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Research Article
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Evaluation of Cytotoxic, Anti-angiogenic and Antioxidant Properties of Standardized Extracts of Strobilanthes crispus Leaves
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N.S. Muslim,
K.W. Ng,
A. Itam,
Z.D. Nassa,
Z. Ismail
and
A.M.S. Abdul Majid
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ABSTRACT
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Strobilanthes crispus (Acanthaceae) is widely used for treatment of cancer in the South Asian region. In this study, validation of GC-TOF mass spectrophotometric methods for quantitative determination of phytoconstituents in methanolic and aqueous extracts of S. crispus was conducted. The cytotoxicity of standardised methanolic and aqueous extracts of S. crispus was assessed against a panel of human cancer cell lines, namely breast carcinoma (MCF7), colon carcinoma (HCT 116), hepatocellular carcinoma (Hep G2), non-small cell lung adenocarcinoma (NCI-H23) and human breast ductal carcinoma (T-47D) cells and one normal colonic fibroblast cell line (CCD-18Co). The cell proliferation assay was performed using tetrazolium (MTT) method. Furthermore, the inhibitory effect of the extracts on angiogenesis was evaluated using ex vivo rat aortic ring assay. Finally, the antioxidant properties of both extracts were studied using DPPH free radical, xanthine oxidase activity and β-carotene-linoleate model system. Aqueous extract was found to be nontoxic towards all cell lines used, while the methanolic extract exhibited cytotoxic response towards the T-47D and MCF7 cells. Both the extracts demonstrated detectable anti-angiogenic activity. The extracts displayed very strong inhibitory activity towards xanthine oxidase enzyme, however, they demonstrated moderate antioxidant properties, which is evidenced by the quenching of DPPH free radical and preventing the bleaching of β-carotene by linoleic acid.
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Received: April 30, 2010;
Accepted: June 10, 2010;
Published: July 14, 2010
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INTRODUCTION
Human mortality is increasing drastically due to the different kinds of cancer
globally. Therefore, the trend now has been shifted towards the natural medicine,
logically based on traditional medicines, in search of better drugs against
cancer (Galal et al., 1991; Hoffmann
et al., 1993). Angiogenesis plays a vital role in the growth and
metastasis of tumours and several chronic inflammatory diseases including rheumatoid
arthritis and proliferative diabetic retinopathy (Folkman,
1995). Since a close relationship between tumor growth and angiogenesis
had been clarified, various angiogenic inhibitors for use in cancer treatment
have been studied. Therefore, the inhibition of angiogenesis emerges as a potent
target for prevention and treatment of cancer (Fadzelly
et al., 2006).
Strobilanthes crispus L. Bremek (family-Acanthaceae) is traditional
medicinal plant native to tropical countries like Madagascar and Malay archipelagos.
The plant is believed to have health-giving properties, so locally it is also
known as pecah beling. Traditionally the medical practitioners in Malaysia and
Indonesia were used to prepare the aqueous extract from leaves of S. crispus
by boiling them and the different forms of the extract were used as diuretic,
antidiabetic, antilytic, laxative and to treat inflammatory diseases and tumors
(Fadzelly et al., 2006). Several scientific studies
have confirmed that infusion of the dried leaves has the antidiabetic, diuretic,
antilithic and laxative properties (Perry and Metzger, 1980).
Ismail et al. (2000) reported that extract of S. crispus showed
antioxidant activity. The hot aqueous-extract of fermented and unfermented leaves
was found to reduce blood glucose in hyperglycemic rats, while unfermented leaves
also reduced glucose level in normal rats. Both fermented and unfermented leaves
also exhibited improved lipid profiles (Fadzelly et al.,
2006). Rahmat et al. (2006) reported that
the methanolic extract displayed modest cytotoxic effect on colon cancer (Caco-2)
and human liver cancer (HepG-2).
In this study we developed the validation of GC-TOF mass spectrophotometric
methods for quantitative determination of phytoconstituents in methanolic and
aqueous extracts of S. crispus. In order to study the bio-pharmacological
properties of methanolic and aqueous extracts of S. crispus leaves, we
assessed the cytotoxic efficacy the standardized extracts against a panel of
human cancer cell lines such as, metastatic breast carcinoma highly invasive
breast carcinoma (MCF7), less invasive breast ductal carcinoma cells (T-47D),
colon carcinoma (HCT 116), hepatocellular carcinoma (Hep G2) and non-small cell
lung adenocarcinoma (NCI-H23) and one normal colonic fibroblast cell line (CCD-18Co).
