Advanced
Pharmaceutical
Bulletin
Adv Pharm Bull, 2014, 4(Supp1), 459-464
doi: 10.5681/apb.2014.068
http://apb.tbzmed.ac.ir
Research Article
Evaluating Antiproliferative and Antioxidant Activity of Marrubium
crassidens
Sanaz Hamedeyazdan1,2, Simin Sharifi1,2, Hossein Nazemiyeh2, Fatemeh Fathiazad3*
Students’ Research Committee, Faculty of Pharmacy, Tabriz University of Medical Sciences, Iran.
Research Center for Pharmaceutical Nanotechnology, Faculty of Pharmacy, Tabriz University of Medical Sciences, Iran.
3
Department of Pharmacognosy, Faculty of Pharmacy, Tabriz University of Medical Sciences, Iran.
1
2
Article History:
Received: 14 April 2014
Revised: 21 May 2014
Accepted: 23 May 2014
ePublished: 10 August 2014
Keywords:
Marrubium crassidens
Lamiaceae
MTT assay
MCF-7 cell line
Free radical scavenger
Phenols
Abstract
Purpose: Naturally occurring substances as novel drugs in cancer therapy, at all times,
represent a challenge to science since medicinal plants are proving to be brilliant sources of
new chemopreventive agents.
Methods: In the present study, methanol extract from aerial parts of Marrubium crassidens
was assessed for its antiproliferative activity in the breast cancer cell line MCF-7 through
MTT bioassay using cell viability and cytotoxicity indices. The antioxidant property of M.
crassidens extract together with its phenolic and flavonoids content were evaluated, as well.
Results: According to data obtained in the study, M. crassidens exhibited antiproliferative
activity with a gradual rise in cytotoxicty effect setting out on 240µg/mL concentration of the
extract. Moreover, the RC50 value for antioxidant activity of the extract was determined as
40µg/mL and values for the total phenolic and flavonoids were calculated as 512.64mg gallic
acid equivalent and 212.73mg quercetin equivalent per 100g of dry plant material.
Conclusion: Generally, the observed antiproliferative and antioxidant properties of M.
crassidens could be certified to the high amounts of phenolic and flavonoid content detected
in the extract.
Introduction
Not surprisingly, the upward desire in capturing the
wisdom of traditional healing systems in management of
different sorts of diseases among the nations, has led to a
renewal of interest in herbal medicines. In this regard,
plants with a wide range of biologically active
constituents, at all times, have been providing scientists
with innovative visions both in their natural forms and
also by templates for novel molecular prototypes of
drugs. Followed by a variety of investigations on a
fundamentally unregulated cell growth, cancer has
remained a major health problem worldwide with a
choice of different expressions and pathologies. Seeing
as the deadly nature of cancer, there exists severe
scientific challenge among the researchers to understand
the disease processes headed for discovery of potential
fresh therapies from natural products. It has been
accepted that an imbalance between the production of
oxidants and frequency of antioxidant defenses namely
oxidative stress could be one of the ensuing factors in
DNA and protein damage, cancer, ageing, lipid
peroxidation and inflammatory activities.1 Following
free radicals that are generated during oxidative stress
with unpaired electrons seek for stability through
electron pairing with biological macromolecules like
proteins, lipids, and DNA of healthy human cells,
bringing in about serious consequences of the oxidative
stress conditions. In this issue, breast cancer developing
from the metastatic progress of primary stage of cell
tumors, has been considered as the prevalent malignancy
among women the foremost cause of cancer related
death.2 Even so, phyto medicines confirming to be
appealing sources of new compounds with new
applications in clinical stages seem to have much to offer
in treatment of cancers bringing about rational
opportunities in this filed.3-5
In spite of the fact that herbal preparations from different
parts of plants belonging to various families have been
regarded as valuable medicinal plants, abundant
members of the family Lamiaceae (particularly the genus
Marrubium) have prominent medicinal properties.
Marrubium (horehound) is a genus of about 40 species
native to temperate regions of Europe and Asia, that are
characterized with some potential therapeutic activities
supported by a choice of reports demonstrating
cytotoxicity,
immunomodulating,
vasorelaxant,
antispasmodic, hypolipidemic, hypoglycemic, and
analgesic properties of this genus in vitro and in vivo.6-14
Further studies on the composition, antimicrobial and
antioxidant activities of essential oils extracted from
genus Marrubium have also been reported.15-26
Moreover, plants from this genus are customarily famous
for producing several classes of compounds including
diterpenes, polyphenols, steroids, phenylpropanoids and
flavonoids, some of which have important biological
*Corresponding author: Fatemeh Fathiazad, Tel: +98 (411) 3372253, Fax: +98 (411) 3344798, Email: fathiazad@tbzmed.ac.ir
©
2014 The Authors. This is an Open Access article distributed under the terms of the Creative Commons Attribution (CC BY), which permits
unrestricted use, distribution, and reproduction in any medium, as long as the original authors and source are cited. No permission is required from
the authors or the publishers.
