Industrial Crops and Products 44 (2013) 566571

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Industrial Crops and Products

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Maximizing total phenolics, total avonoids contents and antioxidant activity

of Moringa oleifera leaf extract by the appropriate extraction method
Boonyadist Vongsak a , Pongtip Sithisarn a , Supachoke Mangmool b ,
Suchitra Thongpraditchote c , Yuvadee Wongkrajang c , Wandee Gritsanapan a,
a
Department of Pharmacognosy, Faculty of Pharmacy, Mahidol University, Bangkok 10400, Thailand
b
Department of Pharmacology, Faculty of Pharmacy, Mahidol University, Bangkok 10400, Thailand
c
Department of Physiology, Faculty of Pharmacy, Mahidol University, Bangkok 10400, Thailand

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

Article history: Moringa oleifera L. (Moringaceae) has been used as traditional medicines in many tropical and sub-
Received 25 July 2012 tropical countries. Having phenolics and avonoids as major constituents, the leaf extract has been
Received in revised form reported to exhibit antioxidant activity both in vitro and in vivo. To obtain the maximum yields of
12 September 2012
these compounds, which consequently inuence the antioxidant activity, varying extraction meth-
Accepted 25 September 2012
ods were examined. Squeezing, decoction, maceration, percolation and soxhlet extraction were used
to extract fresh and dried leaves of M. oleifera. Distilled water was used in squeezing and decoc-
Keywords:
tion, while 50 and 70% ethanol were used in the other methods. The contents of total phenolics and
Antioxidant
Extraction method
total avonoids, free radical scavenging activity and ferric reducing power (FRP) of each extract were
Moringa oleifera quantitatively determined. Quantitative analysis of active compounds was accomplished through high
Total avonoid content performance liquid chromatography (HPLC). Extract from the most effective extraction method was
Total phenolic content then selected for reactive oxygen species assay (ROS) in HEK-293 cells. Maceration with 70% ethanol
of dried leaves promoted the extract with maximum amounts of total phenolics (13.23 g chlorogenic
acid equivalents/100 g extract) and total avonoids (6.20 g isoquercetin equivalents/100 g extract). This
extract also exhibited high DPPH-scavenging activity (EC50 62.94 g/mL) and the highest FRP value
(51.50 mmol FeSO4 equivalents/100 g extract). At the concentration of 100 g/mL, the extract could
signicantly reduce relative amount of intracellular ROS. The contents of major active components,
crypto-chlorogenic acid and isoquercetin, in the dried plant powder were 0.05 and 0.09% (w/w), respec-
tively. Considering various factors involved in the extraction process, maceration with 70% ethanol
was advantageous to other methods with regards to simplicity, convenience, economy, and providence
of the extract containing maximum contents of total phenolics and total avonoids with the highest
antioxidant activity. Maceration and 70% ethanol were recommended as the extraction method and
solvent for high quality antioxidant raw material extract of M. oleifera leaves for pharmaceutical and
nutraceutical development.
2012 Elsevier B.V. All rights reserved.

1. Introduction optimal extraction method should be simple, rapid, economical and

Extraction of plant materials depends on various factors such as
solvents, methods, and extraction time to separate different qual-
ity and quantity of bioactive components in the crude extracts
(Hayat et al., 2009). In addition, the nature of the sample matrix
and the compounds to be extracted also substantially affect the ef-
ciency of extraction (Mustafa and Turner, 2011). Theoretically, the
Corresponding author at: Department of Pharmacognosy, Faculty of Phar-
macy, Mahidol University, 447 Sri-Ayudthaya Road, Ratchathewi, Bangkok 10400,
Thailand. Tel.: +66 2 644 8677x1500/5530; fax: +66 2 644 8701.
E-mail addresses: wandeegrit@yahoo.ac.th, wandee.gri@mahidol.ac.th
(W. Gritsanapan).
applicable to a large scale industry (Pothitirat et al., 2010).
Various parts of Moringa oleifera Lam. (moringa) of the Fam-
ily Moringaceae have been used as herbal medicines in tropical
and subtropical countries such as India, Pakistan, the Philippines,
Thailand and Africa. The leaves, owers and immature pods of this
plant are used as high nutritive supplement with various phar-
macological properties (Anwar et al., 2007; Chumark et al., 2008;
Anjula et al., 2011). Moreover, moringa have long been recognized
in the Ayurvedic and Unani systems of medicine for prevention
and treatment of several diseases, e.g., gastric ulcers, skin diseases,
hay fever, fatigue and bronchitis (Anwar et al., 2007). The leaf
extracts of M. oleifera have been reported to exhibit antioxidant
activity both in vitro and in vivo due to abundant phenolic acids
and avonoids (Chumark et al., 2008; Verma et al., 2009). Some

