CARYOLOGIA
Vol. 63, no. 2: 157-161, 2010
Cytotoxicity of Hibiscus rosa-sinensis flower extract
Özmen* Ali
Adnan Menderes Üniversitesi, Fen-Edebiyat Fakültesi, Biyoloji Bölümü Aydın, Turkey.
Abstract — Many species of Hibiscus are grown for their showy flowers or used as landscape shrubs. Hibiscus
has also medicinal properties. Flowers of these plants are rich in polyphenols, flavonoids and anthocyanins. In
this study flower decoction of Hibiscus rosa-sinensis has been tested for cytotoxic activity. Allium cepa L. has
been used for evaluating cytotoxicity. Decoction of flowers was toxic on root output and length in A. cepa L.
and reduced the mitotic index significantly. It can be suggested that Hibiscus rosa-sinensis flowers contains antimitotic constituents which stop the cell division in anywhere of the cell cycle.
Key words: Allium cepa L., antimitotic, cytotoxicity, Hibiscus rosa-sinensis.
INTRODUCTION
Many species of Hibiscus are grown for their
showy flowers or used as landscape shrubs. Hibiscus has also medicinal properties and takes
part as a primary ingredient in many herbal teas.
One species of Hibiscus, known as Kenaf (Hibiscus cannabinus), is extensively used in paper
making. It has a use as antidote to poisoning
with chemicals (acid, alkali and pesticides) and
venomous mushrooms. This plant has a resistance to fungal pathogens. Its essential oil has
antifungal activity and one of its constituents
was found to be active against human cancer cell
lines in several stages of cellular division (MOUJIR
et al. 2007). Another, Hibiscus sabdariffa is used
as a vegetable and to make herbal teas and jams.
H. sabdariffa flower extract is rich in polyphenol,
flavonoid and anthocyanin. It has been reported
previously that PCA (protocatechuic acid) from
Hibiscus show strong antioxidant and antitumor
promotion effects. H. sabdariffa extract is an apoptotic inducer and a specific activator of JNK/
p38 MAPK pathway. The molecular mechanism
underlying this effect could be described as the
*Corresponding author: phone: ++90 256 21280001869; fax: ++90 256 2135379; e-mail: aozmen@adu.edu.tr.
induction of apoptosis via activation of the p38
MAP kinase that subsequently phosphorylates
the target protein c-jun and trigger the signal to
further activate the apoptotic protein cascades
that contain Fas-mediated signalling. As an outcome, cytochrome c is released from the mitochondria, leading to the cleavage of caspase-3
(LIN et al. 2007; CHANG et al. 2005; TSENG et al.
2000). Hibiscus taiwanensis is a medicinal plant
which is used in Chinese traditional medicine as
anti-inflammatory, antifungal, antipyretic and
anthelmintic agent. Methanolic extract of Hibiscus taiwanensis has cytotoxic activities against
human carcinoma cell lines (WU et al. 2005; WU
et al. 2004). H. syriacus acetone extract was reported antiproliferative against human lung cancer cells and induce apoptosis by activating p53
and AIF in this cell line (CHENG et al. 2008).
The herb Hibiscus rosa-sinensis L. (Malvaceae) is native to China. It is a shrub widely
cultivated in the tropics as an ornamental plant
and has several forms with varying colours of
flowers. The red flowered variety is preferred
in medicine. The leaves and flowers have healing properties (NADKARNI 1954; ALI and ANSARI
1997; KURUP et al. 1979). Flowers have been
found to be effective in the treatment of arterial
hypertension (DWIVEDI et al. 1977) and to have
significant anti-fertility effect (SINGH et al. 1982;
SETHI et al. 1986). Hibiscus rosa-sinensis leaves
and flowers are observed to be promoters of hair
158
growth (ADHIRAJAN et al. 2003).
It has been reported that the plants of the
Hibiscus genus have the potential to provide
biologically active compounds that act as antioxidants, as well as being cardio protective, and
that are able to deter the proliferation of malignant cells. Thus, the Hibiscus genus deserves
additional evaluation as a provider of chemo
preventive agents. Hence, Hibiscus sp. may be a
great natural source for the development of new
drugs and may provide a cost effective mean of
treating cancers and other diseases in the developing world (MAGANHA et al. 2009).
