INTRODUCTION:
Herbal plants are rich in many
pharmacologically active compounds like flavonoids, saponins, alkaloids.
Flavonoids naturally exist in almost all the plant kingdom. Flavonoids can be
found in almost all dietary plants, like fruits and vegetables. Flavonoids were
also present in many medicinal plants, and herbal remedies consists flavonoids
have been used in folk medicine throughout the world. Flavonoids from Sphaeranthus amaranthoides may be usefull chemopreventive agents for cancer. The
polyphenolic compounds from Sphaeranthus
amaranthoides shows a remarkable
broad spectrum of the action on different biological activities, for example,
antianalgesic and anti-inflammatory [1], wound healing [2], antimicrobial
activity.
The plant Sphaeranthus amaranthoides is a weed in the paddy field of
Southern India, particularly in Tanjore, Thirunelveli, Southern Mysore and
Travancore area[3],[4]. In folk medicine, it has been proved that the activity of the plant
is due to the presence of the flavonoids. In this context the current study
concentrates on the cytotoxic activity of flavonoids on the lung cancer cell
lines. Lung cancer is one of the major prevalent life-threatening human cancers
and increasing in terms of morbidity and mortality. The current medication is with synthetic
drugs leads to drastic side effects. The natural drugs play an essential role
to reduce the side effects on the normal cells and increase the cytotoxicity of
the tumor cells.
Chemicals and Reagents:
Quercitin was used as standard and was
obtained from Sigma Aldrich. Methanol and acetonitrile was purchased of
analytical grade. TLC plates of silica gel 60 which are not on fluorescent
indicator were purchased, and the rest of the chemicals used for the anticancer
activity were of kit based.
METHODOLOGY:
Collection of Leaf material: The leaf
material was obtained from Tirunalveli district and it was authenticated by V.
Chelladurai, Rtd Botanist. The collected leaf material was shade dried and
powdered into coarse and preserved for further use. Sample preparation for HPLC: 2.0g of Sphaeranthus amaranthoides dried leaf powder was taken and subjected to
maceration agitation with 10mL of EtOH/ H2O (1:1 v/v). The hydroethanolic extracts were filtered,
and their final volume was adjusted to 20ml with EtOH/ H2O (1:1
v/v). The hydroethanolic extracts were simply filtered through a Millipore
membrane with 0.5µm. For HPTLC and HPLC analysis, the extracts were subjected
to liquid-liquid extraction using 5mL of CHCl3; the hydroethanolic
layer was filtered through 0.5 µm Millipore membrane for the preparation of the
sample for HPLC analysis.
HPLC-UV-Pad analysis: the analysis was
performed in a Shimadzu Prominence_UFLC XR with Phenomenex
Gemini NX C18 column (150×4.6mm, 5µ) injection volume 10 µL wit flow rate 0.5
ml/min. Mobile phase A is 0.01M Ammonium Formate in water and Mobile phase B is
Acetonitrile. The column oven was thermostat controlled at 35oC and
the detection was monitored at 254 and 350nm.
Thin layer chromatography: The hydro ethanolic extract was used
for analyses, TLC was carried out on
silica gel 60 aluminium sheets which are precoated with wit EtOAc/HCOOH/H2O
(6:1:1 v/v). The solvent should be dried after developing plots and the
flavonoids were visualized under UV at 360 NM [5].
TLC a: TLC was performed according to the Brasseur and
Angenot et al 1996. The TLC plates were visualized at 254 NM with the suitable
spray reagent.
Cell Preparation
and Culturing:
A549 lung adenocarcinoma cell line was
procured from National Centre for Cell Science (NCCS), Pune, India with the
passage number 11. Cells were maintained in Dulbecco’s Minimum Essential Media
(DMEM) supplemented with 10% Fetal Bovine Serum (FBS), with 100units/mL penicillin
and 100μg/mL streptomycin. Cells were cultured in a humidified atmosphere
with 5% CO2 at 37 °C. Cells were grown in 75cm2 culture
flask and after a few passages, cells were seeded for experiments. The
experiments were done at 70 to 80% confluence. Upon reaching confluence, cells
were detached using 0.25% Trypsin-EDTA solution.