0926-6690/$ see front matter 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.indcrop.2012.09.021
 B. Vongsak et al. / Industrial Crops and Products 44 (2013) 566571
researchers claimed that moringa leaves were rich in chlorogenic
acid, gallic acid, kaempferol and quercetin glycosides (Bennett et al.,
2003; Brahma et al., 2009).
Recently, moringa leaves have become popular as a dietary sup-
plement and herbal medicine. Therefore, it would be of nutritional
benet to determine the appropriate extraction method and sol-
vent that promote the extract with high biological activities and
high yield of active compounds for nutraceutical production. Thus,
this work aimed to probe the appropriate extraction method and
solvent that would promote M. oleifera leaf extract with the high-
est contents of total phenolics, total avonoids among other major
active compounds, and the highest antioxidant activity. The results
from this experiment could be used as the guidance for further
standardization and application of moringa leaf extract in pharma-
ceutical/nutraceutical production.
2. Materials and methods
2.1. Chemicals
Isoquercetin, crypto-chlorogenic acid, chlorogenic acid, 1,1-
diphenyl-2-picrylhydrazyl (DPPH) radical, fetal bovine serum
(FBS), Dulbeccos modied eagles medium (DMEM), penicillin,
streptomycin, dimethylsulfoxide (DMSO), 3-(4,5-dimethylthiazol-
2-yl)-2,5-diphenyl tetrazolium bromide (MTT), dichloruorescein-
diacetate (DCFH-DA), phosphate buffered saline pH 7.4 (PBS) and
triton X-100 were obtained from SigmaAldrich (St. Louis, MO,
USA). Folin-Ciocalteu reagent, potassium ferricyanide and fer-
ric chloride were obtained from Fluka Biochemika (Steinheim,
Germany). Aluminum chloride, trichloroacetic acid, potassium
dihydrogen phosphate and hydrogen peroxide were purchased
from Merck (Darmstadt, Germany). Sodium bicarbonate, and fer-
rous sulfate were purchased from Ajax Finechem (NSW, Australia),
and Carlo Erba (Val de Reuil, France), respectively. Other chem-
icals and solvents were of analytical grade and purchased from
Labscan Asia (Bangkok, Thailand), except for 95% ethanol which
was obtained from the Excise Department, Bangkok, Thailand and
was distilled before use.
2.2. Plant materials
The mature leaves of M. oleifera were collected from Baan Klang
Sub-District, Muang District, Pathum Thani Province, Thailand in
October 2010. The samples were identied by Dr. W. Gritsana-
pan and the voucher specimens (BVMO011010) were deposited at
Department of Pharmacognosy, Faculty of Pharmacy, Mahidol Uni-
versity, Bangkok, Thailand. The leaves were cleaned by tab water
and a portion was dried in a hot oven at 60 C for 24 h. The dried
samples were ground and passed through a sieve (20 mesh). The
powdered drugs were kept in sealed containers and protected from
light until used. Another portion of fresh sample was used for
squeezing, decoction, and maceration.
2.3. Investigation of suitable method for extracting of M. oleifera
leaves
Several extraction methods were performed using 50 and 70%
(v/v) ethanol as solvents, except for squeezing and decoction in
which distilled water was used. Each extraction was repeated many
times until exhaustion. Each method was done in triplicate.
2.3.1. Squeezing (SZ)
The fresh leaves of M. oleifera were extracted by mincing with
distilled water (1:10, w/v) and the mixture was squeezed and l-
tered through muslin cloth and Whatman No. 1 lter paper. The
567
ltrate was lyophilized to yield a freeze-dried squeezing leaf extract
(SZ).
2.3.2. Decoction of fresh leaves (DF)
The fresh leaves were minced into small pieces, boiled with dis-
tilled water (1:10, w/v) at 100 C for 30 min and ltered through
a Whatman No. 1 lter paper. The marc was repeatedly extracted
until exhaustion.
2.3.3. Decoction of dried leaves (DD)
