Food Chemistry 300 (2019) 125162

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Food Chemistry
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Simultaneous extraction and separation of oil, proteins, and glucosinolates T

from Moringa oleifera seeds
⁎
Rui Chena, Xiu-Juan Wanga, Yao-Yuan Zhanga, Yan Xinga, Liu Yanga, He Nia, Hai-Hang Lia,b,
a
Guangdong Provincial Key Lab of Biotechnology for Plant Development, School of Life Sciences, South China Normal University, Guangzhou 510631, China
b
School of Life Sciences, Huizhou University, Huizhou 516007, China

A R T I C LE I N FO A B S T R A C T

Keywords: Moringa oleifera is a worldwide cultivated edible and medicinal plant. Its seeds are rich in oil, proteins, and
Moringa oleifera seeds glucosinolates. A practical method was developed to simultaneously extract and separate the three groups of
Oil substances from M. oleifera seeds. Smashed seed material was loaded into columns with petroleum ether: ethanol
Proteins 8:2 (PE-ethanol) and eluted sequentially with 4.8-fold PE-ethanol to extract oil, and 10.8-fold water to extract
Glucosinolates
proteins and glucosinolates. More than 95% of oil, proteins, and glucosinolates were extracted. The extracts were
Extraction
Separation
separated automatically into ether (oil) phase and ethanol aqueous phase. The latter was further separated into
proteins and glucosinolates by 70% ethanol precipitation. The main glucosinolate was identified by LC-MS as
GLC (4-α-rhamnopyranosyloxy-benzyl glucosinolate). After purification, 22.3 g refined oil, 33.0 g proteins, and
5.5 g purified GLC from 100 g M. oleifera seeds were obtained. This study provides a simple and high-efficient
method to utilize M. oleifera seeds.

1. Introduction Jusoh, & Endut, 2016; Medeiros et al., 2018). Moringa glucosinolates
are a group of glucosinolats comprising a benzene ring with two
Moringa oleifera is an important woody food and medicinal plant rhamnose moieties attached. There are four kinds of glucosinolates in
cultivated widely in the world (Anwar, Latif, Ashraf, & Gilani, 2007; M. oleifera which are unique and important functional components of
Palafox et al., 2012; Vaknin & Mishal, 2017). The whole plant, in- M. oleifera, the main glucosinolate in M. oleifera seeds is GLC (4-α-
cluding seeds, leaves, stems, and roots, is rich in nutrients and can be rhamnopyranosyloxy-benzyl glucosinolate). Moringa glucosinolates
used for food (Gopalakrishnan, Doriya, & Kumar, 2016; Mahmood, have anti-inflammatory, anticancer, and hypoglycemic potential, and
Mugal, & Haq, 2010). The plant produces more than 5 kg dry seeds per can be used in phytopharmaceutical, nutraceutical and healthy food
plant or 24 tons of seeds per hectare annually (Ndubuaku, Ndubuaku, & products (Bennett et al., 2003; Förster, Ulrichs, Schreiner, Müller, &
Ndubuaku, 2014), with great economical value. Mewis, 2015; Jaja-Chimedza et al., 2018; Ma, Ahmad, Zhang, Khan, &
It is reported that M. oleifera seeds contain 19–47% oil, 10–52% Muhammad, 2019; Melchini et al., 2013; Prakash & Gupta, 2012; Rajan
proteins, and 2.5–20% glucosinolates. Moringa oil, which has more et al., 2016; Santos et al., 2018).
than 70% unsaturated fatty acids and is rich in oleic acid, has been used Several methods are available for Moringa oil extraction, including
as a high-quality vegetable oil and in traditional medicine for the solvent extraction, cold-pressing, supercritical fluid extraction, and
treatment of arthritis, rheumatism, and hypertension (Aviara, Musa, aqueous enzymatic extraction (Abdulkarim, Long, Lai, Muhammad, &
Owolarafe, Ogunsina, & Oluwole, 2015; Bhuptawat, Folkard, & Ghazali, 2005; Bhuptawat et al., 2007; Bhutada, Jadhav, Pinjari,
Chaudhari, 2007; Fernandes et al., 2015; Gopalakrishnan et al., 2016; Nemade, & Jain, 2016; Fakayode & Ajav, 2016; Yusoff, Gordon, &
Mahmood et al., 2010; Ojiako & Okeke, 2013; Palafox et al., 2012; Niranjan, 2015; Yusoff, Gordon, Ezeh, & Niranjan, 2016; Zhao & Zhang,
Rashid, Anwar, Moser, & Knothe, 2008; Ruttarattanamongkol, 2013, 2014; Zhong et al., 2018). Solvent extraction has some ad-
Siebenhandl-Ehn, Schreiner, & Petrasch, 2014). Moringa proteins, vantages, while cold-pressing, supercritical fluid extraction, and aqu-
which have anti-microbial potential, can be made into food, beverage, eous enzymatic extraction are limited due to low yield, high cost, and
and animal feed (Baptista et al., 2017; Bhuptawat et al., 2007; Broin long processing time (Sánchez-Machado et al., 2015; Yusoff et al.,
et al., 2002; Dezfooli et al., 2016; Ghebremichael, Gunaratna, 2015). Proteins in M. oleifera seeds are water soluble and can be ef-
Henriksson, Brumer, & Dalhammar, 2005; Hamid, Lananan, Khatoon, fectively extracted by water and aqueous salts (Baptista et al., 2017;

