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ORIGINAL ARTICLE
The article was published by Academy of Chemistry of Globe Publications
www.acgpubs.org/RNP © Published 04/01/2015 EISSN:1307-6167
Rec. Nat. Prod. 9:3 (2015) 312-322
Chemical Constituents, in vitro Antioxidant and Antiproliferative
Activities of Perralderia coronopifolia Coss. subsp. eu-coronopifolia
M. var. typica M. extract
Sara Boussaha1, Khadidja Bekhouche1, Azzedine Boudjerda1, Francisco Leόn2,
Serkan Koldaş3, Ayse Sahin Yaglioglu3, İbrahim Demirtaş3, Ignacio Brouard2,
Eric Marchioni4, Djamila Zama1, Samir Benayache1 and Fadila Benayache1*
1Unité de Recherche Valorisation des Ressources Naturelles, Molécules Bioactives et Analyses
Physicochimiques et Biologiques (VARENBIOMOL), Université Constantine 1,
25000 Constantine, Algérie
2Instituto de Productos Naturales y Agrobiología, CSIC, Av. Astrofísico Fco.
Sánchez, 3, 38206 La Laguna, Tenerife, Spain
3Plant Research Laboratory, Chemistry Department, Çankırı Karatekin University,
Ballica Campus, 18100 Çankırı, Türkiye
4Equipe de Chimie Analytique des Molécules Bioactives (IPHC-LC4, UMR 7178), Université de
Strasbourg, Faculté de Pharmacie, 74 Route du Rhin, 67400 Illkirch, France.
(Received April 04, 2014; Revised September 28, 2014; Accepted October 09, 2014)
Abstract: Phytochemical investigations of extracts from the aerial parts (leaves and flowers) of Perralderia
coronopifolia Cosson resulted in the isolation of nine secondary metabolites corresponding to three flavonoids:
rhamnazin (1), chrysosplenol D (3), and (2R, 3R) taxifolin (4), two monoterpene glycosides : myrtenol- β-D-
glucopyranoside- 6'-O-acetate (2) and myrtenol β-D-glucopyranoside (7), a disaccharide: sucrose (9) and three di-O-
caffeoylquinic acid derivatives : methyl 3, 5-di-O-caffeoyl quinate (5) and methyl 3,4-di-O-caffeoyl quinate (6) as a
mixture and 1,5-di-O-caffeoylquinic acid (8). The structures were identified by spectroscopic methods such as 1H and
13C NMR, COSY, HSQC and HMBC experiments, HRESI-MS and comparison with literature data. Myrtenol-β-D-
glucopyranoside-6'-O-acetate (2) was isolated in pure and native state for the first time. The other compounds are new
for the genus Perralderia Cosson. The ethyl acetate extract showed a high antioxidant effect, especially DPPH radical
scavenging activity with IC50=7.01±0.28µg/mL) compared to ascorbic acid (IC50= 5±0.1µg/mL. This extract also
showed antiproliferative activity against HeLa (human cervix carcinoma) and C6 (rat brain tumor) cells.
Keywords: Asteraceae; Perralderia coronopifolia; flavonoids; caffeoylquinic acids; terpenoids; antioxidant activity;
antiproliferative activity. © 2015 ACG Publications. All rights reserved.
1. Introduction
The North African genus Perralderia (Asteraceae – Inuleae – Inulinae) includes three species. They
grow in the elevated regions of Morocco, Algeria and Libya in the north-western part of the African
continent, primarily in the mountainous regions. Perralderia paui Font Quer is found in the North of
Morocco, P. garamantum Asch occurs in Libya and P. coronopifolia Coss. which includes two subspecies,
subsp. purpurascens (Coss. ex Batt.) M. and subsp. eu-coronopifolia M. with two varieties, var. typica M.
and var. dessignyana (Hochr.) M. grow in Algeria [1, 2]. As part of our ongoing program of research of
new molecules with potential biological activity from plants [3-10], we report our results on P.
