Cytotoxicity of Hibiscus rosa-sinensis fl ower extract
←
→
Page content transcription
If your browser does not render page correctly, please read the page content below
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 anti-
mitotic constituents which stop the cell division in anywhere of the cell cycle.
Key words: Allium cepa L., antimitotic, cytotoxicity, Hibiscus rosa-sinensis.
INTRODUCTION induction of apoptosis via activation of the p38
MAP kinase that subsequently phosphorylates
Many species of Hibiscus are grown for their the target protein c-jun and trigger the signal to
showy flowers or used as landscape shrubs. Hi- further activate the apoptotic protein cascades
biscus has also medicinal properties and takes that contain Fas-mediated signalling. As an out-
part as a primary ingredient in many herbal teas. come, cytochrome c is released from the mito-
One species of Hibiscus, known as Kenaf (Hi- chondria, leading to the cleavage of caspase-3
biscus cannabinus), is extensively used in paper (LIN et al. 2007; CHANG et al. 2005; TSENG et al.
making. It has a use as antidote to poisoning 2000). Hibiscus taiwanensis is a medicinal plant
with chemicals (acid, alkali and pesticides) and which is used in Chinese traditional medicine as
venomous mushrooms. This plant has a resist- anti-inflammatory, antifungal, antipyretic and
ance to fungal pathogens. Its essential oil has anthelmintic agent. Methanolic extract of Hibis-
antifungal activity and one of its constituents cus taiwanensis has cytotoxic activities against
was found to be active against human cancer cell human carcinoma cell lines (WU et al. 2005; WU
lines in several stages of cellular division (MOUJIR et al. 2004). H. syriacus acetone extract was re-
et al. 2007). Another, Hibiscus sabdariffa is used ported antiproliferative against human lung can-
as a vegetable and to make herbal teas and jams. cer cells and induce apoptosis by activating p53
H. sabdariffa flower extract is rich in polyphenol, and AIF in this cell line (CHENG et al. 2008).
flavonoid and anthocyanin. It has been reported The herb Hibiscus rosa-sinensis L. (Mal-
previously that PCA (protocatechuic acid) from vaceae) is native to China. It is a shrub widely
Hibiscus show strong antioxidant and antitumor cultivated in the tropics as an ornamental plant
promotion effects. H. sabdariffa extract is an ap- and has several forms with varying colours of
optotic inducer and a specific activator of JNK/ flowers. The red flowered variety is preferred
p38 MAPK pathway. The molecular mechanism in medicine. The leaves and flowers have heal-
underlying this effect could be described as the ing 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;
*Corresponding author: phone: ++90 256 2128000- SETHI et al. 1986). Hibiscus rosa-sinensis leaves
1869; fax: ++90 256 2135379; e-mail: aozmen@adu.edu.tr. and flowers are observed to be promoters of hair158 ÖZMEN
growth (ADHIRAJAN et al. 2003). ly. For concretizing the effect of Hibiscus flow-
It has been reported that the plants of the ers Lettuce extract were used in parallel. Allium
Hibiscus genus have the potential to provide cepa L. has been used for evaluating cytotoxicity
biologically active compounds that act as anti- since Allium test fits well in a test battery com-
oxidants, as well as being cardio protective, and posed of prokaryotes and /or other eukaryotes
that are able to deter the proliferation of ma- (FISKESJÖ 1993).
lignant cells. Thus, the Hibiscus genus deserves
additional evaluation as a provider of chemo
preventive agents. Hence, Hibiscus sp. may be a MATERIAL AND METHOD
great natural source for the development of new
drugs and may provide a cost effective mean of Test materials - Hibiscus flowers and Lettuce
treating cancers and other diseases in the devel- were purchased from commercially available
oping world (MAGANHA et al. 2009). herbalist and markets.
Plants are an indispensable source of natural Extraction of Hibiscus flowers and Lettuce - De-
products for medicine. The chemical constitu- coctions were prepared from 15 g of dry and
ents of the plant cell that exert biological ac- milled Hibiscus flowers and Lettuce by boiling
tivities on human and animal cells fall into two for 15 min. in 1000 ml distilled water. After boil-
distinct groups, depending on their relative con- ing it has been cooled at room temperature and
centration in the plant body, as well as their ma- filtered through a filter paper. The applied con-
jor function: primary metabolites, the accumula- centrations (5 g/l and 10 g/l) were prepared by
tion of which satisfies nutritional and structural dilution of these stock solutions.
