Stable, water extractable isothiocyanates from Moringa oleifera leaves attenuate inflammation in vitro

Article abstract of DOI:10.1016/j.phytochem.2014.03.028

Moringa (Moringa oleifera Lam.) is an edible plant used as both a food and medicine throughout the tropics. A moringa concentrate (MC), made by extracting fresh leaves with water, utilized naturally occurring myrosinase to convert four moringa glucosinolates into moringa isothiocyanates. Optimum conditions maximizing MC yield, 4-[(α-L-rhamnosyloxy)benzyl]isothiocyanate, and 4-[(4′-O-acetyl-α-L-rhamnosyloxy)benzyl]isothiocyanate content were established (1:5 fresh leaf weight to water ratio at room temperature). The optimized MC contained 1.66% isothiocyanates and 3.82% total polyphenols. 4-[(4′-O-acetyl-α-L-rhamnosyloxy)benzyl]isothiocyanate exhibited 80% stability at 37 °C for 30 days. MC, and both of the isothiocyanates described above significantly decreased gene expression and production of inflammatory markers in RAW macrophages. Specifically, both attenuated expression of iNOS and IL-1β and production of nitric oxide and TNFα at 1 and 5 μM. These results suggest a potential for stable and concentrated moringa isothiocyanates, delivered in MC as a food-grade product, to alleviate low-grade inflammation associated with chronic diseases.

Full text of DOI:10.1016/j.phytochem.2014.03.028

Phytochemistry 103 (2014) 114–122
Contents lists available at ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Stable, water extractable isothiocyanates from Moringa oleifera leaves
attenuate inflammation in vitro
a
a
a
a
Carrie Waterman a, , Diana M. Cheng , Patricio Rojas-Silva , Alexander Poulev , Julia Dreifus ,
⇑
Mary Ann Lila b, Ilya Raskin a
a Department of Plant Biology & Pathology, Rutgers, The State University of New Jersey, 59 Dudley Road, New Brunswick, NJ 08901, USA
b Plants for Human Health Institute, North Carolina State University, 600 Laureate Way, Kannapolis, NC 28081, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Moringa (Moringa oleifera Lam.) is an edible plant used as both a food and medicine throughout the tro-
pics. A moringa concentrate (MC), made by extracting fresh leaves with water, utilized naturally occur-
ring myrosinase to convert four moringa glucosinolates into moringa isothiocyanates. Optimum
-L-rhamnosyloxy)benzyl]isothiocyanate, and 4-[(40-O-acetyl-
conditions maximizing MC yield, 4-[(a a-
Received 28 November 2013
Received in revised form 1 March 2014
Available online 11 April 2014
L-rhamnosyloxy)benzyl]isothiocyanate content were established (1:5 fresh leaf weight to water ratio
at room temperature). The optimized MC contained 1.66% isothiocyanates and 3.82% total polyphenols.
4-[(40-O-acetyl-
a-L-rhamnosyloxy)benzyl]isothiocyanate exhibited 80% stability at 37 °C for 30 days.
MC, and both of the isothiocyanates described above significantly decreased gene expression and produc-
tion of inflammatory markers in RAW macrophages. Specifically, both attenuated expression of iNOS and
IL-1b and production of nitric oxide and TNFa at 1 and 5 lM. These results suggest a potential for stable
and concentrated moringa isothiocyanates, delivered in MC as a food-grade product, to alleviate low-
Keywords:
Moringa oleifera
Moringaceae
Chronic inflammation
Isothiocyanates
4-[(
benzyl]isothiocyanate
4-[(40-O-acetyl-
-L-rhamnosyloxy)
a-L-rhamnosyloxy)
a
grade inflammation associated with chronic diseases.
benzyl]isothiocyanate
Ó 2014 Elsevier Ltd. All rights reserved.
Introduction
polyphenols and, most interestingly, four unique sugar-modified
aromatic glucosinolates (1–4) (Bennett et al., 2003). In both the
Moringa (Moringa oleifera Lam.) is a fast growing tropical tree
known as the ‘‘drumstick’’ or ‘‘horseradish tree.’’ It is one of 13
species in the monogenic family, Moringaceae, within the order
Brassicales, to which broccoli and the other cruciferous vegetables
belong. Moringa leaves are historically used as nutritious foods
and traditional medicine in Asia and Africa. Elevated nutrient con-
tent in their leaves can partly be attributed to the relatively low
moisture content (ca. 76%) of fresh leaves compared with ca.