The dried powdered leaves was boiled with distilled water
(1:10, w/v) at 100 C for 30 min and then ltered. The marc was
re-extracted until exhaustion.
2.3.4. Maceration of fresh leaves (MF70)
The fresh leaves were minced into small pieces and macera-
ted with 70% ethanol (1:20, w/v) for 72 h at room temperature
(28 2 C) with occasional shaking. The extract was ltered and the
marc was re-macerated with the same solvent until the extraction
was exhausted.
2.3.5. Maceration of dried leaves (MD50 and MD70)
The dried powdered leaves were separately macerated with
50 and 70% ethanol (1:40, w/v) for 72 h at room temperature
(28 2 C) with occasional shaking. The extract was ltered and
the marc was re-extracted by the same process and solvent until
the extraction was exhausted.
2.3.6. Percolation of dried leaves (PD50 and PD70)
The dried powdered leaves were separately mixed with 50 and
70% ethanol (1:5, w/v) and the mixture was allowed to stand for 1 h.
Then the mixture was transferred to a percolator, and 50 and 70%
ethanol was added (nal proportion of 1:10, w/v). The extraction
was done at room temperature with a ow rate of 1 mL/min until
the percolation was exhausted.
2.3.7. Soxhlet extraction of dried leaves (SD50 and SD70)
The dried powdered leaves were separately placed into a thim-
ble and were extracted with 50 and 70% ethanol (1:50, w/v) in a
soxhlet apparatus. Extraction was carried out at ve cycles/h until
exhaustion (20 h).
The combined extract from each extraction method (except
squeezing) was separately ltered through a Whatman No. 1 l-
ter paper. The ltrate was dried under reduced pressured at 50 C
using a rotary vacuum evaporator. The crude extract was weighed
and kept in a tight container protected from light.
2.4. Determination of total phenolic compounds content
The content of total phenolic compounds was determined using
Folin-Ciocalteu procedure (Pothitirat et al., 2009). Each sample
(1000 g/mL), 200 L was mixed with 500 L of the Folin-Ciocalteu
reagent (diluted 1:10 with deionized water) and 800 L of sodium
bicarbonate solution (7.5%, w/v). The mixture was allowed to stand
at room temperature for 30 min with intermittent shaking. The
absorbance was measured at 765 nm using a UVVisible (UVVIS)
spectrophotometer (PerkinElmer, USA). The content of total pheno-
lic compounds was calculated as mean SD (n = 3) and expressed as
grams of chlorogenic acid equivalents (CAE) in 100 g of the extract
and dried powder.
2.5. Determination of total avonoid compounds content
Total avonoids were analyzed using aluminum chloride col-
orimetric method (Pothitirat et al., 2009). Sample (1000 g/mL)
568 B. Vongsak et al. / Industrial Crops and Products 44 (2013) 566571
of 500 L was mixed with 500 L of 2% aluminum chloride solu-
tion. The mixture was allowed to stand at room temperature for
10 min with intermittent shaking. The absorbance of the mixture
was measured at 415 nm against a blank sample without aluminum
chloride using UVVIS spectrophotometer. The content of total
avonoids was calculated as mean SD (n = 3) and expressed as
grams of isoquercetin equivalents (IQE) in 100 g of the extract and
dried powder.
2.6. Determination of antioxidant activity
2.6.1. DPPH-scavenging assay
The free radical scavenging activity of the extracts and of
standard solutions (chlorogenic acid and isoquercetin) were
investigated using 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical
scavenging method (Pothitirat et al., 2009). A total of 750 L of the
extract or standard was added to 750 L of DPPH in methanol solu-
tion (152 M). After incubation at 37 C for 20 min, the absorbance
of each solution was determined at 517 nm using UVVIS spec-
trophotometer. The corresponding blank readings were also taken
and percent inhibition was then calculated as follows:
 
