Food Chemistry 120 (2010) 290–295

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Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Analytical Methods

Effects of binary solvent extraction system, extraction time and extraction

temperature on phenolic antioxidants and antioxidant capacity from
mengkudu (Morinda citrifolia)
Yin Yin Thoo a, Swee Kheng Ho a, Jia Yun Liang b, Chun Wai Ho b,*, Chin Ping Tan a,*
a
Department of Food Technology, Faculty of Food Science and Technology, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia
b
Department of Food Science and Nutrition, Faculty of Applied Sciences, UCSI University, No. 1, Jalan Menara Gading, UCSI Heights, Cheras 56000, Kuala Lumpur, Malaysia

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

Article history: An investigation into the effects of ethanol concentration (0–100%, v/v), extraction time (20–120 min)
Received 18 June 2009 and extraction temperature (25–65 °C) on the extraction of phenolic antioxidants from mengkudu (Mor-
Received in revised form 5 September 2009 inda citrifolia) was performed using a single-factor experiment. Total phenolic content (TPC) and total fla-
Accepted 16 September 2009
vonoid content (TFC) assays were used for determination of phenolic compounds. Antioxidant capacity
was evaluated by measuring the scavenging effect on 2,20 -azino-bis(3-ethylbenzothiazoline-6-sulphonic
acid) (ABTS) and 2,20 -diphenyl-1-picrylhydrazyl (DPPH) radicals. Experimental results showed that
Keywords:
extraction conditions had significant effect on extraction of phenolic compounds and antioxidant capac-
Mengkudu (Morinda citrifolia)
Total phenolic content (TPC)
ities. The optimised conditions were 40% ethanol for 80 min at 65 °C, with values of 919.95 mg GAE/100 g
Total flavonoid content (TFC) DW for TPC, 472.73 mg CE/100 g DW for TFC, 791.71 lmol TEAC/100 g DW for ABTS and 1928.5 lmol
2,20 -Azino-bis(3-ethylbenzothiazoline-6- TEAC/100 g DW for DPPH. TPC was significantly correlated with DPPH under the effects of ethanol con-
sulphonic acid) (ABTS) radical-scavenging centration (r = 0.932) and extraction time (r = 0.938).
capacity Ó 2009 Elsevier Ltd. All rights reserved.
2,2-Diphenyl-1-picrylhydrazyl (DPPH)
radical-scavenging capacity

1. Introduction
Medicinal plants are promising sources of natural antioxidant
compounds, as many of the phytochemicals from plant extract
have been identified to exhibit antioxidant activity. One well-
known medicinal plant is Morinda citrifolia or locally known as
‘mengkudu’ (Zin, Abdul-Hamid, & Osman, 2002). Among the vari-
ous phytochemicals in M. citrifolia fruits and leaves, phenolic com-
pounds, particularly flavonoids, are widely regarded as some of the
major bioactive compounds which have been shown to possess
various therapeutic properties (Deng, West, Jensen, Basar, &
Westendorf, 2009; Müller et al., 2009).
Taking into consideration the compositions of natural sources of
phenolic compounds, as well as the structure and physicochemical
properties of these compounds, a universal extraction protocol is
not conceivable and a definite extraction procedure must be de-
signed and optimised for each phenolic source (Contini, Baccelloni,
Massantini, & Anelli, 2008; Silva, Rogez, & Larondelle, 2007). In the
present study, solvent extraction using binary solvent of ethanol
* Corresponding authors. Tel.: +60 3 89468418; fax: +60 3 89423552 (C.P. Tan),
tel.: +60 3 9101 8880; fax: +60 3 9102 3606 (C.W. Ho).
E-mail addresses: cwho@ucsi.edu.my (C.W. Ho), tancp@putra.upm.edu.my (C.P.
Tan).
and water was adopted from a consideration of health and safety
in handling. Amongst the various factors contributing to the effi-
ciency of the solvent extraction process and the recovery of antiox-
idant compounds from natural materials, ethanol concentration,
extraction time and extraction temperature are often investigated
(Liyana-Pathirana & Shahidi, 2005; Wang, Sun, Cao, Tian, & Li,
2007; Zhang et al., 2007).
The two most commonly used optimisation studies are the sin-
gle-factor experiments and response-surface methodology (RSM).
Single-factor experiments were used here, despite being time-con-
suming and having several drawbacks, such as lack of information
on interaction of the factors (Zhang et al., 2007). However, it was
able to provide fundamental information on the ranges for extrac-
tion parameters (factors) that showed significant effects (p < 0.05)
on the extraction of phenolic compounds from M. citrifolia. The
minimum and maximum values obtained for each extraction
parameters, which can then serve as key information in RSM for
the generation of a central composite rotatable design (CCRD). Cor-
relation analyses on the factors and responses as well as responses
with responses were also important in judging the suitability of the
assays used in the optimisation studies.
The aim of this study was to investigate the effect of ethanol
concentration, extraction time and extraction temperature on the
extraction of phenolic antioxidants (total phenolic content, TPC;