Additionally, the inhibitory effect of methanolic and aqueous extracts on angiogenesis
was evaluated using ex vivo rat aortic ring assay. Finally, the antioxidant
property of the extracts was studied using DPPH free radical, xanthine oxidase
and β-carotene-linoleate models.
MATERIALS AND METHODS Chemicals, cell culture and reagents: Dulbeccos buffered saline phosphate from Sigma-Aldrich, Germany and trypsin was purchased from Gibco, Life Technology, UK. 1, 1-diphenyl-2-pircryldrazyl (DPPH), sodium bicarbonate, Penicillin/Streptomycin (PS) solution, MTT reagent was purchased from Sigma-Aldrich, Germany. Suramin, amphotericin B, aprotinin, 6-aminocaproic acid, L-glutamine, thrombin and gentamicin were obtained from Sigma-Aldrich, Germany. Fibrinogen was obtained from Calbiochem, USA. Matrigel matrix (10 mg mL-1) was obtained from SABiosciences, USA. M199 was obtained from Gibco BRL. Aprotinin, thrombin (EC 3, 4.21.5), foetal bovine serum, amphotericin B (fungizone), gentamycin, ε-aminocaproic acid, phosphate-buffered saline (PBS) and sodium chloride were obtained from Sigma, St Louis, MO. All cancer cell lines used in the study were purchased from American tissue culture collection (Rockvill, MD, USA). The medium used for HCT 116, T47D and NCI-H23 cell lines was RPMI 1640 (Sigma-Aldrich, Germany) containing 10% heat-inactivated FBS and 1% penicillin/streptomycin. The cell lines CCD-18Co, MCF7 and Hep G2 were cultured in Dulbeccos Modified Eagle Media (Gibco, Life Technology, UK) supplemented with 10% heat-inactivated FBS and 1% penicillin/streptomycin. Other chemicals used in this study were analytical grade. Extraction: S. crispus leaves were collected from Padang, West Sumatera Indonesia in March 2009. The plants were identified and deposited in herbarium of School of Biology, Universiti Sains Malaysia with voucher number 10931. This research project was conducted in the School of Pharmaceutical Sciences, Universiti Sains Malaysia, Penang, Malaysia. Briefly, the fresh leaves were washed and dried in the oven at 40°C then powdered mechanically. Five gram of the powdered material was macerated with 200 mL of water and 200 mL of methanol in separate flasks at their boiling points for 6 h. After cooling, the mixtures were filtered then the filtrates were dried under vacuum and stored in airtight containers and used for phytochemical investigations.
Analysis by GC-TOF mass spectrometry: Characterisation of the unknown
chemical species was performed using a Hewlett-Packard 6890 GC coupled with
a LECO Pegasus II time-of-flight mass spectrometer with electron impact ionization
in the reflectron mode. Data analysis of peaks observed was carried out using
the Chrom TOF mass data analysis software packages. Helium was used as carrier
gas at a flow rate of 1 mL min-1 using a DB-WAX fused silica capillary
column (20 mx0.18 mm, I.D; 0.18 μm film thickness). A 1000 μg mL-1
methanolic and aqueous extract of S. crispus solution was used for analysis,
which was prepared by dissolving 10 mg of the extracts in 10 mL of methanol.
The GC open program was set between 50°C to 250°C (5 min hold) at temperature
ramping rate of 20°C min-1, using splitless injection with the
injector temperature being fixed at 200°C. The ion source temperature was
maintained at 180°C with the mass range of detection configured between
50-500 amu. The spectral scan rate was set at 20 spectra/sec and the volume
of injected sample maintained at 1 μL. Identification of the compounds
was carried out by referring to NIST (National Institute for Standard and Technology)
1998 Mass Spectral Database and the Terpene Essential Oil Library (Korytar
et al., 2002).
Determination of antioxidant activity
Inhibition of xanthine oxidase activity: Xanthine oxidase inhibitory activity
of S. crispus extracts were determined according to methods developed
by Sweeney et al. (2001). The 1.3 mL of buffer
phosphate (pH 7.5) and 0.2 mL of 0.2 unit mL-1 xanthine oxidase solutions
were taken into test tubes containing 0.5 mL of 100 μg mL-1
aqueous and methanolic extracts. After 10 min of incubation at room temperature,
1.5 mL of 0.15 mM xanthine substrate solution was added to this mixture. The
mixture was incubated for 30 min at room temperature and the reading were taken
at 293 nm using UV/VIS spectrophotometer against 0.5 mL methanol, 1.3 mL phosphate
buffer (pH 7.5), 0.2 mL xanthine oxidase and 1.5 mL of aqueous as blank. 0.5
mL of methanol, 1.3 mL of phosphate buffer solution (pH 7.5), 0.2 mL of xanthine
oxidase and 1.5 mL of xanthine substrate solution were also used as the control.
