SUPPLEMENTARY MATERIAL
Chemical profile by LC-MS/MS, GC/MS and antioxidant activities of
the essential oils and crude extracts of two Euphorbia species
Abdulselam Ertasa*, Mustafa Abdullah Yilmazb, Mehmet Firatc
a
Department of Pharmacognosy, Faculty of Pharmacy, Dicle University, 21280
Diyarbakir, Turkey
b
Dicle University Science and Technology Research and Application Center, 21280
Diyarbakır, Turkey
c
Department of Biology, Faculty of Education, Yüzüncü Yıl University, 65080 Van,
Turkey
*Corresponding author:Abdulselam Ertaş, Dicle University, Faculty of Pharmacy,
Department of Pharmacognosy,21280 Diyarbakir, Turkey. E-mail:
abdulselamertas@hotmail.com; abdulselam.ertas@dicle.edu.tr, tel: +90 412 248
8030/7536.
1
Abstract
In the current study, it was aimed to investigate the chemical composition and
antioxidant activities of two Euphorbia species. The major component of the
fatty acid compositions obtained from the petroleum ether extracts was
identified as palmitic acid for Euphorbia gaillardotii and E. macroclada. The
main constituents of the essential oils were identified as arachidic acid for E.
gaillardotii and tetratetracontane for E. macroclada. Among the studied
twenty-seven compounds, hesperidin, rutin, hyperoside and quinic, malic,
gallic and tannic acids were found to be the most abundant compounds in two
Euphorbia species. The methanol extracts of E. gaillardotii and E. macroclada
showed strong antioxidant activity in all tested methods. Particularly, IC50
values of E. macroclada methanol extract that was the richest in terms of total
phenolic-flavonoid contents, were found to be lower than α-tocopherol and
BHT (butylated hydroxytoluene) in β-carotene bleaching, DPPH (2,2-diphenyl1-picrylhydrazyl) free and ABTS cation radical scavenging methods.
Keywords: Euphorbia gaillardotii; Euphorbia macroclada; LC-MS/MS;
GC/MS; antioxidant activity.
2
Experimental
Chemicals and instruments
Phenolic, essential oil and fatty acid compositions of Euphorbia species were
determined by using Shimadzu UHPLC ESI MS/MS (Shimadzu, Kyoto, Japan), and
GC/MS instruments, respectively. A Shimadzu UV spectrophotometer and BioTek
Power Wave XS microplate reader (USA) were used for the activity assays. 2,2′azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) (purity:
97.5%), and butylated hydroxytoluene (BHT) (≥99%) were purchased from Merck
(Germany); quercetin (%95), protocatechuic acid (%97), chrysin (%97), rutin (%94),
hesperatin (%95), naringenin (%95), rosmarinic acid (%96), vanillin (%99), pcoumaric acid (%98), caffeic acid (%98), chlorogenic acid (%95), formic acid (≤
100%), 2,2-diphenyl-1-picrylhydrazyl (DPPH) (≥ 95%), -carotene, linoleic acid (≥
93%), Tween 40, pyrocathecol (≥ 99%) and 5,5-dithiobis-(2-nitro benzoic acid)
(DTNB) (≥ 98%), were obtained from Sigma (Germany); -tocopherol (≥ 95.5%)
was from Aldrich (Germany).
Plant material
We collected the whole plants of Euphorbia gaillardotii BOISS. ET BLANCHE and
Euphorbia macroclada BOISS from southeast of Turkey (Diyarbakır) in July 2013 by
Dr. A. Ertaş (Department of Pharmacognosy, Faculty of Pharmacy, Dicle University)
and they were identified by Mehmet Fırat (Department of Biology, Faculty of
Education, Yüzüncü Yıl University). Voucher specimens have been strored in the
Herbarium of Yüzüncü Yıl University(VANF: 30185 for Euphorbia gaillardotii and
VANF: 30186for Euphorbia macroclada).
Preparation of plant extracts for biological activities and GC/MS
Whole plant materials were dried and powdered, 100 g of each plant material were
sequentially macerated with petroleum ether and methanol for 24 hour at 25°C. The
solvents were evaporated after filtration thus crude extracts were obtained. Essential
oils were obtained using a Clevenger apparatus from the aerial parts of plants (100 g),
which were crumbled into small pieces and soaked in distilled water (500 ml) for 3 h.