0308-8146/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodchem.2009.09.064
 Y.Y. Thoo et al. / Food Chemistry 120 (2010) 290–295
and total flavonoids content; TFC) and the free radical-scavenging
capacity of extracts from M. citrifolia for radicals generated by 2,20 -
azinobis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) and 2,2-
diphenyl-1-picrylhydrazyl (DPPH).
2. Materials and methods
2.1. Chemicals
2,20 -Azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) diam-
monium salt (ABTS, 98% purity), 2,20 -diphenyl-1-picrylhydrazyl
(DPPH, 95% purity), potassium persulphate (P98% purity), sodium
nitrite and (+)-catechin hydrate (P98% purity) purchased from Sig-
ma–Aldrich (Steinheim, Germany). Folin–Ciocalteu’s phenol re-
agent, sodium carbonate (P99.9% purity) and sodium hydroxide
solution (1 mol/L, 1 N) purchased from Merck (Darmstadt, Ger-
many). Gallic acid (98% purity) and trolox (97% purity) purchased
from Acros Organics (New Jersey, USA). Absolute ethanol
(P99.4% v/v), denatured ethanol (absolute ethanol 19: methanol
1) and aluminium chloride-6-hydrate (>99 purity) purchased from
Fisher Scientific Co. (Leicestershire, UK). All the reagents were ana-
lytical grade and all stock solutions were prepared by purified
deionised water (MilliQ purification system, Millipore, France).
2.2. Plant material
Powdered dried fruits of M. citrifolia were purchased from a lo-
cal supplier, Ethno Resources Sdn. Bhd (Selangor, Malaysia). M.
citrifolia powder was then vacuum-packaged in nylon-linear low-
density polyethylene pouches and stored in the dark at an ambient
temperature (25 °C) for a maximum of 1 month until used for fur-
ther analysis.
2.3. Extraction of plant material
Dried fruits of M. citrifolia (2 g) were extracted with solvent
(20 mL) in a shaking machine (Model Green SSeriker, Vision, Kor-
ea) or a temperature-controlled water bath shaker (Model WNB
7–45, Memmert, Germany) at a constant speed for the various
times at the required temperature. The crude extracts were then
filtered through Whatman No. 1 filter paper (Whatman Interna-
tinoal Ltd., England) and used directly for estimation of antioxidant
compounds and assessment of antioxidant capacities through var-
ious chemical assays. Each extraction was duplicated and all anal-
ysis was performed in three replications.
2.4. Experimental design
Single-factor experiments were used for solvent extraction in
the present study. A total of three factors were studied, namely,
ethanol concentration, extraction time and extraction temperature.
The levels for each independent variable were chosen based on
four responses of the crude extracts: the total phenolic content
(TPC) and total flavonoids content (TFC) as a priority, followed by
2,20 -azinobis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS)
radical-scavenging capacity and 2,2-diphenyl-1-picrylhydrazyl
(DPPH) radical-scavenging capacity.
(1) Phenolic compounds in M. citrifolia were extracted by using
a binary solvent of ethanol and deionised water at room
temperature (25 °C) for 60 min. The ethanol concentration
varied by adjusting the ethanol composition from 0% (v/v)
to 100% (v/v) in the ethanol–water binary mixture. The best
ethanol concentration was selected based on the values of
the four responses.
291
(2) Phenolic compounds were extracted using the best ethanol
concentration chosen in the first step. In this step, extraction
time varied from 20 to 120 min and the extraction tempera-
