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Apoptotic and Antioxidant Activities of Methanol Extract of Mussaenda roxburghii Leaves
Farhadul Islam 1*, Obayed Raihan2, Dipjoy Chowdhury3, Mahbuba Khatun1, Natasha Zuberi1, Laboni Khatun1,
Afrina Brishti4, Entaz Bahar3 and Jahan Ara Khanam1
1
Department of Biochemistry and Molecular Biology, University of Rajshahi, Rajshahi-6205,
Bangladesh
2
Department of Pharmacy, Jessore Science and Technology University, Jessore, Bangladesh
3
Department of Pharmacy, International Islamic University Chittagong, Chittagong, Bangladesh
4
Department of Pharmacy, University of Rajshahi, Rajshahi-6205, Bangladesh
*Address
for correspondence: Md. Farhadul Islam, Assistant professor, Department of
Biochemistry and Molecular Biology, University of Rajshahi, Rajshahi-6205, Bangladesh
E-mail: [email protected]/[email protected]
Tel. +880-1712974596
Fax: +880-721750064
Abstract
The use of plants and their active substances increases day by day for the discovery of
therapeutic agents due to their versatile applications. Current research is directed towards finding
naturally-occurring antioxidants having anticancer properties since oxidants play a crucial role in
developing various human diseases. The present study was designed to investigate the anticancer
and antioxidant activity of methanol extract of Mussaenda roxburghii (designated as MMR).
Anticancer activity of MMR has been carried out against Ehrlich ascites carcinoma (EAC) cells
with three different doses (20, 40 and 60 mg/kg/day) by monitoring the parameters like tumor
weight measurement, survival time of tumor bearing mice, growth inhibition of cancer cells,
morphological changes and nuclear damage of EAC cells etc. whereas antioxidant capacity was
determined by measuring total antioxidant capacity (TAC), DPPH free radical scavenging
capacity, ferrous reducing capacity assay.
The extract showed highest anticancer activity at the dose of 60 mg/kg/day (i.p.). It caused
81.4% (P < 0.01) cells growth inhibition and reduced tumor burden significantly (78.5%; P <
0.001) when compared with control. It also increased the life span of EAC bearing mice
significantly (73.5 %; P < 0.01). High LD50 value (600 mg/kg) of MMR indicated its low host
toxic effects. MMR treated EAC cells showed membrane blebbing, chromatin condensation,
nuclear fragmentation (apoptotic feature) in Hoechst 33342 staining under fluorescence
microscope. DNA fragmentation assay in agarose gel (1.5%) electrophoresis also rectified that it
causes EAC cells death by apoptosis. MMR also showed moderate antioxidant properties
compare to standard antioxidant catechin and ascorbic acid in a dose dependent manner. M.
roxburghii possess significant anticancer and moderate antioxidant properties. Thus, this plant
may therefore be considering a good source for natural chemopreventive drugs as well as a
possible pharmaceutical supplement.
Key Words: Apoptosis, Intrinsic pathway, anticancer agents, antioxidant, Mussaenda
roxburghii, EAC cells, Chemoprotective drugs etc.
Introduction
Plants have been serving an important role for mankind’s medicine from the very beginning of
human history and there is an incredible historical legacy in folklore uses of plant preparations in
medicine [1]. Scientific research endeavor on plants used in ethno-medicine led to the discovery
of many valuable drugs such as bleomycin, taxol, camptothecin, vincristine and vinblastine [2].
Active phytochemical principle such as flavonoids, polyphenols, proanthocyanidin etc. act as
strong free radical scavenger. Free radicals including superoxide radicals, hydroxyl radicals,
singlet oxygen and hydrogen peroxide (reactive oxygen species; ROS) are often generated as byproduct of biological reactions, or from exogenous factors. These ROS may be very damaging as
they can induce oxidation of lipids causing membrane damage, decrease membrane fluidity and
lead to cancer via DNA mutation [3]. Cancer is a non-communicable disease and due to lack of
effective drugs/treatment modality it is rating the top second cause of death [4]. A potent
scavenger of ROS may serve a possible prevention against the free radicals-mediated diseases
like cancer [5]. Antioxidants are radical scavengers and protect the human body from free
radicals. Antioxidant based drugs or formulations for the prevention and treatment of cancer
engaged many researchers worldwide to explore the therapeutic potential of medicinal plants in
nature.