90% moisture content of most vegetables. Moringa leaves contain
approximately 27% protein by dry weight, and all essential amino
acids. In addition, they contain high levels of vitamins and
beneficial phytoactives (Pandey et al., 2012). The latter include
Brassicaceae and Moringaceae, isothiocyanates (ITCs) are formed
from their glycosylated precursors, glucosinolates (GLSs), via a
reaction carried out by myrosinase (thioglucoside glucohydro-
lase), an enzyme activated during plant tissue wounding or
digestion. Myrosinase cleaves the thio-linked glucose in the GLS,
leaving the aglycone which rearranges quickly to form the active
ITC.
Despite well documented health benefits of ITCs from crucifers,
such as sulforaphane (SF) from broccoli and phenethyl isothiocya-
nate from winter cress on inflammation and cancer, their clinical
and dietary use is somewhat restricted because of their inherent
chemical instability. For example SF, formed from broccoli
glucoraphanin, its GLS precursor, is rapidly converted to several
degradation products, mainly dimethyl disulfide and S-methyl
methylthiosulfinate, making it difficult to formulate and deliver
by means other than eating fresh vegetables (Franklin et al.,
2013). Consuming ITCs from crucifers in their non-active, but more
stable, precursor form as GLSs remains an option. However, GLSs
undergo an uncertain and variable degree of enzymatic conversion
to ITCs by host gut microbiota (Traka and Mithen, 2009).
Abbreviations: ARE, antioxidant response elements; GLSs, glucosinolates; IL-1b,
interleukin-1 beta; IL-6, interleukin-6; iNOS, inducible nitric oxide synthase; ITCs,
isothiocyanates; Keap1, Kelch-like ECH-associated protein 1; MC, moringa concen-
trate; NF-jB, nuclear factor kappa-light-chain-enhancer of activated B cells; Nfr2,
nuclear factor (erythroid-derived 2)-like 2; NO, nitric oxide; SF, sulforaphane; TE,
trolox equivalents; TNF
a
, tumor necrosis factor alpha.
⇑
Corresponding author. Tel.: +1 (848) 932 6363, fax: +1 (732) 932 9441.
E-mail address: waterman@aesop.rutgers.edu (C. Waterman).
http://dx.doi.org/10.1016/j.phytochem.2014.03.028
0031-9422/Ó 2014 Elsevier Ltd. All rights reserved.

C. Waterman et al. / Phytochemistry 103 (2014) 114–122
115
OR3
O
O
OSO3
S
N
HO
OR1
CH3
OR2
OH
OH
O
CH2OH
Moringa glucosinolates (1-4)
OR3
R1 R2 R3
O
O
1, 5
2, 6
3, 7
4, 8
H
Ac
H
H
H
H
H
OR1
CH3
OR2
N
C
Ac
H
H
S
H
Ac
5 8
Moringa isothiocyanates ( - )
Fig. 1. Moringa glucosinolates (GLSs) 1–4 and isothiocyanates (ITCs) 5–8. Molecular masses: 1 = 570, monoacetylated 2–4 = 612, 5 = 353, monoacetylated 6–8 = 311.
