Vol. 9(34), pp.

861-874, 15 September, 2015

DOI: 10.5897/AJPP2015. 4396
Article Number: E7CE67C55310
ISSN 1996-0816
African Journal of Pharmacy and
Copyright © 2015 Pharmacology
Author(s) retain the copyright of this article
http://www.academicjournals.org/AJPP

Full Length Research Paper

In vitro antioxidant, cytotoxic and phytochemical

studies of Clinacanthus nutans Lindau leaf extracts
Intan Soraya Che Sulaiman1*, Mahiran Basri1,2*, Kim Wei Chan3, Siti Efliza Ashari1,2 , Hamid
Reza Fard Masoumi1 and Maznah Ismail3
1
Department of Chemistry, Faculty of Science, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia.
2
Centre of Foundation Studies for Agricultural Science, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor,
Malaysia.
3
Laboratory of Molecular Biomedicine, Institute of Bioscience, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor,
Malaysia.
Received 27 June, 2015; Received 17 August, 2015

An increasing demand for natural additives has drawn more interest from consumers due to their
relatively safe and wide acceptance. The present work examines the potential of different solvent
system of Clinacanthus nutans Lindau leaves as a source of natural antioxidant. We also screened for
its phytochemical constituents and cytotoxicity toward breast cancer cell. In vitro antioxidant activity of
the n-hexane, dichloromethane, ethyl acetate, and ethanol extracts were assessed via 1,1-diphenyl-
picrylhydrazyl (DPPH) radical scavenging activity, oxygen radical absorbance capacity and ß-carotene
bleaching activity assays, whereas the cytotoxic effect was tested on tumorigenic breast cancer
estrogen positive cell (MCF-7) and normal fibroblast cell (3T3) using the tetrazolium assay. Liquid
chromatography mass spectrometry equipped with an electrospray ionization source liquid
chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI/MS) was used to analyze
the amount of targeted phenolic and fatty acids in ethanol and ethyl acetate extracts. Several phenolic
compounds and fatty acids were identified and quantified from ethanol and ethyl acetate extracts of this
plant. Ethanol and ethyl acetate extracts demonstrated stronger antioxidant activity than n-hexane and
dichloromethane extracts (p<0.05). Thus, ethanol and ethyl acetate extracts of C. nutans may be
explored as new sources of antioxidants in herbal medicines research.

Key words: Cytotoxicity, polyphenols, polyunsaturated fatty acid (PUFA), Sabah snake grass, anticancer.

INTRODUCTION

Antioxidant activity is associated to many biological properties of antioxidants is to defend body against free
functions in human body. One of the most important radical damage. Free radicals, especially reactive oxygen

*Corresponding author. E-mail: [email protected], [email protected]. Tel: +60-389-467-266. Fax: +60-389-

466-997.
Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution
License 4.0 International License
862 Afr. J. Pharm. Pharmacol.