coronopifolia Coss. subsp. eu-coronopifolia M. var. typica M. growing in the Algerian Sahara. Our first
selection criterion for this plant was based on the fact that considering the difficult conditions to which
Boussaha et.al., Rec. Nat. Prod. (2015) 9:3 312-322 313
Saharan plants are subjected (hydrous insufficiency and oxidative stress), they are likely to accumulate
specific secondary metabolites to survive, to adapt and to develop. Moreover, in the best of our
knowledge, no reports on the isolation of any secondary metabolites from this plant are available to date. In
the present work on the chemical constituents of the chloroform and ethyl acetate soluble parts of the
aqueous-EtOH extract of the aerial parts (leaves and flowers), an acylated monoterpene glucoside has been
isolated in pure and native state for the first time, together with eight known secondary metabolites. We
report here the chemical study of these compounds, the free radical DPPH scavenging potential, the
antiproliferative activities against HeLa (human cervix carcinoma) and C6 (rat brain tumor) cells of the
ethyl acetate soluble part.
2. Materials and Methods
2.1. General
UV spectra were recorded using a Shimadzu model UV-1700 spectrophotometer. NMR spectra were
obtained by Bruker model Avance 400 and AMX-500 spectrometers with standard pulse sequences,
operating at 400 and 500 MHz for 1H and 100 and 125 MHz for 13C, respectively. MeOH-d4, DMSO-d6 or
CDCl3 were used as solvents and TMS as internal standard. EIMS were taken on a Micromass model
Autospec (70 eV) spectrometer. HRESI-MS was performed with a LCT Premier XE Micromass Waters
spectrometer in positive ionization mode (Waters Corporation). Column chromatography (CC) was carried
out with Si gel Fluka (cat. 60737) (40-63 μm), and column fractions were monitored by TLC Si gel 60
F254, 0.2 mm, Macherey Nagel (cat. 818-333) by detection with a spraying reagent
(CH3COOH/H2O/H2SO4; 80:16:4) followed by heating at 100 °C. Preparative TLC was carried out on Si
gel 60 PF254 + 366 (20 × 20 cm, 1 mm thickness, Analtech cat. 02014).
2.2. Plant materiel and chemicals
The plant material was collected from the area of Taghit in the south-west of Algeria on May 2011 and
authenticated by M. Mohamed Benabdelhakem, director of the nature preservation agency, Bechar. A
voucher specimen (PCA0511-TAG-ALG-52) has been deposited at the Herbarium of the
VARENBIOMOL research unit, Constantine 1 University. The chemicals which were used for the assays
were in analytical grade and obtained from Sigma–Aldrich and Roche.
2.3. Extraction and Isolation
Air-dried leaves and flowers (1400 g) of Perralderia coronopifolia (Asteraceae) were macerated at
room temperature with EtOH–H2O (80:20, v/v) for 48 h, three times. After filtration, the filtrate was
concentrated and dissolved in H2O (650 ml). The resulting solution was extracted successively with CHCl3,
EtOAc and n-butanol. The organic phases were dried with Na2SO4, filtered using common filter paper and
concentrated in vacuum at room temperature to obtain the following extracts: CHCl3 (2 g), EtOAc (7 g) and
n-butanol (40 g).
The CHCl3 extract was fractionated by CC (silica gel; n-hexane/EtOAc step gradients and then with
increasing percentages of MeOH) to yield 24 fractions (F1-F24) obtained by combining the eluates on the
basis of TLC analysis. Fraction F15 (70 mg) (n-hexane/EtOAc, 65:35), was submitted to preparative plates
of silica gel TLC (CHCl3 /EtOAc,1:1) and purified on a Sephadex LH-20 column to give rhamnazin (1) (2
mg) [11]. Fraction F17 (277 mg) (n-hexane/EtOAc, from 55:45 to 45:55) was chromatographed on a silica
gel column (CHCl3 /EtOAc) to give myrtenol-β-D-glucopyranoside-6'-O-acetate (2) (56 mg) [12]. The
EtOAc extract (7 g) was chromatographed on a silica gel column (CH2Cl2/Me2CO step gradients and then
with increasing percentages of MeOH) to yield 25 fractions (Fr1-Fr25) according to their TLC behavior.
