International Journal of Phytomedicine 9 (2017) 43

436-442
http://www.arjournals.org/index.php/ijpm/index

Original Research Article ISSN: 0975-0185

seaweed gracilaria
A study on the ethanolic extracts of red seaweed-
corticata(j.agardh) j. Agardh, to assess the antiproliferative activity and
morphological characterization of apoptosis on hela cell lines
Ashwini S1*, Manjula Shantaram1

*Corresponding author: Abstract

Marine algae are excellent source of bioactive compounds that can be used as an alternative
Ashwini S source for finding novel anti-cancer
anti molecules. Gracilaria corticata, red algae from Surathkal
beach, Karnataka were studied for their anti-proliferative
anti proliferative activities and their morphological
1Department characterization on He La cells were assessed. Cytotoxicity of the algal ethanolic extracts on
of Studies in Biochemistry, Post
HeLa cells
cell were assayed by 3-(4,5-dimethylthiazol-2-yl)-2, 5-- diphenyltetrazolium bromide
Graduate Centre Mangalore University, India
(MTT) reduction method. Morphological assessment was done by examinations with Hoechst
staining and acid vacuoles were determined using acridine orange. Induction of apoptosis was
Received: 11 April 2017 studied using caspase activity.Based on IC50value, further morphological assessment was
Accepted: 10 July 2017 carried out and apoptosis was confirmed using Hoechst 33342 staining and acridine orange
Published: 02 September 2017 staining respectively. Treated cells became round with blebbings with cond
condensed nuclei. Acidic
lysosomal vacuoles formation occurred in treated cells.These red algae were able to suppress
proliferation and promote apoptosis--
apoptosis mediated cell death with induction of initial stages of
apoptosis in HeLa cell lines. Thus, this seaweed can be a potent candidate for isolating new
green drug anticancer molecules. However, further characterization at the molecular and
structural levels are required.
Key
Keywords: HeLa, MTT, Acridine orange, Hoechst 33342, Gracilaria corticata

Introduction composition. They have been used as foodstuff that has been
historically consumed around the globe but is only consumed in
appreciable
ppreciable amounts in certain areas of the world today. Certain
Cancer is one of the most serious threats in human diseases and algae have long been used in traditional Chinese herbal medicine
carcinoma of the uterine cervix is the second most common female in the treatment of cancer [2]. While chemical analysis would
tumor worldwide, surpassed only by breast cancer. Its incidence is suggest a number of nutritional benefits of seaweed for their
disproportionately
ely high (>80%) in the developing world and its richness
hness in polysaccharides and terpenoids, they are considered as
treatment of disease is usually palliative, aimed only at symptom promising bioactive molecules with anticancer activity, that
control [1]. Presently, there is a considerable scientific discovery of seaweeds and their extracts have attracted great interest in the
new anti-cancer
cancer agents from natural products. Due to the pharmaceutical industry as a source of bioactive compounds. From
resistance of cancer cells to antitumor drugs, finding new effective 1998
998 to2006, so far 592 marine natural products were investigated
anticancer compounds with minimal side effects has been an for its activity in pharmacology studies [3].It has been long time that
extraordinary challenge for many scientists. human beings have known about marine algae, its rich source of
Marine organisms are vital and promising resources in cancer pharmacologically active metabolites with antineoplastic,
research and a number of compounds have undergone clinical antimicrobial and antiviral effects [4,5].
trials as antitumor agents. Seaweed is a marine macro alga. They From the past few decades, there has been an upsurge in the
are classified as red algae (Rhodophyta), hyta), brown algae search for new plant-derivedderived drugs. Marine compounds are
(Phaeophyta) or green algae (Chlorophyta), based on the however under-represented
represented in the current pharmacology boom. It
pigmentation and depending on their nutrient and chemical is anticipated that the marine environment will become a valuable