The experiment was carried out in triplicate and the final results averaged
between the three data sets. Inhibition percentage was calculated using the
formulae:
where, As is absorbance of sample and Ac is absorbance of control.
DPPH free radical scavenging activity: DPPH free radical scavenging
activity of extracts was determined according to Akowuah
et al. (2005) and Abdille et al. (2005)
with some modifications. Solutions of 100, 200, 400, 600 and 800 μL of
1000 μg mL-1 extracts were placed in separate tubes and the
amount was made up to 1 mL with methanol. To this mixture, 2 mL of 0.1 mM DPPH
(1,1-Diphenyl-2-picrylhydrazyl) was added. After 60 min of incubation at room
temperature, absorbance was measured at 517 nm using spectrometer UV/VIS Lambda
45, against methanol as the blank. Volume of 1, 2, 4, 10, 20, 40, 60 and 80
μL of 500 μg mL-1 gallic acid, ascorbic acid, quercetin
and Butylated Hydroxyl Anisole (BHA) were used as references and 5 mL of 0.1
mM DPPH was used as the reagent. Two control solutions containing 1 mL of methanol
and 2 mL of 0.1 mM DPPH for S. crispus were used as reference compounds.
The experiments were carried out in triplicate. Free radical scavenging activity
of the extracts and effective concentration 50% (EC50) were determined
according to the following formulae:
where, As is absorbance of sample and Ac is absorbance of control.
Antioxidant assay using β-carotene-linoleate model system: Antioxidant
assay using β-carotene-linoleate model system of S. crispus extracts
was analyzed according to Abdille et al. (2005)
with modifications. 0.2 mg of β-carotene was added to 0.2 mL chloroform,
20 mg of linoleic acid and 200 mg of Tween-40 (polyoxyethylene sorbitan monopalmitate).
Chloroform was removed under vacuum at 40°C and the resulting mixture was
diluted vigorously with 10 mL aqueous. To this emulsion, 40 mL of oxygenated
aqueous was added. 4 mL aliquots of the emulsion were pipetted into separate
test tubes containing 0.2 mL of 200 ppm extracts, quercetin, BHA and BHT in
ethanol. Quercetin, BHA and BHT were used for comparative purposes. A control
containing 0.2 mL of ethanol and 4 mL of the above emulsion was prepared. The
test tubes were placed at 50°C in an aqueous bath and the absorbance was
measured at 470 nm (absorbance at zero time, t = 0) using UV/VIS Lambda 45 spectrophotometer.
The next absorbance was measured after 120 min of incubation. A blank with a
similar mixture was prepared without the presence of β-carotene. All analyses
were carried out in triplicate. The Antioxidant Activity (AA) was evaluated
using the following formula:
where A0 and A0° are the absorbance value measured
at zero time of the incubation for test sample and control, respectively. At
and At° are the absorbance values were measured after incubation
for test sample and control, respectively. The results were expressed in percentage
(%) of bleaching inhibition of β-carotene.
Evaluation of cytotoxicity activity: The MTT viability assay was performed
with slight modifications as previously described by Mosmann
(1983). In brief, cells were seeded at 5000 cell density per well for each
96-well plates in 100 μL medium. After an overnight incubation, 100 μL
of culture medium containing test substance were added into each well to make
the final concentration of 150, 100, 80, 60, 40, 20, 10 and 5 μg mL-1
with 0.01% DMSO in each well. For positive control, the cells were treated with
600 ng mL-1 of vincristine. The untreated cells that received only
the medium in 0.01% DMSO were used as negative control. All the cells were treated
for 72 h. Each experiment was done thrice with four replicates for each concentration.
MTT was first prepared as a stock solution of 5 mg mL-1 in phosphate
buffer saline and was filtered using 0.22 μm syringe filter. At the end
of the treatment period (72 h), 20 μL of MTT solution was added to each
well. After 4 h incubation at 37°C, the medium was removed and 200 μL
of DMSO was added to the well to dissolve the formazon crystal. After 5 min
of shaking, the optical density was recorded using Multiskan Ascent plate reader
(Thermo Scientific, Waltham) at 570 nm for absorbance and 650 nm as reference
filter.