Mussaenda species member of the Rubiaceae (coffee family) are native to the old World tropics,
from West Africa through the Indian sub-continent, South East Asia and into Southern China.
They have been used in Chinese, Fijian and Indian traditional medicine for various ailments such
as diuretic, antiphlogistic, antipyretic and also used to detoxify mushroom poisons and terminate
early pregnancy [6]. Active ingredients from these family plants also have potential cytotoxic,
antibacterial, antiviral, anti-RSV activity [7-10]. Exploration of an indigenous medicinal plant,
Mussaenda roxburghii (family: Rubiaceae) commonly known as Himalayan Mussaenda which is
a shrub and distributed in moist shady places of Bangladesh, Bhutan and Myanmar is under
investigation. Root paste of the plant is applied to the tongue to treat boils [11], and a recent
study showed that it inhibits the growth of both Staphylococcus aureus and Escherichia coli
effectively [12]. As there is no further pharmacological report of this plant elsewhere, we first
here present the anticancer activity against Ehrlich ascites carcinoma (EAC) cells and antioxidant
activity to find out effective chemopreventive drugs.
Materials and Methods
Chemicals and reagents
All the chemicals and reagents used throughout the investigation were of reagent grade and
purchased from BDH, UK, E’MERK, Germany and Sigma Aldrich, US.
Test animals
Adult male Swiss albino mice, five to six weeks old (25±3 gm body weight) were collected from
animal resource branch of the International Centre for Diarrheal Disease Research, Bangladesh
(ICDDR’B) and used throughout the studies. Animals were housed in polypropylene cages
containing sterile paddy husk as bedding material under hygienic conditions with a maximum of
six animals in a cage. They were maintained under controlled conditions (12:12 h light-dark),
temperature (25 ± 5○ C). The mice were fed with standard mice food-pellets (collected from
ICDDR’B) and water was given in ad libitum.
Cell lines
EAC cells were obtained by the courtesy of Indian Institute of Chemical Biology (IICB),
Kolkata, India. The cells were maintained as Ascites tumour in swiss albino mice by
intraperitoneal inoculation (bi-weekly) of 2×106 cells/mouse.
Ethical clearance
Protocol used in this study for the use of mice as animal model for cancer research was approved
by the Institutional Animal, Medical Ethics, Biosafety and Biosecurity Committee (IAMEBBC)
for Experimentations on Animal, Human, Microbes and Living Natural Sources (225/320IAMEBBC/IBSc), Institute of Biological Sciences, University of Rajshahi, Bangladesh.
Plant materials
The collected leaves of the plant were shade dried for several days with occasional sun drying
and reduced to coarse powder. The dried powder was extracted with methanol (yield 8.5 %) at
room temperature for 14 days with occasional shaking and stirring. The combined extract was
filtered through cotton and then Whatman No.1 filter papers and was concentrated with a rotary
evaporator under reduced pressure at 45° C. The crude extract designated (MMR) then dried and
stored in a vacuum container for further use. The crude extract was dissolved in 2% (V/V)
dimethylsulfoxide (DMSO) for the experiments.
Phytochemical screening of crude extract
The phytochemical components of the plant were screened using the standard methods [13]. The
components
analyzed
for
were
saponins,
saponin
glycosides,
steroid,
glycosides,
proanthocyanidin, anthraquinones, tannins, flavonoids, alkaloid, volatile oils, phenols and
balsam (gum).
Determination of median lethal dose (LD50)
Methanol extract of M. roxburghii (MMR) was dissolved in 2 % (v/v) DMSO and were injected
intraperitoneally to seven groups of mice (n=4) at different doses [200mg/kg (i.p.), 400 mg/kg
(i.p.), 500 mg/kg (i.p.), 550 mg/kg (i.p.), 600 mg/kg (i.p.), 650 mg/kg (i.p.), 700 mg/kg (i.p.), and
750 mg/kg (i.p.)]. The LD50 value was then calculated by the procedure described in the literature
[14].