species (ROS), are harmful radicals produced by content (TFC) of the four different extracts were also
physiological and biochemical processes (Cai et al., evaluated.
2004). ROS, when reacted with other substances in the
body, may result into cell or tissue injury. Natural
antioxidant compounds purified from plant products are MATERIAL AND METHODS
gaining more interest than synthetic antioxidants due to
their relatively safe and wide acceptance by consumers. Chemicals
Therefore, attention has been focused toward exploring
All solvents and reagents were of analytical or high performance
natural antioxidants from plant sources. Phenolics are liquid chromatography (HPLC) grade. n-Hexane, dichloromethane,
common compounds which contribute to antioxidant ethyl acetate, ethanol, folin-ciocalteu reagent, gallic acid, 1,1-
properties and also exhibit pro-oxidant properties. Pro- diphenyl-picrylhydrazyl (DPPH), 6-hydroxy-2,5,7,8-tetramethyl-2-
oxidant action of rich phenolic plant may be an important carboxylic acid (Trolox), linoleic acid, rutin, 3-[4,5-dimethylthiazol-2-
character associated with their anticancer properties (Cai yl]-2,5-diphenyltetrazolium bromide, 2,2-Azobis (2-methyl
et al., 2004; Azam et al., 2004; Rodríguez et al., 2007). propionamidine) dihydrochloride) (AAPH), fluorescein sodium salt,
ß-carotene and alpha tocopherol were obtained from Sigma-Aldrich
Thus, phenolic compounds when absorbed into (Germany). Sodium carbonate (Na2CO3), polyvinylpolypirrolidone
bloodstream will undergo chemical modification to result (PVPP), aluminum chloride (AlCl3), acetonitrile, iron chloride (III)
in antioxidant or pro-oxidants. Association of rich phenolic (FeCl3), hydrochloric acid (HCl) and Tween 20 were purchased
plant toward cytotoxicity of tumor cell is due to their pro- from Merck (Darmstadt, Germany).
oxidative activity which accelerated a good effect of
oxidative damage (Gomes et al., 2003; Rodríguez et al.,
2007). Plant
Native to tropical Asian countries, Clinacanthus nutans
Fresh leaves of whole C. nutans were collected from botanical farm
Lindau (C. nutans) (Acanthaceae) is a small shrub,
in Jelebu, Negeri Sembilan, on January, 2014. The plant was
locally known in Malaysia as Sabah Snake Grass or authenticated at Department of Biology, Faculty of Science,
Belalai Gajah. Traditionally, paste from fresh leaves of C. Universiti Putra Malaysia, Malaysia by biologist Associate Prof. Dr.
nutans are being consumed as a remedy for bites and Rusea Go. The voucher specimen number (RG5125) is deposited
stings, especially by snake, mosquitos, millipedes, at the Herbarium Unit of Universiti Putra Malaysia.
catfish, centipedes, hornets, jellyfish, ants, bees,
scorpions and wasps in Thai folk medicine (Sakdarat et Preparation of extracts
al., 2009; P’ng et al., 2012). An ethnobotanical survey in
Singaporean communities revealed that C. nutans fresh The leaves of C. nutans were air-dried under sun shade and
herbs are consumed as a traditional medicine for its grounded to fine powder using lab blender (Waring MX7011S). The
general detoxification purposes (Siew et al., 2014; fine powder was sequentially soaked (3 days, repeated twice) in n-
Uawonggul et al., 2006). Moreover, C. nutans is reported hexane, dichloromethane, ethyl acetate and ethanol. The extracts
in all four solvents were collected separately, filtered through
to exhibit better protection to plasmid DNA against Whatman filter paper No. 1, concentrated by using rotary
riboflavin photoreaction in comparison to green tea evaporator (Rotavapor R-210, Buchi, Switzerland) at approximately
(Yuann et al., 2012). Chloroform extracts of C. nutans are 40°C and stored at -20°С prior to further analysis.
reported to have the anti-proliferative effect on cancer cell
lines of human erythroleukemia (K-56), and human
Total phenolic content (TPC)
Burkitt’s lymphoma (Raji) at 100 µg/ml (Yong et al.,
2013). In Malaysia, local traditions claim that C. nutans Poor solubility of n-hexane and dichloromethane extracts in
has a unique properties to cure cancer (Yong et al., 2013; aqueous reagents limited the measurement of TPC. Therefore, n-
Yuann et al., 2012), hence drawn public interest and its hexane and dichloromethane extracts were purified (Ramadan et
leaves been commercialized as herbal tea. al., 2003) prior to TPC determination. TPC of C. nutans extracts
However C. nutans is well documented for its anti- was determined by a method developed by Negi (2012). An aliquot
of sample was prepared in methanol and pipetted out into a test
inflammatory and anti-herpes simplex virus (Wanikiat et tube and mixed with 2.5 ml of diluted Folin-Ciocalteu’s reagent (10-
al., 2008; Kunsorn et al., 2013; Farooqui et al., 2015). fold). Then, 2 ml of 7.5% of Na2CO3 was added. The test tube was
Thus, cytotoxicity of C. nutans extracts toward cancer allowed to stand for 30 min at room temperature before absorbance
cells and phytochemical assessment are still limited. The was measured at 760 nm using UV-Visible spectrometer Shimadzu
main objectives of this study were to determine the UV-1601. Methanol was used as a blank and gallic acid was used
antioxidant properties of four different solvent extracts as standard. TPC values were determined from a calibration curve
prepared from graph absorbance against a series of gallic acid
from the leaves of C. nutans, screen their phytochemical concentrations (0.01, 0.02, 0.03, 0.04 and 0.05 mg/ml). All
constituents and cytotoxicity toward breast cancer cell measurements were performed in triplicate and the results were
estrogen positive (MCF-7). The total phenolic content expressed as milligram gallic acid equivalent per hundred grams of
(TPC), total tannin content (TTC) and total flavonoid extract (mg GAE/100 g extract).
 Sulaiman et al. 863