DOI:10.5138/09750185.2082

This article is distributed under the terms of the Creative Commons Attribution License,, which permits unrestricted use
and redistribution provided that the original
origina author and source are credited.
 Ashwini et al. International Journal of Phytomedicine 9 (3) 436-442 [2017]

source of novel compounds in the near future, as it represents 95% extract was clear. The resulting pasty extracts were stored in a
of the biosphere. Recent studies have shown that several bioactive refrigerator at 4°C for future use.
compounds obtained from algae induced tumor cell death by
apoptotic mechanism [6-8]. Substances isolated from algae, such Culturing of HeLa Cells
as fucoidan, laminaran and terpenoids have activity against cancer
cell strains [9,10]. Extensive researches is being carried out on the
cellular and molecular basis of the carcinogenesis cascade that Human cervical cancer (HeLa) cell lines obtained from (NCCS)
provides a targeted approach for cancer chemoprevention, which Pune were maintained in RPMI-1640 supplemented with 10%
also aims to halt or reverse the development and progression of FBS(full form), penicillin (100 U/ml) in a humidified atmosphere of
precancerous cells through use of non- cytotoxic doses of nutrients 50 μg/ml CO2at 37°C.
and/or pharmacological agents [11,12].
Natural marine products evidencing apoptotic activity has dragged Cell viability assay
attention as new leads for anticancer alternative and
complementary preventive or therapeutic agents [13]. Nowadays, In vitro assay for cytotoxic activity: Cytotoxicity of the algal
seaweed-related products are used widely, not only as health ethanolic extracts on HeLa cells were assayed by 3-(4,5-
foods, but also in clinical drugs for the prevention and treatment dimethylthiazol-2-yl)-2, 5- diphenyltetrazolium bromide (MTT)
cancer. The protective effects of dietary component in Asian reduction assay [19].The anticancer activity of the seaweed
communities, kelps and other red and green seaweed against extracts was observed at 24hours, 48 hours and 72 hours, in which
mammary [14], skin [15] and intestinal cancer [16] are supported by ethanol extracts of G. corticata showed a greater activity with an
epidemiological data [17] and rodent model studies [18]. As a need IC50 value of 244.7μg/ml at 48hours. However, the drug was found
for a more evidence relating anticancer activity, the present study to be inactive at 72hours.
was conducted on HeLa cell lines. Morphological examinations were conducted by Hoechst 33342
and acridine orange(AO) staining and were performed when the
Chemicals and Reagents cells reached 70–80% of confluence.

HeLa cancer cell lines were purchased from National Centre for Apoptosis detection assay with Hoechst 33342 (Sigma
Cell Science (NCCS), Pune, India. RPMI 1640 medium (#AL199A, B-2262)
Himedia), Fetal Bovine Serum (#RM10432, Himedia), MTT
Reagent (5 mg/ml) (# 4060 Himedia),DMSO (#PHR1309, Sigma), HeLa cells were stained with Hoechst 33342 to study cellular and
Berberine (# B3251, Sigma), D-PBS (#TL1006, Himedia), 96-well nuclear morphology. H33342 binds preferentially to A-T base-pairs
plate for culturing the cells (From Corning,USA),T25 flask (# in DNA. The dye internalizing in the cells is an active transport
12556009, Biolite - Thermo), Paraformaldehyde(PFA) (HiMedia), process which requires physiologic conditions and does not require
Acridineorange(Sigma),EDTA, Aceticacid (Merck), permeabilization for labeling. The condition typically varies among
various cell types. The procedure for staining and
Methodology and Experimental Design analyzing5x106cells/coverslip approximately was seeded in
buffered media, pH 7.2. After overnight i n c u b a t i o n , the cells
were treated with drugs of IC50values and incubated for 48 hours,
Collection of seaweed sample then fixedin 4%PFA. Homogenously aspirated and spent media
was removed and 1 ml of saline was added and centrifuged at
Seaweeds were collected from the rocks of Surathkal beach (13 1500 rpm for 10 minutes. Supernatant was discarded and 2μg/ml
00`34.1" N lat. and 74 47`16.1" E long.), Dakshina Kannada district, of Hoechst stain was added to pellet. Cells were then incubated at
Karnataka. Samples were washed with freshwater to remove 37C for 1 hour. Apoptosis was analyzed under fluorescent
adhering debris and identified as Gracilaria sp. by Dr. C. R. K microscope after incubation. Washing is not recommended.
Reddy, CSIR-Central Salt and Marine Chemicals Research
Institute, Baroda, Gujarat. The collected samples were transferred
to the laboratory in a polythene bag, shade dried and powdered. Morphological studies