3
Esterification of total fatty acids and GC/MS conditions
A hundred milligram of the petroleum ether extract was refluxed in 0.1 M KOH
solution in 2 mL of methanol during 1h, the solution was cooled and 5 mL of water
was added. The aqueous mixture was neutralized with 0.5 mL of HCl solution, it was
extracted with diethyl ether: hexane (1 : 1; 3.5 mL) mixture. The separating organic
phase was washed with 10 mL water, and dried over anhydrous Na2SO4. The solvent
was evaporated under vacuum and then fatty acid methyl esters were obtained. The
analyses were performed using a Thermo Scientific Polaris Q GC-MS/MS. GC/MS
procedure described by Kılıc et al. (2007) was applied.
Preparation and GC/MS conditions for essential oil
Essential oil was obtained using a Clevenger apparatus from the aerial parts of
theplant (100 g), which was crumbled into small pieces and soaked in distilled water
(500 ml) for 3 h. The obtained essential oil was dried over anhydrous Na2SO4 and
stored at +4 C for a sufficient period of time.The essential oil was diluted using
CH2Cl2 (1:3 volume/volume) prior to GC/FID (Gas Chromatography/Flame
Ionization Detector) and GC/MS analysis. GC/FID analysis was performed using
Thermo Electron Trace GC/FID detector and GC/MS analysis was performed using
the same GC and Thermo Electron DSQ MS.
The following GC conditions were applied for both GC/MS and GC/FID
analyses. The GC oven temperature was kept at 60 C for 10 min and programmed to
280 °C at a rate of 4 °C/min and then kept constant at 280 °C for 10 min. A nonpolar
Phenomenex DB5 fused silica column (30 m0.32 mm, 0.25 μm film thickness) was
used with helium at 1 mL/min (20 psi) as a carrier gas. The split ratio was adjusted to
1:50, the injection volume was 0.1 μL, and EI/MS was recorded at 70 eV ionization
energy. The mass range was m/z 35–500 amu. n-Alkanes (C8-C24) and pentacosane,
hexacosane, octacosane, tricontane, and tetracontane from Ultrakkit WRK-101
Hydrocarbons # 2 were used as reference points in the calculation of Kovats Indices
(KI) by the same conditions(Altun et al. 2007; Ertas et al. 2014).
Identification of the compounds was based on comparing their retention times
and mass spectra with those obtained from authentic samples and/or the NIST and
Wiley spectra as well as data from the published literature.
4
Determination of total phenolic and flavonoid contents
The concentrations of phenolic content in the crude extracts were expressed as
micrograms of pyrocatechol equivalents (PEs) (Slinkard & Singleton 1977). 100 μL
solution of the samples in methanol was added to 4.6 mL of distilled water and 100
μL of Folin-Ciocalteu’s Reagent, and mixed thoroughly. After 3 min, 300 μL sodium
carbonate (2%) was added to the mixture and shaken intermittently for 2 h at room
temperature. The absorbance was read at 760 nm. The concentration of phenolic
compounds was calculated according to the following equation that was obtained
from standard pyrocatechol graphic:
Absorbance = 0.0164 pyrocatechol (μg) + 0.0266 (R2 = 0.9969)
Measurement of flavonoid content of the crude extracts was based on the
method described by Moreno et al. with a slight modification and results were
expressed as quercetin equivalents (Moreno et al. 2000). An aliquot of 1 mL of the
solution (contains 1 mg of crude extract in methanol) was added to test tubes
containing 0.1 mL of 10% aluminium nitrate, 0.1 mL of 1 M potassium acetate and
3.8 mL of methanol. After 40 min at room temperature, the absorbance was
determined at 415 nm.The concentration of flavonoid compounds was calculated
according to the following equation:
Absorbance = 0.1519 quercetin (μg) – 0.1294
(R2 = 0.9986)
Antioxidant activity of the extracts
To establish the antioxidant activity, we used the β-carotene-linoleic acid test system,
DPPH free radical and ABTS cation radical scavenging activity methods.
-Carotene bleaching method
0.5 mg of -carotene in 1 mL of chloroform was added into linoleic acid (25 L) and
Tween 40 emulsifier (200 mg) mixture. After evaporating chloroform, 100 mL of
distilled water saturated with oxygen was added followed by shaking, 160 μL of this
mixture was transferred into different test tubes containing 40 μL of the sample
solutions at different concentrations. The emulsion was added to each tube, the zero
time absorbances of the values were read at 470 nm. The mixture was incubated for 2
h at 50 C (Miller 1971).