Total tannin content (TTC) was used for the standard and methanol was used as a blank.
Results were expressed as milligram rutin equivalent per hundred
TTC of CN extract was estimated by using insoluble grams of extract (mg RE/100 g extract).
polyvinylpolypirrolidone (PVPP), a method determined by Makkar
(2003). Aliquot parts (1.0 ml) containing a 10 mg/ml sample was
mixed with 100 mg of PVPP in test tube. The mixture was vortexed Antioxidant assays
and left for 15 min at 4°С, and centrifuged for 10 min at 3000 rpm.
Non-tannin phenolic content appeared as clear supernatant on top 1,1-Diphenyl-picrylhydrazyl scavenging activity
of the test tube, removed by pipetted and determined with the same
method as TPC (Negi, 2012). The difference between TPC and Radical scavenging activity was determined according to Ramadan
non-tannin phenolic content is an estimation of the amount of TTC et al. (2003) by reduction of 1,1-diphenyl-picrylhydrazyl (DPPH)
in the extract. Results are expressed as milligram gallic acid radical in methanol. A methanolic solution of DPPH radical was
equivalent per hundred grams of extract (mg GAE/100 g extract). freshly prepared at concentration 0.2 mM. Initially, an aliquot of
sample was mixed with 2.34 ml of DPPH solution. After being
vortexed for 20 s, the resulting mixture was allowed to stand for 30
Total flavonoid content (TFC) min in the dark. Finally the absorbances of reaction mixture were
recorded at 515 nm using UV-Visible spectrophotometer Shimadzu
Total flavonoid content (TFC) was assessed by aluminium UV-1601. Trolox was used as a standard and the DPPH
colorimetric method (Iqbal et al., 2005). An aliquot of 0.5 mL of scavenging activity of C. nutans extracts was expressed as
sample and 2% AlCl3 were mixed in test tube. The mixture was milligrams trolox equivalent per hundred grams of extract (mg
vortexed and allowed to stand for 10 min at room temperature, and TE/100g extract). The inhibition percent was calculated according
the absorbance of the reaction mixtures was measured at 435 nm to the following equation (Lee et al., 2002).
by using a UV-Visible spectrophotometer Shimadzu UV-1601. Rutin

(Absorbance of control – Absorbance of sample)

%Inhibition = × 100 (1)
Absorbance of control

Oxygen radical absorbance capacity to incubation for 1 h at 50°С. Results were expressed as milligrams
α-tocopherol equivalent per hundred grams of extract (mg Teq/100
The antioxidant properties of the extract of C. nutans were g extract). Percentages of antioxidant activity of C. nutans extracts
measured in vitro using oxygen radical absorbance capacity were calculated by using the formula (El-Ghorab et al., 2007):
(ORAC) method. ORAC was measured using 2,2’-azobis (2-
amidinopropane) dihydrochloride (AAPH) as a peroxyl radicals
source and fluorescein sodium salt as a molecular probe (Huang 100 (DRC - DRS)
and Ou, 2002). ORAC values were calculated using the regression AA% =
equation between trolox concentration and the net area under the
curve (AUC). This dynamic curve combines both inhibition time and
DRC (2)
inhibition percentages of the free radical damage by the antioxidant
into a single value. The assay was analyzed with FLUOstar Where, AA= antioxidant activity; DRc= degradation rate of control=
OMEGA (BMG LABTECH, Germany) microplate reader utilizing [(a/b)/60]; DRs=degradation rate of sample = [(a/b)/60]; a= initial
fluorescence filters for an excitation wavelength 485 nm and absorbance; b= absorbance after incubation.
emission wavelength 520 nm. Results are expressed as micromole
trolox equivalent per hundred grams of extract (µmol TE/100 g
extract).
Liquid chromatography-mass spectrometry (LC-MS) analysis
of phenolic and fatty acid compounds

ß-carotene bleaching activity LC-MS targeted analysis was performed to quantify phenolic and
fatty acid compounds in two selected extract of C. nutans with good
ß-Carotene bleaching activity (BCB) activity was determined antioxidant activity. Liquid chromatography (LC) analysis was
according to the protocol described by Wettasinghe and Shahidi carried out using UPLC Waters ACQUITY UPLC, followed by single
(1999). ß-Carotene-linoleic acid emulsion was prepared by adding quadrupole mass spectrometry equipped with an electrospray
40 mg of linoleic acid and 400 mg of Tween 20 to a test tube ionization source (LC-ESI/MS). For fatty acid analysis, LC analysis
containing 3 ml of ß-carotene solution (5 mg ß-carotene/50 ml was performed with an ACQUITY UPLC BEH C18 column (100 mm
chloroform). The mixture was vortexed and dried under a stream of × 2.1 mm × 1.7 µm), and the mobile phase temperature was set to
nitrogen. After that, 100 ml of distilled water was added to the 30°C. The solvent gradient was 75% A and 25% B in 15 min in the
mixture to form a ß-carotene-linoleic acid emulsion. In a different negative ionization mode. The mobile phase was composed of
test tube, 1.5 ml of prepared emulsion was pipetted to 20 µl of solvent A: acetonitrile (LC-MS grade) and solvent B: 2-propanol
sample (5 mg/ml). Methanol was used as a negative control and (LC-MS grade). A flow rate of 0.15 ml/min was used and 1 µl of
distilled water as a blank. Absorbances at 470 nm UV-Visible sample were injected. For phenolic analysis, liquid chromatography
spectrometer Shimadzu UV-1601 of the mixture were recorded prior mobile phase temperature was set to 35°C. The solvent gradient
864 Afr. J. Pharm. Pharmacol.