In contrast to crucifers, moringa GLSs (1–4) (Fig. 1) contain an
additional sugar moiety in the aglycone/ITC portion of the
molecule. They can be converted in situ to four bioactive and rela-
tively stable moringa ITCs (5–8) (Fig. 1). Of these, compound 5
are to maintain a healthy diet and exercise regime. Unfortunately,
rates of obesity and T2DM, especially among children, continue to
grow. Genetic, social, and economic factors have certainly influ-
enced the epidemic (Wang and Beydoun, 2007). Identifying foods
which can help prevent and mitigate manifestations of these con-
ditions is of great interest. Recently, the search for food compo-
nents that regulate the inflammatory response has attracted
particular attention due to evidence linking chronic low-grade
inflammation to insulin resistance and obesity. Several key bio-
markers of inflammation have been identified as hallmark signs
of the pro-inflammatory response found in obesity-induced diabe-
(4-[(
a
-L-rhamnosyloxy)benzyl]isothiocyanate) and compound 8
(4-[(40-O-acetyl-
a-L-rhamnosyloxy)benzyl]isothiocyanate) are the
most abundant isothiocyanates formed from GLS 1 and 4, usually
making up over 95% of the total ITCs present. In contrast,
compound 6 (4-[(20-O-acetyl-
anate) and 7 (4-[(30-O-acetyl-
a
a
-L-rhamnosyloxy)benzyl]isothiocy-
-L-rhamnosyloxy)benzyl]isothiocy-
anate) are only formed in small quantities from trace amounts of
their respective GLSs precursors, compounds 2 and 3 (Amaglo
et al., 2010; Bennett et al., 2003). Our optimized moringa concen-
trate extract (MC) contained 1.15% of 5, 0.51% of 8 and approxi-
mately 0.06% of 6 and 7 combined. The moringa ITCs are solid
and relatively stable compounds at room temperature, in contrast
to volatile ITCs from crucifers that are mostly viscous liquids. The
retained rhamnose sugar moiety found in moringa ITCs is extre-
mely unique in nature and likely responsible for their high stability
and solid appearance (Brunelli et al., 2010). Previous research with
moringa extracts has predominantly utilized commercially avail-
able dried leaf powder for experimentation. This powder usually
contains much lower levels of ITCs (5–8) due to the destruction
of myrosinase in the drying process. Preparing a MC extract with
high ITCs (5–8) content takes advantage of endogenous myrosinase
in fresh moringa leaves to convert GLSs (1–4) to ITCs (5–8). There
by making MC a useful vehicle for delivering these compounds in
the human diet.
Moringa has been used medicinally throughout the centuries to
treat a multitude of acute and chronic conditions. In vitro and
in vivo studies with the plant have suggested its effectiveness in
treating conditions including inflammation, hyperglycemia, and
hyperlipidemia (Bennett et al., 2003; Mbikay, 2012; Fahey, 2005).
Moringa’s therapeutic effects were linked to the anti-inflamma-
tory, antibacterial and antioxidant properties of its phytochemi-
cals, such as flavonols and phenolic acids (Mbikay, 2012).
However, there has been minimal effort focused on the therapeutic
activity of GLSs and ITCs present in moringa, even though ITCs
from crucifers are some of the most well researched phytoactive
therapeutics in human health.
tes. These include cytokines: tumor necrosis factor alpha (TNFa),
interleukin-1 beta (IL-1b), interleukin-6 (IL-6), as well as inducible
nitric oxide synthase (iNOS), and nitric oxide (NO), an important
cellular signaling molecule in insulin signaling catalyzed by iNOS
(Bhargava and Lee, 2012; Ferrante, 2007; Xu et al., 2003). iNOS
expression and NO overproduction have been implicated in the
pathogenesis of disease states, particularly associated with chronic
inflammation (Hobbs et al., 1999). TNF
rectly interfere with insulin signaling (Hotamisligil et al., 1994).
Interestingly, deregulated overexpression of TNF (Mocellin and
a has been shown to di-
a
Nitti, 2008) and other inflammatory regulators (IL-1b, IL-6, C-reac-
tive protein (CRP), nuclear factor kappa-light-chain-enhancer of
activated B cells (NF-jB), peroxisome proliferator-activated recep-
tor (PPAR), and cyclooxygenase-2 (COX-2)) have been connected to
both metabolic syndrome and cancer development (Bao et al.,
2011; Mirza et al., 2012). For example, SF inhibits NF-
tion in many cancer models (Srivastava and Singh, 2004; Xu et al.,
2005). Activation of NF- B, an upstream regulator of many inflam-
jB transcrip-
j
matory cytokines, has also been well linked to insulin resistance
and diabetes (Mariappan et al., 2010).