ture kept constant at 25 °C. The best extraction time was
again chosen according to the value of the four responses.
(3) Using the best ethanol concentration chosen in first step,
phenolic compounds were extracted at various extraction
temperatures ranging from 25 to 65 °C while holding the
extraction time constant at the best extraction time deter-
mined in the second step. The best extraction temperature
was also chosen according to the value of the four responses.
2.5. Determination of total phenolic content (TPC)
Total phenolic content (TPC) of the extracts was determined
according to Folin–Ciocalteu (FC) procedure (Li, Wong, Cheng, &
Chen, 2008) with slight modifications. Briefly, 1 mL of diluted
crude extracts was mixed with 1 mL of FC reagent (diluted 10-
fold). After 3 min, 800 lL of sodium carbonate anhydrous solution
(7.5%, w/v) was added and vortexed. The absorbance at 765 nm
against blank was determined using UV light spectrophotometer
(Model XTD 5, Secomam, France) after 2 h incubation in dark at
room temperature. Measurements were calibrated to a standard
curve of prepared gallic acid solution (10–70 mg/L) with
y = 0.0396x (R2 = 0.9975) and TPC was then expressed as milligram
of gallic acid equivalents (GAE) per 100 g of dry weight (DW).
2.6. Determination of total flavonoid content (TFC)
Estimation of the TFC in crude extracts was performed accord-
ing to the procedures described by Ozsoy, Can, Yanardag, and Akev
(2007) and Karadeniz, Burdurlu, Koca, and Soyer (2005) with slight
modifications. The reaction mixture contained 0.25 mL of crude ex-
tract, 1.25 mL of deionised water and 75 lL of 5% sodium nitrite.
After 6 min, 150 lL of 10% aluminium chloride-6-hydrate was
added. In the next 5 min, 0.5 mL of 1 M sodium hydroxide solution
and 275 lL deionised water were added and mixed. Immediately,
the reaction mixture absorbance was measured at 510 nm against
a blank and used to calculate TFC using catechin (50–800 lg/mL)
as standard (y = 0.0033x; R2 = 0.9991). The TFC was then expressed
as catechin equivalent (CE), in milligram CE per 100 g DW.
2.7. Evaluation of antioxidant capacity
2.7.1. ABTS radical-scavenging capacity
The ABTS radical-scavenging capacity was determined accord-
ing to Guimaraes et al. (2007) and Surveswaran, Cai, Corke, and
Sun (2007) with some modifications. ABTS radical solution was
produced by gently mixing 10 mL of 7 mM ABTS solution and
10 mL of 2.45 mM potassium persulphate solution and allowed
to stand in dark at room temperature for 12–16 h. This ABTS radi-
cal solution was adjusted with ethanol to an absorbance of 0.7
(±0.02) at 734 nm before usage. One hundred microlitre of crude
extract or ethanol (as control) was added to 3.9 mL of ABTS radical
solution and allowed to react for 6 min. The decrease of absorbance
at 734 nm was measured against a blank (ethanol). The percentage
of ABTS radical-scavenging capacity was calculated as
[1  (Ae  Ac)]  100% (Ae = A517 in the presence of crude extract;
Ac = A517 of negative control solution). The ABTS was then ex-
pressed as micromoles of trolox equivalents antioxidant capacity
(TEAC) using equation obtained from standard curve of prepared
trolox (0.1–0.8 mM) y = 120.1142x, R2 = 0.9984.
2.7.2. DPPH radical-scavenging capacity
Free radical-scavenging capacity using stable DPPH radical was
performed according to the modified method of Miliauskas, Vens-
292 Y.Y. Thoo et al. / Food Chemistry 120 (2010) 290–295