Angiogenesis study: Rat aortic ring explant cultures were prepared by
modification of protocols previously described (Brown et
al., 1996; Zhu et al., 2000). Aortic
rings were prepared from male Sprague Dawley rats. Aortas were sectioned into
1 mm-long cross sections, rinsed several times with Hanks Balanced Salt Solution
containing 2.5 μg mL-1 amphotericin B (Sigma, St. Louis. MO).
The assay was performed in a 48-well tissue culture plate (Coster Corning, USA).
500 μL of 3 mg mL-1 fibrinogen (Calbiochem, USA) in serum free
M199 growth medium (Gibco, UK) was added to each well with 5 mg mL-1
of aprotinin (Sigma-Aldrich, Germany) to prevent fibrinolysis of the vessel
fragments. Each tissue section was placed in the center of the well and 15 μL
of thrombin (50 NIH U mL-1) (Sigma-Aldrich, Germany) in 0.15 M NaCl.
Immediately after embedding the vessel fragment in the fibrin gels, 0.5 mL of
medium M199 supplemented with 20% HIFCS (Gibco, UK), 0.1% έ-aminocaproic
acid (Sigma-Aldrich, Germany), 1% L-Glutamine (Sigma-Aldrich, Germany), 1% amphotericin
(Sigma-Aldrich, Germany), 0.6% gentamicin (Sigma-Aldrich, Germany) were added
to each well. Methanolic and aqueous extracts at varying concentrations ranged
from 6.25 to 100 μg mL-1 was added to the complete growth medium.
Control cultures received medium without the test substances. Suramin (Sigma-Aldrich,
Germany), a well recognized anti-angiogenic agent was used as a positive control
(Sahib et al., 2009). Cultures were incubated
at 37°C for 5 days, in humidified CO2 and the medium was replaced
daily. The magnitude of blood vessel outgrowth on day five of the procedure
was quantified as per the technique developed by Nicosia
et al. (1997) using inverted microscope (Olympus, Japan) supplied
with a digital camera (Lieca CCD, Japan) and Leica QWin computerized imaging
software.
The experimental work was consistent with guidelines of the USM Committee for Animal Care and received approval from the USM Animal Ethical Committee for the present work (Reference Number USM/PPSF/50 (084) Jld.2). Statistical analysis: The results of this experiment are presented as Mean±SD of triplicate experiments analyzed using SPSS 12 (SPSS Inc. Chicago, IL). Differences between mean is evaluated by one-way ANOVA and Tukey's-b multiple comparisons at p<0.05. RESULTS
GC - TOF mass spectrometry analysis: Figure 1a shows
the results of the GC-TOF MS chromatogram of the methanolic extract of S.
crispus and Table 1 reveals the molecular components identified
above 1% detection level.
| Fig. 1: |
(a) GC/TOF-MS chromatograms of the methanol extracts of S.
crispus leaves and (b) GC/TOF-MS chromatograms of aqueous Ew extracts
of S. crispus leaves |
The results show that the sample contains 1-heptatriacotanol (1.03%), 3,5-dithiahexanol
5,5-dioxide (1.04%), 3,7,11,15-Tetramethyl-2-hexadecen-1-ol (3.48%), 3-octadedecyne
(9.25%), 7-hexadecenoic acid, methylester (2.70%), 9,12,15-octadecatrienoic
acid, methylester, (Z, Z, Z)- (1.21%), 9,12-octadecadienoic acid, methylester
(2.24%), α-sitosterol (7.08%), aromadendrene oxide-(2) (1.39%), campesterol
(2.63%), hexadecanoic acid, methylester (12.11%), hydrazine carboxamide (2.03%),
lupeol (3.60%), monoethanolamine (2.52%), n-propyl acetate (2.20%), octadecanoic
acid, methylester (1.14%), phenol,2,4-bis(1,1-dimethylethyl)- (2.60%), phytol
(3.78%) and stigmasterol (7.86%).
Figure 1b shows the results of the GC-TOF MS chromatogram
of the aqueous extract of S. crispus and Table 1 reveals
the molecular components identified. The following chemical constituents were
identified; 1,1-dimethylamino-1-butene (1.13%), 2,5-dimethoxy-4-(methylsulfonyl)amphetamine
(2.55%), 3,5-dithiahexanol 5,5-dioxide (3.09%), benzenemethanol, alpha-(1-aminoethyl)-
(1.37%), cyclobutanol (13.56%), dimethyl sulfoxide (1.57%), hexadecanoic acid,
methylester (1.84%), hydrazine carboxamide (3.32%), monoethanolamine (5.53%),
n-propyl acetate (5.38%) and undecane (2.10%).