Fraction Fr7 (90.4 mg) (CH2Cl2/Me2CO, 75:25) was submitted to preparative silica gel TLC
(CHCl3/MeOH/H2O, 9.8:1.1:0.1) and purified over Sephadex LH-20 column eluted with MeOH to afford
Chemical composition of Perralderia coronopifolia 314
chrysosplenol D (3) (3 mg) [13, 14] and (2R, 3R)-taxifolin or dystilin (4) (4 mg) [15]. Fraction Fr13 (82 mg)
(CH2Cl2/Me2CO, 70:30) was fractionated on a Sephadex LH-20 column using MeOH as eluent to give 7
sub-fractions. Sub-fractions 3 and 4 which had similar composition were combined and submitted to
preparative TLC (CHCl3/MeOH/H2O, 8:2:0.1) and further purified by Sephadex LH-20 column using
MeOH for elution to yield a mixture of methyl 3, 5-di-O-caffeoyl quinate (5) [16, 17] and methyl 3, 4-di-
O-caffeoyl quinate (6) (18 mg) [18]. In this paper, the numbering of the quinic acid ring follows the new
IUPAC nomenclature recommendation. Fraction Fr15 (219 mg) (CH2Cl2/Me2CO, 66:34) was
rechromatographed on a silica gel column (n-hexane/Et2O step gradients and then with increasing
percentages of EtOAc) to give myrtenol β-D-glucopyranoside (7) (9 mg) [19, 20]. Fraction Fr20 (129 mg)
(CH2Cl2/Me2CO, 16:84) was further applied to a Sephadex LH-20 column using MeOH for elution to
obtain 8 sub-fractions. Sub-fraction 6 was purified over Sephadex LH-20 column to afford 1, 5-di-O-
caffeoylquinic acid (8) (13 mg) [21]. Fraction Fr23 (332 mg) (100% Me2CO) gave after purification by
crystallization in MeOH, sucrose (9) (7.5 mg) [22, 23] (Figure 1).
2.4. Cell culture
HeLa (human cervix carcinoma) and C6 (rat brain tumor) cells were grown in Dulbecco's modified
eagle’s medium (DMEM, Sigma), supplemented with 10% (v/v) fetal bovine serum (Sigma, Germany) and
2% Penicillin-Streptomycin (Sigma, Germany) at 37°C in a 5% CO2 humidified atmosphere.
2.5. Antiproliferative Activity
2.5.1. Preparation of Cell Suspension
HeLa cells in the culture flask were detached from bottom of flask by 10 mL Trypsin-EDTA solution.
After detachment, 10 mL of medium was added into the flask and mixed thoroughly. This suspension was
transferred to Falcon tubes and centrifuged at 600 rpm for 5 min. After removing the supernatant, 5 mL of
medium was added to Falcon tube and mixed carefully. Cell concentration of this cell suspension was
measured by CEDEX HiRes Cell Counter which uses Trypan Blue.
2.5.2. Preparation of E-Plate 96
50 µL of medium was added into each well of E-Plate 96. The plate was incubated in the hood then in
the incubator for 15 min in each time. After this period, the E-Plate 96 was inserted to the RTCA-SP station
and a background measurement was performed. Then 100 µL of the cell suspension (2,5x104 cells/100 µL)
was added into wells, except in the last three wells where only 100 µL of medium was added to check if
there would be an increase from culture medium. The plate was left in the hood for 30 min, and then
inserted to xCELLigence station in the incubator. A measurement was performed for 80 min. Extract was
dissolved in DMSO to a final concentration of 20 mg/mL. 25 µL of this solution was mixed with 475 µL of
medium. The extract solutions (50, 20 and 10 µL equivalent to 250, 100 and 50 µg/mL concentrations,
respectively) were added into the wells and the final volumes were completed to 200 µL with medium. No
extract solution was added into control and medium wells. Then the plate was inserted to xCELLigence
station in the incubator and a measurement for 48 h was started.