kutonis, and Beek (2004), Saha et al. (2004), and Cai, Sun, Xing, Luo,
and Corke (2006). Crude extract or absolute ethanol (as control)
(100 lL) was added to 3.9 mL of ethanolic DPPH (60 lM). Soon
after vortexing the reaction mixture for 1 min, the tubes were
placed in dark for 30 min and absorbance was measured at
517 nm. The DPPH radical-scavenging capacity (%) was calculated
as [1  (Ae  Ac)]  100% (Ae = A517 in the presence of crude ex-
tract; Ac = A517 of negative control solution). Measurements were
calibrated to a standard curve of prepared trolox (0–2.5 mM) with
y = 37.284x (R2 = 0.9997) and expressed as micromoles of TEAC per
100 g DW.
2.8. Statistical analysis
Results were expressed as mean ± standard deviation of repli-
cate solvent extractions and triplicate of assays and analysed by
MINITAB (version 14). One-way analysis of variance (ANOVA) with
Tukey’s test was carried out to test significant differences between
levels of treatment. Significant levels were defined using the values
p < 0.05 and p < 0.01. Pearson correlations between variables were
established using MINITAB (version 14).
3. Results and discussion
3.1. Extraction solvent evaluation
As described in Fig. 1a–d, the ethanol concentration showed sig-
nificant effect (p < 0.05) on both phenolic contents (TPC and TFC)
and antioxidant capacities (ABTS and DPPH radical-scavenging
capacities). The yield of TPC was maximised at 40% ethanol then
followed by a considerable drop with further increases in ethanol.
Similarly, ABTS and DPPH radical-scavenging capacities also saw a
decline with further increases in ethanol after the maximum val-
ues at 80% and 40% ethanol, respectively. Conversely, the yield of
TFC increased with increasing ethanol and reached maximum va-
lue at 100% ethanol.
This experimental result was in accordance with previous re-
ports suggesting that a binary solvent system was superior to a
mono-solvent system (water or pure ethanol) in the extraction of
phenolic compounds in regard to their relative polarity (Wang et
al., 2007; Zhang et al., 2007). Although TFC extraction was opti-
mised by the 100% ethanol (mono-solvent system), but this does
not contribute to high antioxidant capacities. This circumstance
may be due to the inability of 100% ethanol to extract phenolic
compounds which are more water-soluble (Chirinos, Rogez, Cam-
pos, Pedreschi, & Larondelle, 2007). In short, a high yield of individ-
ual phenolic compounds necessary will not exhibit a high
antioxidant capacity, which indeed is dependent on the synergistic
effects of the extracted phenolics. Therefore, further study should
be carried out to identify the predominant compounds of the M.
citrifolia with respect to their antioxidant mechanisms and syner-
gistic actions.
The extraction of phenolic compounds from a sample is directly
related to the compatibility of the compounds with the solvent
system according to the ‘‘like dissolves like” principle (Zhang et
al., 2007). Based on the present experimental results, it is predicted
that M. citrifolia contains diverse phenolic compounds with differ-
ent polarity. As solvent polarity increased, accordingly the relative
extractions of TPC and TFC changed; their maximum yield was at
different ethanol concentrations. Thus, there is no single ethanol
concentration able to recover all of the individual phenolic com-
pounds from a sample. In accordance with previous report, we pro-
posed that the TPC which maximised at low ethanol concentration
contain higher proportion of hydrophilic compounds, while TFC
recovered at high ethanol concentration were lipophilic com-
pounds (Durling et al., 2007).
It was also observed that the antioxidant capacity of the crude
extracts was sensitive to the solvent polarity. In particular, DPPH
radical-scavenging capacity of the crude extracts extracted with a
high ethanol concentration decreased considerably after reaching