Plants are an indispensable source of natural
products for medicine. The chemical constituents of the plant cell that exert biological activities on human and animal cells fall into two
distinct groups, depending on their relative concentration in the plant body, as well as their major function: primary metabolites, the accumulation of which satisfies nutritional and structural
needs, and secondary metabolites, which act as
hormones, pharmaceuticals and toxins. Secondary metabolites are compounds belonging to extremely varied chemical groups, such as organic
acids, aromatic compounds, terpenoids, steroids, flavonoids, alkaloids, carbonyls, etc. Their
function in plants is usually related to metabolic
and/or growth regulation, lignifications, colouring of plant parts and protection against pathogen attack. Even though secondary metabolism
generally accounts for less than 10% of the total plant metabolism, its products are the main
plant constituents with pharmaceutical properties (KINTZIOS and BARBERAKI 2004).
Some Asian and African countries up to 80%
of the population rely on traditional medicine
for their primary health care needs (FARNSWORTH
et al. 1985). Herbal medicines can be very lucrative but adulteration or counterfeit herbs can
also be a health hazard. The WHO 2008, also
notes, though, that “inappropriate use of traditional medicines or practices can have negative
or dangerous effects” and that “further research
is needed to ascertain the efficacy and safety”
of several of the practices and medicinal plants
used by traditional medicine systems.
Medicinal properties of Hibiscus species are
evident in literature. Hence these plants have antiproliferative effects on human cancer cell lines.
Related with these primary outputs; flower decoction of Hibiscus rosa-sinensis has been tested
for cytotoxic activity in this study by comparing
Lactuca sativa (Lettuce) extract. Lettuce is a cultivation plant and used in human diet abundant-
ÖZMEN
ly. For concretizing the effect of Hibiscus flowers Lettuce extract were used in parallel. Allium
cepa L. has been used for evaluating cytotoxicity
since Allium test fits well in a test battery composed of prokaryotes and /or other eukaryotes
(FISKESJÖ 1993).
MATERIAL AND METHOD
Test materials - Hibiscus flowers and Lettuce
were purchased from commercially available
herbalist and markets.
Extraction of Hibiscus flowers and Lettuce - Decoctions were prepared from 15 g of dry and
milled Hibiscus flowers and Lettuce by boiling
for 15 min. in 1000 ml distilled water. After boiling it has been cooled at room temperature and
filtered through a filter paper. The applied concentrations (5 g/l and 10 g/l) were prepared by
dilution of these stock solutions.
Antimitotic activity - Allium cepa has been used
for evaluating cytotoxic properties since the early 1920’s (GRANT 1982). Small onion bulbs are
carefully unscaled and cultivated on top of test
tubes filled with different concentrations from
decoction of flowers. Tap water and Lettuce
has been used as control groups. The test tubes
were kept in an incubator at 24±2°C. After 72 h
the roots were counted and their lengths were
measured for each onion. The emerged roots has
been fixed with glacial acetic acid/absolute alcohol (1/3 v/v). For evaluation, the root tips were
put into aceto-orcein dye. Well-stained root tips
were prepared for microscopic observation by
squashing on a slide. MI was expressed in terms
of divided cells/total cells. A statistical analysis
was performed on the collected data by using
Graph Pad Prism 5.0. The means of the controls
and flower extracts were obtained from descriptive analysis and One way ANOVA test has been
performed to obtain P values.
RESULTS AND DISCUSSION
Growth inhibition - After 72 h treatment, root
lengths and numbers has been determined for
controls and for each concentration of Hibiscus
flower extract. The collected data are presented
in Figure 1 and Figure 2.
Extract of Hibiscus flowers has shown a dose
dependent inhibition of root output in Allium
cepa but Lettuce extract does not inhibit the root
output significant (Figure 1 and Table 1).
159
CYTOTOXICITY OF HIBISCUS ROSA - SINENSIS FLOWER EXTRACT
On the other hand Hibiscus flower extracts
reduces the elongation of Allium roots in 72 h. If
compared with Lettuce extract this extract type
is very effective on root growth. This is a predefinition parameter for mitotic index reduce. It
can be suggested that mitotic index is reduced in
shorter roots (Figure 2 and Table 1).
These results show that the extracts from
Hibiscus rosa-sinensis flowers have inhibitory effects on root growth and length in Allium cepa.