Determination of cell growth inhibition
Cells growth inhibition with the extract was performed by the standard method [15]. For this
experiment, five groups of swiss albino mice (n=6) weighing 25±3 gm were used. For
therapeutic evaluation 2×106 EAC cells in every mouse were inoculated into each group on day
“0”. Treatments with MMR and bleomycin (standard clinically used anticancer drug) were
started after 24 hours of tumor inoculation and continued for five consecutive days. Here group
one to three received MMR at the doses of 20 mg/kg (i. p.), 40 mg/kg (i. p.), and 60 mg/kg (i. p.)
per day respectively. Group four treated with bleomycin at the dose of 0.3 mg/kg (i. p.) whereas
group five was used as control receiving solvent only. Mice from every group were sacrificed on
day six and total intraperitoneal tumor cells were harvested by normal saline (0.98%). Viable
tumor cells were first identified with trypen blue and then counted by a haemocytometer under
inverted microscope (XDS-1R, Optika, Italy). Total numbers of viable cells in every animal of
the treated groups were compared with those of control (EAC bearing only) mice.
Bioassay of EAC cells
The effect of MMR on transplantability/bioassay of EAC cells was studied by the method
described in the literature [16]. For this experiment, two groups of mice (n=6) were inoculated
with 17×105 EAC cells on day “0”. Group 1, was treated with MMR at the dose of 60 mg/kg/day
(i.p.) for five consecutive days and group 2 used as control. On day 7, tumor cells from the
treated mice (group 1) were harvested in cold (0.9 %) saline, pooled, centrifuged and reinoculated into two fresh groups of mice (n=6) as before. No further treatment was given on
these mice. On day 5 of tumor re-inoculation, they were sacrificed and viable tumor cells/mouse
were estimated.
Determination of average tumor weight and survival time
A brief description of the method used by Sur et al [15], is given bellow. For this experiment,
five groups of swiss albino mice (6 in each group) were used. On day ‘0’, 2 ×106 EAC cells per
mouse were inoculated to each group of mice for therapeutic evaluation. Treatment with MMR
as well as bleomycin was started after 24 hours of tumor inoculation and continued for 10 days.
Tumor growth was monitored by recording daily weight change. Host survival was recorded and
expressed as mean survival time (MST) in days and percent increase of life span was calculated
by using the following formula:
Ʃ Survival time (days) of each mouse in a group
Mean survival time (MST) =
Total number of mice
Percent increase of life span (ILS) % =
MST of treated group
MST of control group
-1 × 100
Effect of MMR on normal peritoneal cells
Effects of MMR on normal peritoneal cells on normal mice were determined by counting total
peritoneal cells and number of macrophages [17]. Normal mice (n=6) were treated with MMR (i.
p.) at the dose of 20 mg/kg, 40 mg/kg and 60 mg/kg/day for three consecutive days. The
untreated group used as control. After 24 hours of last treatment, the animals were sacrificed
after injecting 5 ml of normal saline (0.98%) into the peritoneal cavity of each mouse. The
number of intraperitoneal exuded cells and macrophages were counted with 1% neutral red by
haemacytometer under microscope.
Morphological changes and nuclear damage of EAC cells
Cellular apoptosis induced by the extract (MMR) was studied by the method described earlier
with little modification [18]. Morphological observation of cells, in absence and presence of
MMR (60 mg/day) were studied using a fluorescence microscope (Olympus IX71, Korea). At
first EAC cells were collected from culture plates receiving MMR and saline (none treated
control plates) and stained with 0.1 µg/ml of Hoechst 33342 at 37oC for 20 min. Then the cells
were washed with phosphate buffer saline (PBS) and re-suspended in PBS for observation of
morphological changes under fluorescence microscopy. In addition, to determine the necrotic or
late apoptotic cell death, EAC cells were further washed by 0.01% Sodiun azide containing 0.9%
NaCl and then stained with Propidium Iodide (PI).
Effect of caspase inhibitors
In order to find out the involvement of caspases in the MMR-induced cell death, the cells were
also incubated in CO2 incubator with Z-DEVD-FMK (caspase-3 inhibitor, 2µmol/ml) and ZIETD-FMK (caspase-8 inhibitor, 2 µmol/ml) for 1 h, and then the cells were treated with the
extract and kept for another 24 h [19].