% Inhibition =
Ablank Asample
100
Ablank
where Ablank is the absorbance of the control reaction (containing all
reagents except the test compound) and Asample is the absorbance
of the test compound. The EC50 value, the concentration of sample
required for 50% scavenging of the DPPH free radical, was deter-
mined from the curve of percent scavenging plotted against the
concentration. Each determination was done in triplicate, and the
average EC50 value was calculated.
2.6.2. Ferric reducing power (FRP) method
The procedure for determination of ferric reducing power was
adapted from Ferreira et al. (2007). Sample extract (500 L) was
mixed with 500 L of 0.2 M sodium phosphate buffer and 500 L
of 1% (w/v) potassium ferricyanide solution and then incubated
for 20 min at 50 C. The mixtures was added with 2 ml of 10%
(w/v) trichloroacetic acid and centrifuged at 650 rpm for 10 min.
The supernatant (500 L) was drawn and mixed with 500 L of
deionized water and 100 L of 0.1% (w/v) ferric chloride solution.
The absorbance of the mixtures was measured at 700 nm using
UVVIS spectrophotometer. The content of Fe2+ was evaluated as
mean SD (n = 3) and expressed as mmol FeSO4 equivalents/100 g
extract.
2.7. Cell culture
The human embryonic kidney cell line (HEK-293) was obtained
from American ATCC Cell Line Center. The cells were grown in Dul-
beccos modied Eagles medium (DMEM) supplemented with 10%
fetal bovine serum, 1% penicillin and streptomycin solutions, at
37 C in 5% CO2 humidied incubator.
2.7.1. Cell viability
Cell viability was evaluated by the MTT assay (Garca-Alonso
et al., 2006). HEK-293 cells (180 L) were plated into a 96-well plate
at a density of 5.0 103 cells/well. Cells were grown overnight and
then 20 L of the extracts, standards (08000 g/mL) and control
(0.2% DMSO) were added to the cells. After 48 h of treatment with
different concentrations, the medium was replaced with 200 L
of 1% DMEM and 50 L of MTT solution (5 mg/mL) and the cells
were further incubated for 4 h. The medium was then removed and
150 L of DMSO was added into each well to dissolve formazan
crystals. The plate was read using a Tecan microplate reader at
570 nm. The percentage of viable cells was determined according
to the following equation.
 Absorbance of treated cells 
Cell viability (%) = 100
Absorbance of control cells
2.7.2. Reactive oxygen species (ROS) assay
DCFH-DA was used to detect the intracellular ROS levels in
HEK-293 cells (Kosem et al., 2007). Briey, HEK-293 cells were
cultured on a 12-well plate at a density of 1.0 105 cells/well in
900 L and then 100 L of the extracts (1000 g/mL), standards
(isoquercetin 100 g/mL and crypto-chlorogenic acid 100 g/mL)
and control (0.2% DMSO) were added to the cells for 24 h. After
that, cells were incubated with 10 M DCFH-DA in 900 L DMEM
at 37 C for 30 min in the dark. After the incubation, 100 L of 1 mM
hydrogen peroxide (H2 O2 ) was added and incubated at 37 C for
1 h. Then, the cells were washed once with phosphate buffered
saline pH 7.4 (PBS) and lysed with 1% triton X-100. Fluorescence
was measured at an excitation and emission wavelengths of 495
and 525 nm, respectively by Gemini XPS uorescence microplate
reader. The percent of ROS production was determined according
to the following equation:
F 
1
Relative amount of intracellular ROS (%) = 100
F0
F0 was the uorescence intensity of the control (cells without the
extract) and F1 was the uorescence intensity in the presence of
the standard compound or the extract.
2.8. Quantitative analysis of major active compounds by HPLC
Standard solutions of crypto-chlorogenic acid and isoquercetin
were prepared at 1 mg/mL in 50% methanol. They were diluted
into seven concentrations (5.0, 2.5, 1.0, 0.5, 0.25, 0.125 and
0.0625 g/mL) for a calibration curve. Each extract was dissolved
in 50% methanol (1 mg/mL). All solutions were ltered through a
0.2 mm membrane lter.
The leaf extract from each extraction method was analyzed by
the validated HPLC method (Vongsak et al., 2012) performed on
a Shimadzu SPD-10A (Japan). The separation was carried out on
a Hypersil BDS C18 -column (4.6 mm 150 mm i.d., 5 mm) with a
C18 guard column. A gradient elution program was used with the
mobile phase, combination of solvent A (1% glacial acetic acid): sol-
vent B (methanol) as follows: 80:2060:40 for 12 min, 60:4045:55
for 20 min, 45:5525:75 for 1 min, held for 10 min, 25:75100%
B for 1 min, held for 10 min. The ow rate was 1.0 mL/min at
ambient temperature. The injection volume for all samples and
standards was 20 L and the UVVIS detector was monitored for
the absorbance at 334 nm.
2.9. Statistical analysis
The results were reported as mean standard deviation (SD)
(n = 3). The average contents of total phenolics, total avonoids and
EC50 of the extracts prepared by the different extraction methods
were statistically investigated using one-way analysis of variance
(ANOVA) with least signicant difference (LSD) by SPSS for Win-
dows 16.0. A statistical probability (p value) less than 0.05 indicated
a statistically signicant difference between groups.
3. Results and discussion
The yields of crude extracts and the contents of total phenolics
and total avonoids are shown in Table 1. Decoction of fresh leaves
(DF) gave the highest yields of crude extracts (60.95% dry weight,
14.66% fresh weight), while squeezing (SZ) gave the lowest yield
Table 1
Yields of crude extracts, contents of total phenolics, total avonoids, crypto-chlorogenic acid and isoquercetin. The bold values indicated the most suitable extraction method of moringa leaves.