needs, and secondary metabolites, which act as Antimitotic activity - Allium cepa has been used
hormones, pharmaceuticals and toxins. Second- for evaluating cytotoxic properties since the ear-
ary metabolites are compounds belonging to ex- ly 1920’s (GRANT 1982). Small onion bulbs are
tremely varied chemical groups, such as organic carefully unscaled and cultivated on top of test
acids, aromatic compounds, terpenoids, ster- tubes filled with different concentrations from
oids, flavonoids, alkaloids, carbonyls, etc. Their decoction of flowers. Tap water and Lettuce
function in plants is usually related to metabolic has been used as control groups. The test tubes
and/or growth regulation, lignifications, colour- were kept in an incubator at 24±2°C. After 72 h
ing of plant parts and protection against patho- the roots were counted and their lengths were
gen attack. Even though secondary metabolism measured for each onion. The emerged roots has
generally accounts for less than 10% of the to- been fixed with glacial acetic acid/absolute alco-
tal plant metabolism, its products are the main hol (1/3 v/v). For evaluation, the root tips were
plant constituents with pharmaceutical proper- put into aceto-orcein dye. Well-stained root tips
ties (KINTZIOS and BARBERAKI 2004). were prepared for microscopic observation by
Some Asian and African countries up to 80% squashing on a slide. MI was expressed in terms
of the population rely on traditional medicine of divided cells/total cells. A statistical analysis
for their primary health care needs (FARNSWORTH was performed on the collected data by using
et al. 1985). Herbal medicines can be very lu- Graph Pad Prism 5.0. The means of the controls
crative but adulteration or counterfeit herbs can and flower extracts were obtained from descrip-
also be a health hazard. The WHO 2008, also tive analysis and One way ANOVA test has been
notes, though, that “inappropriate use of tradi- performed to obtain P values.
tional medicines or practices can have negative
or dangerous effects” and that “further research
is needed to ascertain the efficacy and safety” RESULTS AND DISCUSSION
of several of the practices and medicinal plants
used by traditional medicine systems. Growth inhibition - After 72 h treatment, root
Medicinal properties of Hibiscus species are lengths and numbers has been determined for
evident in literature. Hence these plants have an- controls and for each concentration of Hibiscus
tiproliferative effects on human cancer cell lines. flower extract. The collected data are presented
Related with these primary outputs; flower de- in Figure 1 and Figure 2.
coction of Hibiscus rosa-sinensis has been tested Extract of Hibiscus flowers has shown a dose
for cytotoxic activity in this study by comparing dependent inhibition of root output in Allium
Lactuca sativa (Lettuce) extract. Lettuce is a cul- cepa but Lettuce extract does not inhibit the root
tivation plant and used in human diet abundant- output significant (Figure 1 and Table 1).CYTOTOXICITY OF HIBISCUS ROSA - SINENSIS FLOWER EXTRACT 159
On the other hand Hibiscus flower extracts LIN et al. 2007; MOUJIR et al. 2007; CHANG et al.
reduces the elongation of Allium roots in 72 h. If 2005; WU et al. 2005; WU et al. 2004; TSENG et
compared with Lettuce extract this extract type al. 2000) it was found that Hibiscus rosa-sinensis
is very effective on root growth. This is a pre- flower decoction has also cytotoxic properties in
definition parameter for mitotic index reduce. It plant test systems.
can be suggested that mitotic index is reduced in Some secondary metabolites are considered
shorter roots (Figure 2 and Table 1). as metabolic waste products, for example, alka-
These results show that the extracts from loids may function as nitrogen waste products.
Hibiscus rosa-sinensis flowers have inhibitory ef- However, a significant portion of the products
fects on root growth and length in Allium cepa. derived form secondary pathways serve either as
protective agents against various pathogens (e.g.
Antimitotic activity - In Figure 3 the mitotic in- insects, fungi or bacteria) or growth regulatory
dexes are presented for controls and for Hibiscus molecules (e.g. hormone-like substances that
extracts. It is evident that decoction of flowers stimulate or inhibit cell division). Due to these
reduced the mitotic index significantly but Let- physiological functions, secondary metabolites
tuce extract has a very low effect on cell division are potential anticancer drugs, since either di-
compatible with root length data. rect cytotoxicity is affected on cancer cells or the
In conformity with human cell cytotoxicity of course of tumor development is modulated, and
plants belonging to this genus (CHENG et al. 2008; eventually inhibited. Administration of these
TABLE 1 — The average root lengths and numbers in control and in treatment concentrations after 72 h.