Determination of antioxidant activity
Inhibition of xanthine oxidase assay: Both aqueous and methanolic extracts of S. crispus displayed a strong inhibitory effect on xanthine oxidase activity as presented in Table 2, at 100 μg mL-1 concentration, methanolic extract showed 90.28% and the aqueous extract exhibited about 89.06% xanthine oxidase inhibition. Table 1: |
The quantitative estimation of phytochemicals identified
in the extracts of S. crispus leaves detected using GC-TOF MS |
 |
NA: Not available |
DPPH free radical scavenging activity: Results show that both aqueous
and methanolic extracts were able to quench the stable free radical DPPH to
yellow-coloured 1,1-diphenyl-2-picrylhydrazyl by its hydrogen donating ability.
The scavenging activity of aqueous extract was more than the methanolic extract
of S. crispus as shown in Table 3. The reference standards,
gallic acid, quercetin, BHA and ascorbic acid displayed potent DPPH free radical
scavenging activity, their EC50 values are tabulated in Table
4.
β-carotene-linoleate model system: The result of antioxidant activity
using β-carotene-linoleate model system of methanolic and aqueous extracts
of S. crispus is depicted in Table 5. The extracts
showed weak response in the β-carotene-linoleic acid assay. Whereas, the
standard references, BHA and BHT, demonstrated potent activities in this assay
system with bleaching inhibition percentage about 95.84 and 95.12, respectively
(Table 5).
Table 2: |
Xanthine oxidase inhibition assay |
 |
Data is represented in Mean±SD (n =6), *p<0.05.when
compare to control |
Table 3: |
DPPH free radical scavenging activity of extracts of S.
crispus leaves |
 |
Data is represented in Mean±SD (n = 6) |
Table 4: |
EC50 values of reference standards against DPPH
free radical scavenging activity |
 |
Table 5: |
Antioxidant activity of using β-carotene linoleate model
system |
 |
*Data is represented in Mean±S.D (n = 6), *p<0.05.
when compare to control |
Evaluation of cytotoxicity activity: MTT cell proliferation assay was
conducted. Five human cancer cell lines (MCF7, T-47D, HCT 116, Hep G2 and NCI-H23)
and one normal colonic fibroblast cell line (CCD-18Co) were exposed to the different
concentrations of extracts. However, result showed that, only at their high
concentrations the extracts demonstrated cytotoxicity against all cell lines
except for the T-47D and MCF7 breast cancer cells. Table 6
depicts the IC50 values of the extracts for various cell lines.
Effect of extracts on vessel sprout formation from rat aorta: The effect
of methanolic and aqueous extracts on angiogenesis was studied using an ex
vivo rat aortic ring assay. In this assay, the rat aortic endothelium exposed
to a three-dimensional matrix containing angiogenic factors switches to a microvascular
phenotype generating branching networks of microvessels (Nicosia
et al., 1992). As shown in Fig. 2, microvessels
grew out from the rat aorta in the control (Fig. 3a) when
cultured in the medium.
| Fig. 2: |
Graph showing the extent of angiogenesis inhibition by methanolic
and aqueous extracts of S. crispus with compare to suramin as the
positive control |
Table 6: |
IC 50 value for cytotoxicity on panel of cell
lines for S. crispus aqueous and methanol extracts |
 |
Methanol extracts. Data is represented in Mean±SD (n
= 6), *p<0.05.when compare to control |
| Fig. 3: |
Effect of S. crispus extracts on rat aortic micros
vessel growth. (a) Control, (b) aortic ring treated with methanolic extract,
(c) aortic ring treated with aqueous extract and (d) aortic ring treated
with suramin |
The anti-angiogenic activity was quantified by measuring the cell-sprouting
area around the aortas exposed to the various concentrations of methanolic extract
and the control. The aqueous extract (Fig. 3b) at 100 μg
mL-1 showed moderate activity (16.67±8.11 %) and methanolic
extract (Fig. 3c) exhibited least activity (6.25±3.6
%) at 100 μg mL-1 concentration, whereas, the anti-angiogenesis
effect of suramin (Fig. 3d) on the sprouting of microvessels
from rat aorta was significantly dose dependent (p<0.05) and it showed 69.44±14.24%
inhibition at 100 μg mL-1 concentration.