⁎
Corresponding author at: School of Life Sciences, South China Normal University, Guangzhou 510631, China.
E-mail address: li_haihang@[Link] (H.-H. Li).

[Link]
Received 15 December 2018; Received in revised form 30 June 2019; Accepted 8 July 2019
Available online 09 July 2019
0308-8146/ © 2019 Elsevier Ltd. All rights reserved.
R. Chen, et al. Food Chemistry 300 (2019) 125162

Dezfooli et al., 2016; Ghebremichael et al., 2005). Bennett et al. (2003) 15% separating gels), and electrophoresed at 80 voltage for the first
analyzed glucosinolates in vegetative and reproductive tissues of M. 30 min and then at 120 voltage for another 1.5 h. The proteins on SDS-
oleifera, and Förster et al. (2015) developed a reliable extraction and PAGE gels were stained by Coomassie blue after electrophoresis.
quantification method for the intact glucosinolates in M. oleifera.
However, these studies are mainly for analytic purposes and are for a 2.4. Analyses of glucosinolates
single group of substances, practical methods for resource utilization of
M. oleifera seeds are rarely reported. Moringa glucosinolates were analyzed in Shimadzu SPD-20A HPLC
In this study, we developed a high-efficient column chromato- system (Shimadzu, Japan) with LC-20AT UV detector at 229 nm and
graphic extraction (CCE) method (Han et al., 2014; Hu et al., 2015) YMC-packed ODS column (250 mm × 4.6 mm, 5 μm), according to the
with gradient elution (Han et al., 2014) to simultaneously extract oil, method of Förster et al. (2015), but using a different HPLC system and
proteins, and glucosinolates in M. oleifera seeds for comprehensive analytical conditions (including column and mobile phase). The column
utilization of the medicinal and edible plant resource. The extracts were was eluted at a flow rate of 1.0 mL/min using a binary gradient pro-
simply separated into oil, proteins, and GLC. Purified products of re- gram with solvents A (40% acetonitrile in 0.1 M ammonium acetate)
fined oil, proteins, and GLC from M. oleifera seeds were obtained at very and B (0.1 M ammonium acetate): 0–2 min, 0–1% A; 2–20 min, 1–50%
low costs. A; 20–24 min, 50–100% A; 24–26 min, 100% A; 26–27 min, 100–1% A;
and 27–35 min, 1–0% A. All samples were filtered through a 0.45 μm
2. Materials and methods membrane filter before injection into HPLC.
As Moringa glucosinolate compounds are not available, Sinigrin
2.1. Material and reagents (Yuanye, China), a structural analogue of Moringa glucosinolates from
rapeseed, was used as an external standard for quantitative analysis of
Dried M. oleifera seeds, imported from India, were purchased from Moringa glucosinolates. A HPLC quantitative curve of Sinigrin amount
Qingping Chinese Medicine Market in Guangzhou, China. The seeds to peak area was prepared using different concentrations of Sinigrin
were dried in a 50 °C oven for 4 h, hulled and ground. Material between (see Results and discussion section). Glucosinolate contents in M. olei-
20 and 60 mesh was used for the experiments. fera seeds were determined by peak area based on the Sinigrin standard
Protein molecular marker was purchased from Thermo (Waltham, curves.
MA, USA). Coomassie brilliant blue R-250 and mixed standard of fatty The main glucosinolate from M. oleifera seeds was identified in the
acid methyl ester (FAME) were purchased from Sigma-Aldrich Co. (St. Agilent 6540 UHD Q-TOF LC/MS (Palo Alto, CA, USA). Samples were
Louis, MO, USA). Sinigrin standard was purchased from YuanYe separated in a SB-Aq column (100 mm × 2.1 mm, 1.8 µm, Agilent) at