Method Yield of crude extract Total phenolics Total avonoids Content of crypto-chlorogenic acid (%, w/w) Content of isoquercetin (%, w/w)
(% dry weight)*

(g CAE/100 g (g CAE/100 g (g IQE/100 g (g IQE/100 g In extract* In dry powder* In extract* In dry powder*
extract)* dry powder)* extract)* dry powder)*

SZ 21.96 1.11a (5.28 0.27) 2.35 0.18a 0.25 0.02a 0.95 0.07a 0.10 0.00a 0.0076 0.0004a 0.0017 0.0002a 0.0086 0.0008a 0.0019 0.0003a
DF 60.95 1.57b (14.66 0.38) 4.76 0.04b 1.39 0.04b 0.98 0.04a 0.29 0.02b 0.0074 0.0005a 0.0045 0.0002a Undetectable Undetectable
DD 59.24 1.14b 4.41 0.05b 2.61 0.06c 0.91 0.10a 0.54 0.07c 0.0067 0.0006a 0.0036 0.0001a Undetectable Undetectable
MF70 42.70 2.16c (10.27 0.52) 9.23 0.52c 1.90 0.19d 4.89 0.21b 1.01 0.09d 0.0465 0.0029b 0.0198 0.0016b 0.1447 0.0048b 0.0618 0.0038b
MD70 40.50 1.24c 13.23 0.55d 5.35 0.09e 6.20 0.48 c 2.51 0.11e 0.1317 0.0065c 0.0533 0.0011c 0.2176 0.0098c 0.0880 0.0014c
MD50 38.34 1.17cdg 7.22 0.16e 2.93 0.15c 3.03 0.20d 1.23 0.11f 0.0967 0.0009d 0.0371 0.0011d 0.1892 0.0011d 0.0725 0.0019d
PD70 32.75 1.93efh 10.91 0.30f 3.71 0.36f 5.29 0.05e 1.80 0.23g 0.0956 0.0011d 0.0313 0.0016e 0.0713 0.0011e 0.0233 0.0010e
PD50 34.47 1.41efgh 7.36 0.22e 3.28 0.19c 3.30 0.15d 1.46 0.06h 0.0517 0.0055e 0.0178 0.0007b 0.0211 0.0030a 0.0073 0.0004f
SD70 35.87 1.12dfgh 12.47 0.24g 4.55 0.22g 6.71 0.22f 2.45 0.07e 0.1050 0.0006f 0.0377 0.0014d 0.2354 0.0087f 0.0845 0.0032c

