Food Chemistry 218 (2017) 152–158
Contents lists available at ScienceDirect
Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem
Subcritical ethanol extraction of flavonoids from Moringa oleifera leaf
and evaluation of antioxidant activity
Yongqiang Wang a, Yujie Gao b,c, Hui Ding d,⇑, Shejiang Liu d, Xu Han d, Jianzhou Gui e, Dan Liu e
a
School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China
b
Tianjin Academy of Environmental Sciences, Tianjin 300191, China
c
Tianjin United Environmental Engineering Design Company Limited, Tianjin 300191, China
d
School of Environmental Science and Engineering, Tianjin University, Tianjin 300072, China
e
School of Environmental and Chemical Engineering, Tianjin Polytechnic University, Tianjin 300387, China
a r t i c l e i n f o a b s t r a c t
Article history: A large-scale process to extract flavonoids from Moringa oleifera leaf by subcritical ethanol was developed
Received 27 November 2015 and HPLC–MS analysis was conducted to qualitatively identify the compounds in the extracts. To opti-
Received in revised form 24 August 2016 mize the effects of process parameters on the yield of flavonoids, a Box-Behnken design combined with
Accepted 8 September 2016
response surface methodology was conducted in the present work. The results indicated that the highest
Available online 13 September 2016
extraction yield of flavonoids by subcritical ethanol extraction could reach 2.60% using 70% ethanol at
126.6 °C for 2.05 h extraction. Under the optimized conditions, flavonoids yield was substantially
Keywords:
improved by 26.7% compared with the traditional ethanol reflux method while the extraction time
Response surface methodology
Flavonoids
was only 2 h, and obvious energy saving was observed. FRAP and DPPH assays showed that the extracts
Moringa oleifera leaf had strong antioxidant and free radical scavenging activities.
Subcritical ethanol extraction Ó 2016 Elsevier Ltd. All rights reserved.
Antioxidant property
1. Introduction bioactive compounds makes the animals and humans ingest signif-
icant amounts of flavonoids in their diet (Manach, Scalbert,
Moringa oleifera (M. oleifera) is a widely growing tree in India, Morand, Remesy, & Jimenez, 2004). In human bodies, oxidation is
and also being cultivated in Niger, Haiti, Mexico, China, etc an essential process to product energy, but sometimes the produc-
(Morton, 1991). Recent years, M. oleifera has attracted great tion of oxygen-derived free radicals is uncontrolled and damaging
attention among researchers because of the potential use in many to human cells. Antioxidants such as flavonoids have ability to
fields. Almost every part of the tree can be eaten, and more impor- scavenge these free radicals and reduce the risk of death from coro-
tant thing is that all parts of the tree have great potential as nary heart disease (Hertog, Feskens, Hollman, Katan, & Kromhout,
medicines (Abdulkarim, Long, Lai, Muhammad, & Ghazali, 2005). 1993). Therefore, it is essential to develop and utilize antioxidants
Therefore, many research workers paid great attention to the to protect human body from free radicals (Singh & Rajini, 2004).
medicinal and nutritional uses of M. oleifera (Anwar, Latif, Ashraf, Many new extraction technologies have been developed to
& Gilani, 2007). Meanwhile, environmental uses of M. oleifera seed separate flavonoids from plants (Dai & Mumper, 2010), such as
such as treatment of waste water also aroused scholarly interest ultrasound-assisted extraction (UAE) (Albu, Joyce, Paniwnyk,
(Kansal & Kumari, 2014). M. oleifera leaf contains lots of nutrients Lorimer, & Mason, 2004; Zhang, Yang, Li, & Wang, 2008), subcriti-
which can be absorbed into human body, such as vitamins, cal water extraction (SWE) (Jo et al., 2013; Matshediso, Cukrowska,
minerals, and fatty acids (Moyo, Masika, Hugo, & Muchenje, & Chimuka, 2015), supercritical fluid extraction (SFE) (Maran,
2011). Additionally, the leaf has been certified to contain various Manikandan, Priya, & Gurumoorthi, 2015), microwave-assisted
compounds like flavonoids, phenolics, and carotenoids which can extraction (MAE) (Rostagno, Palma, & Barroso, 2007). Subcritical
be used as antioxidant (Alhakmani, Kumar, & Khan, 2013; extraction does not require an alternative energy source such as
Vongsak, Sithisarn, & Gritsanapan, 2014). microwave and ultrasound. In addition, subcritical water extrac-
Flavonoids are widely distributed in plants and have many roles tion (SWE) is a new and ‘green’ method to extract bioactive com-
and functions. The wide distribution compared with other pounds from plants, and the dielectric constant of subcritical
water is different under different conditions. But SWE needs high
⇑ Corresponding author. temperature to reach the subcritical condition which may destroy
E-mail address: dinghui@tju.edu.cn (H. Ding). the bioactive compounds (Dai & Mumper, 2010), and extraction
http://dx.doi.org/10.1016/j.foodchem.2016.09.058
0308-8146/Ó 2016 Elsevier Ltd. All rights reserved.