Biotechnology Co. (Shanghai, China). HPLC solvents were purchased 40 °C and eluted at 0.4 mL/min solvent flow rate using a binary gra-
from Burdick & Jackson Inc. (Muskegon, MI, USA). Food-grade ethanol dient program with solvents A (acetonitrile) and B (0.2% formic acid
(95%) was used in all experiments. Other solvents and reagents were and 10 mM formic acid, v/v): 0–5 min, 0–10% A; 5–10 min, 10–50% A.
analytical or food grade, and purchased from local suppliers. The MS parameters: skimmer voltage, 65 V; Nozzle voltage, 500 V; V
Cap, −3500 V/4000 V; nebulizer pressure, 45 psi; drying gas flow, 8 l/
2.2. Oil analysis min; gas temperature, 300 °C. MS spectra was recorded from 100 to
1000 m/z.
The amounts of crude and refined oil were weighed by an electronic
analytical balance with 0.1 mg readability. Thin-layer chromatographic 2.5. Selection of extraction conditions for the column chromatographic
(TLC) analysis of Moringa oil was conducted on pre-coated analytical extraction
silica gel plates (0.25 mm thickness). The loaded plates were treated
with petroleum ether: ethyl acetate: glacial acetic acid ([Link].1) and For selection of the best extraction solvent to dissolve oil, proteins,
stained in iodine vapor. Acid value and peroxide value of the refined oil and glucosinolates in M. oleifera seed material, 1.0 g of dried material
were analyzed by the methods of China National Standard GB5009.229- was added into 5.0 mL different extraction solvents. A series of petro-
2016 and GB5009.227-2016, respectively. leum ether: ethanol (PE-ethanol) from 10:0, 8:2, 6:4, 4:6, 2:8 to 0:10
Fatty acid composition of refined oil was analyzed according to the were used to extract oil. And ethanol: water from 10:0, 8:2, 6:4, 5:5,
method of China National Standard GB 5009.168-2016. The fatty acids 4:6, 2:8 to 0:10 were used to extract proteins and glucosinolates. All
of refined oil were converted to their corresponding methyl esters in extracted solutions were incubated at 30 °C water bath for two hours
KOH-methanol solution (Zhong et al., 2018) and analyzed with Agilent with shaking once every 10 min by hands. After centrifugation at 5000g
7890B-5977B GC–MS (Palo Alto, CA, USA) equipped with capillary for 10 min, the solutions were obtained and analyzed.
column (10 m × 0.25 mm, 0.2 µm). The injector and detector tem- The solvent volume for the material fully absorbed was defined as
peratures were 270 °C and 280 °C, respectively. The column tempera- the minimum volume (MV), which was used as a basic volume unit to
ture was set at 100 °C for the first 13 min, then programmed as follows: monitor the extraction process in the column chromatographic extrac-
rose from 100 °C to 180 °C at a rate of 10 °C/min, held at 180 °C for tion (CCE). To determine the MVs solvent for oil extraction and for
6 min, rose from 180 °C to 200 °C at a rate of 1 °C/min, held 200 °C for proteins and glucosinolates extraction, 1.0 g of seed material was added
20 min, rose from 200 °C to 230 °C at a rate of 4 °C/min and held 230 °C into 5.0 mL extraction solvents, incubated at 30 °C water bath for dif-
for 10.5 min. The carrier gas was nitrogen at a flow rate of 1.0 mL/min ferent hours (1–4 h) with shaking once every 10 min by hands, and then
with a split ratio of 100:1. centrifuged at 5000g for 10 min to separate the solvent. The minimum
dissolution time was determined by analyzing target substances in the
2.3. Proteins analysis solvents at different time points.

