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Journal of Traditional and Complementary Medicine 7 (2017) 452e465

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Journal of Traditional and Complementary Medicine

journal homepage: http://www.elsevier.com/locate/jtcme

Original Article

Comparative study of herbal plants on the phenolic and flavonoid

content, antioxidant activities and toxicity on cells and zebrafish
embryo
Hassan Fahmi Ismail a, Zanariah Hashim a, *, Wong Tet Soon a, Nur Syukriah Ab Rahman b,
Ain Nabihah Zainudin b, Fadzilah Adibah Abdul Majid b
a
Department of Bioprocess and Polymer Engineering, Faculty of Chemical & Energy Engineering, Universiti Teknologi Malaysia, 81310, Malaysia
b
Institute of Marine Biotechnology, Universiti Malaysia Terengganu, 21030, Malaysia

a r t i c l e i n f o a b s t r a c t

Article history: Natural antioxidants derived from plants have shown a tremendous inhibitory effect on free radicals in
Received 15 November 2016 actively metabolizing cells. Overproduction of free radicals increases the risk factor of chronic diseases
Received in revised form associated with diabetes, cancer, arthritis and cardiovascular disease. Andrographis paniculata, Cinnamon
26 December 2016
zeylanicum, Curcuma xanthorrhiza, Eugenia polyantha and Orthosiphon stamineus are ethnomedicinal
Accepted 27 December 2016
plants used in the Asian region to treat various illnesses from a common fever to metabolic disease. In
Available online 16 January 2017
this study, we have quantified the total phenolic (TPC) and flavonoid content (TFC) in these plants and its
inhibitory effect on 1,1-diphenyl-2-picrylhydrazyl radical (DPPH) and 2,20 -azinobis(3-
Keywords:
Herbal
ethylbenzothiazoline-6-sulfonic acid) (ABTS) free radicals as well as the cytotoxicity effect on cell lines
Cytotoxicity proliferation and zebrafish embryogenesis. Results showed that Cinnamon zeylanicum and E. polyantha
Embryotoxicity have the highest phenolic and flavonoid content. Furthermore, both herbs significantly inhibited the
Total phenolic content formation of DPPH and ABTS free radicals. Meanwhile, O. stamineus exhibited minimum cytotoxicity and
Antioxidant embryotoxicity on tested models. Good correlation between IC50 of 3T3-L1 cells and LC50 embyrotox-
icity was also found. This study revealed the potent activity of antioxidant against free radical and the
toxicology levels of the tested herbal plants.
© 2017 Center for Food and Biomolecules, National Taiwan University. Production and hosting by Elsevier
Taiwan LLC. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/
licenses/by-nc-nd/4.0/).

1. Introduction antioxidants exhibit tremendous protective responses against ROS

through free radicals scavenging, metals chelating, quenching of
Reactive oxygen species (ROS); superoxide anion (O 2 ), hydroxyl single and breaking the autoxidative chain reaction and restore the
radical (OH) and hydrogen peroxide (H2O2) are byproduct mole- ‘redox homeostasis’ state to its original level.4e9 Evidences showed
cules generated during aerobic cell metabolism. The presence of that natural antioxidants deliver better effectiveness as compared
these molecules is crucial as they modulate diverse physiological to synthetic antioxidants. A study of vitamin E on human plasma
signaling and homeostasis. In turn, overproduction of ROS results in and tissues revealed that naturally derived vitamin E gives twice
oxidative stress and contributes to the development of cancer, higher absorption rate as compared to synthetic vitamin E.10 In
diabetes, aging, inflammation, cardiovascular disease and neuro- addition, dietary supplement from fruits, vegetables, cereals, bev-
degenerative diseases.1e3 Synthetic and naturally derived erages, spices and herbs not only offer targeted beneficiary anti-
oxidant compounds, but a whole range of antioxidants, vitamins
and phytochemicals. Andrographis paniculata (AP), Cinnamon zey-
* Corresponding author. lanicum (CZ), Curcuma xanthorrhiza (CX), Eugenia polyantha (EP)
E-mail addresses: [email protected] (H.F. Ismail), and Orthosiphon stamineus (OS) are commonly used in daily culi-
[email protected] (Z. Hashim), [email protected] (W.T. Soon), nary as well as traditional medicinal preparations in South Asian
[email protected] (N.S.A. Rahman), [email protected]
(A.N. Zainudin), [email protected] (F.A.A. Majid).
region. Rich with aromatic flavors, pharmacological studies
Peer review under responsibility of The Center for Food and Biomolecules, discover that these herbs contained a variety of potent phyto-
National Taiwan University. chemicals that help to treat various illnesses.

http://dx.doi.org/10.1016/j.jtcme.2016.12.006
2225-4110/© 2017 Center for Food and Biomolecules, National Taiwan University. Production and hosting by Elsevier Taiwan LLC. This is an open access article under the CC
BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
 H.F. Ismail et al. / Journal of Traditional and Complementary Medicine 7 (2017) 452e465 453