B. Vongsak et al. / Industrial Crops and Products 44 (2013) 566571

SD50 33.58 1.58efgh 13.25 0.59d 4.46 0.39g 3.77 0.09g 1.27 0.06f 0.1207 0.0001g 0.0405 0.0027f 0.1937 0.0023e 0.0648 0.0037b

Dissimilar letters in the same column indicate signicantly different at p < 0.05 using one-way ANOVA.
SZ, squeezing; DF, decoction of fresh leaves; DD, decoction of dried leaves; MF70, maceration of fresh leaves with 70% ethanol; MD70, maceration with 70% ethanol of dried leaves; MD50, maceration with 50% ethanol of dried
leaves; PD70, percolation with 70% ethanol of dried leaves; PD50, percolation with 50% ethanol of dried leaves; SD70, soxhlet extraction with 70% ethanol of dried leaves; SD50, soxhlet extraction with 50% ethanol of dried leaves;
CAE, chlorogenic acid equivalent; IQE, isoquercetin equivalent.
*
Expressed as mean SD (n = 3), (. . .) % fresh weight.

Table 2
DPPH scavenging assay, FRP assay, cell viability, and ROS production of M. oleifera leaf extracts and standard compounds.

Assay SZ DF DD MF70 MD70 MD50 PD70 PD50 SD70 SD50 CCA IQ

DPPH EC50 (g/mL)* 367.32 7.59a 123.44 8.80b 128.63 1.52b 94.19 5.10c 62.94 7.05d 164.57 13.41e 95.94 3.34f 183.82 5.42g 55.07 4.94d 74.02 1.35h 8.72 0.27 5.91 0.28
FRP Assay (mmol 27.45 0.86a 46.44 0.49b 46.91 0.88b 35.07 1.65c 51.50 0.93d 43.36 0.89e 47.25 0.84b 44.09 0.33e 48.17 0.69b 42.23 2.45e 137.21 0.53 217.74 0.23
FeSO4
equivalent/100 g
extract)*
Cell viability EC50 378.36 29.99 201.01 2.08 30.69 4.14
(g/mL)*
Relative amount of 408.08 12.95 295.22 18.06 108.35 13.94
intracellular ROS
(%)*
Dissimilar letters in the same row indicate signicantly different at p < 0.05 using one-way ANOVA.
SZ, squeezing; DF, decoction of fresh leaves; DD, decoction of dried leaves; MF70, maceration of fresh leaves with 70% ethanol; MD70, maceration with 70% ethanol of dried leaves; MD50, maceration with 50% ethanol of dried
leaves; PD70, percolation with 70% ethanol of dried leaves; PD50, percolation with 50% ethanol of dried leaves; SD70, soxhlet extraction with 70% ethanol of dried leaves; SD50, soxhlet extraction with 50% ethanol of dried leaves;
CCA, crypto-chlorogenic acid; IQ, isoquercetin.
*
Expressed as mean SD (n = 3).