1 2 3 4 5 6 7 8 9 10
Tap Water RN 34 32 28 34 42 38 22 45 37 36
ARL (mm) 22 18 27 33 26 33 25 28 24 23
Lettuce RN 30 34 36 35 44 29 32 36 28 31
(Lactuca sativa) ARL (mm) 26 23 29 31 24 21 28 19 18 35
Decoction RN 7 11 8 10 6 14 18 12 9 13
(5 g/l) ARL (mm) 5 3 3 4 2 3 4 6 5 5
Decoction RN 4 7 5 6 8 3 4 7 2 5
(10 g/l) ARL (mm) 3 4 3 3 2 2 3 2 3 2
RN: root number, ARL: average root lenght
Fig. 1 — The average root numbers in controls and Fig. 2 — The average root lengths in controls and flower
flower extracts after 72 h. extracts after 72 h.160 ÖZMEN
mutations to chromosomal aberrations (LEME
and MORALES 2008). Chromosomal aberrations
are changes in chromosome structure resulting
from a break or exchange of chromosomal ma-
terial. Most of the chromosomal aberrations ob-
served in cells are lethal, but there are many cor-
responding 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 inhibit-
ed spindle formation similar to the effect of col-
chicine (BADR 1983), and induction of C-mitosis
commonly associated with spindle poisons, indi-
cating turbogenic effect (SHAHIN and EL-AMOODI
1991). ODEIGAH et al. (1997) describes the pres-
ence of C-mitoses as a possibly reversible effect
Fig. 3 — Mitotic index (MI) in controls and flower ex- (weak toxic effect). In this study colchicine like
tracts after 72 h. mitosis has been observed by microscopic analy-
sis of Allium roots. Data are not given because
the number of C-mitosis in this study was not
compounds at low concentrations may be lethal important statistically.
for microorganisms and small animals but in In respect of this results, Hibiscus rosa-sinen-
larger organisms they may specifically affect the sis flowers contains anti-mitotic constituents that
fastest growing tissues (KINTZIOS and BARBERAKI can stop the mitosis in anywhere of the cell cy-
2004). Hence in this study the mitotic index may cle. Furthermore these constituents probably af-
be reduced at %5 levels by hormone-like sec- fect the cytoskeleton by tubulin polymerization
ondary metabolites that probably found in flow- or degradation.
er decoction. Reduction in the mitotic activity
could be due to inhibition of DNA synthesis or Acknowledgements — The author is indebted to
blocking in the G2 phase of the cell cycle, pre- Ezgi AKAT for preparing the extracts and helping to
venting the cell from entering mitosis (TÜRKOĞLU count the cells by microscopic observations.
2008).
The species A. cepa also presents other advan-
tages with suitable chromosomal features; this REFERENCES
plant bears large and few chromosomes (2n = 16)
what facilitates the evaluation of chromosome ADHIRAJAN N., RAVI K.T., SHANMUGASUNDARAM N. and
BABU M., 2003 — In vivo and in vitro evaluation
damages or disturbances in cell division cycle. of hair growth potential of Hibiscus rosa-sinensis
This test have proved to be of great value, since Linn. Journal of Ethnopharmacology, 88: 235-
combine a high sensibility to detect mutagens in 239.
different environments and a great capacity to AKINBORO A. and BAKARE A.A., 2007 — Cytotoxic and
evaluate distinct genetic endpoints, from point genotoxic effects of aqueous extracts of five medici-
TABLE 2 — Number of roots and analyzed cells.