MTT Assay:
According to Safadi et al [5] the proliferation of
A549 cells was assessed by MTT assay. The proliferation test is based on the
color reaction of mitochondrial dehydrogenase in living cells by MTT. Cells
were plated in 96-well plate at a concentration of 5 × 104
cells/well 24 h after plating. After 24h of cell incubation, the medium was
replaced with 100µl medium containing flavonoid fraction at different
concentrations (5 – 640µg/ well) and incubated for 24h. Untreated cells served
as controls and received only 0.1% DMSO in which the fraction was prepared. At
the end of treatment period, media from control and flavonoid-treated cells was
discarded and 20μl of MTT (5mg/ml PBS) was added to each well. Cells were
then incubated for 4h at 37°C in CO2 incubator. MTT was then
discarded and the colored crystals of producing formazan were dissolved in
200μl of DMSO and mixed effectively by pipetting up and down.
Spectrophotometrical absorbance of the purple blue formazan dye was measured
using an ELISA reader (BIORAD) at 570nm. The optical density of each sample was
compared with control optical density and graphs were plotted.
Ethidium
Bromide/Acridine Orange (Dual Staining):
Ethidium
bromide/acridine orange staining was carried out by the method of Gohel et al[6]. A549 cells were plated at a
density of 5×104 in 48-well plates. They were allowed to grow at
37°C in a humidified CO2 incubator until they were 70–80% confluent.
Then cells were treated with 40µg/ml and 80µg/ml (selected
based on the IC50 concentration) of the flavonoid fraction for 24h.
The culture medium was aspirated from each well and the cells were gently
rinsed twice with PBS at room temperature. Then equal volumes of cells from
control and drug treated were mixed with 100μl of dye mixture (1:1) of
ethidium bromide and acridine orange) and viewed immediately under Nikon
inverted fluorescence microscope (Ti series) at 10x magnification. A minimum of
300 cells was counted in each sample at two different fields. The percentage of
apoptotic cells was determined by [% of apoptotic cells = (total number of
apoptotic cells/total number of cells counted) ×100].
Assessment of
Nuclear Morphology after Propidium Iodide Staining:
Propidium iodide
staining was performed according to the method of Chandramohan et al[7]. PI was used to quantify
the apoptotic cells. A549 cells were
plated at a density of 5 × 104 in 48-well plates. They were allowed
to grow at 37°C in a humidified CO2 incubator until they are 70–80%
confluent. Then cells were treated with 40µg/ml and 80µg/ml of flavonoid for 24 hrs. The culture medium
was aspirated from each well and the cells were gently rinsed twice with PBS at
room temperature, before fixing in methanol: acetic acid (3:1 v/v) for 10 min,
and stained with 50μg/ml Propidium iodide for 20min before flowcytometry
analysis. Nuclear morphology of apoptotic cells with condensed/ fragmented
nuclei was examined by fluorescence microscopy and at least 1 ×103cells
were counted for assessing apoptotic cell death.
Flow Cytometry:
The effect of flavonoid fraction on the
cell cycle distribution was assessed according to Tu et al [8]. A549 cells (1×105
cells/ml) were treated with 40µg/ml and 80µg/ml cultured for 24h. The treated
cells were harvested, washed with phosphate-buffer saline (PBS) and fixed in
75% ethanol at 4◦C overnight. After washing twice with cold
PBS, cells were suspended in PBS containing 40μg/ml propidium iodide (PI) and
0.1mg/ml RNase A followed by shaking at37◦C for 30min. The
stained cells were analyzed with a flow cytometer (Becton-Dickinson, San Jose,
CA, USA) and the data were consequently calculated using WinMDI 2.9 software
(TSRI, La Jolla, CA, USA).
Statistical Analysis:
Data were
expressed as mean ± S.E.M and analyzed by Tukey’s test to determine the
significance of differences between groups. A p value lower than 0.05, 0.01
or/and 0.001 were considered to be significant.
RESULTS AND
DISCUSSION:
Sample
preparation and optimization of HPLC The flavonoids were isolated using
hydro-ethanolic separation and the isolated flavonoids subjected to HPLC
(fig.1) then to TLC. The extract appeared in two spots with below Rf values to
the quercetin standards in TLC. The orange fluorescent spot indicated the
presence of quercetin derivatives [9].
The
hydro-ethanolic extract is highly efficient in the separation of flavonoids
from Sphaeranthus amaranthoides. The
compound showed with the 9.67 retention time and with 94% of the area will
indicate the purity of the flavonoid (fig 2). Acetonitrile gives better results
than using methanol and was therefore selected as the organic solvent in the
optimized HPLC conditions for the extracts of the Sphaeranthus amaranthoides
[10].