Table 1. Extraction yields of C. nutans in four different systems.

Sample extracts Weight of extracts (g) Percentage of extract (%)

n-Hexane 7.85±0.07 1.78
Dichloromethane 9.18±0.30 2.09
Ethyl acetate 4.36±0.10 0.99
Ethanol 9.30±0.24 2.11
Values are expressed as mean ± standard deviation (n=3).

was initiated with 75% A and 25% B for 8.5 min, and then increased by using four different solvent systems. The yields
to 100% B, held for 11.5 min and reinitialized to 75% A and 25% B obtained were extracted from total dry weight of 440 g C.
in 5 min at both ionization modes. The mobile phase was
nutans. Among the extracts, the highest and the lowest
composed of solution A: Water (LC-MS grade) + 0.1% formic acid
and solution B: Methanol (LC-MS grade) + 0.1 % formic acid. A flow yields were obtained by ethanol and ethyl acetate
rate of 0.15 ml/min was used and 1 µl of sample were injected. extracts, respectively. Total percentage of all extracts
Samples were filtered through a 0.22 µm Millipore filter, type GV obtained is 6.97% from the dry weight C. nutans. The
(Millipore, Bedford, MA) prior to UPLC injection. The mass raw data high yield in ethanol was probably due to the high
was analyzed by Masslynx MS Software version 4.1. Automated solubility of major components of C. nutans such as
quantification with MassLynx targeted quantitative analysis was
performed by QuanLynx (Waters Technologies). The identification
phenolic group in ethanol (Ismail et al., 2010).
of phenolic standard and fatty acid compounds in ethanol and ethyl
acetate extracts of C. nutans were obtained by comparing the
retention times to reference standards. The results are expressed Total phenolic content (TPC), total tannin content
as mg/100 g of extract. (TTC) and total flavonoid content (TFC) analysis

Cytotoxicity assay Phenolic, flavonoid and tannin compounds derived from

plants are small molecules which play important roles as
The two previously selected extracts of C. nutans were tested antioxidants. Phenolic compounds are widely distributed
further for its cytotoxicity activity. Extracts were test on MCF-7 in plants and possess ultraviolet protection, pigmentation,
human breast cancer estrogen positive and 3T3 normal fibroblast and disease resistance (Prior et al., 2005; Maestri et al.,
cells. All cells were maintained in RPMI medium supplemented with
10% fetus calf serum (FCS), 100 unit/ml penicillin and 0.1 mg/ml
2006). Harmful oxidation caused by free radicals in the
streptomycin. Cytotoxicity of the extracts were determined with MTT human body can be blocked by phenoxide ions that are
assay according to protocol by Mosmann (1983) using 3-[4,5- delocalized in these antioxidants, which scavenge the
dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide. Initially, cell free radicals and detoxify the organism (Chang et al.,
culture with the concentration of 1 × 105 cells/ml was prepared and 2001; Maestri et al., 2006). The results obtained from
plated (100 µl/well) onto 96-wellplates. The cell was incubated for TPC in the different extracts were summarized in Figure
24 h at 37°, 5% CO2. After incubation, the diluted ranges of
samples (1.56, 3.125, 6.25, 12.5, 25, 50, and 100 µg/ml) were
1. The highest TPC in C. nutans was quantified in ethanol
added to each well and incubated for another 24 h. Then MTT extracts (1850 mg GAE/100 g extract) whereas the
solution at 2 mg/ml was added to each well and left to stand for 1 h lowest content was measured in n-hexane extracts (60
before measured at 570 nm. The cytotoxicity result was expressed mg GAE/100 g extract) (p < 0.05). TTC (Figure 2) of C.
as IC50, defined as the concentration of sample that caused nutans extracts varied from 55.4 to 1737 mg GAE/100 g
inhibition of 50% cell growth.
extract. The highest tannin content was detected in
ethanol (1737 mg GAE/100 g extract) whereas the lowest
Statistical analysis content was determined in n-hexane (55.4 mg GAE/100 g
extract) (p < 0.05).
Values are expressed as mean ± SD of three replicates. Statistical TPC of C. nutans extracts exhibited good correlation to
analyses were performed by one-way ANOVA with P < 0.05. its TTC. Ethanol had the highest in TPC and TTC
followed by ethyl acetate, dichloromethane and n-hexane
extracts, respectively. These findings are in agreement
RESULTS AND DISCUSSION with the observations of Robards et al. (1999) which
observed that polar solvents such as ethanol are a good
Extraction yield choice for extracting phenolic compounds. Moreover, a
few plant species belong to same family of Acanthaceae
Table 1 presents the extraction yields of C. nutans leaves (Lepidagathis anobrya, Hygrophila auriculata and
 Sulaiman et al. 865