2.6. Cell Proliferation Assay
Cells were plated in 96-well culture plates (COSTAR, Corning, USA) at a density of 30.000 cells
per well. Vehicle (DMSO), 5-FU, cisplatin and several samples in various concentrations (5-100 µg/mL)
were added to each well. Cells were then incubated overnight before applying the BrdU Cell Proliferation
ELISA assay reagent (Roche, Germany) according to the manufacturer’s procedure. Briefly, cells were
pulsed with BrdU labeling reagent for 4 h followed by fixation in FixDenat solution for 30 min at room
temperature. Thereafter, cells were incubated with 1:100 dilution of anti- BrdU-POD for 1.30 h at room
Boussaha et.al., Rec. Nat. Prod. (2015) 9:3 312-322 315
temperature. The amount of cell proliferation was assessed by determining the A450 nm of the culture
media after addition of the substrate solution by using a microplate reader (Ryto, China). Results were
reported as percentage of the inhibition of cell proliferation, where the optical density measured from
vehicle-treated cells was considered to be 100% of proliferation. All assays were repeated at least twice
using against HeLa and C6 cells. Percentage of inhibition of cell proliferation was calculated as follows:
(1-Atreatments /Avehicle control) × 100.
Stock solution of the samples, 5-FU were dissolved in sterile DMSO and diluted Dulbecco's modified
eagle’s medium (DMEM; 1:20). DMSO final concentration is below 1% in all tests.
2.7. Lactate Dehydrogenase (LDH) leakage assay
LDH leakage assay was carried out using LDH cytotoxicity detection kit by Roche Diagnostics
GmbH, Mannheim, Germany according to protocol in the user's manual. At 100 concentrations that was the
highest dose at antiproliferative activity test were determined cytotoxicity (%) against HeLa cell line. 5-FU
were used as positive control. Samples and 5-FU were incubated with 100 µL of C6 cell suspension having
5×103 cells/mL in 96-well plates at 37°C for overnight in 5% CO2 atmosphere. All the control and tested
substances were tested in triplicates and twice and mean ± SEM of the absorbance value were taken to
calculate the percentage of cytotoxicity.
Cytotoxicity % = [(Triplicate absorbance − low control) /(High control − low control)] x 100
2.8. Statistical Analysis
The results of this investigation are the means ± SEM of six measurements for each cell type.
Differences between treatment groups were compared by one-way analysis of variance (ANOVA) and p
values < 0.01 and 0.05 were considered statistically significant.
3. Results and Discussion
3.1. Structure elucidation
Compound 2 was previously found in Platychaete aucheri Boiss. (Compositae) but purified as its
tetraacetate after acetylation reaction [12]. In our study, we obtained it in pure and native state. Its positive
mode HRESI-MS spectrum presented [M+Na] ion at m/z 379.1732 (100%) according to the molecular
formula C18H28O7 (calculated for C18H28O7Na: 379.1733), indicating a compound containing 5
unsaturations. The 13C- and DEPT NMR spectra of this compound confirmed the presence of the 18 carbon
atoms. The 13C NMR spectrum showed six characteristic signals of a hexose group notably: a CH2 at δC
63.6 (δH= 4.34, m, 2H) assigned to C-6', a CH at δC 100.8 (δH 4.28, d, J=7.6 Hz, 1H) assigned to the