Y. Wang et al. / Food Chemistry 218 (2017) 152–158 153
efficiency of subcritical water still needs more investigation. Etha- solvent was evaporated at 50 °C under vacuum, and the residue
nol is a widely used solvent for bioactive compounds extraction was extracted again under the same conditions. The sample was
and relatively safe for human (Shi et al., 2005), and supercritical extracted for 3 times in total. This whole experiment was con-
or subcritical temperature of ethanol is much lower than water. ducted for 3 times for accuracy. Finally the products were analyzed
However, few studies have been done on subcritical ethanol by UV–vis spectrophotometry.
extraction of flavonoids from M. oleifera leaf.
There are many factors influencing the subcritical extraction 2.4. Subcritical extraction of flavonoids
process, and the purpose of the present study was to find an
optimal condition to extract flavonoids by large-scale subcritical 60.0 g dried powder of M. oleifera leaf was dissolved by 1.5 L
ethanol extraction and investigate antioxidant ability of the ethanol. Then the mixture was introduced into a gas-cooled fast
extracts. Response surface methodology (RSM) was used to opti- (GCF) reactor (shown in Fig. S1) to extract flavonoids, and a vac-
mize process conditions. The antioxidant property of the extracts uum pump was used to evacuate the air in the reactor. Heating
was investigated by FRAP and DPPH assay. capacity was fixed at 2000 W to heat up the mixture at the begin-
ning, and then turned to 200 W to maintain the temperature. And
the pressure was approximately equaled to the saturated vapor
2. Experimental procedures
pressure of over-heated ethanol solution due to the high vacuum
at the beginning. The extracts were filtered, and the solvent was
2.1. Materials
evaporated at 50 °C under vacuum. UV–vis spectrophotometry
was used to analyze the products. Three factors were considered
Moringa oleifera leaf was obtained from Guangxi province,
to be the most important in this extraction process. Therefore we
China. Rutin and oligomeric proantho cyanidins (OPC) standards
conducted a set of single factor analysis. Flavonoids were extracted
were purchased from J&K Scientific Co. (Beijing, China), and the
with different concentrations of ethanol (55%, 70%, 85% and 100%)
purity was P98% and 99% respectively according to the manufac-
for a given time ranging from 1 to 2.5 h, while the extraction
turer. 2,4,6-tri(2-pyridyl)-1,3,5-triazine (TPTZ) and 2,2-diphenyl-1-
temperature ranged from 110 to 140 °C.
picrylhydrazyl radical (DPPH) were purchased from J&K Scientific
Co. (Beijing, China). Iron (III) chloride hexahydrate (FeCl36H2O)
2.5. UV–vis spectrophotometry analysis
and ferrous sulfate (FeSO4) were obtained from Bodi Chemical
Reagents Co. (Tianjin, China). All other solvents and chemicals
A standard solution (60 lg/mL) of rutin was prepared. The
were obtained from Jiangtian Chemical Reagents Co. (Tianjin,
solvent used in this process was ethanol–water (60:40, v/v). And
China) and were analytical grade.