Fig 1. TLC of
standard and plant extract showing the Rf value 0.7
The chromatogram
of the Sphaeranthus amaranthoides was showed 8 peaks in that
one major peak with 94.78% of flavonoids and retention time lesser than the
quercetin at 254-350 NM. These isolated and identified flavonoids were
subjected to the anticancer activity in the A549 adenocarcinoma lung cancer
cell lines.
Anticancer activity of flavonoids:
Inhibition of growth and proliferation of
human A549 lung adenocarcinoma cells .The growth inhibitory effect of the
flavonoids against A549 lung adenocarcinoma cells was assessed by MTT assay.
The treatment of A549 cells with the drug (5 - 640µg) resulted in significant
reduction in cell growth ranging from 9.13±0.004 to 71.02±0.002 after 24h. A
dose dependent inhibition in the cell growth was observed (Fig 3) with an IC50value
of 78.19µg.
Fig 3: Cytotoxic effect of the flavonoid fraction treatment on
A549 lung adenocarcinoma cells - % of the inhibition against the concentration.
Chemotherapeutic drugs are considered to
kill tumor cells (Fig 4) by activating a cascade of events resulting in
apoptosis. In agreement with this line of thought, we provided evidence that
flavonoids from Sphaeranthus
amaranthoides were able to induce apoptosis with the classic feature of
apoptosis, including initiator and effector caspase activation and
inter-nucleosomal DNA fragmentation.
Control 0.1% DMSO
20µg/ml
80µg/ml
320µg/ml
Fig 4: Morphological
changes induced by different concentrations of Flavonoids
Dual Staining Assay:
The type of the cell death can
be assessed using dual staining with AO/EB. In this, the morphological changes
after double staining with Acridine Orange/Ethidium Bromide (AO/EB) were
investigated. AO/EB staining uses a combination of two dyes to visualize cells
with aberrant chromatin organization. AO penetrates into living cells, emitting
green fluorescence after intercalation into DNA, but it cannot distinguish
viable from non-viable cells. To achieve this, a mixture of Acridine Orange and
Ethidium Bromide was used. EB can uptake by non living cells so the
differential uptake of these two dyes allows the identification of viable and
non-viable cells, where the second day, EB emits red fluorescence in the cells
with an altered cell membrane (Fig 5).
Control
40µg/ml
80µg/ml
Fig 5: shows induction of apoptosis by the
drug in A549
Cells:
Control group the green colored cells are
live cells, in 40µg/ml showed an intense green colored cells which are early
apoptotic cells and late apoptotic cell number are less. In 80µg/ml the cells
are more in red and orange color which is in late apoptotic cells but the early
apoptotic cells are less.
Table 1. Dual
Staining assay of flavonoid fraction:
|
Treatment groups
|
% No of apoptotic cells
|
|
Control
|
9.83±2.42
|
|
40µg/ml
|
32.15±2.17
|
|
80µg/ml
|
62.83±1.74
|
Fig 6: Bar graph showing the
percentage number of apoptotic cells
Viable cells with intact DNA
and nucleus gave green fluorescence. Early apoptotic cells had fragmented DNA,
which exhibited intense green colored nuclei. Late apoptotic and necrotic
cell’s DNA was fragmented and stained orange and red. Besides, some cells
exhibited typical characteristics of apoptotic cells like plasma membrane
blebbing. From the data it was clear that with increasing concentration of
drug, the number of viable cells decreased tremendously. The percentage of
apoptotic cells after treatment with 40µg and 80µg/ml of the drug was
drastically increased (p< 0.001) to 32% and 62% respectively (Fig 6).
Propidium Iodide
– nuclear fragmentation assay:
Apoptosis was further confirmed by analyzing
the nuclear morphology of flavonoids treated A549 cells. Nuclear morphology was
evaluated with membrane-permeable PI stain (Fig 7). The treated cells contained
a number of apoptotic cells when compared to untreated control cells. There was
characteristic nuclear fragmentation of nuclei in treating A549 cells, whereas
the untreated control cells did not show any nuclear fragmentation. The
apoptotic cells displayed characteristic features of reduced size, intense
fluorescence of condensed nuclear chromatin and formation of membrane blebs.
The percentage of apoptotic nuclei after treatment with 40μg/ml and
80μg/ml of the drug increased enormously (p<0.001) to 41% and 62%,
respectively (Fig 7).
Fig 7 Showing the percentage of apoptotic nuclei
Control
40µg/ml
80µg/ml
Fig 8: shows the
nuclear localization of A549 cells by Propidium iodide staining the cells
showed a characteristic reduction of size with the nuclear fragmentation.