anomeric carbon C-1'. The values of the chemical shift of this carbon and the coupling constant indicated
an O-
-glycosylated compound. In the COSY spectrum, the anomeric proton H-1' led to the assignment of
H-2' as a multiplet at δH 3.39 (δC 73.5). On the same spectrum, H-2' showed a correlation with the proton
corresponding to a triplet at δH 3.55 ppm (J=8.4 Hz) which was attributed to H-3' (δC76.3). The multiplicity
of this signal and the value of the coupling constant indicated axial-axial interactions between H-3'/H-2'
and H-3'/ H-4'. This observation led to a glucosylated compound. Moreover, in the HMBC spectrum, H2-6'
showed correlation with the carbone of the carbonyle at δC 171.5 ppm, which showed a correlation with
the protons of the methyl at δH 2.10 indicating the presence of an acetate group on the C-6 position of the
glucosyle moiety. Considering the molecular formula C18H28O7 and the presence the sugar moiety, the
formula of the aglycone moiety of compound 2 was C10H15. Thus, compound 2 was a monoterpene
glucoside 6'-acetate. Besides the signals of the sugar moiety, the 13C- and DEPT NMR spectra of
compound 2 showed two CH3 (δC 20.9 and 26.2), three CH2 (δC 71.8, 31.3 and 31.4), three CH (δC 40.8,
43.3 and 121.4 and two quaternary carbon atoms (δC 144.0 and 38.0). The values of the chemical shifts of
the carbon atoms indicated that the sugar was attached to the CH2 at δC 71.8 and also indicated the presence
Chemical composition of Perralderia coronopifolia 316
of a double bond and two cycles in the monoterpene moiety. Complete 1H and 13C connectivity was
established by extensive use and interpretation of 2D NMR experiments spectra (COSY, HSQC, HMBC
and ROESY). The proton and carbon signals due to this monoterpene moiety were in good agreement with
those of the aglycone of sacranoside A isolated from the underground part of Rhodiola sacra (Prain ex
Hamet) S. H. Fu (Crassulaceae) [19]. Thus, compound 2 was myrtenol-β-D-glucopyranoside-6'-O-acetate.
The structures of known compounds were established by spectral analysis, mainly the HRESI-MS,
the 1H-, 13C-, and 2D-NMR (COSY, ROESY, HSQC and HMBC) as well as by comparing their
spectroscopic data with those reported in the literature.
Myrtenol-β-D-glucopyranoside-6'-O-acetate (2): yellow oil; 1H NMR (400 MHz, CDCl3): δ (ppm) = 5.55 ( 1H, br
s, H-3), 4.13 (1H, br d, J = 12.4 Hz, H-10a), 4.04 (1H, br d, J = 12.4 Hz, H-10b), 2.39 (1H, m, H-6a), 2.28 (2H, br d
each one, J = 20.8 Hz, H-4a and H-4b ), 2.18 (1H, m, H-1), 2.10 (1H, m, H-5), 1.29 ( 3H, s, H-9), 1.16 (1H, dl, J = 8.
7 Hz, H-7b), 0.82 ( 3H, s, H-8), 4.34 (2H, m, H2-6ꞌ), 4.28 (1H, d, J = 7.6 Hz, H-1ꞌ), 3.55 (1H, t, J = 8.4 Hz, H-3ꞌ),
3.40-3.48 (2H, m, H-4ꞌ, H-5ꞌ), 3.39 (1H, m, H-2ꞌ, 2.10 (3H, s, OAc); 13C NMR (100 MHz, CDCl3): δ (ppm) = 144.0
(C, ,C-2), 121.4 (CH, C-3), 71.8 (CH2, C-10), 43.3 (CH,, C-1), 40.8 ( CH, C-5), 38.0 (C, C-6), 31.4 (CH2, C-4), 31.3 (
CH2, C-7), 26.2 (CH3, C-9), 20.9 (CH3, C-8), 100.8 ( CH, C-1'), 76.2 ( CH, C-3ꞌ), 73.7 (CH, C-5ꞌ), 73.5 (CH, C-2'), 70.1
(CH, C-4'), 63.6 ( CH2, C-6ꞌ), 171.5 ( C, COO), 21.0 (CH3, OAc); HR-ESIMS (+): m/z 379.1732 (calcd. 379.1733 for
C18H28O7Na).