Moringa protein concentration was determined by the method of 2.6. Column chromatographic extraction
Bradford (Bradford, 1976) with Coomassie Plus Protein Assay using
bovine serum albumin as the protein standard. Proteins were analyzed Column chromatographic extraction (CCE) includes dissolving the
by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS- target compounds in the material with the optimal solvent and eluting
PAGE) in a Bio-Rad Mini Gel system, according to the method of them from the material in chromatographic columns (Han et al., 2014;
Laemmli (Laemmli, 1970). Samples were mixed with glycerol and Hu et al., 2015).
bromophenol blue, loaded onto a polyacrylamide gel (5% stacking and For independent CCE, 10 g M. oleifera seed material was loaded into

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R. Chen, et al. Food Chemistry 300 (2019) 125162

a column with 1.0 MV extraction solvent, namely 2.4-fold PE-ethanol

(8:2) for oil extraction or 2.7-fold water for proteins and glucosinolates
extraction, at a diameter to bed height ratio of 1:10, by the wet column
preparation method. After standing for certain hours until target com-
pounds dissolved, the column was eluted with the same solvent as
loading at a flow rate of 1.0 MV/h. The eluent was collected in fractions
until all target compounds were extracted. Target compounds in each
fraction were analyzed.
For gradient CCE to simultaneously extract the three groups of
substances (oil, proteins, and glucosinolates), the column was loaded
and eluted with 2.0 MVs PE-ethanol (8:2) for oil extraction, followed by
4.0 MVs water to extract proteins and glucosinolates. The eluent was
collected together as extracts.

2.7. Separation of Moringa oil, proteins, and glucosinolates

The extracts from simultaneous CCE in Section 2.6 were auto-

matically separated into a petroleum ether phase and an ethanol aqu-
eous fraction. The ether phase, which contained Moringa oil, was va-
cuum-concentrated at 40 °C to recover the ether for reuse and crude
Moringa oil was obtained. The ethanol aqueous phase, which contained
proteins and glucosinolates, was separated by adjusting to 70% ethanol
to precipitate proteins. Crude Moringa glucosinolates were obtained by
vacuum-concentration of the ethanol aqueous solution. After separa-
tion, crude oil, proteins, and glucosinolates were obtained.

2.8. Purification of Moringa oil

Crude Moringa oil was refined by deacidification, degumming, and

decolorization (Sánchez-Machado et al., 2015). The crude oil was dis-
acidified by partitioning with equal volume of 1.0 mol/L NaOH at 80 °C
for 10 min. After separation by centrifugation at 5000g for 10 min, the
oil was degummed by washing with equal volume of water at 70 °C for
10 min, followed by centrifugation at 5000g for 10 min. The degummed
oil was decolorized with activated carbon or activated clay. The deco- Fig. 1. Analysis of oil, proteins, and GLC extracted from M. oleifera seeds. TLC
loration efficiency was detected by visible spectrum analysis, and the analysis of Moringa oil extracted with different solvents (A) and before and
yield was calculated. The physicochemical properties and fatty acid after its purification (B); SDS-PAGE analysis of proteins (C); HPLC analysis of
composition of refined oil were analyzed as described in Section 2.2. GLC and its UV spectrum (D); LC-MS spectrum of GLC from M. oleifera seeds,
the main glucosinolate in M. oleifera seeds was identified as GLC (4-α-rham-
nopyranosyloxy-benzyl glucosinolate) (E).
2.9. Purification of Moringa glucosinolates