Even though most herbs and spices are generally recognized as stamineus (OS) (whole plant) were purchased from NatureMedic
safe, but adverse events sometimes occur after consuming herbal Supply (Terengganu, Malaysia) and taxonomically confirmed by
products. In 2013, statistical evaluation by Malaysian Adverse Drug Prof Dr. Fadzilah Adibah Abdul Majid. All specimens were deposited
Reaction Advisory Committee (MADRAC), National Pharmaceutical at Tissue Culture Engineering Research Group, Universiti Teknologi
Control Bureau and Ministry of Health showed that 11,473 cases of Malaysia under vouchers no AP-TCERG-2013, CZ-TCERG-2013, CX-
adverse drug reactions were reported and 0.2% were caused by TCERG-2013, EP-TCERG-2013 and OS-TCERG-2013. Raw materials
herbal products.11 In most countries, safety and toxicology evalu- were ground into coarse form and extracted with filtered water by
ation are not a mandatory requirement for herbal based product the ratio 1.5: 10 for 3 h at 60  C. Spray drying were carried out in
registration. In addition, lack of policies focusing on herbal product Institute of Bioproduct Development (IBD) (Johor, Malaysia). Ex-
production contributes to low quality, ineffective and potentially tracts were stored at 4  C until future purposes.
hazardous consumption. ‘Natural’ term has misled the consumers'
perceptions especially on the possible adverse effects that might 2.3. Biomarker fingerprinting and chromatography technique
arise from inappropriate usage of herbal medications. Adverse ef-
fect events may cause from several factors including the side effects 2.3.1. FTeIR detection
of active compounds, contamination or substitution with toxic Samples were subjected to IR spectroscopic study using Perkin
herbs, heavy metal contamination and herbs-drugs interactions.12 Elmer FTeIR spectrophotometer by employing standard potassium
These factors responsible for causing liver, kidney and lung fail- bromide (KBr) pellet technique. Samples were ground with KBr in
ure, high blood pressure, heart attack and stroke have been the ration of 1:100. Mixture was placed in the mold and pressed.
reported.12e16 The pellet was scanned over the wavelength ranged from
Current safety and toxicology testing for preclinical studies 4000e370 cm1. FTIR spectrums were expressed as percent
comprise of in vitro and in vivo models. Typically, these tests are transmission (%T).
time consuming, expensive and required a large number of animals.
Potentially, zebrafish (Danio rerio), an emerging model in the early 2.3.2. GSeMS analysis
drug discovery and toxicological screening offers several advan- Extracts were analyzed by gas chromatographyemass spec-
tages on physiological, biological and molecular alteration.17e19 Its trometry (GCeMS) (Shimadzu QP2010 Ultra) equipped with a
low maintenance, high fertility, ex utero development and trans- BP5MS capillary column (30 m length, 0.25 mm in diameter,
parent eggs offer clear visualization in all stages of organogenesis 0.25 mm film thickness). Analyses were carried out using a pro-
monitoring. Fish embryo acute toxicity test (FET) is designed by the grammed temperature from 50  C to 300  C and helium as a carrier
OECD as a guideline to evaluate the embryotoxicity effects of gas. Compound identification was established based on the com-
certain compounds on the early 96 h of developmental stages of parison of the GC retention factors with standards and the com-
embryos.20 The use of zebrafish is not only limited to toxicity parison of the mass spectra with the Wiley 138 library data of the
screening. Over the past years, scientists have developed transgenic GCeMS system.
zebrafish through gene alteration and targeted mutation for spe-
cific diseases such as cardiovascular, neurogenesis, digestive sys- 2.3.3. Selected biomarkers identification and quantification
tem, muscles, cancer, immunology, diabetes and One hundred mg of AP, CX, CZ, EP and OS were dissolved in
inflammation.21e26 25 ml of methanol, vortexed for 5 min to ensure dissolution and
In the present study, the water extracts of AP, CX, CZ, EP and OS filtered through a 0.45 mm nylon filter. Standard stock solutions of
were examined for its total phenolic and flavonoid content as well biomarkers were prepared by dissolving 1 mg of biomarkers in 1 ml
as their antioxidant inhibitory effect on DPPH and ABTS radicals. of methanol and sonicated for 5 min. Quantification of androgra-
Furthermore, the toxicity effects of these herbs were evaluated pholide, curcumin, catechin, gallic acid and rosmarinic acid were
using 3T3-L1, 1.1B4 and WRL-68 cell lines and compared with determined by HPLC fingerprinting detection; Waters 2690 Alli-
zebrafish embryotoxicity assay. ance Separation Module with LiChrospher® 100 RP-18 endcapped,
Merck column cartridge (250  4.6 mm, 5 mm). The chromatograms
2. Materials and methods were monitored at wavelengths 340 nm (rosmarinic acid), 272 nm
(gallic acid), 223 nm (andrographolide), 280 nm (cathechin) and
2.1. Materials 420 nm (curcumin).

0.2 N Follin-Ciocaltue reagent, sodium carbonate (Na2CO3), 2.4. Total phenolic content
gallic acid, sodium nitrite (NaNO2), aluminum chloride (AlCl3), so-
dium hydroxide (NaOH), 2,2-diphenyl-1-picrylhydrazyl (DPPH), Quantification of total phenolic content (TPC) was carried out by
potassium persulphate (K2O8S2), were purchased from Sigma FolineCiocalteu method27 with slight modifications. Briefly, 100 ml
Aldrich (St. Louis, MO, USA). Ascorbic acid and methanol was pur- of extract (1 mg/ml diluted in distilled water) was mixed with
chased from Qre €c™ (Selangor, Malaysia). Mouse 3T3-L1 pre- 100 ml of 0.2 N FolineCiocalteu reagents. After 5 min, 80 ml of 7.5%
adipocyte and normal liver cells WRL-68 were purchased from sodium carbonate (Na2CO3) solution was added and incubated for
American Type Culture Collection, ATCC, Manassas, USA and 2 h at RT. The absorbance was measured at 750 nm against the
pancreatic beta cells 1.1B4 from Public Health England, Salisbury, blank. The calibration curve was prepared using the standard gallic
UK. DMEM, RPMI, fetal bovine serum (FBS), fetal calf serum (FCS) acid solution. The total phenolic content was expressed in mg of
and penicillin strep (PS) were purchased from Gibco, Life Tech- Gallic acid equivalent (GAE/100 g of sample).
nologies (Rockville, MD, USA). Adult, wild type, zebrafish were
obtained from commercial supplier, Jurassic Fish, Johor, Malaysia. 2.5. Total flavonoid content

2.2. Herbs and extraction Total flavonoid content (TFC) was determined using aluminum
chloride colorimetric method28 with slight modifications. Briefly,
A. paniculata (AP) (leaves), Cinnamon zeylanicum (CZ) (bark), C. 200 ml of extract was mixed with 12 ml NaNO2 and 12 ml AlCl3. After
xanthorrhiza (CX) (rhizome), E. polyantha (EP) (leaves) and O. 5 min incubation at RT, 80 ml of NaOH was added and re-incubated
454 H.F. Ismail et al. / Journal of Traditional and Complementary Medicine 7 (2017) 452e465