Quantification of the cell death was
estimated using PI staining [11]. The A549 cells have been selected for the
distribution of the cells into three major phases of the cell cycle and make it
possible to detect the apoptotic cells with DNA traces when treated with the
flavonoids from Sphaernathus amaranthoides are being processed
through the FACS for the viability of the cells when tagged through the
Propidium iodide stain with the extract of concentration of about 40 µg/ml and
80 µg/ml. Tremendously, there was a gradual increase in the PI uptake with an
increase in the concentration of the flavonoids. This explains that, with
respective to the concentration of the flavonoids, the number of cells
accumulated G0/G1 was increased. The S phase showed rapid
accumulation of cells at the 40 µg/ml and at this concentration in G0/G1
phase, there is a linear decrease in the accumulation of the cells.
However, the maximum accumulation of the cells at 80 µg/ml was observed in G1
phase. Hence, the above result proves that the increasing in the
concentration of the flavonoids increases the anticancer activity this was
calculated through the FACS in the analysis of the cytometer (Fig 8).
CONCLUSION:
Result in this
study concludes that Flavonoids from Sphaeranthus
amarnathoides possess anticancer
activities and may contribute in the therapeutic effect of this medicinal herb.
However, there is a need of detailed scientific study on traditional medical
practices to ensure that valuable therapeutic knowledge of the plant is preserved
and also to provide scientific evidence for its efficacies.
ACKNOWLEDGEMENT:
The authors thank
Sathyabama University for the kind support and infrastructure.
REFERENCES:
1. Thanigavelan. V, V. Lakshmanakumar, V. Kaliyamurthi,
M. Pitchiah Kumar, and G. Victor Rajamanickam. Pharmacological Study of a
Siddha Holistic Herb Sivakaranthai - Sphaeranthus
amaranthoides Burm for Analgesic and Anti-Inflammatory activities
, Journal of Applied Pharmaceutical Science 2012; 02 (01): 95-101.
2. Geethalakshmi. R, Sakravarthi. C, Kritika. T, Arul
Kirubakaran. M and D. V. L. Sarada. D.V.L
Evaluation of Antioxidant and Wound Healing Potentials of Sphaeranthus amaranthoides Burm. F. BioMed Research International Volume,2014, 2013;1-7.
3. Nadkarne
KM . Indian Materia Medica Vol 1. 3rd Ed. M/S Popular Prakashan, 2010, Mumbai;
1162.
4.
Murugesha Modular KS. Siddha Materia
Medica (Medicinal Plants Division). Dept of, Indian Medicine and Homeopathy,
2008: 228-229.
5.
Safadi FF, Xu J, Smock SL, Kanaan RA,
Selim AH, Odgren PR, Marks SC Jr, Owen TA, Popoff SN. Expression of connective tissue growth factor
in bone: its role in osteoblast proliferation and differentiation in vitro and
bone formation in vivo” J. Cell, 2003, Physio 196:51–62
6. Gohel
A, McCarthy MB, Gronowicz G.
Endocrinology 140; 5339–5347
7. Chandramohan
KVP, Gunasekaran P, Varalakshmi E, Hara Y, Nagini S (2007). Cell Biol. Int 1999; 31: 599–608.
8.
Tu. L.C., Chou. C.K., Chen. C.Y., Chang.
Y.T., Shen. Y.C., Yeh, S.F.
“Characterization of the cytotoxic mechanism of Mana-Hox, an analog of
manzamine pomegranate seeds”, Biochimica et Biophysica Acta. 2004;
1672:148–156.
9. P.
Poukens-Renwart, M. Tits, J. N. Wauters, and L. Angenot. “Densitometric evaluation of spiraeoside
after derivatization in flowers of Filipendula ulmaria (L.)Maxim,” Journal of Pharmaceutical and Biomedical Analysis 1992; 10;
10–12:1085–1088.
10.
Cristina A.
Diagone, Renata Colombo, Fernando M. Lanc¸as, and Janete H. Yariwake. CZE/PAD
and HPLC-UV/PAD Profile of Flavonoids from Maytenus
aquifolium and Maytenus ilicifolia
“Espinheira santa” Leaves Extracts Chromatography Research
International Volume 2012; Article ID
691509,
11. Nuria
S, Violeta GV, Isabel M, Angel M, Thomas GC.
Oxidative stress-induced
apoptosis in retinal photoreceptor cells is mediated by cabins and caspases and
blocked by the oxygen scavenger CR-6. J Biol Chem 2004; 279:39268 – 78.