Hamedeyazdan et al.
properties.27-31 Nonetheless, in order to provide an
efficient herbal remedy it would be of utmost importance
to associate between chemical constituents of a natural
product with its biological properties. Accordingly, in
this study M. crassidens endemic to Armenia,
Azarbaijan, Turkey and Iran was selected to search for
its possible anti proliferative activity against MCF-7
human breast cancer cell line along with its antioxidant
activity in relation to the phenolic and flavonoid contents
of the herbal extract.
Materials and Methods
Materials
In this study, 3(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium
bromide
(MTT),
2,2-diphenyl-1picrylhydrazyl (DPPH), quercetin, gallic acid, and FolinCiocalteu reagent, aluminum chloride, streptomycin,
penicillin G, all from Sigma Aldrich chemical company
Germany and fetal bovine serum (FBS) from Gibco, UK
were used. All other reagents and chemicals were of
analytical grade.
Plant material, extraction and preparation
Marrubium crassidens was collected during the
flowering stage from Chichaklou in East Azarbaijan
province, Iran, in June 2011. A voucher specimen of the
plant (Tbz-Fph-719) representing this collection has been
deposited at the Herbarium of the Faculty of Pharmacy,
Tabriz University of Medical science, Iran. The air-dried
and well grounded aerial parts of M. crassidens (0.5kg)
were extracted with solvents of increasing polarity,
petroleum ether (40–60°C), dichloromethane and
methanol (5L of each solvent, thrice every 48h) by
maceration at room temperature. Afterwards, for further
analysis upon methanol extract, concentration was
qualified under reduced pressure via a rotary evaporator
at 40°C, up to obtain a dried powdered extract.
MTT bioassay
Cytotoxic effect of M. crassidens methanol extract was
assessed by MTT bioassay in MCF-7 human breast
cancer cell line. In the MTT assay reduction of
mitochondrial succinate dehydrogenase converts yellow
dye to a blue formazan product, which shows the normal
activity of mitochondria and thus the cell viability.32 The
MCF-7 cell line was established from National cell bank
of Iran (Pasteur institute, Iran), and cultivated in RPMI
1640 medium (Sigma Aldrich Co. Germany)
supplemented with 10% fetal bovine serum (FBS), 100
mg/ml streptomycin and 100 units/ml penicillin G. The
MCF-7 cells were cultivated at 37 °C in a 5% CO2
incubator. Then, cells with ~90% confluency were
detached by 0.05% trypsin/EDTA. Cell suspension was
distributed into 96- well microtitre plate (200μl/well)
with concentration of 15×103 cells/well. After 24 hours
the cultivated cells were treated with different amount of
methanolic extract (1, 0.75, 0.5, 0.25, 0.1, 0.075, 0.05,
0.025, 0.01mg/ml) dissolved in 1% dimethyl sulfoxide
(DMSO) and were incubated for 24, 48 and 72 hours.
460 | Advanced Pharmaceutical Bulletin, 2014, 4(Supp1), 459-464
Control groups received the same amounts of DMSO
with four wells remained untreated. Moreover, the MTT
reagent was prepared at 2mg/ml in PBS. The normal
culture medium was replaced with 150μl fresh media
plus 50μl of MTT reagent (2mg/ml in PBS), excluding
the cell-free blank control wells. Cells were incubated in
37°C, 5% CO2 and full humidity for 4h. Consequently,
the MTT solution was exchanged by 200μl of DMSO
and 25μl sorenson buffer (0.1M NaCl, 0.1M glycine
regulated to pH: 10.5 with 1M NaOH). The plate was
shaken for 15 min at 37 °C, later optical density (OD) of
the wells were determined at 570 nm using a
spectrophotometric plate reader (SUNRISE TECAN,
Austria). Eventually, the viability and growth of tumoral
cells were calculated via the below formula:
Additionally, cytotoxicity of the M. crassidens extract
was defined by plotting of the percent cytotoxicity index,
CI % = [1- (optical density of sample/optical density of
control)] × 100, versus concentrations of the methanolic
extract of M. crassidens.