2μg/ml of Hoechst 33342 staining was used to study cellular and

Seaweed extraction nuclear morphology, to stain the nuclei for 15 minutes in dark at
room temperature. Acridine orange staining was done to study the
The powdered seaweed was extracted successively using Soxhlet formation of acidic vacuoles. After 48 hours of ethanolic extract
extractor sequentially with different solvents of increasing polarity treatment, cells were additionally incubated in fresh media
namely: chloroform, acetone, methanol, ethanol, and water until the containing acridine orange (1μg/ml) for 15 minutes without fixation.
PAGE | 437 |
 Ashwini et al. International Journal of Phytomedicine 9 (3) 436-442 [2017]

After the staining procedures, extra stain was drained out, washed bodies of various sizes containing well-preserved but compacted
briefly in phosphate buffered saline (PBS)and mounted in PBS cytoplasmic organelles and/or nuclear fragments, which are the
containing 10% glycerol, 2% N-propylgallate. Cells were visualized typical characteristics of apoptosis [24].Several cellular and
under both bright field and fluorescence for molecular, biological features in apoptotic cells, such as cell
Hoechst(excitation356nm,emission 465nmand excitation 488nm, shrinkage, DNA fragmentations, and activation of the caspase
emission 530 (green) ,650(red) for acridine orange in a fluorescence cascade are exhibited. The series of photographs in figures 3A,3B
microscope(Olympus). & 3C illustrates some of the possible morphologies seen with
inverted microscopy, Hoechst 33342 nuclear staining and AO
Caspase Assay staining and fluorescence microscopy. Thus, apoptotic
characteristics such as cell membrane blebbing, shrinkage of cells
/reduction, and separated apoptotic bodies were observed and the
The protease activity of caspases-3, -8,and -9 in HeLa cells was results also suggested that there should be active substances
performed using colorimetric assay kit(GenScript kit,) that is based interacting with special cancer-associated receptors or cancer cell
on spectrophotometric detection of the caspase enzymes after special molecule, thus triggering some mechanisms that cause
cleavage from the labeled substrate. About 2 × 106HeLa cells were death of cancer cell.
treated with ethanolic extract at IC50 and incubated for 48hours, Cytotoxicity of marine algal extracts was evaluated by growth
while untreated cells incubated for 48 hours acted as control. Then, inhibition assay (Figure 1).When the growth inhibited cells were
the cells were centrifuged for 5 min at 2000 rpm to remove the stained with AO and Hoechst 33342 apoptotic cell death was
media. Followed by wash with PBS twice and centrifuged at 2000 observed in time- and dose-dependent manner in all cultures
rpm for 5minutes. The cell pellets were later lysed by the addition of (Figure 2). These results suggested that ethanolic extract caused
50 L cold prepared lysis buffer containing 0.5 L dithiothreitol irreversible cell damages in cultured cells. Intact membranes allow
mixed well, and incubated on ice for exactly 1 hour. The tubes were the uptake of AO, which binds to double-stranded DNA and
vortexed. The resulting cell lysate was centrifuged for 1minute at fluorescence green under 488 nm excitation. In apoptotic cells,
10000 rpm at 4C, and the supernatant was collected. Then, 50 L condensed chromatin material resulted in clumps of intense green
2x reaction buffer containing 0.5 L dithiothreitol were added to fluorescent spots within the cell while untreated cells show a diffuse
50 L supernatant containing 200 g protein in each tube, to which green fluorescence. The characteristic condensation patterns were
5 L caspase substrate was added, transferred to 96-well plate, observed.
wrapped, and incubated at 37C for 2 hours away from light. At the Acridine orange stains both cytoplasm and nucleic acids in
end of the incubation period, the samples were read at 405 nm in uncharged form, which fluoresce bright green, whereas in
ELISA microplate reader. Data was presented as optical density protonated form, it accumulates in the lysosomal acidic vacuoles,
(OD) (405 nm; mean SD) and graph was plotted. Active caspase forms aggregates and fluoresce bright red. When studied under a
cleaves the peptide and releases the chromophore pNA that can be fluorescence microscope, it was observed that in the non-treated
detected spectrophotometrically at optical density 405 nm. and control sets, a minimal red fluorescence was found, whereas,
Theoretically, the apoptotic cell lysates containing active tested increased red fluorescence were observed in the treated cells. In
caspases should yield a considerable emission compared with the ethanolic extract treated cells, almost the whole cytoplasm be
non-apoptotic cell lysates. came red (Figure 3) indicating the merging of all the acidic
vacuoles.
Results and Discussion The induction of apoptosis essentially needs the activation of
caspases. These enzymes are the group of intracellular proteases
which are responsible for planning the cell into apoptotic bodies
The results of the cytotoxicity tested by observing the cellular during apoptosis. Caspases are present as inactive proenzymes
morphological change showed that ethanolic extracts in dose- and form, that are activated by proteolytic cleavage. Caspases-8,-9,
time dependently inhibited the proliferation of the HeLa cancer cell and -3 are located at key junctions in apoptosis pathways.
lines. Induction of apoptosis is a useful approach in cancer As shown in Figure 4, ethanolic extracts increased the activity of
therapies. Kerr & Wyllie (1972) were the first authors that described caspases-8, -9, and -3 in a time-dependent manner. According to
morphological features such as morphology of cells and induction the studies concerning caspase activities, several distinct pathways
of cell death [20]. Several species of algae have been found to be exist resulting in the induction of apoptosis by seaweed. In this
sources of metabolites with anti-tumoral and immune- stimulant work, we noted that ethanolic extracts activate caspase-3 in a time-
activities [21]. response fashion (Figure 4).
Apoptosis is a normal physiologic process that plays a vital role in Marine algae contain many unidentified bioactive components that
homeostasis and growth of the normal and cancer cells, dys are physiologically active substances. Several researchers have
regulation of apoptosis is usually considered as a major cancer reported the studies on bioactivity of marine algae against cancer
property[22,23].The nuclei with condensed chromatin and apoptotic cell lines where their findings have brought great promise to the
PAGE | 438 |
 Ashwini et al. International Journal of Phytomedicine 9 (3) 436-442 [2017]