The main glucosinolate in M. oleifera seeds is GLC (Bennett et al., purification (Fig. 1B). The oil showed a clear spot and was separated
2003; Förster et al., 2015). GLC in aqueous solution was purified by two from other compounds on the TLC, which was used for qualitative
steps, fractionating with N-butanol to remove liposoluble impurities, analysis of the oil during the processes of extraction, separation, and
followed by silica gel column chromatography. The silica gel column purification. As stated in Section 2.2, the amounts of crude and refined
was prepared with ethyl acetate: methanol (10:1). After loading the oil were detected by weighing the samples.
sample (dissolved in minimum volume methanol), the column was The protein concentration was determined based on the quantitative
eluted sequentially with ethyl acetate: methanol 10:1, 5:1, and 2:1, equation (Y = 0.6588x + 0.003, R2 = 0.9972), which had a good
each with 2.0 bed volume (BVs). The eluents were collected in 1.0 BV linear relationship between protein concentration from 0.1 to 1.0 mg/
fractions and analyzed by HPLC. Solutions with purified GLC were mL and A595 absorbance. SDS-PAGE analysis indicated that the proteins
combined together (ethyl acetate: methanol 5:1 and 2:1 eluents) and extracted from M. oleifera seeds were consisted of five main protein
vacuum-concentrated as purified GLC. bands. Most of Moringa proteins had relatively low molecular weights
between 3.4 and 20 kDa (Fig. 1C). This result was consistent to the
2.10. Statistic analysis of results report of Baptista et al. (2017) which indicated 16.5% proteins within
0.9–12.4 kDa and 78.0% proteins with lower than 0.9 kDa in M. oleifera
All quantitative experiments were repeated independently at least seeds.
three times, and the yield or extraction rate of Moringa oil, proteins, As shown in Fig. 1D, the extracts of Moringa glucosinolates had a
and GLC was calculated. The results are given as the averages of three significant UV absorption peak at 229 nm which was used for HPLC
independent repeats ± standard deviation. detection of glucosinolates. HPLC analysis indicated that there was only
one dominant peak in the glucosinolate extracts (Fig. 1D), which had a
3. Results and discussion [M−H]− of m/z 570 and was identified as GLC by LC-MS (Fig. 1E). The
result was consistent to the reports of Bennett et al. (2003) and Förster
3.1. Analyses of Moringa oil, proteins, and glucosinolates et al. (2015), GLC was the main glucosinolate in M. oleifera seeds and
other three glucosinolates were undetectable because of low content.
Fig. 1A and B showed TLC analysis of oil extracted by different The amounts of GLC were determined by peak area based on Sinigrin
ratios of PE-ethanol solvents (Fig. 1A), and the oil before and after standard curves (Y = 3.772X + 0.1651, R2 = 0.9982), which had a

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R. Chen, et al. Food Chemistry 300 (2019) 125162

Fig. 2. Determination of optimal conditions for CCE extraction of Moringa oil (A, C, and E), proteins, and GLC (B, D, and F) from M. oleifera seeds. A and B: solvents
selection for extraction of Moringa oil (A), proteins, and GLC (B); C and D: determination of times for materials to be fully absorbed in PE-ethanol 8:2 (C) and water
(D); E and F: determination of dissolving times of oil in PE-ethanol 8:2 (E) and proteins and GLC in water (F).