DNA fragmentation assay
DNA fragmentation assay in agarose gel electrophoresis was determined by the method
described previously [20]. EAC cells obtained from mice treated with and without extract (1×
106/ml) at the dose 60 mg/kg/day for five consecutive days. The cells were washed with PBS and
re-suspended again in PBS. The total DNA was isolated by using a DNA extraction kit
(Promega, USA) and analyzed by electrophoresis on 1.5% agarose gel containing 0.1 µg/ml
ethidium bromide and visualized under UV illuminator.
Determination of antioxidant capacity
Antioxidant potentiality of methanol extract of the M. roxburghii was evaluated by determining
total antioxidant capacity, determining DPPH radical scavenging activity and ferrous reducing
capacity assays.
Determination of total antioxidant capacity
Total antioxidant capacity (TAC) of MMR and catechin (CA) was determined by the standard
method with some modifications [21]. For this experiment, 0.5 ml of MMR/CA (standard) at
different concentrations was mixed with 3 ml of reaction mixture containing 0.6 M sulphuric
acid, 28 mM sodium phosphate and 1% ammonium molybdate into the test tubes. The test tubes
were incubated at 950C for 10 minutes to complete the reaction. The absorbance was taken at
695 nm using a spectrophotometer against blank after cooling at room temperature. A typical
blank solution contained 3 ml of reaction mixture and the appropriate volume of the same
solvent used for the MMR/CA were incubated at 950C for 10 minutes and the absorbance was
taken at 695 nm. Increased absorbance of the reaction mixture indicated increase total
antioxidant capacity.
Ferrous reducing antioxidant capacity assay
The ferrous reducing antioxidant capacity (FRAC) of MMR/Ascorbic acid (AA) was measured
by the method of Oyaizu [22]. For this purpose, 0.25 ml MMR/AA solution at different
concentrations, 0.625 ml of potassium buffer (0.2 M) and 0.625 ml of 1% potassium ferricyanide
[K3Fe (CN)6] solution were added into the test tubes. The reaction mixture was incubated for 20
minutes at 500 C to complete the reaction. Then 0.625 ml of 10% Trichloro acetic acid (TCA)
solution was added into the test tubes. The total mixture was centrifuged at 3000 rpm for 10
minutes. After which, 1.8 ml supernatant was withdrawn from the test tubes and was mixed with
1.8 ml of distilled water and 0.36 ml of 0.1% FeCl3 solution. The absorbance of the solution was
measured at 700 nm using a spectrophotometer against blank. Increased absorbance of the
reaction mixture indicated increase reducing capacity.
DPPH radical scavenging assay
Free radical scavenging activity was determined by DPPH radical scavenging assay (DRSA) as
reported by Choi et al [23]. For this experiment, a solution of 0.1 mM DPPH in methanol was
prepared and 2.4 ml of this solution was mixed with 1.6 ml of extractives in methanol at different
concentrations. The reaction mixture was vortexed thoroughly and left in the dark at room
temperature for half an hour. The absorbance of the mixture was taken spectrophotometrically at
517 nm. AA was used as reference standard. Percentage DPPH radical scavenging activity (%
DRSA) was calculated by the following equation: (% DRSA) = {(Ao – A1)/Ao} × 100
Where, A0 is the absorbance of the control, and A1 is the absorbance of the extract/standard.
Then % of inhibition was plotted against concentration, and from the graph IC50 was calculated.
Statistical Analysis
The experimental results are expressed as the mean ± SEM (Standard Error of Mean). Data have
been calculated by one way ANOVA followed by Dunnett ‘t’ test using statistical package for
social science (SPSS) software of 10 version.
Results
Phytochemical screening of crude extract
Phytochemical screening of crude extracts of M. roxburghii indicated that the plant had
polyphenols, flavonoids, steroids, glycoside and also alkaloids. The components; anthraquinones,
hydrolysable tannin, saponins, glycosides etc. were not detected in the crude methanol extracts of
the plant (Table 1).
Determination of median lethal dose (LD50)
Lethal dose of MMR was found to be 600 mg/kg, for intraperitoneal treatment in male Swiss
albino mice. The experimental animals showed toxicity at this dose regarding body weight, food
intake as well as general appearance.