5
Free radical scavenging activity method
0.1 mM, 160 µL of DPPH solution in methanol was added to 40 µL of sample
solutions in methanol at different concentrations. After 30 min. the absorbance values
were read at 517 nm. The DPPH free radical scavenging potential was calculated
using the following equation:
DPPH scavenging effect (Inhibition %) =
Acontrol  Asample
Acontrol
 100
AControl is the initial concentration of the DPPH•
ASample is the absorbance of the remaining concentration of DPPH• in the presence of
the extracts or positive controls (Blois 1958).
ABTScation radical decolorization assay
Seven milimolar ABTS in H2O was added to 2.45 mM potassium persulfate to
produce ABTS•+and solution was stored in the dark at 25 C for 12 h. The prepared
solution was diluted with ethanol to get an absorbance of 0.700 ± 0.025 at 734 nm.
ABTS•+ solution (160 µL) was added to each sample solution at different
concentrations. After 30 min, the percentage inhibition at 734 nm was read for each
concentration relative to a blank absorbance (methanol). The following equation was
used to calculate the scavenging capability of ABTS•+ (Re et al. 1999; Ertas et al.
2014):
ABTS•+ scavenging effect (Inhibition %) =
Acontrol  Asample
Acontrol
× 100
Identification and Quantitation of Phenolic Compounds
Preparation of plant extracts for LC-MS/MS
The air-dried and powdered plant materials (10g) were extracted three times with 100
ml of methanol for 24 hours at room temperature. The solvents were removed from
the filtered extract solutions under vacuum at 30 ºC in a rotary evaporator, until dry
methanol extracts were obtained. Dry filtrates were diluted to 250 mg/L and fitrated
with 0.2 μm microfiber filter prior to LC-MS/MS analysis.
Instruments and Chromatographic Conditions
LC-MS/MS analysis of the phenolic compounds were performed by using a Nexera
model Shimadzu UHPLC coupled to a tandem MS instrument. The liquid
chromatograph was equipped with LC-30AD binary pumps, DGU-20A3R degasser,
6
CTO-10ASvp column oven and SIL-30AC autosampler.The chromatographic
seperation was performed on a C18 reversed-phase Inertsil ODS-4 (150 mm×4,6 mm,
3µm) analytical column. The column temperature was fixed at 40˚C. The elution
gradient consisted of mobile phase A (water, 5mM ammonium formate and 0.1%
formic acid) and mobile phase B (methanol, 5mM ammonium formate and 0.1%
formic acid). The gradient program with the following proportions of solvent B was
applied t (min), %B: (0, 40), (20, 90), (23.99, 90), (24, 40), (29, 40). The solvent flow
rate was maintained at 0.5 mL/min and injection volume was settled as 4 µL.
MS Instrumentation
MS detection was performed using Shimadzu LCMS 8040 model triple quadrupole
mass spectrometer equipped with an ESI source operating in both positive and
negative ionization modes. LC-MS/MS data were collected and processed by
LabSolutions software (Shimadzu, Kyoto, Japan). The multiple reaction monitoring
(MRM) mode was used to quantify the analytes: the assay of investigated compounds
was performed following two or three transitions per compound, the first one for
quantitative purposes and the second and/or the third one for confirmation.
Optimization of the LC-MS/MS Method
Subsequent to several combinations of trials, a gradient of methanol (5mM
ammonium formate and 0.1% formic acid) and water (5mM ammonium formate and
0.1% formic acid) system was concluded to be the best mobile phase solution. For
rich ionization and the seperation of the molecules, the mentioned mobile phase was
proved to be the best of all. ESI source was chosen instead of APCI (Atmospheric
Pressure Chemical Ionization) and APPI (Atmospheric Pressure Photoionization)
sources as the phenolic compounds were small and relatively polar molecules.
Tandem mass spectrometry was decided to be used for the current study since this
system is commonly used for its fragmented ion stability (Ertas et al. 2014). The
working conditions were determined as interface temperature; 350 C, DL
temperature; 250 C, heat block temperature; 400 C, nebulizing gas flow (Nitrogen);
3L/min and drying gas flow (Nitrogen); 15L/min.