Studies of moringa ITCs (5–8) show their pharmacological sim-
ilarity to well-studied crucifer ITCs. For example, compound 5 was
a stronger inhibitor of NF-jB expression and myeloma growth in
nude mice than SF (Brunelli et al., 2010). Compounds 5–8 also re-
duced NO formation at low micromolar concentrations in macro-
phages (Cheenpracha et al., 2010). Isothiocyanate 6 specifically
attenuated NO formation more effectively than SF and benzyl
isothiocyanate (Park et al., 2011). However, these publications only
examined the activity of purified moringa ITCs. This study
describes a simple reagent-free method for bioconversion of
moringa GLSs (1–4) into ITCs (5–8) and preparation of a stable
isothiocyanate-enriched, food-grade extract from moringa leaves
(MC). Additionally evidence of in vitro anti-inflammatory activity
of MC, 5, and 8 is presented.
We are particularly interested in the activity of MC and ITCs
related to type 2 diabetes mellitus (T2DM) and other chronic con-
ditions associated with metabolic syndrome (MetS). These condi-
tions pose serious and growing health concerns worldwide
(Alberti et al., 2009). Effective approaches to combat MetS/T2DM

116
C. Waterman et al. / Phytochemistry 103 (2014) 114–122
Once MC preparation had been optimized over temperature
Results and discussion
Optimization of extract
and solvent ratio, larger scale production of MC required the
use of a blender instead of a coffee grinder. This unintended
parameter of scaling up the extraction resulted in a significant
increase in ITC content. Under the same conditions, 1:5 solvent
ratio at 22 °C, the coffee grinder produced a lower amount of 5
and 8, approximately 1.00% total in MC while the blender in-
creased the concentration of total ITCs to 1.66% (1.15% 5 and
0.51% 8) of MC. This was likely due to finer crushing of the leaves
and the presence of water at the time of blending rather than
grinding prior to combining with water in the case of the coffee
grinder. Preparation of MC using the blender was performed with
three separate batches of moringa leaves and subjected to ITC
quantification by LCMS to ensure reproducibility of the extraction
method. The content of ITCs in MC (1.66%) is approximately 3
times higher than the SF content delivered in broccoli sprouts
(calculated using a reported 61% conversion rate of glucorapha-
nin to SF and converted to dry weight factoring 89% moisture
content (Force et al., 2007; Pereira et al., 2002; Song and
Thornalley, 2007)). MC, prepared in large batches, was
subsequently evaluated for chemical stability of ITCs, total poly-
phenol content, oxygen radical absorbance capacity and
anti-inflammatory activity in vitro.
Experiments were performed to optimize in situ biotransforma-
tion of moringa GLSs (1–4) into ITCs (5–8) by myrosinase and to
maximize the yield of MC from fresh leaves. The solvent ratio
(weight of fresh leaves to volume of water) and temperature (22
to 100 °C) were tested to determine the optimal conditions for
MC yield and isothiocyanates (determined by quantifiying 5 and
8). The solvent ratio affected both the concentrations of ITCs 5–8
and the percent yield (Fig. 2A and B). The 1:2 solvent ratio resulted
in a lower average of 5 content (0.45% of MC) compared with the
1:5 and 1:10 dilution, (0.59% and 0.62% of MC, respectively). The
amount of 8 was higher in the 1:5 and 1:10 dilutions, but not
statiscally significant (0.12% of MC compared with 0.20% and
0.19%, respectivly). Larger dilutions resulted in a proportional per-
cent yield increase of MC: (1:2) 5.47%, (1:5) 6.13%, (1:10) 7.87%.
The 1:5 dilution factor was selected as optimum to maximize the
amount of compounds 5–8 captured in MC, while minimizing the
amount of water used for extraction.
The 1:5 solvent ratio was used to evalute the effect of water
temperature on ITC concentration and percent yield. There was a
significant difference in the amount of ITCs (5–8) extracted at
water temperatures between 22 to 80 °C (5: 0.49 to 0.72% of MC,
8: 0.19 to 0.21% of MC) with undetectable amounts at 100 °C
(Fig. 2C). The LCMS mass chromatogram of both GLSs (1–4) and
ITCs (5–8) in a 22 and 100 °C extraction shows heat sensitivity of
myrosinase activity at high temperatures (Fig. 3). At 22 °C, myrosi-
nase converted GLSs (1–4) to ITCs (5–8) by thio-glucose cleavage.