development of cancer treatment activities [25, 26].These

26]. algae [28]. Induction of apoptosis by seaweed via the activation of
can be potent candidates for isolating new lead anticancer caspase-33 was reported previously to be mediated through a
molecules. Similar
ilar studies using apoptotic parameters revealed mitochondrial pathway [29]. Previously
reviously the cold water extract of red
apoptotic mechanisms with total methanolic extract of red algae, alga, Gracilaria corticata, possessing biological activity against
Gracilaria tenuistipitata [27]. Antiproliferative activity of algal tumor cells replication was reported by Zandi et al[30]. However,
extracts in cancer cell lines, may be due to water-soluble
water there is a need for further charac
characterization at both molecular and
polysaccharides, such as laminarans and fucoidans, which are structural levels.
representative anticancer substances extracted from seaweeds

a b c
Figure:1 Morphological changes of HeLa cells under inverted microscope a) Treated with ethanolic extract b) Berberine c) Untreated

a b c
Figure: 2-HeLa cells grown on sterile coverslips were treated with IC50doses of the ethanolic extracts for 48 hours. Then, they were stained with
Hoechst 33342 to study the nuclear morphology under fluorescence microscope. a) 5-Flurouracil
5 Flurouracil b) Ethanolic extract c) Untreated. (arrows show
nuclei with deformity)

PAGE | 439 |
 Ashwini et al. International Journal of Phytomedicine 9 (3) 436-442 [2017]

Phase Green Red Merge

Figure:3 HeLa cells grown on sterile coverslips were treated with IC50 doses of ethanolic extracts for 48 hours. Then they were stained with
acridine orange(1μg/ml) to study the formation of acidic vacuoles.