a
ABTS (µmol TEAC/ 100 g DW)

900 850 ab
a ab
TPC (mg GAE/ 100 g DW)

800 d b b ab ab b
c 800
700
600
750
500 e
400
700
300
200 650
100
(a) (c)
0 600
0 20 40 60 80 100 0 20 40 60 80 100
Ethanol Concentration (%) Ethanol Concentration (%)

400 2500
a a
DPPH (µmol TEAC/ 100 g DW)

a a
TFC (mg CE/ 100 g DW)

350
bc b 2000 b b
300
c
250 1500
d
200 d
1000
c
150

100
500
50
(b) (d)
0 0
0 20 40 60 80 100 0 20 40 60 80 100

Ethanol Concentration (%) Ethanol Concentration (%)

Fig. 1. Effect of ethanol concentration on (a) TPC, (b) TFC, (c) ABTS and (d) DPPH assays from M. citrifolia (n = 2)x. Values are presented as means ± SD of six measurements.
Values marked with the different lower case letters (a–e) are significantly (p < 0.05) different. xReplication of solvent extractions. Note: The error bars represent the standard
deviation.
 Y.Y. Thoo et al. / Food Chemistry 120 (2010) 290–295 293

880 a 840

ABTS (µmol TEAC/ 100 g DW)

a
TPC (mg GAE/ 100 g DW)

860 820 a
b a
840 b b 800 ab
b b ab
820 780
b
800 760
780 740

760 720
(a) (c)
740 700
20 40 60 80 100 120 20 40 60 80 100 120
Extraction Time (min) Extraction Time (min)

450 2100

DPPH (µmol TEAC/ 100 g DW)

a a a a a a
TFC (mg CE/ 100 g DW)

400 a a 2000 a
350
a
ab
300 1900 b
250
1800
200
150 1700
100
1600
50 (b) (d)
0 1500
20 40 60 80 100 120 20 40 60 80 100 120
Extraction Time (min) Extraction Time (min)

Fig. 2. Effect of extraction time (a) TPC, (b) TFC, (c) ABTS and (d) DPPH assays from M. citrifolia (n = 2)x. Values are presented as means ± SD of six measurements. Values
marked with the different lower case letters (a–b) are significantly (p < 0.05) different. xReplication of solvent extractions. Note: The error bars represent the standard
deviation.

80% ethanol (Fig. 1d). A similar phenomenon also reported in mul- antioxidant capacity, 80 min was chosen, as a compromise to the
berry leaves (Kim et al., 2007). Thus, it is believed that the effective best extraction time for phenolic compounds and antioxidant
phenolic compounds in the crude extract, to which are attributed capacity.
the antioxidant capacities, were intermediately polar and their sol-
ubility was very sensitive to the solvent polarity. 3.3. Extraction temperature evaluation
By compromising between the high extraction of phenolic com-
pounds and high antioxidant capacity, a moderate ethanol concen- The impact of extraction temperature on the phenolic com-
tration (40%) was selected as the most appropriate solvent to pounds and antioxidant capacity were investigated in the range
optimise the subsequent extraction parameters. from 25 to 65 °C. Linear relationships were observed between
extraction temperature and recovery of TPC, TFC and ABTS
3.2. Extraction time evaluation (Fig. 3a–c). Meanwhile, DPPH radical-scavenging capacity as a
function of extraction temperature showed a parabolic shape
Extraction time is crucial in solvent extraction for phenolic (Fig. 3d) with a maximum value at 45 °C.
compounds, where phenolic compounds may be governed by the Heat has been found to enhance the recovery of phenolic com-
equilibrium concentrations for phenolic compounds reached be- pounds as described by Durling et al. (2007) and Silva et al. (2007).
fore their corresponding apparent reduction (Spigno, Tramelli, & Increased temperature promotes solvent extraction by enhancing
Faveri, 2007). Hence, excess extraction time indeed reduced the both diffusion coefficients and the solubility of polyphenol content
yield of phenolic compounds. Fig. 2a–d showed the effect of (Al-Farsi & Lee, 2008). Increased solubility of polyphenol contents
extraction time on phenolic compounds and antioxidant capacity. was also reported by Wang et al. (2007), who found increasing
Overall, the experimental results showed that extraction time temperature favored the release of bound polyphenol in a sample
(20–120 min) had significant effect on TPC, ABTS and DPPH but with the breakdown of cellular constituents of plant cells which
not on TFC (p < 0.05). leads to increased cell membrane permeability. Moreover, it is also
The difference in optimum extraction times for TPC and TFC believed that release of these bound polyphenols could further re-
may be due to different degrees of phenolic polymerisation, solu- duce the chances of polyphenols coagulating with lipoprotein,
bility of phenolics and interaction of phenolics with other food thereby enhancing solubility of the polyphenols and diffusion
constituents which then leads to the difference time needed to increasing polyphenol yield (Al-Farsi & Lee, 2008; Zhang et al.,
reach equilibrium between the solution in the solid matrix (M. 2007).
citrifolia) and in the bulk solution (ethanol) (Silva et al., 2007). It In regard to the equilibrium principle, elevated temperature
is also observed that the optimum extraction time for antioxidant could increased the extraction rate and thus reduce the extraction
compounds varies with antioxidant capacity. This phenomenon time to reach maximum polyphenol content recovery. However,
has been postulated that the estimation of ABTS and DPPH radi- elevated temperature may not suitable for all kinds of phenolic
cal-scavenging capacities are not solely dependent on a single compounds. Liyana-Pathirana and Shahidi (2005) reported that
group of antioxidant compounds; indeed it is based on the ability the rate of extraction of thermally stable antioxidants at elevated
of any compounds present that could scavenge ABTS or DPPH rad- temperature is higher than the rate of decomposition of less solu-
icals (Prior, Wu, & Schaich, 2005). ble antioxidants. Thus, only samples with higher proportions of
By taking consideration into practical and economic aspects as thermally stable polyphenols are more appropriate to extract
well in optimising the recovery of phenolic compounds and their under elevated temperature, such as M. citrifolia which has
294 Y.Y. Thoo et al. / Food Chemistry 120 (2010) 290–295