At 100 °C, myrosinase was inactivated and GLSs were not
converted to their respective ITCs. Although the isoform of the
myrosinase enzyme in moringa may be different than the form
in broccoli, the thermal stability of moringa myrosinase is similar
to broccoli myrosinase, with a reported thermal stability at
50–60 °C (Eylen et al., 2008) and complete destruction of the
enzyme above 80 °C (Gallaher et al., 2012). Extraction with the
1:5 solvent ratio at room temperature (22 °C) was adopted to
maintain full enzymatic conversion of GLSs into ITCs.
Stability of compounds
When MC was stored at room temperature, 5 and 8 exhibited
exceptional stability of 70% and 100% respectively, over 30 days.
The accelerated stability studies of MC at 37 °C for 30 days
showed approximately 80% and 20% degradation of 5 and 8
respectively (Fig. 4). The higher stability of 8 is possibly due to
the monoacetylation at the 40 position of the rhamnose sugar.
Greater acetylation is well known to increase the stability of
glycosylated molecules such as anthocyanins (Giusti and
Wrolstad, 2003). Both 5 and 8 demonstrated superior stabil-
ity compared to reported values of SF, the main broccoli
isothiocyanate, which has been reported to degrade by 75% after
6 days at 37 °C (Franklin et al., 2013). SF is a volatile, viscous
liquid, whereas moringa ITCs, purified by HPLC, appear as solids
C
0.8
A
0.8
MIC-1
5
b
8
MIC-4
b
0.6
0.4
0.2
0.0
0.6
0.4
0.2
0.0
a
a,A
1:2
1:5
1:10
22
40
60
80
100
B
10
D
10
b
8
6
4
2
0
8
6
4
2
0
a, b
a
1:2
1:5
1:10
22
40
60
80
Fresh leaves (g): water (mL)
Temperature (°C)
Fig. 2. Effect of dilution factor and temperature on concentration of 5 and 8 and percent yield of MC. (A) Effect of dilution ratio of fresh leaves (g):H2O (mL) on ITC
concentration (mg of ITC/100 mg of MC). (B) Effect of dilution ratio on MC percent yield (mg of MC/100 mg of fresh leaves). All extractions in A and B were performed at room
temperature (22 °C). (C) Effect of temperature on ITC concentration (mg of ITC/100 mg of MC). (D) Effect of temperature on MC percent yield (mg of MC/100 mg of fresh
leaves). All extractions in C and D were performed in a dilution ratio of 1:5. Graphs represent the mean SEM (n = 3). Comparisons were made by a 1-way ANOVA followed by
Tukey’s posthoc test. Significant differences (p < 0.05) between sample sets are signified by letters; different letters indicate significant difference between sample sets, while
the same letter or absence of a letter indicates no difference.

C. Waterman et al. / Phytochemistry 103 (2014) 114–122
117
Fig. 3. Mass chromatogram of GLSs and ITCs after extraction at 22 °C (A) and 100 °C (B). m/z ions for 1 (570) [MꢀH]ꢀ, 2, 3, 4 (612) [MꢀH]ꢀ, 5 (370) [M (311) + C2H4O2
(60)ꢀH]ꢀ and 6, 7, 8 (420) [M (311) + C2H4O2 (60)ꢀH]ꢀ were selected for in both figures. (A). MC prepared at 22 °C (0.1 mg MC injected) showing the presence of: a (5), b
(overlapping peaks of 6 & 7) and c (8) and the absence of any ion trace for GLSs. (B) MC prepared at 100 °C (0.6 mg MC injected) showing the presence of d (1), e (overlapping
peaks of 2 & 3), and f (4), and the absence of any ion trace for ITCs.