for 30 min. The absorbance was read at 510 nm. The calibration analyzed at 570 nm. Samples were run in 6 replicates. Untreated
curve was prepared using standard catechin solution. The total cells were used as a control.
flavonoid content was expressed in mg of g catechin equivalent (CE/
100 g of sample). 2.7.3. Animal test
The study design was approved by the Universiti Kebangsaan
2.6. Antioxidant assay Malaysia Animal Ethics Committee (UKMAEC) (Approval Number:
UTM/FKK/2013/Fadzilah/15-May/506-June-2013-June-2016).
2.6.1. DPPH radical scavenging activity assay Maintenance and breeding procedures were followed the standard
The DPPH radical scavenging activity was determined according OECD guidelines.20 Fish were maintained in 1 ft  1 ft aquaria with
to Guilong Yan protocol with modifications.29 Briefly, 0.2 mM of 12 h light:12 h dark cycle at ±28  C and fed twice daily with dry
DPPH-methanol solution was added to equal volume of extract flake food.
dissolved in methanol at various concentrations (5 mge500 mg/ml).
The mixture was vortexed vigorously and incubated for 30 min at 2.7.4. Egg production, collection
RT. The absorbance was spectrophotometrically measured at Embryos were obtained from spawning zebrafish adults in
517 nm. Ascorbic acid was chosen as positive control. The scav- breeding tanks with sex ratio 3 male:5 female. Spawning was
enging activity was calculated using the following equation: induced in the morning and after 2 h, embryos were collected and
washed using embryo medium. Fertilized and dead embryos were
Ac  As separated according to the descriptions of Kimmel.
DPPH scavenging effectð%Þ ¼  100
Ac
2.7.5. Embryo short-term toxicity test (FET)
where Ac is the absorbance of reaction without samples and As is Toxicity test was referred to the OECD guideline; No. 236: Fish
the absorbance of tested extract. The antioxidant activity was Embryo Acute Toxicity (FET) Test.20 Fertilized embryos were
expressed as median effective concentration (EC50) where the transferred into 24 well plates (30 embryos/well) containing a se-
concentration caused 50% reduction of DPPH. ries of diluted extract ranging from 0.005 mg/ml to 10 mg/ml.
Exposure involved semi-static condition, ±28  C and 12 dark: 12
2.6.2. ABTS radical scavenging activity light cycle period. Plates were sealed with parafilm to minimize
ABTS inhibition was measured based on Iqbal method with evaporation. Embryo development was monitored 24 h interval for
modifications.30 ABTS stock solution was dissolved in water to a 96 h. Four morphological characteristics were evaluated including
7 mM concentration. ABTS radical cation was produced by reacting coagulation of eggs, tail detachment, present of heart beat and
ABTS stock solution with 2.45 mM potassium persulfate and the hatching rates using Carl-Zeiss Inverted Microscope (F51) equipped
mixture was allowed to stand in dark at room temperature for with phase contrast function, camera and software for image
12e16 h. Prior to use, ABTS radical cation was diluted in methanol optimization. Experiments were conducted in triplicates.
until the absorbance at 0.7 ± 0.05. ABTS solution (300 ml) was added
to 30 ml sample and incubated for 6 min at RT. The absorbance was 2.7.6. Calculation of IC50, LC50
read at 750 nm. Ascorbic acid was chosen as positive control. The Median inhibitory concentrations (IC50) and median lethal
radical-scavenging activity was expressed as inhibition percentage, concentration (LC50) were statistically analyzed by GraphPad Prism
and calculated using the following formula. Version 6 software. Non-linear regressions of log (inhibitor) vs.
response with four parameters was selected for IC50 and LC50
Ac  As estimation. The bottom and top constrain were 0% and 100%
ABTS Radical scavenging activity ¼  100
Ac respectively.

where Ac is the absorbance of reaction without samples and As is

2.8. Statistical analysis
the absorbance of tested extract. The antioxidant activity was
expressed as median effective concentration (EC50) where the
All data were expressed in mean ± SEM. Statistical analysis was
concentration caused 50% reduction of ABTS.
performed using SPSS program with one way ANOVA and Tukey
test. Significant difference was considered as p < 0.05.
2.7. Cytotoxicity assay and embryotoxicity assay
3. Results
2.7.1. Cell culture
3T3-L1, 1.1B4 and WRL-68 cells were cultured according to the 3.1. Functional group and biomarker fingerprinting
protocol provided by the depositor. 3T3-L1 and WRL-68 were
grown in DMEM supplemented with 10% FCS and 10% FBS respec- 3.1.1. FTIR spectra analysis
tively with additional 1% PS at 37  C under a humidified atmo- Characterization of active components based on their respective
sphere of 5% CO2. 1.1B4 was cultured in RPMI supplemented with functional groups can be predicted by Fourier transform infrared
10% FBS and 1% PS at 37  C under a humidified atmosphere of 5% spectroscopy (FTIR). Fig. 1 demonstrated the spectra peak stretched
CO2. from 4000 to 370 cm1 for AP, CX, CZ, EP and OS. Similar trends
were observed in all extracts suggesting the existence of alkanes,
2.7.2. MTT cytotoxicity assay alkenes, aromatic rings, hydroxyl compounds, carbonyl com-
Cells were seeded in 96 well plate and incubated overnight. pounds, amino acid, aldehydes, amines, nitriles, amides and ethers
Samples treatment was carried out by replacing spent medium groups as listed in Table 1.
with various concentrations (0.005 mg/ml to 10 mg/ml) of extract In the alkane vibration range, all extracts displayed the C-H
diluted in medium. After 24 h of incubation, 20 ml of 3-(4,5- stretching (3000e2800 cm1) vibration, but only CZ and EP con-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) so- tained both C-H stretching and bending (1500e1440 cm1) vibra-
lution was added to each well and incubated for 4 h. Developed tion. For the alkene group, no CeH stretch (3200e3000 cm1) was
MTT formazon was dissolved in DMSO. Color development was detected, instead the C]C (1680e1600 cm1) stretch were present
 H.F. Ismail et al. / Journal of Traditional and Complementary Medicine 7 (2017) 452e465 455

Fig. 1. FTIR spectra (4000 cm1 to 370 cm1) of Andrographis paniculata (AP), Cinnamon zeylanicum (CZ), Curcuma xanthorrhiza (CX), Eugenia polyantha (EP) and Orthosiphon
stamineus (OS) water extracts. Similar transmission trends were detected in all extracts.