Method Validation Parameters for LC-MS/MS
In the current study, twenty-four phenolic compounds (flavonoids, flavonoid
glycosides, phenolic acids, phenolic aldehyde, coumarin) and three non-phenolic
7
organic acids which are widespread in edible plant materials were qualified and
quantified in two Euphorbia species. In the chromatographic analysis of phenolic
compounds, gradient seperation was applied. Linear regression equations of the
phenolic compound standards wererepresented in Table S3. The linearity of the
phenolic standards was affirmed in the following ranges: 0,025-1 mg/L, 0,01-4 mg/L,
0,25-10 mg/L (Table S3). The regression coefficient of each calibration graph was
found to be higher than 0.99. The limit of detection (LOD) and limit of quantitation
(LOQ) of the method reported in this study were dependent on the calibration curve
established from six measurements. LOD and LOQ of the method were determined by
using the equations 3S/N and 10S/N respectively (S/N refers to the Signal to noise
ratio) (Table S3). Fordifferent compounds, LOD ranged from 0.05to 25.8 µg/L and
LOQ ranged from 0.17to 85.9 µg/L (Table S3). Furthermore, the recovery of the
phenolic compounds standards ranged from 97% to 106.3%.
Statistical analysis
The results of the antioxidant activities and total phenolic-flavonoid contents were
expressed as means  SEM. The results were evaluated using an unpaired t-test and
ANOVA variance analysis with the NCSS statistical computer package. The
differences were considered statistically significant at p 0.05.
8
A
B
C
Figure S1.LC-MS/MS chromatograms of A: 250 ppb standard mix, B: E. gaillardotiimethanol extract,
C: E. macrocladamethnol extract. 1: Quinic acid, 2: Malic acid, 3: tr-Aconitic acid, 4: Gallic acid, 5:
Chlorogenic acid, 6: Protocatechuic acid, 7: Tannic acid, 8: tr- caffeic acid, 9: Vanillin, 10: p-Coumaric
acid, 11: Rosmarinic acid, 12: Rutin, 13: Hesperidin, 14: Hyperoside, 15: 4-OH Benzoic acid, 16:
Salicylic acid, 17: Myricetin, 18: Fisetin,19: Coumarin, 20: Quercetin, 21: Naringenin, 22: Hesperetin,
23: Luteolin, 24: Kaempferol, 25: Apigenin, 26: Rhamnetin, 27: Chrysin.
9
Table S1.GC-MS analysis of the petroleum ether extracts of E. gaillardotii (EGP) and E. macroclada
(EMP).
Rt (min)a
12.00
14.39
18.60
25.27
30.64
30.77
30.86
31.54
37.38
39.36
43.82
Constituentsb
Lauric acid
10-Undecenoic acid
Myristic acid
Palmitic acid
Linoleic acid
Oleic acid
Linolenic acid
Stearic acid
Arachidic acid
Docosane
Behenic acid
Composition (%)c
EGP
EMP
1.7
1.3
0.6
5.8
4.5
32.5
33.3
14.5
26.6
21.3
14.2
14.0
11.0
4.2
4.2
4.5
1.4
1.1
1.3
1.3
Total
99.8
99.5
a
Retention time (as minutes).
b
A nonpolar Phenomenex DB-5 fused silica colum.
c
Percentage of relative weight.
Table S2. Chemical composition of the essential oils fromE. gaillardotii (EGEss) and E. macroclada
(EMEss).
RIa
Constituentsb
Composition (%)c
1188
1368
1377
1409
1455
1484
1498
1528
1583
1654
1677
1800
1986
2156
2171
2185
2259
2366
2852
2900
3094
3508
3600
4400
α -Terpineol
Mint furanone
α-Copaene
Caryophyllene
α-Humulene
Valencene
α-Silenene
α-Muurolene
Caryophyllene oxide
Eudesmol
Cadalene
Octadecane
Hexadecanoic acid
1-Nonadecanol
Butyl phthalate
Z-8-Octadecen-1-ol acetate
2,5-Di-tert octyl-p-benzoquinone
Arachidic acid
1-Hexacosanol
Nonacosane
Ethyl iso-allocholate
17-Pentatriacontene
Hexatriacontane
Tetratetracontane
Total
EGEss
0.5
8.4
0.4
0.3
0.3
5.2
0.8
4.0
0.3
5.6
8.0
3.3
3.0
3.2
32.0
3.0
3.1
8.7
6.2
96.3
EMEss
0.7
6.0
0.8
0.5
0.4
7.3
1.5
1.2
0.6
1.4
1.9
1.9
4.0
3.2
2.2
1.9
2.2
2.0
12.0
42.7
93.4
a
RI Retention indices (DB-5 column)
A nonpolar Phenomenex DB-5 fused silica column
c
Percentage of relative weight.