Caspase activity
0.8
O.D for caspase 9
O.D Value at 405 nm

O.D for caspase 3

0.6
O.D for caspase 8

0.4

0.2

0.0
l

st
ro

te
nt

s
co

hr
s

48
hr
48

Time (Hours)
Figure: 4 Activity of caspases-3, -8, and -9.The
9.The total cell lysates from He La cells treated with ethanolic extracts for 48 hours was analyzed using
caspase colorimetric protease assay kits

PAGE | 440 |
 Ashwini et al. International Journal of Phytomedicine 9 (3) 436-442 [2017]

Conclusion cancer cell line. Thus, this can emerge as a novel candidate as a
potential natural anti-cancer agent. However, further molecular and
Our in vitro study demonstrated that ethanolic extracts of Gracilaria structural level characterization has to be studied.
corticata (J. Agardh) J. Agardhhas cytotoxic activity on HeLa
human cervical cancer cell line. Its ability to induce apoptosis was
also understood with increased caspase activity on human cervical

References

[1]. Costa LS, Telles CB, Oliveira RML, Korean brown seaweed Undaria G2/M arrest and inhibits
Nobre T, Dantas-Santos N, Camara pinnatifida. Carbohyd Polym. 2010; 81: cyclooxygenase-2 activity in human
RB, et al. Heterofucan from Sargassum 41-48. hepatocarcinoma HepG2 cells.
filipendula induces apoptosis in HeLa [10]. Gerwick WH, Bernart MW. Eicosanoids Phytother. Res. 2007; 21:52–57.
cells. Mar. Drugs. 2011; 9, 603–614. and related compounds from marine [19]. Plumb JA. Cell sensitivity assays: the
[2]. YamamotoiI, Takahashi M, Tamura E, algae In: Zaborski OR, Attaway DH MTT assay. Methods Mol Med. 2004;
Maruyama H, MoriI H. Antitumor (org) Marine Biotechnology. New York: 88:165-169.
activity of edible marine algae: Effect of Plenum Press, 1993; 101-152. [20]. Kerr JF, Wyllie AH and Currie AR.
crude fucoidan fractions prepared from [11]. Gamal-Eldeen AM, Ahmed FE, Abo- Apoptosis: A Basic Biological
edible brown seaweeds against L-1210 Zeid MA. In vitro cancer chemo- Phenomenon with Wide-Ranging
leukemia. Hydrobiologia. 1984; 116- preventive properties of polysaccharide Implications in Tissue Kinetics. British
117. extract from the brown alga, Journal of Cancer. 1972; 26: 239-257.
[3]. Glaser KB and Mayer AM. A Sargassum latifolium. Food Chem [21]. Konig GM, Wright AD. Marine natural
Renaissance in Marine Pharmacology: Toxicol. 2009; 47: 1378-1384. products research: Current directions
From Preclinical Curiosity to Clinical [12]. Theisen C. Chemoprevention: what’s in and future potential. Planta Med. 1995;
Reality. Biochemical Pharmacology. a name? J NatlCancer Inst. 2001; 93: 62: 193-211.
2009; 78: 440-448. 743-774. [22]. Asadi H, Orangi M, Shanehbandi D,
[4]. Faulkner DJ. Marine natural products. [13]. Kim SK, Wijesekara I. Development Babaloo Z, Delazar A,
Nat Prod Rep. 2000; 17: 7-55. and biological activities of marine- Mohammadnejad L, et al.Methanolic
[5]. Tzivelekala, Vagias C, Roussis V. derived bioactive peptides: A review. J. fractions of ornithogalum cuspidatum
Natural products with anti-HIV activity Funct. Foods, 2010;2: 1–9. induce apoptosis in pc-3 prostate
from marine organisms. Curr Top Med [14]. Funahashi H, Imai T, Mase T, cancer cell line and wehi-164
Chem. 2003; 3: 1512-1535. SekiyaM, Yokoi K, Hayashi H, et al. fibrosarcoma cancer cell line. Adv
[6]. Park EJ and Pezzuto JM. Antioxidant Seaweed prevents breast cancer? Jpn. Pharm Bull.2014; 4(1):455-458.
Marine Products in Cancer J. Cancer Res. 2001; 92: 483–487. [23]. Aghbali A, Moradi Abbasabadi F,
Chemoprevention. Antioxid Redox [15]. Higashi-Okai K, Otani S, Okai Y. Delazar A, Vosough Hosseini S, Zare
Signal. 2013;19, 115-138. Potent suppressive effect of a Shahneh F, Baradaran B, et al.
[7]. Zhang Z, Teruya K, Yoshida T, Eto H. Japanese edible seaweed, Induction of apoptosis and cytotoxic
and Shirahata S. Fucoidan Extract Enteromorphaprolifera (Sujiao-nori) on activities of Iranian orthodox black tea
Enhances the Anti-Cancer Activity of initiation and promotion phases of extract (bte) using in vitro models. Adv
Chemotherapeutic Agents in MDA-MB- chemically induced mouse skin Pharm Bull .2004;4(3):255-260.
231 and MCF-7 Breast Cancer Cells. tumorigenesis. Cancer Lett.1999;140: [24]. Doonan F, Cotter TG. Morphological
Marine Drugs. 2013;11: 81-98. 21–25. assessment of apoptosis. Methods.
[8]. Kim AD, Lee Y, Kang SH, Kim GY, Kim [16]. Lee EJ, Sung MK. Chemoprevention of 2008; 44: 200–204.
HS and Hyun JW. Cytotoxic Effect of azoxymethane induced rat colon [25]. Albano RM, Pavao MSG, Mourao PAS,
Clerosterol Isolated from Codium carcinogenesis by seatangle, a fiber- Mulloy B. Structural studies of a
Fragile on A2058 Human Melanoma rich seaweed. Plant Foods Hum. sulfated L-galactan from Styela plicata
Cells. Marine Drugs, 2013; 11: 418- Nutr.2003; 58: 1–8. (Tunicate): Analysis of the Smith-
430. [17]. Carper J. Seaweed or kelp. In The degraded polysaccharide.
[9]. Synytsya A, Kim WJ, Kim SM, Pohl R, Food Pharmacy; Bantam Book: New Carbohydrate Research. 1990;208:
Synytsya A, Kvasnicka F, et al. York, NY, USA, 1989; 264–268. 163-174.
Structure and antitumor activity of [18]. Bae SJ, Choi YH. Methanol extract of [26]. Berlinck RGS, Ogawa CA, Almeida
fucoidan isolated from sporophyll of the seaweed Gloiopeltisfurcata induces AMP, Sanchez MAA, Malpezzi ELA,
PAGE | 441 |
 Ashwini et al. International Journal of Phytomedicine 9 (3) 436-442 [2017]