DISCUSSION
Highly reactive free radicals induce oxidative damage in lipids, proteins and
other biomolecules. They are the main responsible elements for numerous degenerative
diseases including cancer (Aruoma, 1994; Ray
and Husain, 2002). Free radical scavengers function as inhibitors at both
initiation and promotion/propagation stages of degenerative diseases and consequently,
antioxidants play an important role in the protection of human body against
oxidative damage (Govindarajan et al., 2005).
Antioxidants serve as potent inhibitors of angiogenesis and thus curb the neoplastic
processes (Bagchi et al., 1997). The consumption
of edible plants, fruits and herbs has been proven to play the prophylactic
role in a number of diseases in humans and animals. Vegetables, fruits and herbs
are rich sources of antioxidants, protease inhibitors and also compounds that
might protect the organism against free radical induced injury and diseases
(Tapiero et al., 2002; Harborne
and Williams, 2000).
In the present study, validation of GC-TOF mass spectrophotometric methods
for quantitative determination of phytoconstituents in methanolic and aqueous
extracts of S. crispus was carried out. The result of the mass spectrometric
analysis reveals the difference in composition of two different organic solvent
extracts of S. crispus. The phytol is a decomposition product of chlorophyll
(Windholz et al., 1976), which is found in the
methanolic extracts (3.78 %) however it failed to appear in the aqueous extract.
Stigmasterol and α-sitosterol are found in significant quantities in the
methanol extract (stigmasterol, 7.89 %; α-sitosterol, 7.08 %), but none
was detected in the aqueous extract. Steroids and lipids are non-polar compounds;
hence their solubility is higher in methanol than aqueous. Stigmasterol and
sitosterol are widely distributed in the plant kingdom synthesised via the mevalonic
acid pathway (Torssell, 1993). Researchers have indicated
that stigmasterol and sitosterol may be useful in prevention of certain cancers,
including ovarian, prostate, breast and colon cancers and may help to reduce
cholesterol level. Rahmat et al. (2006) reported
that the methanolic extract of S. crispus containing these compounds
displayed strong cytotoxic activity on colon cancer (Caco-2). Results of the
present study appear to be synchronized even with the different human colon
carcinoma cell types (HCT 116 and CCD-18Co) used in the study. In addition the
methanolic extract found to be significant cytotoxic against human non-small
cell lung adenocarcinoma cell (NCI-H23) and human breast ductal carcinoma cell
(T-47D). The process of carcinogenesis is a multistep event and oxidative damage
can lead to the development of tumours through several distinct pathways. Polyphenols
such as quercetin exhibits significant antioxidant, anti-inflammatory and antiproliferative
activity. Polyphenols present in plants and because their anti-oxidation ability
they have been shown to regulate cell proliferation and induce apoptosis (Chu
et al., 2002; Sun et al., 2002). In
the present study, for the first time the anti-angiogenic effect of S. crispus
extracts is being reported. The rat aortic ring assay showed detectable
levels of antiangiogenic properties of methanolic and aqueous extract of S.
crispus when compared to the control. The antioxidants are well known to
have potent anti-angiogenic activity (Losso, 2003).
The extracts of S. crispus are rich in antioxidant phytosterols, such
as α-sitosterol, campesterol, phytol and stigmasterol. Thus the strong
antioxidative properties of methanolic and aqueous extract of S. crispus
are defined by the xanthine oxidase inhibition assay, β-carotene-linoleic
acid assay and the DPPH scavenging activity.
CONCLUSIONS
The stark contrast to the findings made by other researchers highlights the
importance of standardisation of plant extract to ensure reliability and repeatability
of research outcome. The methanolic extract exhibited a moderate level of cytotoxic
activity in human adenocarcinoma cell lines. The aqueous extract showed considerable
antiangiogenic activity compared control. The cytotoxic and antiangiogenic properties
of S. crispus may probably due to the presence of potential antioxidants.
These findings suggest that S. crispus could be a source of promising
cytotoxic agents and angiogenesis inhibitors.
ACKNOWLEDGMENT We thank Mr. Noramin from Kulim Hi-Tech for the assistance provided with the Mass Spectrometry studies. This work was funded by the FRGS, grant number 203/PFARMASI/671154, the Malaysian Ministry of Science, Technology and Innovation (MOSTI), grant number 305/PFARMASI/613219: E Science fund and by the research University (RU), grant number 1001/PFARMASI/81144, School of Pharmaceutical Sciences, Universiti Sains Malaysia. Last but not least, the first author would like to thank the USM Fellowship which has supported this research project.
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