Used onions Evaluated roots Evaluated cells Dividing cells Mitotic index
Tap Water 10 100 10000 2878 %29
Lettuce
(Lactuca sativa) 10 100 10000 2742 %27
Decoction
(5 g/l) 10 100 10000 987 %10
Decoction
(10 g/l) 10 100 10000 436 %4CYTOTOXICITY OF HIBISCUS ROSA - SINENSIS FLOWER EXTRACT 161 nal plants on Allium cepa Linn. Journal of Eth- R.M., PEGAS HENRIQUES O.A., LIA DE PAULA RA- nopharmacology, 112: 470-475. MOS A.L., SAFFI J., 2009 — Pharmacological evi- ALI M., ANSARI, S.H., 1997 — Hair care and herbal dences for the extracts and secondary metabolites drugs. Indian Journal of Natural Products, 13: from plants of the genus Hibiscus. Food Chemis- 3-5. try, doi: 10.1016/j.foodchem.2009.04.005. BADR A., 1983 — Mitodepressive and chromotoxic ac- MOUJIR L., SECA A.M.L., SILVA A.M.S., LÓPEZ M.R., tivities of two herbicides in A. cepa. Cytologia, 48: PADILLA N., CAVALEIRO J.A.S. and NETO C.P., 2007 451-457. — Cytotoxic activity of lignans from Hibiscus can- CHANG Y.C., HUANG H.P., HSU J.D., YANG S.F. and nabinus. Fitoterapia, 78: 385–387. WANG C.J., 2005 — Hibiscus anthocyanins rich NADKARNI A.K., 1954 — Indian Materia Medica. extract-induced apoptotic cell death in human pro- Bombay, 631 pp. myelocytic leukemia cells. Toxicology and Applied ODEIGAH P.G.C., NURUDEEN O. and AMUND O.O., Pharmacology, 205: 201-212. 1997 - Genotoxicity of oil field wastewater in Ni- CHENG Y.L., LEE S.C., HARN H.J., HUANG H.C., and geria. Hereditas, 126: 161-167. CHANG W.L., 2008 — The extract of Hibiscus syri- SETHI N., NATH D. and SINGH R.K., 1986 — Terato- acus inducing apoptosis by activating p53 and AIF logical study of an indigenous antifertility medi- in human lung cancer cells. American Journal of cine, Hibiscus rosa-sinensis in rats. Arogya Journal Chinese Medicine, 36: 171-184. of Health Science, 12: 86-88. DWIVEDI R.N., PANDEY S.P. and TRIPATHI, V.J., 1977 SHAHIN S.A. and EL-AMOODI K.H.H., 1991 — Induc- — Role of japapushpa (Hibiscus rosa-sinensis) in tion of numerical chromosomal aberrations during the treatment of arterial hypertension. A trial study. DNA synthesis using the fungicides nimrod and Journal of Research in Indian Medicine, Yoga & rubigan-4 in root tips of Vicia faba L. Mutat. Res., Homeopathy, 12: 13-36. 261: 169-176. FARNSWORTH N.R., AKERELE O.O., BINGEL A.S., SOE- SINGH M.P., SINGH R.H. and UDUPA, K.N., 1982 — JARTA D.D. and ENO, Z., 1985 — Medicinal plants Antifertility activity of a benzene extract of Hi- in therapy. Bulletin World Health Organisation, biscus rosa-sinensis flowers on female albino rats. 63: 965-981. Planta Medica, 44: 171-174. FISKESJÖ G., 1993 — Allium Test 1:A 2-3 day plant test TSENG T.H., KAO T.W., CHU C.Y., CHOU F.P., LIN for toxicity assesment by measuring the mean root W.L. and WANG C.J., 2000 — Induction of Ap- growth of onions (Allium cepa L.). Environmental optosis by Hibiscus Protocatechuic Acid in Human Toxicology and Water Quality, 8: 461-470. Leukemia Cells via Reduction of Retinoblastoma GRANT W.F., 1982 — Chromosome aberration assays (RB) Phosphorylation and Bcl-2 Expression. Bio- in Allium. Mutation Research, 99: 273-291. chemical Pharmacology, 60: 307-315. KINTZIOS S.E. and BARBERAKI M.G., 2004 — Plants TÜRKOĞLU Ş., 2008 — Evaluation of genotoxic effects That Fight Cancer. By CRC Press LLC, USA. of sodium propionate, calcium propionate and po- KURUP P.N.V., RAMDAS V.N.K. and JOSHI P., 1979 — tassium propionate on the root meristem cells of Handbook of Medicinal Plants. New Delhi, 86 Allium cepa. Food and Chemical Toxicology, 46: pp. 2035-2041. LEME D.M. and MARIN-MORALES M.A., 2008 — Chro- WHO Fact sheet N° 134, 2008 — Traditional medi- mosome aberration and micronucleus frequencies cine. in Allium cepa cells exposed to petroleum polluted WU P.L., CHUANG T.H., HE C.X. and WU T.S., 2004 water- A case study Mutation Research, 650: 80- — Cytotoxicity of phenylpropanoid esters from the 86. stems of Hibiscus taiwanensis. Bioorganic & Me- LIN H.H., CHENB J.H., KUO W.H. and WANG C.J., dicinal Chemistry, 12: 2193-2197. 2007 — Chemopreventive properties of Hibiscus WU P.L., WU T.S, HE C.X., SU C.H. and LEE K.H., sabdariffa L. on human gastric carcinoma cells 2005 — Constituents from the Stems of Hibiscus through apoptosis induction and JNK/p38 MAPK taiwanensis. Chem. Pharm. Bull., 53(1): 56-59. signaling activation. Chemico-Biological Interac- tions, 165: 59-75. Received July 22th 2009; accepted February 8th 2010 MAGANHA E.G., DA COSTA HALMENSCHLAGER R., ROSA
You can also read
