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Synthesis, characterization, cytotoxic activity of half-sandwich rhodium(III), and iridium(III) complexes with curcuminoids
Arabian Journal of Chemistry (2017) 10, S2547–S2553
King Saud University
Arabian Journal of Chemistry
www.ksu.edu.sa
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ORIGINAL ARTICLE
Cytostatic activity of Geranium robertianum L.
extracts processed by membrane procedures
Elena Neagu a, Gabriela Paun a,*, Daniel Constantin a
Gabriel Lucian Radu b
a
Centre of Bioanalysis, National Institute for Research-Development of Biological Sciences, 296 Spl.Independentei, PO
Box 17-16, 060031 Bucharest 6, Romania
b
Faculty of Applied Chemistry and Materials Science, Politehnica University of Bucharest, 313 Spl.Independentei, 060042
Bucharest, Romania
Received 15 January 2013; accepted 17 September 2013
Available online 27 September 2013
KEYWORDS
Geranium robertianum;
Ultrafiltration;
Cytostatic activity;
HEp-2p cells;
Potential antitumor agents
Abstract In the present study the antioxidant and cytostatic capacities of some 8% Geranium robertianum (geranium) aqueous extracts processed by membrane procedures (micro- and ultrafiltration) were determined. The extracts were purified by microfiltration and then concentrated by
successive ultrafiltrations using Millipore membranes with 10,000 and 1000 Da cut-off. Two methods were used to establish the extracts’ antioxidant activity: DPPH and ABTS; the cytostatic capacity was evaluated on HEp-2p cell lines, by a qualitative method (cytochemical stain with Giemsa
solution) and quantitative one (MTT test). Four concentrations of Geranium aqueous extracts were
used to test cell viability: 100, 500, 1000 and 2000 lg/mL, 24 and 48 h, against the control culture
(100% viability). The concentrated samples had the highest content of total polyphenols and the
strongest antioxidant effect, having also the biggest impact upon cell viability.
ª 2013 Production and hosting by Elsevier B.V. on behalf of King Saud University. This is an open access
article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).
1. Introduction
The plant natural compounds represent some of the most
important sources for research in the pharmaceutic industry.
In the last years several studies were directed toward
traditional medicines in order to identify natural compounds
* Corresponding author. Tel./fax: +40 21 2200900.
E-mail address: gpaunroman@gmail.com (G. Paun).
Peer review under responsibility of King Saud University.
Production and hosting by Elsevier
with therapeutic properties (Hamburger and Hostetmann,
1991; Weisburger et al., 1996).
Pharmaceutic and pharmacodynamic researches demonstrated that the drugs of vegetal origin are biological products
that are more accessible to human metabolism than the synthetic ones which, sometimes, cause, in addition, poisonous
side effects.
Geranium robertianum L. (Geraniaceae family) has its origin
in Asia, Central and Meridional Europe and it is frequently
used in traditional medicine to treat inflammatory diseases
and cancer. The Geraniaceae family consists in about 11 genera
with almost 800 species spread in temperate and subtropical
areas of both hemispheres. Some species belonging to Gera-
http://dx.doi.org/10.1016/j.arabjc.2013.09.028
1878-5352 ª 2013 Production and hosting by Elsevier B.V. on behalf of King Saud University.
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).
S2548
E. Neagu et al.
nium genus are used in traditional medicine, while others are
cultivated in ornamental scope or for their aromatic oils
(Camacho-Luis et al., 2008).
The active principles identified in geranium are polyphenolic compounds of glycosylated flavonoid type (quercetin,
kaempferol, rhamnetin) and phenolic acids (Miliauskas
et al., 2004a,b). As it has already been reported, the flavonoids
exercise multiple biological effects due to their antioxidant
capacity and ability to neutralize free radicals (Papuc et al.,
2009, 2007).
The flavonoids have a significant role in the plant biochemistry
and, at physiological level, they have hepatoprotective,
antithrombotic, anti-inflammatory, antiviral, antiallergic, antiproliferative, anticancer and immunostimulant actions
(Harborne, 1988; Saija et al., 1995). A series of studies reported
the antibacterial, antifungal, antiallergic actions of flavonoids,
as well as, their protective role in heart diseases, cancer and different pathological states (Martinez et al., 2002; Matsuda et al.,
2003).