Total polyphenol content
MIC-1 @ 25 C
5 at 25
C
1.6
1.4
1.2
1
8 at 25
C
MIC-4 @ 25 C
Total polyphenol (TP) content of MC, determined by the Folin–
Ciocalteu method (Singleton and Rossi, 1965) was 3.82 mg of gallic
acid equivalents/100 mg of MC ( 0.22), which is similar to reported
TP of dried moringa leaves (3.6 to 4.5% DW) (Sreelatha and Padma,
2009). This suggests that the aqueous MC extraction we developed
captured the majority of polyphenols present in fresh leaves. Pre-
dominant polyphenols identified in moringa include rutin, chloro-
genic acid, and quercetin-malonyl-glucoside (Amaglo et al., 2010;
Bennett et al., 2003). The molecular weights of these compounds
were detected in our LCMS analysis of MC, but quantification of
specific polyphenols was not performed.
5 at 37
MIC-4 @ 37 C
C
MIC-1 @ 37 C
8 at 37
C
0.8
0.6
0.4
0.2
0
Oxygen radical absorbance capacity
0
10
20
30
Days
The oxygen radical absorbance capacity (ORAC) of MC was
3.6 mmol Trolox equivalents (TE)/g of MC ( 0.69 SEM), suggesting
it has direct antioxidant potential. The ORAC of MC is greater than
reported values on spices with high ORAC, such as dried cinnamon
powder (2.6 mmol TE/g) (Wu et al., 2004). Fresh and dried moringa
leaf extracts were previously reported to contain high levels of
antioxidant compounds, including phenolics, flavonols, carote-
noids and ascorbic acid (Siddhuraju and Becker, 2003; Dillard
and German, 2000). Antioxidants in various moringa leaf extracts
Fig. 4. Effect of storage of MC at 25 °C and 37 °C on ITC stability and photograph of
purified 8 at 10X magnification (insert). Each bar represents the mean SEM (n = 3).
at room temperature (Fig. 4 insert shows > 98% pure 8 as a
solid material). This is likely due to the higher molecular weight
and unique rhamnose substitution compared with other
isothiocyanates.

118
C. Waterman et al. / Phytochemistry 103 (2014) 114–122
(total polyphenols, total flavonoids) have been shown in vitro to
possess free radical scavenging activity and ferric reducing power
(Vongsak et al., 2012). Reported ORAC data do not preclude the
possibility of indirect antioxidant activity of polyphenols and
moringa ITCs in MC. Flavonoids and isothiocyanates have been
well documented for their indirect antioxidant activity by activat-
ing the Keap1/Nrf2/ARE pathway and inducing production of pro-
tective phase 2 enzymes (Dinkova-Kostova and Talalay, 2008;
Tsuji et al., 2013). The direct, and possible indirect, antioxidant
capacities of secondary metabolites present in MC may contribute
to the observed reduction of pro-inflammatory markers in our
in vitro studies (see below).
inhibited TNF
pounds 5 and 8 at 5
27%, respectively. The enhanced anti-inflammatory activity of MC
compared with ITCs alone needs further investigation. Improved
a
production by 70% compared to the control. Com-
M reduced TNF production by 20% and
l
a
reduction of TNF
a by MC may be explained by the presence of
polyphenols in combination with compounds 5–8, including less
abundant, but perhaps, highly active 6 and 7. The stability and bio-
accessibility of bioactive compounds may also be improved when
delivered in a complex mixture. These results demonstrate the
plausible advantage of delivering compounds 5–8 in a food-grade
product, such as MC. TNF
inhibited by MC, 5, or 8, suggesting that moringa phytochemicals
may inhibit TNF production at the translational level or at the
level of TNF turnover.
a mRNA expression was not significantly
a
a
Anti-inflammatory activity
MC, 5, and 8 inhibited the production of NO significantly (Fig. 6
A & B). This is consistent with previously reported NO inhibition by
MC demonstrated a dose dependent inhibitory effect on iNOS
and IL-1b gene expression (Fig. 5A). Tested concentrations of MC
5 and 8 (IC50 of 14.43 and 2.71
et al., 2010). MC, at 100 g/mL, was able to inhibit NO formation
by 90%. ITCs 5 and 8 are at least partially responsible for this effect,
because they inhibited NO formation at 5 M by 69% and 39%,
lM, respectively) (Cheenpracha
l
ranged from 5 to 100
The molar concentration of total ITCs in MC at the various doses
ranged from approximately 0.28 M to 5.1 M. Almost complete
suppression of iNOS and IL-1b gene expression was observed at
100 g/mL of MC (3.7 M 5 and 1.4 M 8).