Determination of cell growth inhibition
Effects of MMR and bleomycin on EAC cells growth on day six of tumor inoculation are shown
in Figure 1. Treatment with MMR resulted in maximum cell growth inhibition at the doses of 60
mg/kg (i. p.) and 40 mg/kg as evident from 81.4% and 68.7% respectively whereas bleomycin
showed cell growth inhibition by 86.62% (0.3 mg/kg/day).
Bioassay of EAC cells
Transplantability of EAC cell receiving MMR decreased remarkably as 51.4% reduction of
EAC cell growth was observed when EAC cells from MMR treated mice (at the dose 60 mg/kg
i. p.) were re-inoculated into fresh mice and sacrificed and compared with control on day 5.
Determination of average tumor weight and survival time
All effective anticancer drugs show a significant impact on survival time of tumor bearing mice.
The effect of MMR and bleomycin on survival time at different doses has been shown in Figure
2. It has been found that tumor induced mice treated with MMR at doses 20mg/kg, 40mg/kg and
60mg/kg resulted a significant increased of life span, which were 41%, 60.3%, 73.5%
respectively compared to that of control mice. On the other hand, bleomycin increased life span
by 88.7 % when compared to that of control. Effect of MMR at doses 20mg/kg (i.p.), 40mg/kg
(i.p.), 60mg/kg (i.p.) and the antitumor drug bleomycin (0.3 mg/kg) on the average tumor weight
owing to tumourgenesis is shown in Figure 3. Supplementation of the extract of mice previously
inoculated with EAC cells resulted in the inhibition of tumor burden. In the case of control (EAC
bearing) group, the body weight increased by 68.5% on 20th day when compared to the normal.
Mice treated with MMR at doses 20 mg/kg (i.p.), 40 mg/kg (i.p.), 60 mg/kg (i.p.) the body
weight increased by 37.6%, 28.52% and 21.5% respectively on the 20th day. In contrast the use
of bleomycin as standard at the doses of 0.3 mg/kg (i. p.) the body weight was increased by 19.3
% on the 20th day.
Effect of MMR on normal peritoneal cells
The average number of peritoneal exudates cells of normal mice receiving MMR were found to
be (13 ± 1.2) × 106 whereas the macrophage count was (3.6 ± 0.5) × 106 at 60 mg/kg/day
respectively. Treatment with the extract at a dose of 60 mg/kg/day for three consecutive days
significantly enhanced the number of macrophages. Results are shown in Figure 4.
Morphological changes and nuclear damage of EAC cells
Morphological changes of EAC cells were examined by Hoechst 33342 staining after culturing
the cells with MMR and without extract (60 µg/ml) for 24 hrs. EAC nuclei were round, regular
and homogeneously stained with Hoechst 33342 in control group as shown in Figure 5A.
Whereas MMR treated EAC cells showed manifest fragmented DNA in nuclei as shown in
Figure 5B. Apoptotic morphologic alterations such as membrane and nuclear condensation were
also observed clearly by fluorescence microscopy. These results indicate that MMR could induce
apoptosis of EAC cells. Necrotic or late apoptotic cell death caused by extract was also observed
by staining with PI as shown in Figure 5C. Here the numbers of necrotic cells were found to be
very low.
Effect of caspase inhibitors
Caspase inhibitors Z-DEVD-FMK (caspase-3 inhibitor) and Z-IETD-FMK (caspase-8 inhibitor)
were used to understand which pathway was switched on with the MMR treatment. The
cytotoxicity of extract towards Z-IETD-FMK-pretreated EAC cells was significantly reduced to
56.95 %, whereas Z-DEVD-FMK-pretreated cells did not any reduction of cytotoxicity in
comparison to control (Figure 6). The activation of the endogenous Ca2+/Mg2+-dependent
endonuclease is the most distinctive biochemical hallmark of apoptosis.
DNA fragmentation assay
This activated endonuclease-mediated the cleavage of inter-nucleosomes and generate
oligonucleotide fragments of about 180-200 bp length or their polymers. A DNA ladder bands
were obtained in agarose gel electrophoresis of DNA preparation extracted from MMR treated
EAC cells which is characteristic feature of apoptosis induction, whereas in the control group,
smear-like DNA degradation was obtained, which was shown in Figure 7.