Assay for antioxidant activity
The free radical scavenging capacity of the extract was
measured from the bleaching of the purple-colored
methanolic solution of DPPH. The stock concentration of
the M. crassidens methanol extract (1mg/mL) was
prepared followed by dilution to reach for concentrations
5×10-1, 2.5×10-1, 1.25×10-1, 6.25×10-2, 3.13×10-2 and
1.56×10-2 mg/mL of the extract. The acquired
concentrations in the same volumes of 2mL were added
to 2mL of a 0.004% of DPPH solution. Later than a 30
min of incubation at 30 ºC, the absorbance of each
solution was read against a blank sample at 517 nm. The
average absorption value was noted for each sample after
the test was carried out in triplicate. Moreover, as the
positive control the same procedure was gone over with
quercetin. The inhibition percentage of DPPH free
radicals of by the methanol extract was calculated as
follows:
Herein, “A blank” stands for the absorbance value of the
control reaction and “A sample” is the absorbance value
for each sample. In addition, RC50 value, the
concentration of the extract reducing 50% of the DPPH
free radicals, was calculated from the graph of inhibition
percentages versus concentrations of M. crassidens
extract in mg/mL.
Assay for total phenolics content
Total phenolic constituents of the M. crassidens
methanol extract was verified by assigning FolinCiocalteu reagent and Gallic acid as the standard
compound for phenolics, the same procedure as given in
the literature.33-35 Briefly, 0.5mL of methanol solution of
the extract was mixed with 5mL of folin Ciocalteu
Antioxidant and antiproliferative activity of Marrubium crassidens
reagent (a 10% v/v in distilled water) with 4mL of 1M
aqueous Na2CO3 after 5min and the mixture was allowed
to stand for 15 min with intermittent shaking. The
absorbance of the blue color produced by the reaction
was measured using a UV/ Visible spectrophotometer
(Shimadzu 2100 - Japan) at 765 nm. The standard curve
was prepared using 25-300 µg/mL solutions of gallic
acid in methanol: water (50:50). Eventually, the value for
total phenol content of the M. crassidens extract was
represented in terms of gallic acid, equivalent (mg/100g
of powdered dry plant material) which is a common
reference compound.
Assay for total flavonoids content
Determination of the total flavonoid content of the M.
crassidens methanol extract was carried out according to
the colorimetric aluminum chloride method.33-35
Concisely, 0.5mL solution of the extract was mixed with
1.5mL of methanol, 0.1mL of 10% aluminum chloride,
0.1mL of 1M potassium acetate and 2.8mL of distilled
water, which were left at room temperature for 30 min.
Next, the absorbance of the reaction mixture was
measured at 415 nm, spectrophotometrically. After all,
the total flavonoids content of the M. crassidens
methanol extract was calculated as equivalent of
quercetin as the standard compound for flavonoids from
a calibration curve (mg/100g of powdered dry plant
material). The standard curve was managed and
evaluated by different concentrations of quercetin in
methanol 31.25-250 µg/mL, as well.
Results
Antiproliferative activity
Antiproliferative activity of M. crassidens extract on
MCF-7 cell line was quantified by MTT method showing
the time and dose dependent effects through plots of
viability and cytotoxicity index percentages versus
different concentrations of the extract in Figure 1.
According to the findings, the highest descent in cells
viability reached to 21.7% by 1mg/mL of the extract
after incubation of 48 h compared to the control group of
untreated cells. Moreover, in the viewpoint for
cytotoxicity indices, a gradual increase in cytotoxicty
activity had been detected setting out on concentrations
of 240µg/mL plant extract, reaching up to 70% of
cytotoxicity index at 1mg/mL for the MCF-7 cell lines.
Figure 1. Effect of M. crassidens methanol extract on cell proliferation of MCF-7 cell lines presented as percentage of, a) cell viability and
b) cytotoxicity index, versus concentration of the extract.
Antioxidant activity of the M. crassidens extract
Assay for the free radical scavenging activity of the
extract was accomplished via the DPPH method, which
has been generally developed for detection of the
antioxidative properties of the materials in a fairly short
time.36-38 Regarding the results for antioxidant assay, M.
crassidens extract exhibited pleasant antioxidant activity
with RC50 values of 40µg/mL for the extract and 3µg/mL
for the control quercetin.
Total phenols content
The amount of total phenolic compounds present in the
M. crassidens methanol extract was calculated as gallic
acid equivalents ascertained by Folin Ciocalteu method.
The following equation obtained from the standard gallic
acid graph was applied in calculation of the phenolic
compounds concentration.
Sample absorbance = 0.0067 × gallic acid (µg) + 0.0132, (R 2: 0.987)
Correspondingly, the content for M. crassidens total
phenolics showed the value 512.64mg of gallic acid
equivalent in 100g of dry plant material.