1000 a 800 a
c a

ABTS (µmol TEAC/ 100 g DW)

900
TPC (mg GAE/ 100 g DW)

b 790
800 d bc b
780 c bc
700
600 770

500 760
400 750
300
740
200
100 730
(a) (c)
0 720
25 35 45 55 65 25 35 45 55 65
Extraction Temperature (°C) Extraction Temperature (°C)

600 2200
a a

DPPH (µmol TEAC/ 100 g DW)

a ab
TFC (mg CE/ 100 g DW)

500 b 2100
b
2000
ab
400 c
d d 1900
300
1800
200
1700
100 1600
(b) (d)
0 1500
25 35 45 55 65 25 35 45 55 65
Extraction Temperature (°C) Extraction Temperature (°C)

Fig. 3. Effect of extraction temperature on (a) TPC, (b) TFC, (c) ABTS and (d) DPPH assays from M. citrifolia (n = 2)x. Values are presented as means ± SD of six measurements.
Values marked with the different lower case letters (a–d) are significantly (p < 0.05) different. xReplication of solvent extractions. Note: The error bars represent the standard
deviation.

increased phenolic compounds yield with increasing extraction 3.4. Pearson correlation analysis
temperature.
Losses in antioxidant capacity of plant samples are often re- The correlation levels between the level of phenolic com-
ported following a thermal treatment, likely due to the degradation pounds and their antioxidant capacities with regard to the differ-
of polyphenols which were previously mobilised at lower temper- ent extraction conditions are interesting aspect. From the positive
ature (Chan et al., 2009; Liyana-Pathirana & Shahidi, 2005). It is significant (p < 0.05) correlation between TPC and DPPH (0.932)
interesting to note that in the present study, increasing extraction shown in Table 1, we deduce that TPC may responsible for DPPH
temperature had a positive effect to both ABTS and DPPH radical- capacity. Also, it suggests that TPC extraction influenced by etha-
scavenging capacities. From this scenario, it is believed that the nol concentration, may mostly contribute bioactive phenolic com-
phenolic compounds present in M. citrifolia are thermally stable pounds of low-molecular weight, as the DPPH is known to react
and that the extraction time selected in the second stage is suitable preferentially with low-molecular weight phenolic compounds
for both moderate to high temperature without leading to unfa- (Paixão, Perestrelo, Marques, & Câmara, 2007). The positive corre-
vourable degradation. lation between ABTS and DPPH radical-scavenging capacity as-
Taking into consideration the industrial requirements (high says (0.843) indicate they were significantly (p < 0.05) correlated
extraction temperature for a shorter time) as well as making a with each other. We propose that these methods showed similar
compromise between the recovery of TPC and TFC and their anti- predictive ability for the antioxidant capacities of crude extract
oxidant capacities (ABTS and DPPH radical-scavenging capacities), from M. citrifolia obtained with regard to ethanol concentration.
a temperature of 65 °C was selected as the best extraction temper- The present experimental results were consistent with a number
ature in this step. Although 65 °C was high, it did not lead to a sig- of previous studies which found significant (p < 0.05) positive cor-
nificant (p < 0.05) reduction in antioxidant capacities. relations between ABTS and DPPH in crude extracts from plants