O
O
OH
OH OH
CH3O
CH3
O
1
O
O
OH
OH
OH
CH
3
O
CH
3
O
CH
3
O
3
O
O
OH
OH
OH
OH
OH
4
O
O
OH
OH
OH OH
OOH
OH
OH
OH
9
OO
OH
OH
OH
OR 1
2
3
4
5
6
7
8
10
1'
2'
4'
3'
6'
9
2 R = Ac
7 R = H
OR1
OR3
OR2
OR4
ROOC
O
OH
OH
CH3
CH3
H
H
1
2
3
6
R1
R2 R3 R4
5
6
8
H
A
A
H
A
A
A H H A
A =
R
H
Figure 1. Structures of the isolated compounds 1-9
Boussaha et.al., Rec. Nat. Prod. (2015) 9:3 312-322 317
3.2. Biological activities
3.2.1. Antioxidant activity
The free radical scavenging activity of ethyl acetate extract was evaluated through its ability to quench
2, 2-diphenyl-1-picrylhydrazyl (DPPH) radical [24]. Many studies showed that chemical structure has an
important impact on radical scavenging activity. The high antioxidant activity depends on their molecular
structure, the number and the position of the hydroxyl groups [25]. Effective antioxidant activities were
observed in comparison to ascorbic acid. Comparing IC50 values, we observed that ethyl acetate extract of
P. coronopifolia showed a high scavenging effect against DPPH radicals with IC50=7.01±0.28µg/mL
compared to ascorbic acid (IC50= 5±0.1µg/mL (Figure 2). This activity is possibly due to the presence of
the flavonoids: taxifolin [26], rhamnazin [27] and caffeoylquinic acid derivatives found in this plant [28].
Figure 2. DPPH scavenging activity of ethyl acetate extract and vitamin C. Values are mean ± SD of
three samples analyzed, individually in triplicate
3.2.2. Anticancer activity
Anticancer activity is highly studied for the medicinal plants and other natural sources [29]. However,
phytochemical and cytotoxic, antitumor/anticancer investigations on P. coronopifolia have not been studied
so far. The anticancer activity of ethyl acetate soluble part of the aqueous-EtOH extract of the aerial parts
(leaves and flowers) of P. coronopifolia against HeLa (human cervix carcinoma) and C6 (rat brain tumor)
cells was tested using xCELLigence RTCA instrument. As shown in Figures 3 and 4, the extract exhibited
different profiles on different concentrations. It has the most activity with the concentration of 250 µg/mL
and it was about to reach to medium after 48 h post-treatment, while other concentrations [100 and 50
µg/mL] showed very low activity, nearly same with control. The profiles also showed differences at
different time points. Low concentrations showed some activity against HeLa cells at 15th h (Figure 3) and
against C6 cells at 3th h (Figure 4) after post-treatment, but after few hours they lost their capacity of
inhibition. This may be caused because of the low ratio of bioactive molecules in the extract and within the
time, cells started to proliferate after a short time of inhibition.
Chemical composition of Perralderia coronopifolia 318
Figure 3. Anticancer activity of the ethyl acetate extract of P. coronopifolia against HeLa (2,5x104
cell/well) cell line. Each substance was tested twice in triplicates against cell lines using xCELLigence
RTCA instrument. As seen in the diagram, several extract concentrations were applied to the cells and each
color represents a different concentration.
Figure 4. Anticancer activity of the ethyl acetate extract against C6 (2,5x104 cell/well) cell line. Each
sample was tested twice in triplicates against cell lines using xCELLigence RTCA instrument. As seen in
the diagram, several extract concentrations were applied to the cells and each color represent a different
concentration.
3.2.3. Cell proliferation assay
The antiproliferative activities of samples and standard compound were determined against C6 and
HeLa cell lines using BrdU cell proliferation ELISA assay [30, 31]. 5-FU was used as positive control. The
antiproliferative activity of samples and positive control were investigated on eight concentrations (5, 10,
20, 30, 40, 50, 75 and 100 µg/mL). IC50 and IC75 values of the extracts and 5-FU were identified using ED50
plus v1.0 programs (Table 1).