The plant polyphenols’ role in cancer chemoprevention led
to the development of an interesting research domain. Some
polyphenols and their derivates present selective cytotoxicity
against various tumoral cells, much greater than upon normal
cells (Salucci et al., 2002; Serrano et al., 1998). Such compounds could serve as antitumor agents or as a basis for the
synthesis of new drugs by targeted chemical modification (Pellegrinaa et al., 2005; Yang et al., 2000).
In the present study the membrane procedures, micro- and
ultrafiltration, were used to process some aqueous G. robertianum extracts, in order to purify and concentrate them. Previous studies evidenced the ultrafiltration performances to
concentrate some extracts of medicinal plants (Neagu et al.,
2011, 2010; Paun et al., 2011; Paun-Roman et al., 2009).
The membrane procedures of separation and concentration
might have an improved efficiency and lower operation costs
in comparison to the traditional concentration processes used
in the pharmaceutical industry (Shi et al., 2006).
The chromatographic analyses of G. robertianum aqueous extracts were performed by the HPLC–DAD method. The chromatographic measurements were carried out using a HPLC
SHIMADZU system with following components: LC-20AD
sp Pump, the Kromasil C18 column for polyphenol–polycarboxylic acids, DGU-20As Degasser, CTO-20AC thermostat
column, detector SPD-M20A diode array Shimadzu, LCMS
– Shimadzu solution software.
The used standards were: elagic acid, rutin, luteolin, quercetin, kaempferol, gallic acid, cafeic acid, coumaric acid, and
ferulic acid. The used mobile phase had two components:
(A) water, acidulated with 1% formic acid and (B) acetonitril
with 1% formic acid.
2. Materials and methods
2.6. Testing the cytostatic activity of extracts
2.1. Extract obtain
The cytostatic activity of G. robertianum extracts processed by
micro- and ultrafiltration was tested on the Hep-2 (human epidermoid carcinoma of larynges) cell line. The neoplastic cells
were maintained in MEM (Sigma–Aldrich) culture medium
supplemented with 10% fetal bovine serum (Biochrom), 1%
PSN (penicillin, streptomycin, neomycin; Sigma–Aldrich) in
an incubator at 37 C, in wet atmosphere with 5% CO2. The
extracts’ antitumor activity was evaluated both by qualitative
(cytochemical stain with Giemsa solution) and quantitative
(MTT test) methods.
The G. robertianum extract was prepared through maceration,
using the bidistilled water as solvent. The plant leaves were,
previously, finely grinded by means of a GRINDOMIX
GM200 grind mill; the maceration lasted 24 h, at 20 C,
mechanical stirring was sporadically performed; the plant mass
concentration was of 8%.
2.2. Extract concentration
The extract was firstly filtrated through a filter paper, and then
purified by microfiltration using a Millipore membrane of
regenerated cellulose with 0.45 lm pores. The extract concentration was realized by successive ultrafiltrations using Millipore membranes with 10,000 and 1,000 Da cut-offs. The
concentration ratio (expressed as volumes’ ratio between permeate and concentrate) was of 3:1. A KMS Laboratory Cell
CF-1 installation, purchased from Koch Membrane firm –
Germany, was used for both microfiltration and ultrafiltration.
2.3. Determination of polyphenol content
Determination of polyphenol content was done by the Folin–
Ciocalteu method (Folin and Ciocalteu, 1927). The polyphenol
concentration in the sample was calculated based on an etalon
curve of 10–100 lg/mL gallic acid (GAE).
2.4. Determination of antioxidant activity
Determination of antioxidant activity was done with a spectrophotometer, by evaluation of Trolox Equivalent Antioxidant
Capacity (TEAC) with two methods:
– One based on the decrease of the DPPH maximal absorbance, at 519 nm in the presence of antioxidants (Bondet
et al., 1997; Litescu et al., 2000).
– Other based on the decrease of the ABTS maximal absorbance, at 731 nm in the presence of antioxidants (RiceEvans and Miller, 1994).