then 1, 2, 3, 4 and 5 mL rutin solutions were removed in five
volumetric flasks (10 mL) respectively. Next, we added 2 mL of
2.2. HPLC–MS conditions for identification of flavonoids AlCl3 (0.1 mol/L) solution and 3 mL of CH3COONa (1 mol/L) solu-
tion, waiting for 5 min, followed by adding ethanol–water (60:40,
Before the HPLC–MS analysis, a purification process was con- v/v) solvent to the scale. The sample solution without coloration
ducted by the following procedure to enrich the antioxidant com- was used as a reference. Determination wavelength of 420 nm
pounds. A column (30 mm 500 mm) was packed with 150 g was used to analyze the samples. Results were used to draw the
D101 macroporous adsorbent resin, and washed with 300 mL rutin standard curve.
ethanol, 5% HCl, 2% NaOH respectively. After each wash, water 1 mL of the extracts were removed and diluted to 10 mL in a
was used to eliminate the residual ethanol, HCl or NaOH. An volumetric flask (10 mL) by ethanol–water (60:40, v/v) solvent.
extract tank containing 600 mL extracts was fixed on the top of Then 1 mL of this solution was colorated and analyzed, using the
the column to prepare the purification process. When the purifica- method stated above. The extraction yield Y1 (mg RE/g) which
tion was performed, a pump was used to supply the extracts to the meant milligrams of rutin equivalent from 1 g M. oleifera leaf was
tank and keep the volume of extracts fixed in the tank, and the flow calculated as the following Eq. (1), where C (lg/mL) was the flavo-
rate of extracts was 1.5 mL/min. The purification process was noid concentration calculated by rutin standard curve, V (mL) was
lasted for 8 h, and then the purified extracts were analyzed by the volume of the extracts, M (g) was the mass of M. oleifera leaf
the HPLC–MS analysis. used in extraction process. And the extraction yield Y (%) could
The compounds were separated on a C18 column be calculated as the following Eq. (2). For simplicity, we used Y
(150 mm 4.60 mm) operated at 25 °C with the elution solvents as the extraction yield in the following text.
A (0.1% formic acid in water) and B (0.1% formic acid in acetoni-
trile). In addition, a flow-rate of 1.5 mL/min for the following
Y 1 ¼ ð100 C V 103 Þ=M ð1Þ
gradient: 10–30% B in 20 min and 30% B in 20–40 min was
performed to conduct the HPLC separation.
To identify the separated compounds, an electrospray ion mass Y ¼ Y 1 103 100% ð2Þ
spectrometer (ESI-MS) was used under positive ion mode and
scanned from m/z 100 to 1000. Detailed conditions were as follows,
2.6. Experimental design and statistical analysis
needle voltage at 3.5 kV, capillary temperature at 350 °C, nitrogen
as the drying gas at 12 L/min and 350 °C and nebulizer pressure at
In the present study, we used Design Expert Version 8.0 soft-
40 psi.
ware as a design and analysis tool to conduct experiments. Regres-
sion coefficients, significance of the process variables, conformity
2.3. Reflux extraction of flavonoids of the experimental data to models and optimal response variables
can be obtained by using this software. Response variable was
A round-bottom flask and an attached reflux condenser were predicted by a quadratic model shown as the following Eq. (3)
used to conduct this experiment. 10.0 g dried powder of M. oleifera
leaf was extracted with 200 mL of 90% ethanol for 3 h at a X
3 X
3 X
2 X
3
controlled temperature, and heating capacity was fixed at 500 W Y ¼Aþ Bi X i þ C ii X 2i þ C ij X i X j ð3Þ
i¼1 i¼1 i¼1 j¼iþ1
to maintain the temperature. Then the extracts were filtered, the
154 Y. Wang et al. / Food Chemistry 218 (2017) 152–158
where Y was the predicted dependent variable, Xi were the indepen- where A was the absorbance of the sample, C (lg/mL) was the
dent variables, A was the constant coefficient, Bi were the linear concentration of rutin. And R2 of this equation was 0.9997.
regression coefficients, Cij were the interaction effect terms, and
Cii were the quadratic effect terms, respectively. A ¼ 33:6574 C 0:45632 ð5Þ
A three-factor RSM was conducted in this study to investigate The extraction yield was calculated by Eq. (1), and the volume
the relationship between the response variables and process of extracts was about 100 mL in the reflux extractions while
variables, and optimize the extraction process conditions. 10.67 g M. oleifera leaf was used. And the absorbances of the three
Concentration of ethanol (X1: 55%–85%), extraction temperature experiments were 0.666, 0.667 and 0.664. According to the Eqs. (5)
(X2: 120–140 °C) and extraction time (X3: 1.5–2.5 h) were chosen and (1), the average extraction yield which was 20.6 mg RE/g
as independent or process variables, while response variable was (2.06%) could be obtained.
the extraction yield (Y) of flavonoids. After optimization, each vari-
able was coded at three levels of 1, 0, +1 as shown in Table S1.