3.2.3.1. Antiproliferative activity of samples against HeLa cell.
The antiproliferative activity of the ethyl acetate extract was determined against HeLa cell. This
extract has shown to increase the activity depending to the dose (Figure 5). The obtained results indicated
▬250 ▬100 ▬50 ▬Control ▬Medium
▬250 ▬100 ▬50 ▬Control ▬Medium
Boussaha et.al., Rec. Nat. Prod. (2015) 9:3 312-322 319
that the lower concentrations (5 and 10 µg/mL) showed the proliferative activity and the higher
concentrations (50, 75 and 100 µg/mL) showed high antiproliferative activity against HeLa cells compared
to 5-FU.
The potency of inhibition at 100 µg/mL was: 5-FU > EtOAc extract.
Figure 5. Antiproliferative activity of the EtOAc extract and 5-FU against HeLa cell line. Each sample was
tested twice in triplicates against HeLa cell lines. Data showed average of 2 individual experiments
(p<0.01).
3.2.3.2. Antiproliferative activity of sample against C6 cell.
The antiproliferative activity of the ethyl acetate extract of P. coronopifolia was determined against
C6 cell. The extract has shown to increase the activity as dose dependent manner. However, it was
observed to have higher antiproliferative activity than 5-FU at 40-75 µg/mL (Figure 6).
The results of the present work are in agreement with previous evidences, which suggested that
plant extracts are likely sources of substances potentially useful for the development of new drugs
[32].
Figure 6. Antiproliferative activity of the EtOAc extract and 5-FU against C6 cell line. Each substance was
tested twice in triplicates against cell lines. Data showed average of 2 individual experiments (p<0.01)
As shown in Figures 5 and 6, there is no proliferative activity even in lower concentrations against C6
cell lines as seen in lower concentrations against HeLa cell lines. The highest activities were observed
against C6 cell lines than 5-FU in high concentrations, but not the same observations were obtained against
HeLa cell lines.
Chemical composition of Perralderia coronopifolia 320
The antiproliferative activity of ethyl acetate extract is expressed in terms of IC50 and IC75 and given
in Table 1. The comparison of the IC50 and IC75 values for the two cell lines used in this study indicated
that the extract showed a higher activity against C6 cell lines than HeLa cell lines.
Table 1. IC50 and IC75 values of EtOAc extract of P. coronopifolia and 5-FU, presented by
antiproliferative assay against HeLa and C6 Cell Lines.
HeLa cell
C6 cell
IC50
IC75
IC50
IC75
EtOAc extract
45.49
63.71
11.81
44.64
5-FU
*
*
*
12.03
*<5 µg/mL.
3.2.4. Lactate Dehydrogenase (LDH) leakage assay
Cytotoxicity % = 0% No cytotoxic effect was observed
Lactate dehydrogenase (LDH) is a stable cytoplasmic enzyme present in all cells. It is rapidly released
into the cell culture supernatant or blood when the plasma membrane of cells is damaged. For this reason,
the LDH assay is a widely used quantitative test for the toxicity of various molecules and drugs cells [33].
As shown in Table 2, the present study demonstrated that the EtOAc extract of P. coronopifolia has no
cytotoxic effect.
Table 2. The percentage of cytotoxicity (Cytotoxicity %) of EtOAc extract of P. coronopifolia and 5-FU
against C6 Cell Line using Lactate Dehydrogenase (LDH) leakage assay.
Sample Name
Cytotoxicity (%)
EtOAc extract
0
5-FU
19
In conclusion, this study showed that, the ethyl acetate soluble part of P. coronopifolia aqueous-EtOH
extract had an antioxidant activity. This activity is possibly due to the flavonoids chrysosplenol D, taxifolin
and caffeoylquinic acid derivatives which are the major components of this extract. A preliminary
evaluation of the antiproliferative activity of this extract against HeLa and C6 cells revealed that it is quite
promising for the cancer cells studied. They were tested by xCELLigence RTCA instrument and BrdU cell
proliferation ELISA assay, respectively. 5-Fluorouracil was used as positive control. No cytotoxic effect
was observed in this extract.
The relatively high content of bioactive compounds, positive antioxidant and antiproliferative
properties of P. coronopifolia may justify the use of this plant as a new source of valuable antioxidants. In
vivo studies are needed to confirm this pharmacological efficacy.
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