good linear relationship between Sinigrin concentration from 0.5 to As shown in Fig. 2C and D, the MVs of PE-ethanol 8:2 for oil ex-
3.0 mg/mL and peak area. traction and water for proteins and GLC extraction were determined to
be 2.4-fold and 2.7-fold of the seed material (v/w), respectively. The
3.2. Determination of extraction conditions of the CCE method times for target substances in the seed material to be fully dissolved
(reached a dynamic equilibrium) were 1.0 h for Moringa oil and 2.0 h
The CCE method includes two steps. One is to select the best solvent for proteins and GLC in their corresponding extraction solvents (Fig. 2E
to dissolve target compounds, determine its minimum volume for the and F).
material to be fully absorbed and the time for the target compounds From the results above, the optimal solvents and compound dis-
fully dissolved. The next step is to elute the target compounds from the solving times were 2.4-fold PE-ethanol (8: 2) for 1.0 h for oil extraction,
material in a chromatographic column. As shown in Fig. 2, the ex- and 2.7-fold water for 2.0 h for proteins and GLC extraction from M.
traction solvents, the solvent volume for the material fully absorbed, oleifera seed material.
and the substance dissolution time for Moringa oil, proteins, and GLC in
M. oleifera seeds were independently optimized. Moringa oil was best 3.3. Column chromatographic extraction of oil, proteins, and GLC
extracted with PE-ethanol of 8:2 in a series of PE-ethanol from 10:0,
8:2, 6:4, 4:6, 2:8 to 0:10 (Fig. 2A). Pure water and different ethanol to First, M. oleifera seed material was extracted independently by PE-
water ratios were tested to extract Moringa proteins and GLC (Fig. 2B). ethanol (8:2) for oil and by water for proteins and GLC to determine the
Water was the best solvent to extract proteins. In a series of solvents conditions of simultaneous column chromatographic extraction. For oil
with different ethanol to water ratios, the yield of Moringa GLC in- extraction with PE-ethanol (8:2), the seed material was loaded into a
creased from 31 mg/g to 39 mg/g with the increase of ethanol pro- column with 1.0 MV (or 2.4-fold) PE-ethanol, and eluted with the same
portion from 0% to 40%, but no more increased when ethanol pro- solvent. Four MVs of eluent were collected, and the oil extracted in each
portion was higher than 40%. As GLC was extracted at a relative high MV eluent was analyzed. As shown in Fig. 3A, the total oil extracted
level by water, water was selected to extract both proteins and GLC. was 34.1 g in the 4.0 MVs eluent from 100 g seed material. The oil
Therefore, the best extraction solvents were determined to be PE- extraction efficiency reached 93.8% or 97.6% when 1.0 or 2.0 MVs of
ethanol of 8:2 for Moringa oil, and water for Moringa proteins and GLC. eluent were collected. To decrease the use of extraction solvent, the oil

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R. Chen, et al. Food Chemistry 300 (2019) 125162

Fig. 3. Extraction of oil, proteins, and GLC from M. oleifera seeds by the CCE method. A, B, and C: Independent CCE extraction of the three groups of substances
independently using PE-ethanol (8:2) for oil (A) and water for proteins and GLC (B and C); D, E, and F: simultaneous extraction of the three groups of substances from
M. oleifera seeds by the CCE method sequentially eluted with PE-ethanol for oil (D) and water for proteins and GLC (E and F).