The accuracy of the model was investigated by the regression 3.2. Single factor analysis
analysis (R2). And F-test was conducted to analyze the significance
of the model terms. The response surface plots and contour plots There are many factors affecting the extraction yield, among
were used in combination to show how the process variables affect which the ethanol concentration, extraction time, and extraction
the response variables. temperature are the main factors. Single factor analysis was
performed with one factor changed and the others kept unvaried.
2.7. Antioxidant assay The extraction by different ethanol concentrations (55%, 70%,
85%, 100%) was investigated, while the other conditions were kept
The total antioxidant activity of the extracts and standards was the same (extraction temperature was 140 °C, and extraction time
determined by ferric reducing antioxidant power (FRAP) assay, and was 2 h). And the extraction by extraction time of 1, 1.5, 2 and 2.5 h
FeSO4 solution was used as a standard to compare with the was investigated, while the other conditions were kept the same
extracts. The antioxidant activity was also determined by (extraction temperature was 130 °C, and ethanol concentration
2,2-diphenyl-1-picrylhydrazyl radical (DPPH) assay to investigate was 100%). Finally, the extraction by extraction temperature of
the free radical scavenging activity of the extracts, and the 110, 120, 130 and 140 °C was investigated, while the other condi-
oligomeric proantho cyanidins (OPC) was used as a standard to tions were kept the same (extraction time was 2 h, and ethanol
compare with the extracts. concentration was 100%). The detail experimental conditions and
results were shown in Table. S2.
As shown in Fig. 1, the extraction efficiency was the highest
2.7.1. FRAP assay
when the extraction temperature was 130 °C. When the tempera-
FRAP reagent included 50 mmol/L acetate buffer which con-
ture was higher, the extraction efficiency decreased. It could
tained 20.4 g C2H3NaO2 and 80 mL C2H4O2 per liter; 10 mmol/L
speculate that some of the heat-sensitive components in M. oleifera
TPTZ (2,4,6-tripyridyl-s-triazine) solution in which 40 mmol/L
leaf decomposed at higher temperature. The extraction efficiency
HCl solution was used as the solvent; 20 mmol/L FeCl36H2O. FRAP
increased following the increase of extraction time and reached a
reagent was obtained by mixing 100 mL acetate buffer, 10 mL TPTZ
peak at 2 h, and then significantly decreased. A long treatment
solution, and 10 mL FeCl36H2O solution (Benzie & Strain, 1996).
time was also adverse to this process. We found that there was also
0.1 mL different amounts of FeSO4 solutions or extracts were
a best ethanol concentration existing in this extraction process,
added into 6 mL FRAP reagent, and then put in a 37 °C water bath
which was 70%. Therefore, center point (all variables were
for 30 min. Determination wavelength of 593 nm was used to
coded as zero) of RSM was 130 °C (extraction temperature), 2 h
monitor the absorbance of samples.
(extraction time), 70% (ethanol concentration).
2.7.2. DPPH assay
60 lM DPPH was dissolved in 3 mL ethanol, and then 0.5 mL
different amounts of extracts or OPC were added. Blank experi-
ments which contained 0.5 mL of 99% ethanol instead of the
extract were also conducted. The absorbances were monitored at
517 nm (Cervato et al., 2000). The inhibition rate of DPPH free
radical (IR) was calculated by Eq. (4) (Guil-Guerrero, Martínez-
Guirado, del Mar Rebolloso-Fuentes, & Carrique-Pérez, 2006),
where A0 was the absorbance of the blank experiments and As
was the absorbance in the presence of the samples.