Table 1 identified in all extracts suggesting the amino group exists in every
List of chemical functional groups present in AP, CX, CZ, EP and OS.
extracts. Meanwhile, the C^N stretch (2500e2000 cm1) was
Functional group Vibration Range AP CX CZ EP OS detected in OS, AP, CX, and CZ. The presence of amide functional
(Stretch/Bend) groups (NeH Stretch, C]O Stretch, NeH Bend) was also detected in
Alkanes CeH stretch 3000e2800 ✓ ✓ ✓ ✓ ✓ all extracts. The presence of ether functional group
CeH bend 1500e1440 e e ✓ ✓ e (1040e1260 cm1) was also detected in OS, AP, and CX.
Alkenes CeH stretch 3200e3000 e e e e e
C]C stretch 1680e1600 ✓ ✓ ✓ ✓ ✓
Aromatic rings (sp2) ¼ CeH 3200e3000 e e e e e 3.1.2. GCeMS detection
stretch
Extracts were further analyzed by GC-MS to identify the pres-
C]C stretch 1600e1400 ✓ ✓ ✓
Hydroxyl OeH stretch 3600e3200 ✓ ✓ ✓ ✓ ✓ ence of volatile compounds. Table 2 summarizes the five major
compounds compounds detected in each extract. These compounds have
Carbonyl C]O stretch 1610e1550 ✓ e e e e shown beneficial pharmacological activities as anti-viral, anti-in-
compounds flammatory, antioxidant, anticancer, antimicrobial, anti-fungal,
Amino acid C]O stretch 1600e1660 ✓ ✓ ✓ ✓ ✓
antibacterial, anti-acne, anti-hyperglycemic, anti-hypertensive,
Aldehydes C]O stretch 1750e1625 e ✓ e e ✓
CeH stretch 2700e2850 e e e e e antiulcer, anti-arthritis, anti-coagulant, anti-tubercular, anti-
of C]O convulsant, anti-obesity, anti-tumor, anti-HIV agents, CNS-active
Amines NeH stretch 3500e3100 ✓ ✓ ✓ ✓ ✓ compounds, nephroprotective and hepatoprotective activities.
NeH Bend 1640e1550 ✓ ✓ ✓ ✓ ✓
Octadecanoic acid methyl ester detected in all extracts is acting as
Nitriles C^N stretch 2500e2000 ✓ ✓ ✓ ✓
an anti-viral and anti-microbial properties against measles virus.31
e
Amides NeH stretch 3500e3100 ✓ ✓ ✓ ✓ ✓
C]O stretch 1670e1600 ✓ ✓ ✓ ✓ ✓ Meanwhile coumarin identified in CX and OS is an anti-
NeH bend 1640e1550 ✓ ✓ ✓ ✓ ✓ inflammatory, anti-coagulant, antibacterial, anti-fungal, anti-viral,
Ethers CeO stretch 1040 & 1260 ✓ ✓ e e ✓ anti-hypertensive, anti-tubercular, anti-convulsant, anti-obesity,
anti-hyperglycemic, antioxidant, and neuroprotective, anti-tumor,
anti-HIV agents, CNS-active compounds.32e34 Other important
in all extracts. In the aromatic ring region, the existence of compounds were xanthorhixol and cinnamadehyde detected in CX
(sp2) ¼ CeH stretches were not detected. However, most extracts and CZ respectively. Both compounds showed multiple biological
have C]C stretch in the region 1600e1400 cm1 with exception to effects including as antioxidant, anti-microbial, anti-inflammatory,
OS and AP. The absorbance was quite strong in the region anti-arthritis, anti-cancer, anti-hyperglycemic, nephroprotective
3600e3200 cm1 and all extracts exhibit the behavior of OeH and hepatoprotective agents.35,36
group.
The existence of carbonyl compound (1610e1550 cm1) was 3.1.3. Identification and quantification of active biomarkers by
detected only in AP which signifying the existence of ketone group. HLPC
In amino acid C]O region (1600e1660 cm1), all extracts exhibited Five biomarkers were selected for HPLC fingerprinting including
similar vibrations trends, confirming the existence of this func- andrographolide, curcumin, catechin, gallic acid and rosmarinic
tional group. Absorbance in the aldehyde range, C]O acid as previously detected in AP, CX, CZ, EP and OS
(1750e1625 cm1) stretch was detectable in CX and OS. However, respectively.37e41 Quantification of biomarkers (Table 3) showed
the C-H stretch (2700e2850 cm1) for the aldehyde functional that 17.78 mg/mg of andrographolide was detected in AP, 4.65 mg/
group was not detected at all. The absorbance of NeH bend mg of curcumin in CX, 4.29 mg/mg of catechin in CZ, 12.08 mg/mg of
(3500e3100 cm1) and stretches (1640e1550 cm1) were gallic acid in EP and 6.39 mg/mg of rosmarinic acid in OS.
456 H.F. Ismail et al. / Journal of Traditional and Complementary Medicine 7 (2017) 452e465

Table 2
List of five major compounds, chromatogram percentage area, height, retention time and biological activity of AP, CX, CZ, EP and OS detected by GCeMS.

Herb Compound Area% Height% Retention Biological Activity

time

AP Octadecanoic acid methyl ester 22.27 27.95 36.809 Anti-viral, anti-microbial31