Figure 1. Total phenolic content (TPC) of C. nutans leaves from four different solvent systems. Error bars show
the variations of three determinations in terms of standard deviation (p<0.05).

Figure 2. Total tannin content (TTC) of C. nutans leaves from four different solvent systems.
Error bars show the variations of three determinations in terms of standard deviation (p<0.05).

Nelsonia canescens) had also been reported with higher Figure 3 summarized TFC of C. nutans from four different
of phenolic content (Sawadogo et al., 2006). Flavonoids solvents. In this study, TFC of C. nutans extracts was
are widely distributed in fruits and vegetables. The determined through a linear rutin standard curve: (y =
2
antioxidant capacities of flavonoids are related to their 15.32x – 0.0014; R = 0.9977). The values range from
degree of unsaturation and oxidation of the three-carbon 16.6 to 306 mg RE/100 g extract. The highest was
segment (Chang et al., 2001; Robards et al., 1999). recorded in ethanol (306 mg RE/100 g extract) followed
866 Afr. J. Pharm. Pharmacol.

Figure 3. Total flavonoid content (TFC) of C. nutans leaves from four different solvent systems.
Error bars show the variations of three determinations in terms of standard deviation (p<0.05).

by ethyl acetate (203 mg RE/100 g extract), n-hexane the absorbance of the reaction mixture. DPPH assay
(27.3 mg RE/100 g extract), and dichloromethane involving multiple mechanisms monitoring DPPH color
extracts (16.6 mg RE/100 g extract). Flavonoid content in loss can be attributed to either SET or HAT as well as
polar solvents correspond with work reported by Teshima unrelated reactions and steric accessibility. In this study,
et al. (1997) which isolated six C-glucosyl flavones DPPH activity for CN extracts was highest in the ethyl
(vitexin, isovitexin, shaftoside, isomollupentin-7-O-ß- acetate and dichloromethane extracts (semi polar
glucopyranoside, orientin and isoorientin) from methanol extracts) followed by ethanol and n-hexane extracts.
extracts of C. nutans stems and leaves. Solubility of extracts in different testing systems and
stereoselectivity of the radicals may contribute to different
antioxidant activities (Prior et al., 2005). A recent report
Antioxidant assays by Yong et al. (2013) also documented that a chloroform
extract (semi-polar) of C. nutans leaves exhibited highest
DPPH scavenging activity, ORAC and ß-carotene antioxidant activity in a DPPH assay.
bleaching inhibitory activity The ability of C. nutans extracts to inhibit the
discoloration of ß-carotene was spectrophotometrically
Antioxidant assays from DPPH, ORAC and ß-carotene measured by comparing the results to the standard
bleaching (BCB) assay can be based on two main reference of alpha-tocopherol equivalent (Teq) (y =
2
reactions: single electron transfer (SET) and hydrogen 0.9733x + 2.9587; R = 0.9801). The presence of
atom transfer (HAT). Antioxidants in the single reaction antioxidants from extracts may hinder ß-carotene
system may involves multiple mechanisms or different bleaching by neutralizing the linoleic free radical
single mechanisms that respond various ways to different (Perumal et al., 2012). In general, the antioxidant activity
radical sources (Prior et al., 2005). The results obtained of C. nutans extracts ranged from 120 to 720 mg Teq/100
for DPPH and BCB are shown in Figure 4. DPPH assay g extract. n-Hexane extract exhibited the highest
is a decoloration assay using stable organic nitrogen antioxidant activity (720 mg Teq/100 g extract) through
radical 1, 1-diphenyl-1-picrylhydrazyl (DPPH) that β–carotene bleaching (BCB) assay, whilst ethanol extract
changes color from purple to yellow. The assay is showed the least antioxidant activity (120 mg Teq/100 g
commonly used in antioxidant screenings (Sharma and extract) in the bleaching of ß-carotene. Although
Bhat, 2009). Its antioxidant activity is measured phenolic, flavonoid and tannin contents are lower in
spectrophotometrically through the ability of antioxidant nonpolar extracts (n-hexane), the oxidation of linoleic
compounds to reduce the DPPH radical by decreasing acid may cause by other secondary metabolites such as
 Sulaiman et al. 867