IR ¼ ðA0 As Þ=A0 100% ð4Þ
When the IR is 50%, the corresponding concentration is called
IC50. Therefore, the value of IC50 was obtained by fitting the sample
concentration and the inhibition rate (Song, Zhang, Zhang, & Wang,
2010).
3. Results and discussion
3.1. Standard curve of rutin and results of reflux extraction
The rutin standard curve was drawn by plotting the concentra-
tion of rutin standard solution versus the corresponding absor- Fig. 1. Effects of extraction temperature, extraction time and ethanol concentration
bency, as shown in Fig. S2. The regression equation was Eq. (5), on yield of flavonoids.
Y. Wang et al. / Food Chemistry 218 (2017) 152–158 155
Table 1 3.3. Extraction model and statistical analysis
Analysis of variance (ANOVA) for the developed second order polynomial models.
Source Sum of squares Mean square F-value P-value Box-Behnken design (BBD) used in the present work was a three
Model 1.06 0.12 26.23 0.0004 factorial design with three levels that consisted of 16 runs to obtain
X1 0.018 0.018 4.03 0.0915 an optimal process condition. The experimental conditions and
X2 0.12 0.12 27.06 0.0020 flavonoids extraction yield results were shown in Table S3.
X3 0.037 0.037 8.19 0.0288 Additionally, an analysis of variance (ANOVA) was conducted and
X1X2 0.11 0.11 25.27 0.0024
X1X3 0.034 0.034 7.66 0.0325
the regression model was summarized in Table 1. By multiple
X2X3 0.0094 0.0094 2.09 0.1982 regression analysis, the following second-order polynomial could
X21 0.16 0.16 34.77 0.0011 be obtained.
X22 0.33 0.33 74.26 0.0001
X23 0.24 0.24 52.73 0.0003 Y ¼ 2:59 0:048X 1 0:12X 2 þ 0:068X 3 0:17X 1 X 2
þ 0:093X 1 X 3 þ 0:049X 2 X 3 0:2X 21 0:29X 22 0:24X 23 ð6Þ
Fig. 2. Three-dimensional (3D) response surface and contour plot curve illustrating combined effects of (a) extraction time and ethanol concentration (b) extraction time and
extraction temperature (c) extraction temperature and ethanol concentration on extraction yield.
156 Y. Wang et al. / Food Chemistry 218 (2017) 152–158
As we can see in Table 1, F value of the model was 26.23 while Table 2
the P value was only 0.0004, which showed a high significance of Identified flavonoid compounds of Moringa oleifera leaf extracts.
the model. For a good accuracy of a model, R2 must be more than Peak tR (min) MS (m/z) MS fragment Identities
75% (Chauhan & Gupta, 2004). Thus the model stated above with ion (m/z)
a relatively high coefficient of determination value (R2 = 0.9752) a 13.4 595.2 303 Quercetin-diR
illustrated that almost all extraction data could be explained by b 18.8 465.1 303 Quercetin-G
this model. Therefore, using this model to predict the influence c 21.0 449.1 287 Kaempferol-G
d 21.6 507.1 303 Quercetin-G-Ac
of the process variables on the extraction yield was reasonable e 23.4 507.1 303 Quercetin-G-Ac
and reliable. The quadratic variable X22 was statistically very signif- f 24.4 491.1 287 Kaempferol-G-Ac
icant because the P value was lower than 0.0001; two-variable g 27.1 533.1 303 Quercetin-Xyl/Api-S
interaction X1X2, linear variable X2, quadratic variables X21 and X23 h 27.8 517.1 287 Kaempferol-Xyl/Api-S
had significant influences (P < 0.01) on the extraction yield of flavo- diR: dirhamnosyl, G: Glucosyl/Galactosyl moiety, Ac: Acetyl, Xyl: Xylosyl, Api:
noids; linear variable X3 and two-variable interaction X1X3 had Apiosyl, S: Succinoyl.
influences (P < 0.05) on the extraction process, whereas the linear
variable X1 and two-variable interaction X2X3 had no significant
influence (P > 0.1) on the extraction yield of flavonoids. The linear
and quadratic coefficients of each process variable indicated that glycosides in the Moringa oleifera leaf extracts are quercetin and
extraction temperature had more influence on the extraction yield kaempferol. According to Figs. S3 and S4, eight flavonoid
than extraction time, while extraction time was more significant compounds were observed in the extracts, and the details of the
than ethanol concentration. compounds were listed in Table 2.