Other names: Stearic acid methyl ester, Methyl stearate
Mol. Formula: C18H38O2
1,2-Cyclopentanedione 8.50 12.79 6.863 NA
Other names: -
Mol. Formula: C5H6O2
Octadecanoic acid, 6.82 4.90 46.755 Co-solvents, oil carrier, antioxidant,
2,3-dihydroxypropyl ester anti-acne66
Other names: -
Mol. Formula: C21H42O4
n-Undecane 6.47 4.70 11.827 NA
Other names: Hendecane
Mol. Formula: C11H24
Tetraacetyl-d-xylonic nitrile 3.97 1.98 25.861 Antioxidant, anti-cancer67
Other names: (2,3,4-triacetyloxy-5-cyano-5-oxopentyl)
acetate
Mol. Formula: C14H17NO9
CX Octadecanoic acid, methyl ester 19.56 20.78 36.798 Anti-viral, anti-microbial31
Other names: Stearic acid methyl ester, Methyl stearate
Mol. Formula: C18H38O2
Xanthorrhizol 18.12 19.29 29.201 Antioxidant, anti-microbial, anti-inflammatory,
Other names: 2-methyl-5-[(2r)-6-methylhept-5-en-2-yl]phenol anti-hyperglycemic, nephroprotective and
Mol. Formula: C15H22O hepatoprotective35
Benzene, 1-(1,5-dimethyl-4-hexenyl)-4-methyl- 6.43 7.37 22.690 Antioxidant, anti-viral, anti-ulcer68
Other names: Curcumene, Ar-Curcumene,
2-Methyl-6-p-tolyl-2-heptene, Alpha-Curcumene
Mol. Formula: C15H22
4-vinylquaiacol 4.62 5.08 18.069 Antioxidant, anti-inflammatory69
Other names: 2-Methoxy-4-vinylphenol
Mol. Formula: C9H10O2
beta.-Elemenone 4.39 4.51 25.716 NA
Other names: e
Mol. Formula: C15H22O
CZ Coumarin 21.14 21.93 21.505 Anti-inflammatory, anti-coagulant, antibacterial,
Other names: 2H-Chromen-2-one anti-fungal, anti-viral, e anti-hypertensive,
Mol. Formula: C9H6O2 anti-tubercular, anti-convulsant, anti-obesity,
anti-hyperglycemic, anti-oxidant, and
neuroprotective, anti-tumor, anti-HIV agents,
CNS-active
compounds32,33,50,51
Octadecanoic acid, methyl ester 14.92 17.64 36.797 Anti-viral, anti-microbial31
Other names: Stearic acid methyl ester, Methyl stearate
Mol. Formula: C18H38O2
n-Undecane 5.16 3.35 11.824 NA
Other names: Hendecane
Mol. Formula: C11H24
Cinnamaldehyde 4.20 4.64 16.889 Anti-microbial, anti-inflammatory, anti-arthritis,
Other names: Trans-Cinnamaldehyde anti-cancer, anti-oxidant, anti-hyperglycemic,
Mol. Formula: C9H8O anti-obesity, neuroprotective,43,52
Formic acid, 2-propenyl ester 3.26 2.46 8.471 NA
Other names: Formic acid, allyl ester, Allyl formate
Mol. Formula: C4H6O2
EP Octadecanoic acid, methyl ester 22.09 15.67 36.734 An-tiviral, anti-microbial31
Other names: Stearic acid methyl ester, Methyl stearate
Mol. Formula: C18H38O2
Coumarin 20.56 14.56 21.515 Anti-inflammatory, anti-coagulant, antibacterial,
Other names: 2H-Chromen-2-one anti-fungal, anti-viral, e anti-hypertensive,
Mol. Formula: C9H6O2 anti-tubercular, anti-convulsant, anti-obesity,
anti-hyperglycemic, anti-oxidant, and
neuroprotective, anti-tumor, anti-HIV agents,
CNS-active compounds32,33,50,51
Octadecanoic acid, 2,3-dihydroxypropyl ester 7.23 6.43 46.697 Co-solvents, oil carrier, antioxidant, anti-acne66
Other names: -
Mol. Formula: C21H42O4
Eugenol 3.87 3.21 4.562 Anti-cancer, antioxidant, anti-mutagenic,
Other names: 2-Methoxy-4-(2-propenyl)phenol, Eugenic acid, anti-genotoxic, anti-inflammatory6,53
Mol. Formula: C10H12O2
n-Undecane 3.55 2.45 11.854 NA
Other names: Hendecane
Mol. Formula: C11H24
OS Octadecanoic acid, methyl ester 11.08 15.95 36.811 Anti-viral, anti-microbial31
Other names: Stearic acid methyl ester, Methyl stearate
Mol. Formula: C18H38O2
 H.F. Ismail et al. / Journal of Traditional and Complementary Medicine 7 (2017) 452e465 457

Table 2 (continued )

Herb Compound Area% Height% Retention Biological Activity

time

2-hydroxy-4-methylbenzaldehyde 8.27 8.36 21.922 NA

Other names: 4-Methylsalicylaldehyde
Mol. Formula: C8H8O2
Butyrate, 3-methyl-, 3-butenyl-3-methyl- 7.37 7.76 11.833 NA
Other names: 3-methylbut-3-enyl butanoate
Mol. Formula: C9H16O2
Octadecanoic acid, 2,3-dihydroxypropyl ester 7.33 4.60 46.760 Antioxidant, anti-acne66
Other names: -
Mol. Formula: C31H62O4
Coumarin 7.22 2.75 21.512 Anti-inflammatory, anti-coagulant, anti-bacterial,
Other names: 2H-Chromen-2-one anti-fungal, anti-viral, e anti-hypertensive,
Mol. Formula: C9H6O2 anti-tubercular, anti-convulsant, anti-obesity,
anti-hyperglycemic,
anti-oxidant, and neuroprotective, anti-tumor,
anti-HIV agents, CNS-active compounds32,33,50,51

NA Not available.

Table 3
Amount of selected biomarkers in AP (andrographolide), CX (curcumin), CZ (catechin), EP (gallic acid) and OS (rosmarinic acid) quantified by HPLC fingerprinting.

Biomarkers Andrographolide (mg/mg) Curcumin (mg/mg) Catechin (mg/mg) Gallic acid (mg/mg) Rosmarinic acid (mg/mg)
(RT: 2.940) (RT: 2.631) (RT: 4.674) (RT: 2.814) (RT: 3.326)

AP 17.78 e e e e
CX e 4.65 e e e
CZ e e 4.29 e e
EP e e e 12.08 e
OS e e e e 6.39

3.2. Total phenolic and flavonoid content capacities were arranged in the following manner;
CZ > EP > OS > AP > CX. The correlation between phenolic content
Table 4 summarizes the TPC and TFC in AP, CX, CZ, EP and OS. and antioxidant activities were determined by plotting the TPC and
The TPC values varied widely, ranging from 20.1 mg GAE/100 g TFC values against the values of DPPH and ABTS. With reference to
samples to 269.9 mg GAE/100 g samples. CZ exhibited the highest Table 5, significant positive correlations in all herbs extracts were
phenolic content at 269.9 mg GAE/100 g sample followed by EP quantified.
(219.4 mg GAE/100 g sample), OS (94.9 mg GAE/100 g sample), AP
(21.2 mg GAE/100 g sample) and CX (20.1 mg GAE/100 g sample).
3.4. Cytotoxicity effects on cell proliferations
Meanwhile, the TFC detected varied from 21.1 CE/100 g sample to
62.1 mg CE/100 g sample. The highest flavonoid content was in CZ
The cytotoxicity effects of AP, CX, CZ, EP and OS on cells pro-
at 62.1 mg CE/100 g sample followed by OS (29.3 mg CE/100 g
liferations were assessed via MTT assay. Figs. 3e5 illustrate the
sample), AP (24.5 mg CE/100 g sample), CX (21.1 mg CE/100 g
dose-dependent effect of extracts on 1.1B4, 3T3-L1 and WRL-
sample) and EP (20.33 mg CE/100 g sample).
68 cells viability during 24 h of treatment. Fig. 3 (AeE) showed
significant decrease (p < 0.05) on 1.1B4 cells viability starting from
3.3. DPPH and ABTS radical scavenging activities 0.5 mg/ml for AP and EP, 1.0 mg/ml for CZ and 5.0 mg/ml for CX and
OS. In addition, the IC50 values (Table 6) were arranged based on
Investigation on the DPPH and ABTS radical scavenging of AP, decreasing toxicity levels as follow; EP (0.365 mg/ml) > AP
CX, CZ, EP and OS have demonstrated dose-dependent radical (2.345 mg/ml) > CX (3.099 mg/ml) > CZ (4.692 mg/ml) > OS
scavenging activities as presented in Fig. 2. The EC50 calculated for (9.934 mg/ml).
DPPH (Table 4) were 11.03 mg/ml, 15.48 mg/ml, 53.51 mg/ml, Referring to Fig. 4 (AeE), 0.5 mg/ml of AP, EP and CZ significantly
143.7 mg/ml and 326.3 mg/ml for CZ, EP, OS, AP and CX respectively. (p < 0.05) inhibited 3T3-L1 viability. Meanwhile CX and OS caused
Meanwhile, a similar trend was observed in ABTS in which the EC50 significant (p < 0.05) cytotoxicity at 5.0 mg/ml. The IC50 of extracts
for CZ was 78.26 mg/ml followed by EP (94.27 mg/ml), OS (284.9 mg/ for 3T3-L1 cells were arranged as follow; AP (0.6498 mg/ml) > CX
ml), AP (915.0 mg/ml) and CX 1075 mg/ml. Therefore, the antioxidant (3.786 mg/ml) > CZ (3.928 mg/ml) > OS (6.335 mg/ml) > EP