extraction was determined to collect 2.0 MVs eluent. CCE. The results indicated that more than 95% of the three main groups
To extract proteins and GLC, 1.0 MV (or 2.7-fold of the material of substances in the M. oleifera seed material can be simultaneously
weight, v/w) water was loaded with the seed material into a column. extracted by the CCE method with gradient elution when the material
The column was eluted with 4.0 MVs water, the proteins and GLC in in the column was eluted with 2.0 MVs of PE-ethanol (for oil), followed
each MV eluent were analyzed. The total amounts of 37.5 g proteins by 4.0 MVs of water (for proteins and GLC). The simultaneous extrac-
and 6.4 g GLC were obtained in the 4.0 MVs eluent from 100 g seed tion of the three groups of substances was enlarged 300 times to a la-
material. This result was close to Förster et al. (2015), which reported boratory production level of 3.0 kg seed material per extraction, the
glucosinolate content of M. oleifera leaf was 111 µmol/g dw. The pro- same extraction efficiencies were obtained (data not shown).
teins extraction efficiency reached 94.1% and 99.1% when collected 1.0
and 2.0 MVs of eluent, respectively (Fig. 3B). However, as stated in
3.4. Separation of Moringa oil, proteins, and GLC
section 3.2 that water was not the best solvent to extract GLC and its
extraction efficiency by water was relatively low. It needed to collect
The extraction solution of M. oleifera seeds by the simultaneous CCE
3.0 or 4.0 MVs of eluent to reach 91.3% or 95.5% efficiency, respec-
was automatically separated into an ether phase containing oil and an
tively (Fig. 3C). The results indicated that proteins can be extracted
ethanol aqueous phase containing proteins and GLC. The ether phase
with 2.0 MVs water (99.1% extracted), but it needed to collect 3.0 or
was vacuum-evaporated at 40 °C to recover the solvent and crude oil
4.0 MVs eluent for efficient extraction of GLC.
was obtained. Isoelectric precipitation and ethanol precipitation were
Based on the results of independent extraction with PE-ethanol (8:2)
tested to separate the proteins and GLC in the ethanol aqueous phase.
and water above, the three groups of substances in M. oleifera seeds
Ethanol precipitation showed better results and was used to separate
were simultaneously extracted by the CCE method with sequential
proteins and GLC. As shown in Fig. 4A and B, the proteins precipitated
elution of 2.0 MVs PE-ethanol for oil and 4.0 MVs water for proteins
increased with ethanol concentrations and the precipitation time. The
and GLC. As shown in Fig. 3D, E, and F, oil was extracted by 97.6% by
optimal conditions were precipitated in 70% ethanol for 3.0 h. Under
2.0 MVs PE-ethanol (Fig. 3D). Proteins were extracted by 99.2% by the
these conditions, 88.1% proteins were collected in the pellet, while
first 2.0 MVs of water and there was less than 1.0% of proteins in the
89.8% GLC was remained in the solution. By this way, the proteins and
next 2.0 MVs of water eluent (Fig. 3E). GLC was extracted by 78.8% and
GLC in the ethanol aqueous solution were effectively separated. The
95.4% when collected 2.0 and 4.0 MVs of water eluent, respectively
proteins were dried in an oven and 33.0 g protein powder was obtained
(Fig. 3F). The extraction efficiencies of the simultaneous CCE with
from 100 g M. oleifera seed material. The dried proteins were dissolved
gradient elution reached the same high levels as in the independent
in water and analyzed by SDS-PAGE electrophoresis (Fig. 1C). The

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R. Chen, et al. Food Chemistry 300 (2019) 125162

Fig. 4. Separation and purification of Moringa oil, proteins, and GLC. A and B, separation of proteins and GLC in the ethanol aqueous phase by ethanol precipitation
in different ethanol concentration (A) and for different hours (B); C and D, decolorization efficiency (C) and oil recovery (D) of crude oil by different activated carbon
or activated clay (1, 3% charcoal; 2, 20% clay; 3, 6% charcoal; 4, 2% charcoal and 1% clay; 5, 10% clay); E to G, HPLC analysis of GLC in crude extracts (E), partially
purified after N-butanol fractionation (F), and purified by silica gel column chromatography (G).