Experiment under the predicted optimal conditions was con-
3.4. Response surface analysis ducted for three times to obtain a mean value of extraction yield.
By comparing the experimental and predicted extraction yield,
The effects of the process variables and their mutual interac- the model could be verified. Adjusted extraction conditions were
tions on the extraction yield can be investigated by the response 70% of ethanol concentration, 126.6 °C of extraction temperature
surface plots and their contour plots. And the interactions between and 2.05 h of extraction time. The obtained result (2.60%) was
the process variables are significant while the shape of contour closed to the above prediction and showed that the experimental
plots is elliptical (Muralidhar, Chirumamila, Marchant, & Nigam, values had a great agreement with the predictive values. Therefore,
2001). the optimal extraction conditions obtained by RSM were accurate,
The effects of extraction time and ethanol concentration on
extraction yield were shown in Fig. 2(a) while extraction tempera-
ture was set at 130 °C. Fig. 2(a) indicated that the interaction
between extraction time and ethanol concentration had a great
impact on the yield of flavonoids, and extraction time had a more
significant influence than ethanol concentration. The flavonoids
yield increased as the extraction time increased from 1.5 to
approximately 2.1 h, and then dropped as the extraction time
increased from about 2.1–2.5 h, when ethanol concentration was
at a certain value (70%). It was not significant that the increase of
the ethanol concentration affected the extraction yield at a certain
extraction time, but there was still a peak of the extraction effi-
ciency while ethanol concentration was about 70%.
As shown in Fig. 2(b), in which ethanol concentration was 70%,
both extraction time and extraction temperature had obvious
impact on extraction efficiency, but the interaction had no signifi-
cant impact. When extraction time was 2 h, Fig. 2(c) showed
extraction yield obviously increased with temperature raised while
ethanol concentration had no significant effect on the yield. In
addition, the interaction between ethanol concentration and
extraction temperature had a significant impact on the yield of fla-
vonoids. All the results were in good agreement with our findings
in the ANOVA.
3.5. Optimization of extraction and identification of flavonoids
According to the analysis of response surface, the optimum con-
dition was obtained: ethanol concentration, 69.9%; extraction tem-
perature, 126.6 °C; extraction time, 2.06 h. Under the above
condition, the estimated value for Y was obtained, which was
2.61%. The extracts obtained from the optimum condition were
purified by D101 macroporous adsorbent resin as stated in the
chapter 2.2, and analyzed by HPLC–MS. The chromatogram of
HPLC–MS was shown in Fig. S3 while the MS spectrums were
shown in Fig. S4. In Fig. S4, two main fragment ion peaks (287,
303) were obviously observed which represent kaempferol and
quercetin glycosides respectively. Therefore, the main flavone Fig. 3. Total antioxidant activity of (a) extracts and (b) FeSO4 solutions.
Y. Wang et al. / Food Chemistry 218 (2017) 152–158 157
reliable, and efficient. Extraction yield of subcritical extraction (IR%) and IC50 to characterize the free radical scavenging activity
increased about 26.70% compared with the traditional ethanol of extracts. The value of IR increased with the concentration added.
reflux method. Most importantly, this subcritical extraction pro- For extracts from M. oleifera leaf, the IC50 value was 0.7440 mg/L,
cess only needed 4 h (including heating time) to reach the optimal while the IC50 value of OPC was 0.0195 mg/L. Therefore, the free
yield which was 2.60% while the traditional method needed nearly radical scavenging activity of extracts from 1 mg M. oleifera leaf
11 h to reach a yield of 2.06%. And we also observed the power was approximately equivalent to that of 0.026 mg OPC.
consumption of traditional method totaled 4.6 kWh while
2.4 kWh in the subcritical ethanol extraction process.