Table 4
Mean of TPC, TFC and EC50 of DPPH and ABTS radical scavenging activity.

Herbs Total phenolic content Total flavonoid content EC50 e DPPH RSA EC50 e ABTS RSA
(mg GAE/100 g sample) (mg CE/100 g sample) (mg/ml) (mg/ml)

AP 21.2 ± 1.345 24.5 ± 0.741 143.7 915.0

CX 20.1 ± 0.712 21.1 ± 0.217 326.3 1076.0
CZ 269.9 ± 28.6 62.1 ± 3.064 11.03 78.26
EP 219.4 ± 9.586 20.3 ± 0.512 15.48 94.27
OS 94.9 ± 0.310 29.3 ± 1.271 53.51 284.9
Ascorbic acid e e 2.563 34.58

Each value in the table is represented as mean (n ¼ 3).

458 H.F. Ismail et al. / Journal of Traditional and Complementary Medicine 7 (2017) 452e465

Fig. 2. Antioxidant activities of AP, CX, CZ, EP and OS at various concentrations ranging from 5 to 5000 mg/ml. a) DPPH radical scavenging activities. b) ABTS radical scavenging.
Ascorbic acid was used as a standard. Results were expressed in mean ± SEM (n ¼ 3).

Table 5 IC50-1.1B4, IC50-3T3-L1 and IC50-WRL-68 (Eq. 1.1, 1.2, 1.3).

Correlation coefficient (R2) between TPC and TFC against antioxidant activities.
Interestingly, better correlations were calculated for LC50-48 h
Positive correlations were determined for all extracts.
(Fig. 9) (Eq. 2.1, 2.2, 2.3). Prediction between LC50-48 hpf and
Herbs TPC TFC IC50-3T3-L1 was the closest correlation as represented in Eq. 2.2
DPPH ABTS DPPH ABTS (R2 ¼ 0.6077).
AP 0.8441** 0.6859*** 0.8391**** 0.6786***
CX 0.8012**** 0.6546** 0.7990**** 0.6524** 1.1B4; Log (LC50 mg/ml) ¼ 0.4620 log (IC50 mg/ml) þ 0.6697
CZ 0.8227**** 0.7545*** 0.8030**** 0.8068**** [R2 ¼ 0.3444] 1.1
EP 0.7756*** 0.7448*** 0.7308*** 0.8300****
OS 0.7850**** 0.7372*** 0.8043**** 0.7930***
3T3-L1; Log (LC50 mg/ml) ¼ 0.1138 log (IC50 mg/ml) þ 0.4606
** Significant at p < 0.01. [R2 ¼ 0.03492] 1.2
*** Significant at p < 0.001.
**** Significant at p < 0.0001.
WRL-68; Log (LC50 mg/ml) ¼ 0.04306 log (IC50 mg/ml) þ 0.3146
[R2 ¼ 0.0053] 1.3
(6.690 mg/ml). The cytotoxicity effect on WRL-68 (Fig. 5AeE) dis-
played significant reduction (p < 0.05) in viability starting from 1.1B4; Log (LC50 mg/ml) ¼ 1.151 log (IC50 mg/ml) þ 0.4748
0.5 mg/ml for AP, 0.1 mg/ml of CZ, EP and OS. Meanwhile CX has [R2 ¼ 0.1642] 2.1
only showed to be toxic to WRL-68 at 5.0 mg/ml. Based on IC50
values, the cytotoxicity effect of extracts on WRL-68 were arranged 3T3-L1; Log (LC50 mg/ml) ¼ 1.712 log (IC50 mg/ml) þ 0.6064
as follows: AP (0.440 mg/ml) > EP (2.213 mg/ml) > CX (3.406 mg/ [R2 ¼ 0.6077] 2.2
ml) > CZ (3.691 mg/ml) > OS (4.014 mg/ml).
WRL-68; Log (LC50 mg/ml) ¼ 1.581 log (IC50 mg/ml) þ 0.0.4155
3.5. Fish embryo acute toxicity (FET) test on zebrafish [R2 ¼ 0.5495] 2.3