Determination of total antioxidant capacity
The total antioxidant capacity (TAC) of MMR was shown in Figure 8. The plant M. roxburghii
showed potent antioxidant activity like reference standard CA at all the concentrations. The
absorbance of MMR and standard CA were 1.95and 2.82 respectively at 800 mg/ml. The extract
was found to increase the total antioxidant activity with the increasing concentration.
Ferrous reducing antioxidant capacity assay
Figure 9 represent the reductive capabilities of the plant extract compared to Ascorbic acid which
was determined using the potassium ferricyanide reduction method. The reducing power of the
extract was moderately strong while increasing dose it showed remarkable increment.
DPPH radical scavenging assay
The DPPH radical scavenging activity of M. roxburghii is shown in Figure 10. This activity was
found to increase with increasing concentration of the extract. The DPPH radical contains an odd
electron, which is responsible for the absorbance at 515–517 nm and also for a visible deep
purple color. The IC50 value of the extract was 120.4μg/ml while the IC50 value of ascorbic acid
was 18.52μg/ml. These results indicate the moderate antioxidant activity of the methanol extract
of M. roxburghii.
Discussion
Reduction of tumor burden, enhancement of life span of cancer bearing mice, tumor cell growth
inhibition is the important contributing factors of a potential anticancer agent. The efficiency of
MMR as anticancer agent was compared with data obtained by running parallel experiments
with a known clinically used anticancer drug, bleomycin at the dose of 0.3 mg/kg (i.p.) and also
with those found in similar type of extracts available in the literature [4, 24]. The average tumor
weight/burden reducing capacity of this extract has been examined. For tumor bearing mice,
body weight was found to be increased gradually with time period. Treatment of such mice with
the extract reduced the grown up remarkably. The extract, MMR also inhibited the cell growth
rate effectively; as more than 71 % inhibition was found at the dose of 60 mg/kg (i.p.) which is
quite comparable to that of bleomycin (76.6%; at 0.3 mg/kg).
Supplementation of MMR increased the life span of tumor bearing mice very effectively and the
potency was found to increase with the enhancement of dose. In the present experimental design,
a dose up to 60 mg/kg (i.p) was used and further enhancement of the life span will therefore be
expected using higher doses. It is noted that the enhancement of life span has been assigned as a
very important parameter for judging the suitability and efficacy of a compound as anticancer
agent [25]. The high LD50 value (600 mg/kg) of this crude extract indicated little toxicity to the
host.
It has been found that the extract of M. roxburghii has significant effect on the enhancement of
normal peritoneal cells in normal mice. Treatment with MMR at a dose of 60 mg/kg (i.p)
increased the number of macrophages to some extent. This is being considered to be a very
important parameter for acquiring self destroying ability of the animals or living beings towards
cancer cells [26].
It is highly desirable to have compounds that can cause cancer cell death via apoptosis.
Apoptosis eliminates malignant or cancer cells without damaging normal cells and surrounding
tissues [27]. Apoptosis is characterized by cell morphological changes, chromatin condensation,
DNA cleavage, and nuclear fragmentation. Typical morphological features of apoptotic cells can
be observed through microscopic studies such as those using the inverted phase contrast and
fluorescence microscope. Other features such as chromatin condensation and nuclear
fragmentation can be better observed through the double staining with Hoechst 33342 and
Propidium Iodide (PI) using fluorescence microscopic analysis. This is a convenient and rapid
assay, widely used to identify live and dead cells. Hoechst 33342 is a blue fluorescing dye that
stains chromatin DNA. The red fluorescing dye PI is only permeable to dead cells and cannot
enter the intact plasma membrane of living cells. Thus, the staining pattern which resulted from
the simultaneous use of these dyes makes it possible to distinguish normal, apoptotic, and dead
cells population by fluorescence microscopy [28-31]. EAC cells treated with extract (MMR)
shown nuclear condensation, fragmentation, cell membrane blebbing, apoptotic bodies etc. under
the fluorescence microscope which implies that the extract induce EAC cells apoptosis. PI
staining of EAC cells also indicates the late phase apoptosis of induction after treatment with the
extract. The integrity of the DNA was also assessed by agarose gel electrophoresis. DNA
isolated from cells showed a ‘‘ladder’’ pattern in apoptosis [20]. Genomic DNA isolated from
treated and untreated cells shown apoptotic pattern in agarose (1.5 %) electrophoresis. This
characteristics ladder like DNA band in the gel, further conform the induction of apoptosis in
EAC cells to the treatment of MMR.