569
570 B. Vongsak et al. / Industrial Crops and Products 44 (2013) 566571
Fig. 1. Structure of crypto-chlorogenic acid.
Fig. 2. Structure of isoquercetin.
of crude extract (21.69% dry weight, 5.28% fresh weight). There
is no signicant difference in total phenolics contents between
the extracts from dried and fresh leaves of moringa prepared by
decoction method. However, the dried leaves signicantly pro-
moted the crude extract with higher total avonoid content. Among
various extraction methods, maceration of the dried leaves with
70% ethanol (MD70) provided the highest yield of the extract
(40.50%, w/w) with the maximum contents of total phenolics and
Fig. 3. Effect of MD70, crypto-chlorogenic acid, and isoquercetin on ROS production in HEK-293 cells as determined using DCFH-DA uorescent probe. Control was cultured
without extract and H2 O2 exposure. MD70, maceration with 70% ethanol of dried leaves; CCA, crypto-chlorogenic acid; IQ, isoquercetin. The relative amount of ROS (%) is
expressed as mean SD (n = 3). Dissimilar letters in the same column indicate signicantly different at p < 0.05 using one-way ANOVA.
total avonoids (13.23 g CAE/100 g extract and 6.20 g IQE/100 g
extract, respectively). This extract also promoted high DPPH scav-
enging activity (EC50 62.94 g/mL) and the highest FRP value
(51.50 mmol FeSO4 equivalent/100 g extract). The results of ferric
reducing power assay of the decoction extracts did not correlate
with the DPPH scavenging activity or their total phenolics and total
avonoids contents. Many reports demonstrated the differences
between radical scavenging activity and reducing power of hot
water tea extracts, Camellia sinensis and Ixora coccinea (Farhoosh
et al., 2007; Torey et al., 2010) that due to the different mecha-
nisms of each assay. It could be inferred that aside from free radical
scavenging activity through proton donating ability in DPPH assay,
the antioxidant activity of M. oleifera leaf extracts could be accom-
plished through reductones, which exert activity by breaking the
free radical chain suggested in FRP assay.
For quantication analysis, crypto-chlorogenic acid and iso-
quercetin, the main active substances which were previously
isolated from M. oleifera leaves in our laboratory (Vongsak et al.,
2012), were utilized as marker compounds (Figs. 1 and 2).
The contents of crypto-chlorogenic acid and isoquercetin in the
dried leaves were 0.00170.0533% (w/w) and 00.0880% (w/w),
respectively (Table 1). Compared with the extracts from other
extraction methods, MD70 contained the highest contents of
crypto-chlorogenic acid and isoquercetin (0.0533 and 0.0880%,
w/w, respectively). It was indicated that the extracts with high
contents of total phenolics and total avonoids contained high
amounts of crypto-chlorogenic acid and isoquercetin, which cor-
responded to their high antioxidant activities.
To determine optimal concentrations of the MD70 extract
and standards crypto-chlorogenic acid and isoquercetin, MTT
dye was used to determine the cell viability. Median effective
dose (EC50 ) of the MD70 extract was found to be 378.36 g/mL
while crypto-chlorogenic acid and isoquercetin exhibited EC50
at 201.01 2.08 and 30.69 4.14 g/mL, respectively (Table 2).
As a result, reactive oxygen species (ROS) was measured at
concentration of 100 g/mL for the extracts and 10 g/mL for
the standards to provide more than 80% survival of HEK-
293 cells. In the HEK-293 cells, treatment with 1 mM H2 O2
resulted in 5-fold increase of the level of ROS compared to
that of the control (Fig. 3). Pretreatment with the MD70
extract signicantly reduced the H2 O2 -induced ROS production
in HEK-293 cells (Fig. 3). Crypto-chlorogenic acid and iso-
quercetin, the major active compounds found in the extract,
 B. Vongsak et al. / Industrial Crops and Products 44 (2013) 566571
provided strong inhibition of H2 O2 -induced ROS production
(Table 2 and Fig. 3).
Some modern extraction methods such as microwave-assisted
extraction (Hayat et al., 2009), ultrahigh pressure extraction (Xi
et al., 2011) and supercritical carbon dioxide extraction (Bimakr
et al., 2011) have been utilized for the preparation of plant extracts.
These methods have various benets such as improved penetra-
tion of solvent into plant particles, low extraction temperature, and
reduced extraction time, but maceration is simpler, more conve-
nient and less costly in terms of instrumentation (Sithisarn et al.,
2006). Therefore, it is more applicable in small and medium enter-
prises (SMEs) and in developing countries. Maceration and 70%
ethanol are the recommended extraction method and solvent for
the preparation of the most effective antioxidant M. oleifera leaf
extracts for further pharmaceutical and/or nutraceutical products
development.
4. Conclusions
Maceration with 70% ethanol is the most suitable extraction
method of the dried leaves of M. oleifera. It promoted high yield
of the crude extract, the highest contents of total phenolics, total
avonoids, major active compounds, and the most potent antioxi-
dant activity.
Acknowledgments
This work is a part of the Ph.D. thesis of Mahidol University and
was nancial supported by NRCT (the National Research Council of
Thailand). The authors would like to thank Mr. Panupon Khumsu-
pan for his kind proofreading of the manuscript.
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