4. Conclusions
3.6. Antioxidant activity In the present study, RSM in combination with three-factor and
three-level BBD was successfully applied to study and optimize the
3.6.1. FRAP assay process variables for the subcritical ethanol extraction of flavo-
As shown in Fig. 3, FeSO4 solution was used as a standard to noids from M. oleifera leaf. According to the HPLC–MS analysis,
evaluate the total antioxidant activity of extracts. The figure quercetin and kaempferol glycosides were found in the extracts.
showed a linear correlation between the concentration of FeSO4 The experiment results showed that, extraction time and extrac-
solution or extracts and the absorbance, and the concentration of tion temperature had significant effects on the extraction yield.
extracts was expressed as milligrams of M. oleifera leaf per liter. Analysis of variance (ANOVA) showed a high coefficient of deter-
Furthermore, the total antioxidant activity of extracts from 1 mg mination value (R2 > 0.95). Therefore, the mathematical model
M. oleifera leaf approximately equaled the total antioxidant activity developed by Box-Behnken design can be used to predict the
of 0.95–1.35 mmol FeSO4. extraction efficiency of flavonoids. Under the optimal conditions
(extraction temperature: 126.6 °C, extraction time: 2.05 h, ethanol
3.6.2. DPPH assay concentration: 70%), the experimental result was 2.60% and shown
As a stable and well-characterized solid radical source, DPPH is to be in agreement with the predicted one. And the subcritical
a traditional and perhaps the most popular free radical used for extraction used only 4 h to reach the optimal result, while the
free radical scavenging activity assay (Arabshahi-Delouee & traditional ethanol reflux method needed nearly half a day to
Urooj, 2007). OPC, with the strong ability to scavenge free radical, obtain a yield of 2.06%. The traditional method spent twice as
was used as the standard to evaluate the free radical scavenging much energy as subcritical extraction did. Subcritical ethanol
activity of extracts. As shown in Fig. 4, we used inhibition rate extraction will have great potential use in industry. In addition,
antioxidant assays showed that the extracts had strong antioxidant
ability, and extracts from 1 mg M. oleifera leaf approximately
equaled 0.95–1.35 mmol FeSO4 or 0.026 mg OPC.
Acknowledgement
This work was financially supported by the National Natural
Science Foundation of China (Grant No. 21376166).
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.foodchem.2016.
09.058.
References
Abdulkarim, S. M., Long, K., Lai, O. M., Muhammad, S. K. S., & Ghazali, H. M. (2005).
Some physico-chemical properties of Moringa oleifera seed oil extracted using
solvent and aqueous enzymatic methods. Food Chemistry, 93(2), 253–263.
Albu, S., Joyce, E., Paniwnyk, L., Lorimer, J. P., & Mason, T. J. (2004). Potential for the
use of ultrasound in the extraction of antioxidants from Rosmarinus officinalis
for the food and pharmaceutical industry. Ultrasonics Sonochemistry, 11(3–4),
261–265.
Alhakmani, F., Kumar, S., & Khan, S. A. (2013). Estimation of total phenolic content,
in-vitro antioxidant and anti-inflammatory activity of flowers of Moringa
oleifera. Asian Pacific Journal of Tropical Biomedicine, 3(8), 623–627. discussion
626–627.
Anwar, F., Latif, S., Ashraf, M., & Gilani, A. H. (2007). Moringa oleifera: A food plant
with multiple medicinal uses. Phytotherapy Research, 21(1), 17–25.
Arabshahi-Delouee, S., & Urooj, A. (2007). Antioxidant properties of various solvent
extracts of mulberry (Morus indica L.) leaf. Food Chemistry, 102(4), 1233–1240.
Benzie, I. F., & Strain, J. J. (1996). The ferric reducing ability of plasma (FRAP) as a
measure of ‘‘antioxidant power”: The FRAP assay. Analytical Biochemistry, 239
(1), 70–76.
Cervato, G., Carabelli, M., Gervasio, S., Cittera, A., Cazzola, R., & Cestaro, B. (2000).
Antioxidant properties of oregano (Origanum vulgare) leaf extracts. Journal of
Food Biochemistry, 24(6), 453–465.
Chauhan, B., & Gupta, R. (2004). Application of statistical experimental design for
optimization of alkaline protease production from Bacillus sp. RGR-14. Process
Biochemistry, 39(12), 2115–2122.