Figure 4. DPPH scavenging activity and BCB activity of C. nutans from

four different solvent systems. Error bars show the variations of three
determinations in terms of standard deviation (p<0.05).

Figure 5. ORAC antioxidant activity of C. nutans from four different solvent

systems. Error bars show the variations of three determinations in terms of
standard deviation (p<0.05).

carotenoids, vitamins and oils (Perumal et al., 2012, 114.3 to 229.5 mMol TE/100 g extract was observed
2013). (Figure 5). The ethanol extract of C. nutans had highest
ORAC assay based on HAT is a considerable antioxidant capacity (229.5 mMol TE/100 g extract)
antioxidant capacity assay that is the most appropriate to followed by ethyl acetate (181.6 mMol TE/100 g extract),
measure in vitro and in vivo action (Prior et al., 2005; dichloromethane (115.5 mMol TE/100 g extract) and n-
Mariod et al., 2010). In this study, a variation in hexane (114.3 mMol TE/100 g extract). The results
antioxidant capacity with ORAC assays ranging from showed that, TPC, TFC and TTC of C. nutans extracts
868 Afr. J. Pharm. Pharmacol.

Table 2. Quantitative analysis major phenolic and fatty acids compounds identified in ethanol and ethyl acetate
extracts of C. nutans.

Retention Sample extracts (mg/100 g extract)

Standard references
time (min) Ethanol Ethyl acetate
Alpha tocopherol 14.02 439.7±30.0 1166.0±81.1
Kaempherol 6.75 0.1±0.01 N.D
Sinapic acid 4.38 38.8±2.5 154.2±12.3
Vanillin 4.06 7.6±0.3 N.D
Quercetin 6.06 N.D 65.5±9.0
Rutin trihydrate 4.69 23.5±6.9 21.2±1.7
Syringic acid 3.68 24.7±0.7 24.1±2.2
Protocatechuic acid 1.38 56.0±9.0 66.0±11.2
4-Hydrophenylacetic acid 1.53 48.0±9.2 326.0±23.0
Gentisic acid 1.70 18.0±4.3 70.0±13.0
Cinnamic acid 2.88 18.0±2.7 84.4±12.7
Caffeic acid 1.53 N.D 21.2±0.6
4-Hydroxybenzoic acid 1.64 85.0±13.0 169.4±23.0
Coumalic acid 1.88 79.0±8.8 270.0±25.0
p-Coumaric acid 5.79 110.0±18.4 2.0±0.3
3,4-Dimethoxybenzoic acid 7.08 22.0±3.3 46.0±7.5
Catechin hydrate 2.06 N.D 50.0±6.9
Quercetin hydrate 9.09 54.0±6.8 202.0±21.0
Epicatechin gallate 5.18 N.D 16.0±2.7
Myricitrin 7.99 N.D 10.0±0.2
trans-Ferulic acid 6.52 70.0±13.0 14.0±0.3
Vanilic acid 3.68 118.0±17.0 28.0±3.2
Chlorogenic acid 2.51 52.0±9.1 N.D
Linoleic acid 2.75 65.0±10.0 510.0±17.3
Stearic acid 4.4 585.0±50.0 2535.0±40.0
Oleic acid 3.36 67.5±5.0 387.5±15.0
Palmitic acid 3.44 370.0±20.0 1297.5±25.0
Myristic acid 2.82 10.2±0.6 120.2±20.6
Values are ±SD (n=3), N.D: Not detected

