formed on the half volume of water extract It was heated under cooler with barium hydroxide – Ba(OH)2 – in pH of 12, for 1 h Next, the pH was slowly adjusted to 1.5 with the addition of 10% and later the 96% solution of sul-phuric acid The extracts were moved to the separating funnel and were rinsed five times with 20 mL portions of diethyl ether Next, the ether ex-tracts were collected separately from water exex-tracts and rinsed five times with 5% solution of sodium hydrocarbonate (portions of 10 mL)

Carbonate extracts were adjusted to the pH of 3 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 trans-ferred 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, car-bonate extracts were transferred into a separating funnel and washed with five portions of diethyl ether Ether extracts contained phenolic acids un-leashed after hydrolysis

Identification

Two-Dimensional Thin Layer Chromatography 2D-TLC Profiling

Twenty milligrams of each extract were dissolved in 2 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 3 (CAMAG, Muttenz, Switzerland)

The plate was dried in the air and conditioned for approximately

10 min 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

verti-cally over 9 cm in a flat bottom chamber (CAMAG) at 21 °C and with rela-tive humidity of 57% in the following solvent system: toluene–acetic acid–

acetonitrile (75.5:10:7.5 v/v/v) After drying at room temperature for 1 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, coumaric, protocatechuic, syringic, vanillic, and p-hydroxybenzoic acids

HPLC Profiling

Ten milligrams of each extract (EV-FPA, EV-B, EV-C) were redissolved in

2 mL of methanol (HPLC grade), filtered through a 0.45-μm nylon mem-brane filter (Waters,) and subjected to RP-HPLC–DAD analysis The Agilent

1100 system (Santa Clara, USA) coupled with an autosampler and a diode-array 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) Dis-covery-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 min A–B (80:20); in 60 min A–B (60:40); in 70 min A–B (95.5:0.5); running time: 75 min; and in 15 min conditioning of the column The flow rate was set at 1 mL min−1, and the chromatograms were recorded at 260, 280, and 365 nm by monitoring

spec-tra 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 ap-plied in this study was a modification of the methods of Lee et al and Ku-kula-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 2 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 so-lution 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 so-lution each time The reaction mixtures were subsequently incubated in a

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

compari-son 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

con-centration of sample required to scavenge 50% DPPH free radicals

DPPH-scavenging 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

calcu-lated 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 EV-C

Concentra-tion a

% b IC50 c

Concentra-tion a

% b IC50 c

Concentra-tion a

% b IC50 c

25 18.1

290

25 2.7

205

25 21.8

400

Standard

IC 50 of

a Extracts’ dilution (μg mL −1 ).

b Percent of scavenged radicals in comparison with the DMSO-treated blind probe.

c Concentration of extract scavenging half of radicals (μg mL −1)

Results and Discussion

Development of the Extraction Method

According to Cha et al [21], methanolic extracts from Euonymus spp

con-tain the highest concentration of phenolics Therefore, the authors of this

paper chose this extrahent to obtain the best possible results of the

pre-sented antioxidant assay

Minut es

0 5 10 15 20 25 30 35 4 0 45 50 55 60 65 70 7 5

- 250

0

250

500

750

1 000

1 250

1 500

1 750

2 000

2 250

2 500

- 250 0 250 500 750 1000 1250 1500 1750 2000 2250 2500

PD A-28 0n m

vbW C

Fraction C

2

3 4

5 6

8

7

Minut es

0

100

200

300

400

500

600

700

0 100 200 300 400 500 600 700

PD A-28 0n m

vbW B

1

7

3

2

5

6

Fraction B

Minut e s

-50

0

50

100

150

200

250

300

350

400

- 50 0 50 100 150 200 250 300 350

400

PD A- 280n m

v bW A

2

3

8

7

6

5

Fraction A

Fig 1 HPLC chromatograms of fraction A (VE-FPA), fraction B (VE-B), and fraction C (VE-C)

recorded at 280 nm (1 – gallic acid, 2 – protocatechuic acid, 3 – p-hydroxybenzoic acid, 4 – vanillic

acid, 5 – caffeic acid, 6 – syringic acid, 7 – p-coumaric acid, 8 – ferulic acid, and 9 – m-coumaric acid)

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 1

The retention times of all traced phenolic acids together with their UV

max-ima obtained for methanol are listed in Table II

Table II Retention times and UV maxima of traced phenolic acids

Identification

Phenolic Composition and Identification of Phenolic Compounds by 2D-TLC

The phenolic contents of the obtained extracts of E verrucosus Scop –

EV-FPA, EV-B, and EV-C – were analyzed by 2D-TLC, and their identification was accomplished by the comparison of retention times with those of re-spective standards The quantitative compositions of phenolic compounds

on TLC plates are shown in Fig 2

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

sy-ringic acid – 7.6

Fraction A Fraction B

8

7 6

3

2

8

7 6

3 2

Fraction C

3

2

Fig 2 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,

3 – p-hydroxybenzoic acid, 6 – syringic acid, 7 – p-coumaric acid, 8 – ferulic acid)

Thanks to the two-dimensional separation, ferulic, p-coumaric, and sy-ringic 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 pheno-lic constituents of the extracts could be identified in a quick and cheap way

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 1

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,

in-cluding gallic acid, protocatechuic acid, p-hydroxybenzoic acid, vanillic

ac-id, syringic acac-id, caffeic acac-id, p-coumaric acac-id, ferulic acac-id, and

m-coumaric acid

Fraction EV-FPA (fraction A) rich in phenolics did not contain gallic

ac-id 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

differ-ences 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 ele-vated temperature did not lead to the decomposition of phenolic acids The IC50 value of EV-PTB (205 ± 8 µg mL−1) remains higher than that of EV-FPA (290 ± 5 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 1 are

relevant to the results of the performed DPPH assay