Dai, J., & Mumper, R. J. (2010). Plant phenolics: Extraction, analysis and their
Fig. 4. Scavenging activity of (a) extracts and (b) OPC on DPPH free radical. antioxidant and anticancer properties. Molecules, 15(10), 7313–7352.
158 Y. Wang et al. / Food Chemistry 218 (2017) 152–158
Guil-Guerrero, J. L., Martínez-Guirado, C., del Mar Rebolloso-Fuentes, M., & Moyo, B., Masika, P. J., Hugo, A., & Muchenje, V. (2011). Nutritional characterization
Carrique-Pérez, A. (2006). Nutrient composition and antioxidant activity of 10 of Moringa (Moringa oleifera Lam.) leaf. African Journal of Biotechnology, 10(60),
pepper (Capsicum annuum) varieties. European Food Research and Technology, 12925–12933.
224(1), 1–9. Muralidhar, R. V., Chirumamila, R. R., Marchant, R., & Nigam, P. (2001). A response
Hertog, M. G., Feskens, E. J., Hollman, P. C., Katan, M. B., & Kromhout, D. (1993). surface approach for the comparison of lipase production by Candida cylindracea
Dietary antioxidant flavonoids and risk of coronary heart disease: The Zutphen using two different carbon sources. Biochemical Engineering Journal, 9(1), 17–23.
Elderly Study. Lancet (London, England), 342(8878), 1007–1011. Rostagno, M. A., Palma, M., & Barroso, C. G. (2007). Microwave assisted extraction of
Jo, E.-K., Heo, D.-J., Kim, J.-H., Lee, Y.-H., Ju, Y.-C., & Lee, S.-C. (2013). The effects of soy isoflavones. Analytica Chimica Acta, 588(2), 274–282.
subcritical water treatment on antioxidant activity of golden oyster mushroom. Shi, J., Nawaz, H., Pohorly, J., Mittal, G., Kakuda, Y., & Jiang, Y. (2005). Extraction of
Food and Bioprocess Technology, 6(9), 2555–2561. polyphenolics from plant material for functional foods—Engineering and
Kansal, S. K., & Kumari, A. (2014). Potential of M. oleifera for the treatment of water technology. Food Reviews International, 21(1), 139–166.
and wastewater. Chemical Reviews, 114(9), 4993–5010. Singh, N., & Rajini, P. S. (2004). Free radical scavenging activity of an aqueous extract
Manach, C., Scalbert, A., Morand, C., Remesy, C., & Jimenez, L. (2004). Polyphenols: of potato peel. Food Chemistry, 85(4), 611–616.
Food sources and bioavailability. American Journal of Clinical Nutrition, 79(5), Song, H., Zhang, Q., Zhang, Z., & Wang, J. (2010). In vitro antioxidant activity of
727–747. polysaccharides extracted from Bryopsis plumosa. Carbohydrate Polymers, 80(4),
Maran, J. P., Manikandan, S., Priya, B., & Gurumoorthi, P. (2015). Box-Behnken 1057–1061.
design based multi-response analysis and optimization of supercritical carbon Vongsak, B., Sithisarn, P., & Gritsanapan, W. (2014). Simultaneous HPLC quantitative
dioxide extraction of bioactive flavonoid compounds from tea (Camellia sinensis analysis of active compounds in leaves of Moringa oleifera Lam.. Journal of
L.) leaf. Journal of Food Science and Technology-Mysore, 52(1), 92–104. Chromatographic Science, 52(7), 641–645.
Matshediso, P. G., Cukrowska, E., & Chimuka, L. (2015). Development of pressurised Zhang, H.-F., Yang, T.-S., Li, Z.-Z., & Wang, Y. (2008). Simultaneous extraction of
hot water extraction (PHWE) for essential compounds from Moringa oleifera leaf epimedin A, B, C and icariin from Herba Epimedii by ultrasonic technique.
extracts. Food Chemistry, 172, 423–427. Ultrasonics Sonochemistry, 15(4), 376–385.
Morton, J. (1991). The horseradish tree, Moringa pterygosperma (Moringaceae)—A
boon to Arid Lands? Economic Botany, 45(3), 318–333.