Total flavonoids content
The total flavonoids content in M. crassidens methanol
extract via aluminum chloride as the shift reagent was
determined according to the equation obtained from the
standard quercetin graph:
Absorbance = 0.008 × Quercetin (µg) - 0.0683 (R2: 0.9999).
With reference to the relative standard curve, amounts
for the flavonoid contents was calculated as 212.73mg
quercetin equivalent in 100g of powdered plant material,
comparing the absorbance values for methanolic extract
solution with the standard solutions of quercetin.
Discussion
As far as we know, plants had been used for various
medicinal purposes long before recorded history. Even
so, advances in clinical studies is evidence for the
implication of herbal medicine in the treatment and
management of different sorts of diseases so far as
improvements in analysis and quality control of
medicinal plants come about. In this regard, the obtained
data in our study strongly suggest the methanolic extract
Advanced Pharmaceutical Bulletin, 2014, 4(Supp1), 459-464 | 461
Hamedeyazdan et al.
of M. crassidens is moderately cytotoxic to MCF-7 cells
in a dose and time dependent manner. In consistence to
our lately published paper on other species of genus
Marrubium, M. persicum, disclosing its anti proliferative
activity,39 the present study strengthen the concept that
incorporation of this plant in herbal remedy as a potential
novel cancer chemopreventive agent might help prevent
or downgrade the chance of breast cancer or other
oxidative stress related diseases, as well.
Surveys in this filed, such as that conducted by Alkhatib
et al. have shown a common natural source for the hemisynthesis of future ladanein-derived flavones was found
to be Marrubium vulgare which possessed moderate
antileukemic activity on K562, K562R (imatinibresistant), and 697 human leukemia cell lines.30
Elsewhere, some phenylethanoid derivatives isolated
from aerial parts of M. deserti de Noé were undertaken
through antigenotoxic analyses by Zaabat et al. The
findings of their study showed that the isolated
compounds were able to significantly inhibit βgalactosidase induction caused by the mutagen agent
nitrofurantoin along with potent antioxidant capacity
even more than positive control trolox.31 It is of note to
mention the diversity of compounds present in different
species of genus Marrubium like diterpenoid contents
that could be responsible for the chemopreventive effects
of the extract, according to the available reports
describing the protective role of these compounds in a
range of oxidative stress related diseases.
The imperative role of exogenous antioxidants such as
natural herbal antioxidant compounds like phenolic
acids, polyphenols and flavonoids, plus endogenous
antioxidants produced by the human body in scavenging
peroxide, hydroperoxide or lipid peroxyl free radicals
leading degenerative diseases could not be
disclaimed.40,41 Concerning the results of the study,
significant antioxidant activity with regard to the
potential radical scavenging ability was detected by the
methanol extract of M. crassidens. Although the
potential antioxidant activity of this plant could be linked
to the complex mixtures of different compounds that are
present in most botanicals and herbs, the conventional
role of flavonoids and other phenolics present in the
methanolic extract of M. crassidens donating a hydrogen
atom for scavenging the stable DPPH radical is
inevitable. Many researchers believe there is a linear
relation between antioxidant activity and phenolic
contents such as polyphenols, flavonoids and catechins
that had been recognized to be connected with anticancer
activity of various plants.42-44 On the whole, it was
established that the increase in the phenolics and
flavonoids content of M. crassidens methanol extract
brought about higher radical scavenging activity in line
with enhanced cytotoxicity activity of the extract in
MCF-7 cells.
Conclusion
The present study puts forward the antioxidant activity of
M. crassidens extract together with its cytotoxic activity
462 | Advanced Pharmaceutical Bulletin, 2014, 4(Supp1), 459-464
could be helpful in prevention of some serious diseases
like breast cancer. It is of value to indicate the potential
role of some known phenolics in inhibition of the
transformed or malignant cells growth via initiation of
programmed cell death or apoptosis. Therefore, it seems
requisite to search for the distinctive mechanism of
action in the relative antiprolifreative activity of M.
crassidens methanolic extract through investigation on
cell cycle analysis. Although many phytomedicines from
herbal products exert their beneficial effects through the
additive or synergistic action of various chemical
compounds, additional bioassay-guided fractionation
approaches on M. crassidens extract might be worthy in
purifying and identifying the foremost active constituents
in charge of proliferation inhibition of MCF-7 cells,
since it is the very first report in this regard.
Acknowledgments
The authors would like to thank the Research ViceChancellor of Tabriz University of Medical Sciences for
financial support of this study. This article was written
based on a data set of PhD. thesis, registered in Tabriz
University of Medical Sciences (5/4/6651- NO. 71).
Conflict of interest
The authors report no conflict of interests.
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