There are two main apoptotic pathways-the extrinsic (death receptor) pathway is activated by
binding of tumor necrosis factor (TNF) ligands such as TRAIL to corresponding cell surface
receptors, for example, TRAIL receptors, triggering the assembly of the death-inducing signaling
complex (DISC) that drives caspase-8 activation[27]. The intrinsic (mitochondrial) pathway
involves mitochondrial outer membrane permeabilization accompanied by the release of
cytochrome C and second mitochondria-derived activator of caspases (Smac) into the cytosol104.
Cytochrome C initiates the formation of a caspase-9, caspase-3 activation platform, that is, the
apoptosome, while Smac relieves the inhibition of caspases by neutralizing inhibitor of apoptosis
(IAP) proteins[32]. To understand the involvement of specific signaling pathway trigger by
MMR in EAC cells, the specific inhibitors of candidate molecules such as caspase-8 and
caspase-3 were used. Data obtained from our experiment shows that EAC cells pretreated with
caspase-8 inhibitor (caspase-8 blocked/inactivated and caspase-3 remain active) showed 56.95 %
growth inhibition in comparison to control whereas, cells pretreated with caspase-3(caspase-3
inactive and caspase-8 active) shown very little or no cells growth inhibition. This result leads
one to conclude that caspase-3 mediated signaling pathway is involved in EAC cells apoptosis
induced by the treatment of M. roxburghii.
Our present study results also reflected that the same extract of M. roxburghii showed moderate
antioxidant activity. Numerous animal studies have been published demonstrating decreased
tumor size and/or increased longevity with the combination of chemotherapy and antioxidants
[33]. Resistance to chemotherapeutic agents is thought to be due to reduced accumulation into
the tumor cell [34]. Recent research has focused on the ability of flavonoids type antioxidant
compound to increase the concentration of chemotherapeutics in tumor cells. New findings
within the past few years have revealed that the binding of selective glycosides compound to
Na+, K + -ATPase results in complex but well-documented changes in cell signaling events [35].
This “signalosome” complex includes the enzyme, Na+, K + -ATPase as well as Src,
phosphoinositide-3 kinase (PI3K), and phospholipase C each of which, in turn, sets into action
complex signaling events that can result in tumor cell death through either apoptosis or
autophagy-related mechanisms [36-37]. Apoptotic cell death mediated by glycosides has been
demonstrated in a number of cell lines [38]. 2006) for example oleandrin produced an increase in
expression of Fas and TumorNecrosis Factor Receptor 1 (TNFR1), resulting in potentiation of
apoptosis in tumor cells but not in normal primary cells. As such, they represent a promising
form of targeted cancer chemotherapy [39]. On the other hand, alkaloids derived from plants;
block cell division by preventing microtubule function. There is increasing evidence showing
that even minor alteration of microtubule dynamics can engage the spindle checkpoint, arresting
cell cycle progression at mitosis and eventually leading to apoptotic cell death. Though the
presence of flavonoids, glycosides and alkaloids of the extract could be attributed to the
anticancer effect of M. roxburghii through apoptosis but still there is no consensus about which
substances are exactly responsible for these effects. So much more investigation including
identification specific active compound involved has to be carried out against various other
cancer cell lines and higher animal models, in order to confirm the plant as a potent anticancer
drug reservoir.
Conclusions
In the light of above observations, it can be concluded that the methanol extract of M. roxburghii
showing potential anticancer activity through intrinsic mitochondrial pathway and might be
considered as one of the promising resources in cancer chemotherapy. It also showed moderate
antioxidant activity which might be providing save guard against damaging oxidants as well as
boost up chemo-preventive potential.
Acknowledgement
The authors are grateful to IICB, Kolkata, India, for providing the EAC cells, central science
laboratory of Rajshahi University, Bangladesh for giving support in fluorescence microscopy and
also to ICDDRB, for supplying the experimental mice standard mice pellets.
Conflict of interest
We have no conflict of interests.