Costa LV, et al. Chemical and Oral Cancer Cells Involves Apoptosis, from brown seaweeds inhibit
pharmacological characterization of DNA Damage, and Oxidative Stress. proliferation of melanoma cells and
halitoxin from Amphimedon viridis BMC Complementary and Alternative induce apoptosis by activation of
(Porifera) from the southeastern Medicine. 2012;12: 142. caspase-3 in vitro. Marine Drugs. 2011;
Brazilian coast. Comparative [28]. Misurcova L, Skrovankova S, Samek 9(12): 2605–2621.
Biochemistry and Physiology Part C: D, Ambrozova J and Machu L. Health [30]. Zandi K, Tajbakhsh S, Nabipour I,
Pharmacology, Toxicology and benefits of algal polysaccharides in Rastian Z, Yousefi F, Sharafiah S, et
Endocrinology. 1996;115: 155-163. human nutrition. Adv in Food and Nutr al.In vitro antitumor activity of Gracilaria
[27]. Yeh CC, Yang JI, Lee JC, Tseng CN, Res. 2010; 66:75-145. corticata (a red alga) against Jurkat
Chan YC, Hseu YC, et al. Anti- [29]. Ale M T, Maruyama H, Tamauchi H, and molt-4 human cancer cell lines.
Proliferative Effect of Methanolic Mikkelsen JD, Meyer AS. Fucose- African Journal of Biotechnology.
Extract of Gracilaria tenuistipitata on containing sulfated polysaccharides 2010;9: 6787-6790.

PAGE | 442 |