Compounds 5 and 8, tested at 1 and 5 M, also showed signifi-
lg/mL (0.08% to 1.66% total ITC content).
l
l
l
respectively. Reduced iNOS expression and NO production by
inducers of the phase 2 response, such as the isothiocyanate SF,
have been correlated to suppression of inflammation (Liu et al.,
2008). The improved reduction by MC treatment over individual
ITCs was also evident in NO production, but to a lesser degree than
l
l
l
l
cant reduction of mRNA expression of iNOS and IL-1b (Fig. 5B).
These concentrations were much lower than effective concentra-
observed in the TNFa production experiments. Again, this may be
explained by the trace amounts of 6 and 7, approximately 0.06% of
MC. These two compounds have been reported to inhibit NO for-
tions reported for polyphenols, such as quercetin (100
quired to inhibit iNOS gene expression in a similar experimental
design (Wadsworth and Koop, 1999). Additionally, MC at 100 g/mL
(Fig. 5C) and 5 at 5 M (Fig. 5D) were able to decrease IL-6 gene
expression significantly. However, no reduction of TNF gene
lM), re-
l
mation at low micromolar concentrations (IC50 of 1.67
lM and
l
2.66 M, respectively (Cheenpracha et al., 2010)). MC, 5, and 8
l
a
showed no signs of cytotoxicity at the concentrations tested in
anti-inflammatory assays, as demonstrated in 3-(4,5-dimethylthia-
zol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)-based cell
viability assays (data not shown).
expression was seen at any of the concentrations of MC and ITCs
and tested.
NO and TNF
a
cytokine production were reduced by MC, 5 and 8
g/mL, containing 1.15% 5 and 0.51% 8,
(Fig. 6A and B). MC at 100
l
Fig. 5. Anti-inflammatory effects of MC, 5 and 8 on LPS-induced iNOS, IL-1b, IL-6 and TNF
MC, 5, or 8 and then induced with LPS for 6 h. Values show relative gene expression compared to vehicle with LPS control determined by comparative DDCt analysis. (A) Effect
of MC on iNOS and IL-1b. (B) Effect of 5 and 8 on iNOS and IL-1b. (C) Effect of MC on IL-6 and TNF . (D) Effect of 5 and 8 on IL-6 and TNF . Each bar represents the mean SEM
(n = 4), except for TNF
in D where n = 2. ⁄⁄⁄ = p < 0.001, ⁄⁄ = p < 0.01, ⁄ = p < 0.05. Comparisons to controls were made by Dunnett’s test for iNOS measurements or Wilcoxon’s
test in all other experiments.
a gene expression in RAW 264.7 macrophage cells. Cells were pretreated for 2 h with
a
a
a

C. Waterman et al. / Phytochemistry 103 (2014) 114–122
119
Fig. 6. Anti-inflammatory effect of MC, 5, or 8 on LPS-induced NO and TNF
and then induced with LPS for 6 h. Values show relative fold change in production of NO or TNF
a
protein production in RAW 264.7 macrophage cells. Cells were pretreated for 2 h with MC or ITCs
protein measured by the Greiss method and ELISA, respectively. (A) Effect of
a protein production. (B) Effect of ITCs on NO and TNFa
protein production. Each bar represents the mean SEM (n = 3). ⁄⁄⁄ = p < 0.001, ⁄⁄ = p < 0.01,
a
MC on NO and TNF
⁄ = p < 0.05. Comparisons to vehicle with LPS controls were made by a Dunnett’s test.
Conclusion
Experimental
A rapid and efficient method was developed for bioconversion
of moringa GLSs into ITCs and optimization of conditions for pro-
ducing an aqueous moringa leaf concentrate (MC) containing
1.15% of 5 and 0.51% of 8. The ORAC value for MC was relatively
high, even when compared to spices such as oregano, thyme, and
cinnamon, often considered gold standards as direct antioxidants.