Table 1
Correlation between assaysa under influence of different extraction conditions (n = 2).b

rc Ethanol concentration Extraction time Extraction temperature

TPC TFC ABTS TPC TFC ABTS TPC TFC ABTS
TFC 0.655 0.164 0.763
ABTS 0.657 0.253 0.383 0.143 0.741 0.810
DPPH 0.932** 0.465 0.843* 0.938** 0.033 0.531 0.396 0.763 0.073
a
TPC, total phenolic content; TFC, total flavonoid content; ABTS, ABTS radical-scavenging capacity; DPPH, DPPH radical-scavenging capacity.
b
Replication.
c
r, correlation coefficient.
*
Significant level at p < 0.05.
**
Significant level at p < 0.01.
 Y.Y. Thoo et al. / Food Chemistry 120 (2010) 290–295
(Tabart, Kevers, Pincemail, Defraigne, & Dommes, 2009; Teow
et al., 2007).
However, with respect to extraction time, phenolic compounds
are either weakly positively correlated or negatively correlated
with antioxidant capacity. Only one substantial negative signifi-
cant correlation was found between TPC and DPPH (0.938) at
p < 0.05. This strong negative relationship suggested they might
have different behaviour when subjected to increasing extraction
time. This phenomenon is consistent with that reported by Liya-
na-Pathirana and Shahidi (2005) where prolonged extraction time
may lead to decomposition of bioactive compounds. In the present
study, TPC, which here is believed to be composed of phenolic
compounds of low-molecular weight, is more susceptible to
decomposition due to the prolonged exposure of phenolic com-
pounds to temperature, light and oxygen with increasing extrac-
tion time, which then leads to declining antioxidant capacity.
Therefore, the type of active compounds that contribute to the
antioxidant capacity in a crude extract is crucial in selecting the
optimum extraction time.
Strong correlations were found between ABTS with TPC and TFC
as well as DPPH with TFC under the influence of extraction temper-
ature (Table 1). From these experimental results, we propose that
the phenolic compounds are thermally stable. These outcomes
are consistent with the results shown in Fig. 1b, which suggest that
the TFC present in M. citrifolia possesses a lipophilic character and
so is more thermally stable, having a strong correlation with the
ABTS and DPPH radical-scavenging capacity assays. At the same
time, a poor negative correlation was found between ABTS and
DPPH radical-scavenging capacity assays, with no significant dif-
ference at p < 0.05 (Table 1). This result was consistent with a pre-
vious report showing that some of bioactive compounds which
have ABTS radical-scavenging capacity may not show DPPH radi-
cal-scavenging capacity (Wang et al., 1998).
4. Conclusions
An effective solvent extraction system for extracting phenolic
antioxidants from M. citrifolia was determined using single-factor
experiments. The optimal solvent extraction conditions, chosen
as a compromise between the yield of phenolic compounds (TPC
and TFC) and their antioxidant capacities (ABTS and DPPH radi-
cal-scavenging capacities), were 40% ethanol for 80 min at 65 °C.
Significant correlation (p < 0.05) was found for yields with solvent
concentration and extraction time but not with extraction temper-
ature. TPC was significantly correlated with DPPH with respect to
ethanol concentration (r = 0.932) and extraction time
(r = 0.938). This study provides constructive information for fur-
ther optimisation on the extraction of phenolic compounds from
M. citrifolia using response-surface methodology (RSM).
Acknowledgement
Financial support of this work by Universiti Putra Malaysia
through Research University Grant Scheme (05-01-09-0747RU) is
gratefully acknowledged. The authors would like to thank UCSI
University for the laboratory facilities.
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