correspond to their ORAC antioxidant capacity, which nutans. Phenolic are antioxidant compounds which act as
indicates that the main compounds that contribute to agents to neutralize harmful free radicals (ROS). Table 2
antioxidant activity may come from these groups. The summarizes the analysis of phenolic and fatty acid
relationship between Folin-ciocalteu and ORAC are in quantitative analysis of ethanol and ethyl acetate extracts
good agreement with what was obtained by Prior et al. of C. nutans through LC-ESI/MS. Saturated,
(2005) and Bedawey et al. (2010). Since one antioxidant monounsaturated and polyunsaturated fatty acids are
capacity assay would not be comprehensive, various good for the health (Piorkowski and McClements, 2014).
methods are required to measure total antioxidant Long chain omega-3 or -6 polyunsaturated fatty acids
capacity and none of them are an ideal reference method (PUFAs) can lower the production of reactive oxygen
(Erel, 2004; Prior et al., 2005). species (ROS), thus decreasing the risk of various
diseases and improves. The effect of omega-3 long chain
LC-MS analysis of phenolic and fatty acid PUFAs on ROS is stronger than saturates,
compounds monounsaturates and polyunsaturates of the omega-6
series. These two types of fatty acids, also known as
Several phenolic and fatty acid compounds were essential fatty acids (EFA) are important in human diet
identified in ethanol and ethyl acetate extracts of C. because they cannot be produced by the body and must
 Sulaiman et al. 869

1 2 3

4 5 6

7 8 9

10

Figure 6. Structure of major phenolic and fatty acid compounds identified in ethyl acetate and ethanol extracts of C. nutans.

be obtained from food (Richard et al., 2008; Simopoulos, ethanol extract discovered to have intense of p-coumaric
1991). acid (7) (110 mg/100 g) and vanilic acid (8) (118.0
Among of 23 tested phenolic and fatty acid compounds mg/100 g).
(Figure 6), alpha tocopherol (1) was the most abundant Mustapa et al. (2015) have reported that polar extract
compound in both extracts, 439.7 mg/100 g extract of C. nutans with microwave-assisted extraction (MAE)
(ethanol) and 1166.0 mg/100 g extract (ethyl acetate). and soxhlet extraction have discovered to have plenty of
Ethyl acetate extract was quantified to have high content phytol compound, compared to supercritical fluid
of sinapic acid (2) (154.2 mg/100 g), 4-hydrophenylacetic extraction (SFE) which have major of palmitic acid.
acid (3) (326.0 mg/100 g), 4-hydroxybenzoic acid (4) Palmitic acid is one of the fatty acid quantified in the
(169.4 mg/100 g), coumalic acid (5) (270 mg/100 g) and present study in both ethyl acetate and ethanol extracts.
quercetin hydrate (6) (202.0 mg/100 g). Meanwhile, The fatty acid profile of both extract contain of linoleic
870 Afr. J. Pharm. Pharmacol.

Extract concentration (µg/ml)

Figure 7. Toxicity effect of ethyl acetate extracts of C. nutans at various concentrations (3.125 to 100
µg/ml) on tumorigenic cell MCF-7. Data represented in percentage (%) of cell viability as mean ±
standard deviation (n=3) (p< 0.05).

acid (polyunsaturated), stearic acid, oleic acid (NCI), a crude extract may be considered as active for an
(monounsaturated), palmitic acid and myristic acid. Both IC50 <30 µg/ml. Both tested extracts demonstrated
extracts discovered to have an abundance of stearic acid cytotoxic on the breast cancer estrogen positive (MCF-7)
(9) that is, 585.0 mg/100 g extract and 2535.0 mg/100 g cell. As shown in Figures 7 and 8, the IC50 value for ethyl
extract respectively in ethanol and ethyl acetate extracts. acetate extracts was 24.04 ± 1.7 µg/ml, whereas ethanol
Long chain polyunsaturated fatty acid (LC-PUFAs) extract of CN was 28.90 ± 2.1 µg/ml (Figure 8). This
linoleic acid (10) was detected in both extracts; 65.0 mg/g finding is in agreement with Yong et al. (2013) suggested
extract and 510.0 mg/g extract ethanol and ethyl acetate that semi polar extract (chloroform) of C. nutans
extracts, respectively. Linoleic acid is an omega-6 fatty demonstrated higher antiproliferative activity than polar
acid, required for proper skin function. Increasing levels extract (methanol). Quantitative evaluation in present
of EFAs (omega-3 and omega-6) can increase cell study revealed that certain targeted phenolics and fatty
membrane fluidity, enhance barrier function and repair, acids (alpha tocopherol, quercetin, 4-hydrophenylacetic
decrease trans-epidermal water loss, moisturize, improve acid, coumalic acid, linoleic acid, stearic acid, and
cell immunity, and acts as an anti-inflammatory. In palmitic acid) found to be abundant in ethyl acetate
pharmaceutical applications, EFAs can enhance the compare to ethanol extract. In agreement with Ramadan
absorption of bioactive and can also be used as carrier et al. (2003) antioxidant activity also was affected by the
oils (Prottey et al., 1975; Simopoulos, 1991). level of PUFA content. This may be explained by the
cytotoxicity activity of ethyl acetate extract which is
stronger than ethanol extract on tumorigenic cell MCF-7.
Cytotoxicity assay Furthermore, squalene and several long fatty acids have
been reported in this plant previously (Mustapa et al.,
According to the standards of National Cancer Institute 2015). Squalene compound, which is good in antioxidant
 Sulaiman et al. 871