2.5. HPLC analysis of the obtained extracts
2.6.1. MTT test
The cell viability was determined by the MTT test. The spectrophotometer method is based on the conversion of dimetiltiazol-2-difeniltetrazolium bromide (MTT) in purple
crystals of formazan under the action of mitochondrial dehydrogenases in live cells. The formed formazan amount is proportional to the number of live cells and is appraised with a
spectrophotometer after the crystals’ dissolution into isopropylic alcohol.
Cytostatic activity of Geranium robertianum L. extracts processed by membrane procedures
Table 1
S2549
Polyphenol content in Geranium robertianum extracts.
Type of membrane
Polyphenols (lg/mL)
Millipore 10,000 Da
Millipore 1000 Da
Sample
Permeate
Concentrate (UF1)
Permeate
Concentrate (UF2)
Concentrate UF1 + UF2 (50:50)
6% aqueous extract
675.04 ± 6.3
745.61 ± 9.1
536.89 ± 5.3
763.89 ± 9.3
695.55 ± 6.2
Table 2
Antioxidant activity of analyzed extracts.
Sample
TEACDPPH (lmol Trolox/g)
TEACABTS (lmol Trolox/g)
6% Geranium robertianum aqueous extract_MF
6% Geranium robertianum extract perm_UF1
6% Geranium robertianum extract conc_UF1
6% Geranium robertianum extract perm_UF2
6% Geranium robertianum extract conc_UF2
6% Geranium robertianum extract conc_UF1 + UF2 (50:50)
169.02 ± 6.86
151.12 ± 7.85
180.91 ± 6.04
176.87 ± 1.71
216.39 ± 6.64
182.57 ± 6.38
539.56 ± 11.21
390.00 ± 4.29
573.20 ± 9.29
452.54 ± 9.76
1286.96 ± 3.89
1123.85 ± 13.04
Figure 1
DPPH inhibition.
The cells were inoculated at 3.5 · 104 cells/ml density on
plates with 24 wells and incubated at 3700 BAC in wet atmosphere with 5% CO2, 24 h, to allow cell adherence. After incubation, the medium was carefully removed and replaced with
fresh culture medium supplemented with different extract concentrations. The culture medium was removed 24 and 48 h
after the addition of the extract, and the MTT solution
(0.25 mg/ml) was added. The culture was incubated at 37 C,
3 h. Then, the obtained formazan crystals were solubilized
with isopropanol and the colorant absorbance was measured
at 570 nm by means of a Tecan Microplate Reader.
2.6.2. Cell morphology
In order to observe their morphology, the cells were inoculated
on plates with 24 wells at a density of 3.5 · 104 cells/ml. 24 and
48 h after the addition of the extract, they were washed with
PBS, fixed with cold methanol ( 20 C), stained with Giemsa
solution (15 min) and photographed with a Zeiss Axio Observer optic microscope.
3. Results and discussions
3.1. Total polyphenol content
In the fractions obtained during the ultrafiltrations, respectively the permeates and concentrates are presented in Table 1.
The highest polyphenol content was found in the concentrated extracts, especially in extract UF2 processed by Millipore membrane with 1000 Da cut-off.
In the G. robertianum extracts obtained and processed by
micro- and ultrafiltration the antioxidant activity was determined by two (with DPPH and ABTS) spectrophotometric
S2550
Table 3
E. Neagu et al.
Content in active substances of interest, expressed in lg/g vegetal mass in Geranium robertianum extracts.
Sample
Elagic acid Rutin Luteolin Quercetin Kaemfoerol Gallic acid Cafeic acid Ferulic acid Cumaric acid
(lg/g vegetal mass)
6% Aqueous extract_MF
744.15
Conc_UF1 (Millipore 10,000 Da) 779.63
Conc_UF2 (Millipore 1,000 Da) 900.13
Conc_UF1 + Conc_UF2 (1:1)
729.13
Table 4
1.73
3.83
4.935
5.41
3.56
4.03
8.16
5.01
0
0
0
0
1.86
0
0.964
0
978.86
1056.79
1070.78
958.43
0
0
0
0
0
0
0
0
0
0
0
0
Cell viability in Hep-2p cultures under the action of 8% Geranium robertianum aqueous concentrated extracts.