FET test performed on zebrafish revealed to be more toxico-

sensitive towards the embryos development. Measurement on 4. Discussions
survival rate and embryogenesis end points were carried out every
24 h by optical monitoring. Fig. 6 (AeE) demonstrated the dose- For centuries, herbaceous plants have been frequently used for
dependent effects on embryos mortality and survival curves dur- their scent and flavor in daily culinary as well as dietary supple-
ing the 96 h of extracts treatment. With reference to Table 7, CZ ment for health promotion. The present study examined the
exhibited the highest toxicity with calculated LC50 of 0.0508 mg/ inhibitory capacity of water extracts of A. paniculata (AP), Cinnamon
ml, followed by EP (0.06039 mg/ml), AP (0.5256 mg/ml), CX zeylanicum (CZ), C. xanthorrhiza (CX), E. polyantha (EP) and O.
(0.7037 mg/ml) and OS (1.685 mg/ml). stamineus (OS) on the formation of DPPH and ABTS free radicals.
Assessment of the morphological aspects revealed a straight Furthermore, the toxicity effect of extracts was measured under
spine, normal body shape and heart rate, round yolk sac and specific condition via in vitro cytotoxicity assay and zebrafish em-
pigmentation on the body and eyes for the vehicle control (Fig. 7A) bryo acute test.
and non-toxic concentrations (Fig. 7B). In contrast, teratogenic The phenolic compounds are predominant constituents in
concentration (Fig. 7C), the concentration that cause abnormal or- plants and categorized as phenolic acid, flavonoids, tannins, cur-
gan development demonstrated bent spine, enlarged yolk sac, cuminoids, coumarins, lignins and quinones. Our study revealed
pericardial edema, slow heartbeat and delayed hatching (>72 hpf). that CZ and EP contained the highest phenolic and flavonoid sub-
Exposure on the toxic concentration (Fig. 7D) exhibited egg coag- stances. However, these values were relatively low in comparison
ulation and undeveloped organs, especially on the formation of the to previous reports carried out using different types of extraction
spine, tail, cartilage, heart and jaw. processes and solvents. Microwave assist extraction (MAE) of CZ
produced 1679 mg CAE/100 g DW of phenolic content which was 6
3.6. Cytotoxicity and embryotoxicity correlation fold higher than our results.42 Meanwhile, an advance subcritical
water extraction process produced ±17,500 mg GAE/100 g DW of
The correlation between cytotoxicity and embryotoxicity was phenolic content from cinnamon bark.43 Extraction solvent also
expressed in logarithmic IC50 versus the corresponding LC50. affected the yield of phenolic content. 80% ethanol (50  C/2 h)
Fig. 8 demonstrated that LC50-96 hpf was weakly correlated with produced 1460 mg GAE/100 g DW of EP.44
 H.F. Ismail et al. / Journal of Traditional and Complementary Medicine 7 (2017) 452e465 459

Fig. 3. 1.1B4 cell proliferation during AP, CX, CZ, EP and OS treatment for 24 h. Different levels of cytotoxicity effect were identified in each herb. Significant decrease (p < 0.05) in
viability was observed starting from 0.5 mg/ml for AP (Fig. 3A) and EP (Fig. 3D), 1.0 mg/ml for CZ (Fig. 3C) and 5.0 mg/ml for CX (Fig. 3B) and OS (Fig. 3E). AP and EP, 500 mg/ml for CZ
and 1000 mg/ml for CX and OS.

Two approaches have been developed particularly to quantify presence of phytochemical compounds such as andrographolide,
the antioxidant ability to suppress or inhibit the formation of ROS; curcumin, catechin, gallic acid, rosmarinic acid, coumarin, cinna-
1) radical scavenging activity (RSA) of compounds (DPPH, ABTS, maldehyde and eugenol might play important roles on inhibitory
ORAC) and 2) reduction process of samples (FRAP, CUPRAC).45 effect of ROS. Coumarin detected in CZ and EP is a naturally
Scavenging of DPPH radicals is mechanized by the donation of occurring polyphenol and a remarkable antioxidant agent.32,49e51
hydrogen atom to the unpaired electron of nitrogen bridge causing Meanwhile, cinnamaldehyde, an active compound in CZ signifi-
the purple color turn to yellowish. Meanwhile, the ABTSþ radical cantly reduced the formation of lipid peroxide in STZ-induced rat
cation undergoes the reduction process by hydrogen donating plasma, thus prevent the glycation of free radical scavenging en-
antioxidant and can be spectrophotometrically measured. It is well zymes (SOD, CAT and CPx) in pancreatic beta cells.52 Eugenol, a
accepted that the phenolic and flavonoid content significantly in- prime component in EP, played tremendous beneficial effects as
fluence the antioxidant activities.46e48 In agreement, our findings anti-cancer, antioxidant and anti-inflammatory agent.6,53
showed that radical scavenging of DPPH and ABTS were positively In vitro cytotoxicity assay is performed to quantify toxicity ef-
correlated to the phenolic and flavonoid content of herbs. The fect of tested compounds towards the basic cellular functions via
460 H.F. Ismail et al. / Journal of Traditional and Complementary Medicine 7 (2017) 452e465

Fig. 4. 3T3-L1 cell proliferation during AP, CX. CZ, EP and OS treatment for 24 h. Significant decrease (p < 0.05) in viability was observed starting from 0.5 mg/ml for AP (Fig. 4A), CZ
(Fig. 4C) and EP (Fig. 4D) and 5.0 mg/ml for CX (Fig. 4B) and OS (Fig. 4E).

ATP, MTT, MTS, LDH or NRU assay.54e56 In this study, water extracts ROS and LDH, reduction in MMP, increasing of cell membrane
of AP, CX, CZ, EP and OS resulted in a dose-dependent cytotoxicity permeability and CYP C. The activation of downstream caspase
effect on 1.1B4, 3T3-L1 and WRL-68 cell line, but with different (Caspase 8 and 9), the important markers in apoptotic pathways
IC50 values. OS exhibited the lowest cytotoxicity effect to all cells, were elevated during AP treatment.57 On the contrary, WOS dis-
meanwhile AP demonstrated highest cytotoxicity effects. Previous played the lowest cytotoxicity effect with IC50 of 9.934 mg/ml,
study on IC50 of AP aqueous partition on WRL-68, a normal liver 6.335 mg/ml and 4.014 mg/ml for 1.1B4, 3T3-L1 and WRL-68
cells gave value greater than 100 mg/ml whereas for butanol, respectively. According to Al-Saude, the partition B2 of OS super-
chloroform and hexane partition, the values ranged from 52 to critical carbon dioxide extraction demonstrated selective cyto-
62 mg/ml.57 In another study, 500 mg/ml aqueous and ethanol toxicity effect on PC3 and MDA-MB cancer cells. Interestingly,
extract of AP was non-toxic to the human normal lung fibroblast MCF7, MDA-MB, HCT116 and CCD-12 CO were shown to have mild
cell line Hs888Lu.47 Moreover, AP exhibited greater sensitivity and negligible toxic towards all partitions of OS supercritical car-
towards cancer cells. IC50 of ethanol partition of AP on human bon dioxide extract.58
hepatocarcinoma HePG2 were between 17e23 mg/ml. AP showed Early development of embryos is the most sensitive phase to-
an increase of apoptotic event in HePG2 through the elevating of ward external stimuli; i.e. toxicant, chemicals and mechanical
 H.F. Ismail et al. / Journal of Traditional and Complementary Medicine 7 (2017) 452e465 461

Fig. 5. WRL-68 cell proliferation during AP, CX. CZ, EP and OS treatment for 24 h. Significant decrease (p < 0.05) in viability was observed starting from 0.5 mg/ml for AP (Fig. 5A),
1.0 mg/ml for CZ (Fig. 5C), EP (Fig. 5D) and OS (Fig. 5E). CX (Fig. 5B) was shown to inhibit cell proliferation at 5.0 mg/ml.