results indicated that the proteins were a mixture of multiple low mo- Table 1
lecular proteins between 3.4 and 20 kDa (Fig. 1C), which was also Fatty acid composition and relative percentage of Moringa refined oil.
consistent to the reports of Dezfooli et al. (2016) and Ghebremichael Fatty acid Relative percentage (%)
et al. (2005). The ethanol solution with GLC in it was vacuum-con-
centrated to dry at 60 °C to recover ethanol for reuse and crude GLC was Oleic acid (C18:1) 72.30
Behenic acid (C22:0) 6.50
obtained.
Palmitic acid (C16:0) 5.80
Stearic acid (C18:0) 5.80
Arachidic acid (C20:0) 3.70
3.5. Purification and analysis of Moringa oil, proteins, and GLC products
Eicosenoic acid (C20:1) 1.90
Palmitoleic acid (C16:1) 1.20
The crude oil was refined by deacidification (79.8% recovery) and Tetracosanoic acid (C24:0) 0.92
degumming (92.1% recovery) based on the method by Sánchez- Linoleic acid (C18:2) 0.68
Machado et al. (2015), followed by decolorization. Different amounts of Linolenic acid (C18:3) 0.17
Myristic acid (C14:0) 0.11
activated carbon, activated clay or their combinations were used to Heptadecanoic acid (C17:0) 0.086
decolorize the oil. The results showed that 10% activated clay had the Erucic acid (C22:1) 0.073
best performance to remove the color substances in the oil based on the Heneicosanoic acid (C21:0) 0.067
visible spectrum analysis at 400–680 nm (Fig. 4C), which resulted in Docosadienoic acid (C22:2) 0.065
bright yellow refined oil at the decolorization recovery of 89.1%
(Fig. 4D). The refined oil had an acid value of 0.39 mg/g and peroxide
chromatography, which provided a simple and practical method for the
value of 0.022 g/100 g, which were much lower than the limits of
purification of GLC.
1.0 mg/g and 0.13 g/100 g, respectively, according to the China Na-
Overall, this work developed a simple procedure to comprehen-
tional Standards of Food Oils GB15196-2015. Analysis of fatty acid
sively utilize M. oleifera seeds (Fig. 5). Three main groups of valuable
composition of the refined oil (Table 1) indicated that the refined oil
substances, oil, proteins, and GLC, were effectively extracted by the
contained 76.3% unsaturated fatty acids, and among them 72.3% was
simultaneous CCE method with two steps elution, at more than 95%
oleic acid. The result was consistent to the reports of Sánchez-Machado
extraction efficiencies. The extracts were simply separated and purified
et al. (2015) and Zhong et al. (2018). They reported that Moringa oil
at high recoveries. Purified substances were obtained at the yields of
contained more than 70% unsaturated fatty acids and had good anti-
22.3 g oil, 33.0 g proteins, and 5.5 g GLC from 100 g M. oleifera seeds,
oxidant capacity.
and the organic solvents (petroleum ether and ethanol) used can be
The crude GLC was dissolved in water and purified by fractionation
recovered for reuse. The procedure of simultaneous extraction and se-
with N-butanol to remove the liposoluble impurities (Fig. 4E and F),
paration of oil, proteins, and GLC from M. oleifera seeds was not re-
followed by silica gel column chromatography (Fig. 4G). After pur-
ported before. The purified products of Moringa oil, proteins, and GLC
ification and dried, GLC powder with 91.7% purity was obtained at the
can be further developed as healthy food or medicine. This work pro-
yield of 5.5 g from 100 g M. oleifera seeds, and purified GLC was a
vided a simple way for comprehensive utilization of M. oleifera seeds.
yellowish-brown, viscous, hygroscopic substance. The overall recovery
rate of GLC from the extraction to purified GLC reached 85.9%. Förster
et al. (2015) reported that Moringa glucosinolates were simply purified 4. Conclusion
by barium acetate precipitation. In our work, GLC with high purity was
obtained by N-butanol fractionation and silica gel column M. oleifera is a worldwide edible and medicinal plant with high yield

6
R. Chen, et al. Food Chemistry 300 (2019) 125162

Fig. 5. Diagram of the extraction, separation and purification of oil, proteins, and GLC from M. oleifera seeds. A, refined oil; B, proteins; C, purified GLC.

of seeds which are rich in oil, proteins, and glucosinolates. This work Technology Programs (2014A010107026), the China Central
developed a high-efficient method for simultaneous extraction and se- Government Agricultural Technology Promotion (2015-60-43) and the
paration of oil, proteins, and glucosinolates from M. oleifera seeds. The Natural Science Funds of Guangdong Province (2017A030310031).
seed material was loaded into a column, and the three groups of sub-
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