Antimitotic activity - In Figure 3 the mitotic indexes are presented for controls and for Hibiscus
extracts. It is evident that decoction of flowers
reduced the mitotic index significantly but Lettuce extract has a very low effect on cell division
compatible with root length data.
In conformity with human cell cytotoxicity of
plants belonging to this genus (CHENG et al. 2008;
LIN et al. 2007; MOUJIR et al. 2007; CHANG et al.
2005; WU et al. 2005; WU et al. 2004; TSENG et
al. 2000) it was found that Hibiscus rosa-sinensis
flower decoction has also cytotoxic properties in
plant test systems.
Some secondary metabolites are considered
as metabolic waste products, for example, alkaloids may function as nitrogen waste products.
However, a significant portion of the products
derived form secondary pathways serve either as
protective agents against various pathogens (e.g.
insects, fungi or bacteria) or growth regulatory
molecules (e.g. hormone-like substances that
stimulate or inhibit cell division). Due to these
physiological functions, secondary metabolites
are potential anticancer drugs, since either direct cytotoxicity is affected on cancer cells or the
course of tumor development is modulated, and
eventually inhibited. Administration of these
TABLE 1 — The average root lengths and numbers in control and in treatment concentrations after 72 h.
Tap Water
Lettuce
(Lactuca sativa)
Decoction
(5 g/l)
Decoction
(10 g/l)
RN
ARL (mm)
RN
ARL (mm)
RN
ARL (mm)
RN
ARL (mm)
1
34
22
30
26
7
5
4
3
2
32
18
34
23
11
3
7
4
3
28
27
36
29
8
3
5
3
4
34
33
35
31
10
4
6
3
5
42
26
44
24
6
2
8
2
6
38
33
29
21
14
3
3
2
7
22
25
32
28
18
4
4
3
8
45
28
36
19
12
6
7
2
9
37
24
28
18
9
5
2
3
10
36
23
31
35
13
5
5
2
RN: root number, ARL: average root lenght
Fig. 1 — The average root numbers in controls and
flower extracts after 72 h.
Fig. 2 — The average root lengths in controls and flower
extracts after 72 h.
160
ÖZMEN
Fig. 3 — Mitotic index (MI) in controls and flower extracts after 72 h.
compounds at low concentrations may be lethal
for microorganisms and small animals but in
larger organisms they may specifically affect the
fastest growing tissues (KINTZIOS and BARBERAKI
2004). Hence in this study the mitotic index may
be reduced at %5 levels by hormone-like secondary metabolites that probably found in flower decoction. Reduction in the mitotic activity
could be due to inhibition of DNA synthesis or
blocking in the G2 phase of the cell cycle, preventing the cell from entering mitosis (TÜRKOĞLU
2008).
The species A. cepa also presents other advantages with suitable chromosomal features; this
plant bears large and few chromosomes (2n = 16)
what facilitates the evaluation of chromosome
damages or disturbances in cell division cycle.
This test have proved to be of great value, since
combine a high sensibility to detect mutagens in
different environments and a great capacity to
evaluate distinct genetic endpoints, from point
mutations to chromosomal aberrations (LEME
and MORALES 2008). Chromosomal aberrations
are changes in chromosome structure resulting
from a break or exchange of chromosomal material. Most of the chromosomal aberrations observed in cells are lethal, but there are many corresponding aberrations that are viable and can
cause genetic effects, either somatic or inherited
(AKINBORO and BAKARE 2007). My results did not
show induction of chromosome or chromatid
type of aberration in the treated cells.
C-mitosis indicated that the chemical inhibited spindle formation similar to the effect of colchicine (BADR 1983), and induction of C-mitosis
commonly associated with spindle poisons, indicating turbogenic effect (SHAHIN and EL-AMOODI
1991). ODEIGAH et al. (1997) describes the presence of C-mitoses as a possibly reversible effect
(weak toxic effect). In this study colchicine like
mitosis has been observed by microscopic analysis of Allium roots. Data are not given because
the number of C-mitosis in this study was not
important statistically.
In respect of this results, Hibiscus rosa-sinensis flowers contains anti-mitotic constituents that
can stop the mitosis in anywhere of the cell cycle. Furthermore these constituents probably affect the cytoskeleton by tubulin polymerization
or degradation.
Acknowledgements — The author is indebted to
Ezgi AKAT for preparing the extracts and helping to
count the cells by microscopic observations.
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Received July 22th 2009; accepted February 8th 2010