Sample
% Cell viability
24 h
P1-MF
P2-Conc UF1
P3-Conc UF2
P4 = P2 + P3 (50:50)
48 h
100 lg/mL
500 lg/mL
1000 lg/mL
2000 lg/mL
100 lg/mL
500 lg/mL
1000 lg/mL
2000 lg/mL
90.7
60.7
60.0
61.2
91.1
54.0
60.9
56.4
38.7
36.2
36.0
39.0
6.8
5.9
5.9
10.9
89.7
71.5
70.4
70.5
70.0
50.3
56.0
56.3
23.5
20.9
21.6
30.2
2.8
3.1
2.8
7.6
methods, and also appraised the DPPH inhibition percent. The
results are shown in Table 2 and Fig. 1.
A correlation between the polyphenol amount of studied
extracts and their antioxidant capacity could be observed,
the highest antioxidant capacity being found in the concentrated extracts, especially in the UF2 extract, in which the
highest amount of total polyphenols was also recorded, in correlation with literature data (Andarwulan et al., 2010; Velioglu
et al., 1998).
It was revealed that the highest value (81.02%) of DPPH
inhibition was found in the same UF2 concentrated extract.
3.2. HPLC analysis of obtained extracts
The optimum analysis conditions were established using the
standards specific to the compounds of interest.
The samples of plant extracts were analyzed by the interpolation of the areas obtained for each compound. The results
were expressed in lg/g dry matter and presented in Table 3.
The main polyphenols identified in the analyzed extracts
were gallic and elagic acids – the highest amounts, while rutin
and luteolin – lower amounts; after the extract concentration,
the gallic and elagic acid amounts were preponderant in the
UF2 concentrate.
Figure 2
Control cells – 24 h.
Figure 3
Control cells – 48 h.
3.3. Testing the extracts’ cytostatic activity
The number of viable cells was calculated by reference to control (cells cultivated in the absence of extracts) considered as
having 100% viability (Table 4).
It was observed that:
– Cell viability decreased as the extracted dose increased
and it at the same time decreased with the exposure
duration (preponderantly from the second concentration – 500 lg/mL).
– P2 and P3 concentrated samples similarly affected the
viability of Hep-2 cell cultures, having the strongest
effect.
Cytostatic activity of Geranium robertianum L. extracts processed by membrane procedures
Figure 4
P1–500 lg – 48 h.
Figure 5
P1–1000 lg – 48 h.
S2551
Figure 7
P2–1000 lg – 48 h.
Figure 8
P3–500 lg – 48 h.
covered by 1–3 nucleoli, and had fine cytoplasm. Here and there
some dividing cells were observed. The culture was dense; the
cells covered more than 95% of the well surface (Figs. 2 and 3).
The treated cultures, starting with the 500 lg/mL dose,
showed a substantially modified morphology, the cells were
smaller, the nuclei were condensed, the cytoplasm was granular and here and there vacuoles were observed. The cells were
rare, covering about 40% of the well surface. The dividing cells
were not observed (Figs. 4–11).
4. Conclusions
Figure 6
P2–500 lg – 48 h.
In order to exemplify the geranium extracts’ cytotoxic effect
upon cell cultures we present also some microscopic images of
control and treated (500 and 1000 lg/mL, 48 h, P1–P4 Geranium extracts) cell cultures (Figs. 2–11).
The untreated cells revealed morphology typical of a culture
of tumoral cells: they were polygonal or fusiform, with big nuclei,
In the present work, purified and concentrated G. robertianum extracts were obtained and analyzed from the
point of view of polyphenolic compounds;
The antioxidant capacity of the studied extracts
showed the highest TEAC values in case of extracts
concentrated especially through the ultrafiltration
membrane with 1000 Da cut-off (UF2). The same
extract had the highest percentage of DPPH inhibition;
The complex in vitro evaluation of the cytostatic activity revealed the significant potential of G. robertianum
concentrated extracts;
S2552
E. Neagu et al.
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Figure 9
P3–1000 lg – 48 h.
Figure 10
P4–500 lg – 48 h.
Figure 11
P4–1000 lg – 48 h.
The purified and concentrated G. robertianum extracts,
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be considered potential chemotherapeutic agents.
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