Table 6 of extracts into the embryos hence delayed the effect of toxicant
IC50 of extracts that cause 50% inhibition on 1.1B4, 3T3-L1 and WRL-68 cell viability. until hatching. Dechorionation of embryos is one step to overcome
this problem by; 1) enzymatic softening of chorion using proteo-
Herbs 1.1B4 (mg/ml) 3T3-L1 (mg/ml) WRL-68 (mg/ml)
lytic proteinase enzymes 2) mechanical dechorionation using fine
AP 2.345 0.6498 0.440
forceps or automated machines59 and 3) microinjection through
CX 3.099 3.786 3.406
CZ 4.692 3.928 3.681 penetrating the chorion to the perivetilline space. The delayed in
EP 0.365 2.430 2.213 hatching is also suspected due to the absence of hatching enzymes
OS 9.934 6.335 4.014 secreted by hatching gland of zebrafish such as zebrafish hatching
enzyme 1 (ZHE1). Evidences on the effect of compounds causing
hatching gland dysfunction are well documented during exposure
of nanoparticles and methylmercury.60,61 Furthermore, the
stress.59 Our results evidenced a significant increment of toxicity development of heart and blood circulation is completed by
effect after 48 hpf particularly for CZ and EP, causing the decrease approximately 24e48 hpf and it is the first internal organ to form
of the survival rate, malformation of organs, abnormal heart beat and function properly. Consequently, toxic compounds flow to the
and delayed in hatching rates. Results suggest that the chorion specific affected organ thus affecting the later stages of embryo
substantially protected the embryos by slowing down the diffusion development. Several reports have displayed the sensitivity of
462 H.F. Ismail et al. / Journal of Traditional and Complementary Medicine 7 (2017) 452e465

Fig. 6. Mortality and survival curves of 96 hpf zebrafish embryos during AP, CX. CZ, EP and OS treatment. At a concentration of 0.05 mg/ml, CZ (Fig. 6C) and EP (Fig. 6D) caused
significant mortality. Meanwhile, AP (Fig. 6A) and CX (Fig. 6B) caused mortality at 0.5 mg/ml and OS (Fig. 6E) at 5 mg/ml.

Table 7
LC50 of extracts at 48 hpf and 96 hpf of FET test. embryo was 1.61 g/l.63 High concentration of seed cake showed to
Herbs LC50-48 hpf (mg/ml) LC50-96 hpf (mg/ml) induce yolk sac and pericardial edema in 72 hpf zebrafish embryo.
Comparison study between cytotoxicity and embryotoxicity was
AP 0.5255 0.5256
CX 0.7486 0.7037 previously conducted using polymeric nanoparticles,64 poly-
CZ 0.9858 0.05058 amidoamine (PAMAM) and polypropylenimine (PPI) and the result
EP 0.9212 0.06039 showed higher sensitivity of embryos compared to cell lines.
OS 1.685 1.685
Similar results were found in our study in agreement with the
previous study. The LC50 of zebrafish embryotoxicity is signifi-
zebrafish embryo on plant extracts. A study on the cardiovascular cantly lower as compared to the IC50 of cytotoxicity assay per-
system (HBR and blood flow) using an ethanolic extract of Cynodon formed in HepG2 and DU145 cell lines which both derived from
dactylon and Sida acuta showed an altered heart beat rate and the human. In contrast, in a study on the toxicity of insecticide pro-
blood velocity during cardiac cycle in 3 hpf zebrafish embryos.62 poxur, insignificant difference between the embryotoxicty LC50-
The LC50 for C. dactylon and S. acuta were 32.6 mg/ml and 96 hpf and IC50 of zebrafish gill cell value was calculated perhaps
20.9 mg/ml respectively. Another study on seed cake of Jatropha due to the similarity of models sources, providing a similarity on
curcus, a biodiesel production waste, the LC50 for zebrafish the toxicity mechanism itself.65
 H.F. Ismail et al. / Journal of Traditional and Complementary Medicine 7 (2017) 452e465 463

Fig. 7. Morphological effect of extracts on zebrafish embryogenesis after 96 h of exposure (hpf). (AeB) 96 hpf embryo during control and non-toxic concentration exposure
demonstrated straight spine, normal body shape, normal heartbeat, round yolk sac and high intensity of pigmentation on the body and eye. (C) 96 hpf embryos after exposed to
teratogen concentration exhibited morphological defects such as bending spine, pericardial edema, enlarged yolk sac and hatching delayed. (D) Coagulated eggs with organ
malformation after exposure to toxic concentration.

Fig. 8. Correlation between LC50-96 hpf and IC50 of three tested cells. 1.1B4 displayed Fig. 9. Correlation between LC50-48 hpf and IC50 of three tested cells. Better re-
the closest relation between cytotoxicity and embryotoxicity with R2 ¼ 0.3444. Linear lationships were observed between LC50-48 hpf and IC50 as compared to LC50-96 hpf
regression indicated a correlation between LC50-96 hpf and IC50 with positive slope of and IC50 for 3T3-L1 cells and WRL-68. 3T3-L1 exhibited the highest correlation with
0.4620 and y-intercept at 0.6697. LC50-48 hpf with R2 ¼ 0.6077. The slope was 1.712 and y-intercept at 0.6064.

Funding
To summarize, the present study demonstrated that phenolic
and flavonoid content varied between the tested extracts. In addi- This research was supported by Research University Grant,
tion, the DPPH and ABTS scavenging activities were found to be Universiti Teknologi Malaysia (RUG: 10H27); NRGS, Ministry of
significantly correlated with the amount of TPC and TFC. Cinnamon Agriculture, Malaysia (4H016); and Fundamental Research Grant
zeylanicum and Eugenia paniculata significantly inhibited ROS for- Scheme (FRGS), Ministry of Higher Education, Malaysia (59424).
mation in both DPPH and ABTS assay at low concentration. Our
findings also suggested that OS produced the lowest toxicity effect Conflict of interest
on cell lines and zebrafish embryos. Moreover, a good correlation
was determined between this two toxicity models. The authors declare that they have no conflict of interest.
464 H.F. Ismail et al. / Journal of Traditional and Complementary Medicine 7 (2017) 452e465

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