b
10
Table S3. Analytical parameters of LC- MS/MS method
Analyte
no
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
Analytes
Quinic acid
Malic acid
tr-Aconitic acid
Gallic acid
Chlorogenic acid
Protocatechuic acid
Tannic acid
tr- caffeic acid
Vanillin
p-Coumaric acid
Rosmarinic acid
Rutin
Hesperidin
Hyperoside
4-OH Benzoic acid
Salicylic acid
Myricetin
Fisetin
Coumarin
Quercetin
Naringenin
Hesperetin
Luteolin
Kaempferol
Apigenin
Rhamnetin
Chrysin
RT
3.36
3.60
4.13
4.25
5.29
5.51
6.30
7.11
8.57
9.17
9.19
9.67
9.69
9.96
11.38
11.39
11.42
12.10
12.18
13.93
14.15
14.80
14.84
14.85
16.73
18.41
20.60
Equation
f(x)=33.6626*x+25132.9
f(x)=93.6102*x-5673.77
f(x)=79.2908*x-28416.2
f(x)=358.069*x+26417.5
f(x)=48.9828*x+26779.7
f(x)=36.8568*x+6197.38
f(x)=90.2704*x+30233.2
f(x)=1585.16*x+83957.5
f(x)=44.5478*x-574.867
f(x)=73.5303*x+27064.3
f(x)=18.0298*x-1149.86
f(x)=51.8835*x+3841.66
f(x)=195.773*x+105641
f(x)=0.978146*x+827.221
f(x)=635.003*x+54284.6
f(x)=915.178*x+72571.4
f(x)=54.2823*x+5414.67
f(x)=331.870*x+34409.0
f(x)=236.639*x+34370.3
f(x)=206.102*x+1693.14
f(x)=1100.55*x+39055.7
f(x)=160.323*x+6545.07
f(x)=111.474*x+3057.10
f(x)=20.9677*x+571.241
f(x)=543.793*x+18525.6
f(x)=110.091*x+632.444
f(x)=698.787*x+23531.7
R2a
RSD%b
0.9927
0.9975
0.9933
0.9901
0.9932
0.9991
0.9955
0.9942
0.9995
0.9909
0.9992
0.9971
0.9973
0.9549
0.9925
0.9904
0.9991
0.9988
0.9924
0.9995
0.9956
0.9961
0.9992
0.9917
0.9954
0.9994
0.9965
0.0388
0.1214
0.3908
0.4734
0.1882
0.5958
0.9075
1.0080
0.4094
1.1358
0.5220
0.8146
0.1363
0.2135
1.4013
0.6619
2.8247
2.4262
0.4203
4.3149
2.0200
1.0164
3.9487
0.5885
0.6782
2.5678
1.5530
Linearity
Range
(mg/L)
250-10000
250-10000
250-10000
25-1000
250-10000
100-4000
100-4000
25-1000
250-10000
100-4000
250-10000
250-10000
250-10000
100-4000
25-1000
25-1000
100-4000
100-4000
100-4000
25-1000
25-1000
25-1000
25-1000
25-1000
25-1000
25-1000
25-1000
LOD/LOQ
(µg/L)
Recovery
(%)
22.3 / 74.5
19.2 / 64.1
15.6 / 51.9
4.8 / 15.9
7.3 / 24.3
25.8 / 85.9
10.2 / 34.2
4.4 / 14.7
10.1 / 33.7
15.2 / 50.8
10.4 / 34.8
17.0 / 56.6
21.6 / 71.9
12.4 / 41.4
3.0 / 10.0
4 / 13.3
9.9 / 32.9
10.7 / 35.6
9.1 / 30.4
2.0 / 6.8
2.6 / 8.8
3.3/ 11.0
5.8 / 19.4
2.0 / 6.6
0.1 / 0.3
0.2 / 0.7
0.05 / 0.17
103.3
101.4
102.8
102.3
99.7
100.2
97.8
98.6
99.2
98.4
101.7
102.2
100.2
98.5
106.2
106.2
106.0
96.9
104.4
98.9
97.0
102.4
105.4
99.1
98.9
100.8
102.2
a
RT: Retention time
R2: coefficient of determination
c
RSD: relative standard deviation
d
LOD/LOQ(µg/L): Limit of deteection/Limit of quantification
b
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