Extract concentration (µg/ml)

Figure 8. Toxicity effect of ethanol extracts of C. nutans at various concentrations (3.125 to 100
µg/ml) on tumorigenic cell MCF-7. Data represented in percentage (%) of cell viability as mean ±
standard deviation (n=3) (p< 0.05).

activity, also claimed to have anti-cancer, anti-tumor and Conclusion

chemo-preventive properties (Reddy and Couvreur,
2009; Ezhilan and Neelamegam, 2012). Ethanol and ethyl acetate extracts of C. nutans exhibited
Highest antioxidant activity of ethyl acetate and ethanol the highest antioxidant activity amongst all the C. nutans
extracts in present study may significantly contribute to its extracts. Higher content of phenolic in ethyl acetate and
cytotoxic effect. This is because extract with antioxidant ethanol extracts than other extracts have contributed to
can act by scavenging reactive oxygen species (ROS) in its stronger antioxidant activity and cytotoxicity against
human body. ROS are highly reactive and can be tumor cell. In addition, antioxidant activity of extract is
categorized into two groups: free oxygen radicals and also affected by higher level of PUFA. Phenolic, flavonoid
non-radical. ROS participate in the two stage of and tannin are very soluble in the high polarity solvents.
carcinogenesis, inducing cancer and carcinogens. Further studies are recommended to be conducted on
−
Among ROS, superoxide (O2• ), hydrogen peroxide potential used of these extracts as anticancer developed
(H2O2) and hydroxyl radicals (•OH) are the most found in and pharmaceutical applications.
cancer (Feng et al., 2015). Therefore by reducing the
level of the ROS can effectively prevent and suppress the
occurrence of cancer. Moreover both extract showed not ACKNOWLEDGMENT
active against normal fibroblasts cell 3T3 with IC50 values
53.06±3.8 µg/ml (Figure 9) and 73.84 ± 5.4 µg/ml (Figure This project was funded by Universiti Putra Malaysia
10) for ethyl acetate and ethanol extracts, respectively. (Vote. No GP-IPS/2014/9438735). The authors would like
Thus, both extract showed a good selectivity effect on to acknowledge Mrs. Azney Zuhaily Md Taib from
cancer cell (MCF-7) and can potentially be used as a Malaysia Genome Institute and Mrs. Nooraini Mohd. Ain
good source of anticancer and antioxidant. from Laboratory of Cancer Research UPM-MAKNA for
872 Afr. J. Pharm. Pharmacol.

Extract concentration (µg/ml)

Figure 9. Toxicity effect of ethyl acetate extracts of C. nutans at various
concentrations (3.125 to 100 µg/ml) on normal fibroblasts cell 3T3. Data represented
in percentage (%) of cell viability as mean ± standard deviation (n=3) (p< 0.05).

Figure 10. Toxicity effect of ethanol extracts of CN at various concentrations (3.125-

100 µg/mL) on normal fibroblasts cell 3T3. Data represented in percentage (%) of cell
viability as mean ± standard deviation (n=3). P< 0.05.
 Sulaiman et al. 873

providing technical supports as well as Ministry of Higher protein concentrates from defatted kenaf seed. Food Chem. 123:747-
752.
Education, Malaysia for providing the financial assist Mosmann T (1983). Rapid colorimetric assay for cellular growth and
under MyBrain15 Postgraduate Scholarship Programme. survival: application to proliferation and cytotoxicity assays. J.
Immunol. Methods 65:55-63.
Mustapa AN, Martin Á, Mato RB, Cocero MJ (2015). Extraction of
phytocompounds from the medicinal plant Clinacanthus nutans
Conflicts of interest
Lindau by microwave-assisted extraction and supercritical carbon
dioxide extraction. Ind. Crops Prod. 74:83-94.
Authors have none to declare. Negi PS (2012). Plant extracts for the control of bacterial growth:
efficacy, stability and safety issues for food application. Int. J. Food
Microbiol. 156:7-17.
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