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Table 1. Phytochemical components of crude methanol extract of M. roxburghii
Phytochemical components
E. camadulensis
Alkaloids
+
Saponins
-
Saponin glycosides
-
Tannins
-
Hydrolysable tannins
-
Phlobatannins
-
Anthraquinones
-
Glycosides
+
Cardiac Glycosides
-
Flavonoids
+
Steroid
+
Volatile oils
-
PolyPhenols
+
+: Present;
-: Absent
Figure 1. Effects of MMR on EAC cells growth inhibition
100
***
**
*
% of cell growth inhibition
80
60
40
20
0
Bleomycin (0.3mg/k)
MMR (60mg/kg)
MMR (40mg/kg)
MMR (20mg/kg)
Results are shown as mean ± SEM (Standard Error of Mean), where significant values are * P <
0.05, ** P < 0.01 and *** P < 0.001 when (EAC+ MMR) treated mice compared with EAC
bearing control mice (EAC bearing only).
Figure 2. Effects of MMR on survival time of tumor bearing mice
50
***
**
Meas Survival Time (MST) in days
40
**
*
30
20
10
0
Control
Bleomycin (0.3 mg/kg)
MMR (20mg/kg)
MMR (40mg/kg)
MMR (60mg/kg)
Results are shown as mean ± SEM (Standard Error of Mean), where significant values are * P <
0.05, ** P < 0.01 and *** P < 0.001 when (EAC+ MMR) treated mice compared with EAC
bearing control mice (EAC bearing only).
Figure 3. Tumor weight of EAC bearing mice treated with MMR and bleomycin
25
20
Tumor weight in gm
15
*
10
**
***
5
***
0
0
5
10
15
Control
Bleomycin (0.3 mg/kg)
(40mg/kg)
MMR -5
MMR (60mg/kg)
20
25
MMR (20mg/kg)
Results are shown as mean ± SEM (Standard Error of Mean), where significant values are * P <
0.05, ** P < 0.01 and *** P < 0.001 when (EAC+ MMR) treated mice compared with EAC
bearing control mice (EAC bearing only).
Figure 4. Effects of MMR on the enhancement of macrophages and peritoneal cells
16
*
Number of cells (10 6)
12
***
*
8
**
**
4
*
0
Control
MMR (20mg/kg)
Total peritoneal cell
MMR(40mg/kg)
MMR (60mg/kg)
Macrophage
Results are shown as mean ± SEM (Standard Error of Mean), where significant values are * P <
0.05, ** P < 0.01 and *** P < 0.001 when (Normal+ MMR) treated mice compared with control
mice.
Figure 5. Fluorescence microscopic view of control and treated EAC cells
B
A
C
A) EAC of normal mice shown no apoptotic feature. B) EAC cells treated with extract shown
nuclear condensation, fragmentation (black arrow), cell membrane blebbing (red arrow), and
apoptotic bodies (white arrow) etc. C) Cells undergone late apoptosis (black arrow) and normal
dead (white arrow) shown in PI staining.
Figure 6. Effect caspases inhibitors on EAC cells
Results are shown as mean ± SEM (Standard Error of Mean).
Figure 7. In vivo effects of extract on DNA fragmentation of EAC cells
B
A
DNA run and detected on 1.5% agarose gel electrophoresis. A) DNA from control EAC cells, B)
DNA from MMR treated EAC cells (DNA fragmentation detected from treated EAC cells).
Figure 8. Determination of total antioxidant capacity of M. roxburghii
3.5
3
Absorbance (700 nm)
2.5
2
1.5
1
0.5
0
0
100
200
300
400
500
600
Concentration (m g/m l)
MMR
CA
Results are shown as mean ± SEM (Standard Error of Mean).
700
800
Figure 9. Determination of ferrous reducing antioxidant capacity of M. roxburghii
3.5
Absorbance (695 nm)
3
2.5
2
1.5
1
0.5
0
0
200
400
600
Concentration (m g/m l)
MMR
AA
Results are shown as mean ± SEM (Standard Error of Mean).
800
Figure 10. Determination of DPPH radical scavenging activity of M. roxburghii
100
90
% of scavenging
80
70
60
50
40
30
20
10
0
0
50
100
150
200
250
Concentration (microgram/ml)
MMR
AA
Results are shown as mean ± SEM (Standard Error of Mean).
300
350
400