MC, 5, and 8 demonstrated anti-inflammatory activities at the gene
level using an in vitro murine neoplastic mononuclear cell model of
lipopolysaccharide (LPS)-induced inflammation. Compounds 5 and
8 were isolated from MC and shown to be relatively stable, solid
compounds, likely due to the presence of rhamnose residues.
Moreover, 8 demonstrated high stability when compared to vola-
tile isothiocyanates from Brassicaceae. This stability presumably
arises from the monoacetylation at the 4’ position of the rhamnose
sugar. The mediation of selected anti-inflammatory biomarkers by
MC, and its stable ITC constituents, provides preliminary support
for the use of MC as a food ingredient in the prevention and treat-
ment of conditions associated with chronic inflammation. Moringa
ITCs (5–8) appear to have similar pharmacological potential as
well-known ITCs from crucifers. However, moringa ITCs (5–8) offer
two key advantages over cruciferous ITCs such as SF. Firstly, they
are concentrated in MC to much higher concentrations than other
ITCs in foods, such as SF in broccoli sprouts. Secondly, solid ITCs
possess superior chemical stability and shelf life than liquid and
volatile ITCs from crucifers.
Plant material and sample preparation
Fresh leaves and seeds from M. oleifera (Indian PKM-1 variety)
were shipped overnight from Moringa Farms, Sherman Oaks, CA.
The leaves were extracted on the day of arrival to produce MC.
Moringa seeds were cultivated at Rutgers University greenhouse
until the plants flowered. A voucher specimen (CW1) was prepared
and deposited at the Chrysler Herbarium of Rutgers University
(CHRB).
Fresh M. oleifera leaves were blended (Vitamix 5200 Blender,
Cleveland, OH) thoroughly with room temperature Millipore H2O
in a ratio of leaves (1 g) to H2O (5 mL) for batch reproducibility
of MC used in stability tests and all biological assays. Micro
preparation of MC for temperature/dilution optimization was per-
formed by grinding fresh leaves in a coffee grinder (Krups, Cuisin-
art Model DCG-20N Series, East Windsor, Millville, NJ) and then
placing them in H2O. Leaf extracts (either prepared with the
blender or coffee grinder) were placed on a shaker (Lab Line Orbit
Shaker, Kochi, India) for 30 min at room temperature. In tempera-
ture experiments, the extracts were placed in water baths at desig-
nated temperatures for 30 min. Following incubation, the extracts
were filtered through Miracloth (Calibiochem, Billerica, MA) and
centrifuged for 10 min at 3200g and 4 °C. The supernatants, which
appeared as transparent brown ‘‘teas,’’ were individually decanted
and lyophilized to produce MCs. In some cases, particularly with

120
C. Waterman et al. / Phytochemistry 103 (2014) 114–122
larger batches, centrifugation was repeated in order to clear all
solid materials from the supernatant.
the gradient were 95% A and 5% B. The gradient progressed linearly
to 5% A and 95% B over 30 min and then remained isocratic for the
next 8 min. During the following 4 min, the ratio was brought to
initial conditions linearly. An 8 min equilibration interval was
included between subsequent injections. 1H NMR spectra were
recorded in MeOH-d4 on a 500 Varian VNMRS 500 MHz.
Extraction and isolation
Compounds 5 and 8 were extracted from fresh moringa leaves
using a modified approach to previously published methods
(Cheenpracha et al., 2010). Briefly, both were initially extracted
from ground leaves (200 g) in MeOH (400 mL) for 4 h at room
temperature.
The MeOH extract was evaporated to dryness under vacuum
and partitioned in H2O and hexanes (1:1 v/v) three times with
200 mL of each solvent. Equal volumes of EtOAc were then added
to the H2O fraction and partitioned. The EtOAc fraction was dried
down and resuspended in CH3CN–H2O (1:1), sonicated briefly,
Optimization and reproducibility of extraction
MC was prepared in ratios of 1:2, 1:5, and 1:10 (g of fresh
leaves: mL H2O) for optimization of ITC content and percent yield.
Triplicate samples of fresh moringa leaves (8 g) were ground in a
coffee grinder, diluted accordingly in H2O at room temperature,
and mixed for 30 min. For temperature experiments, fresh moringa
leaves (8 g) were ground in a coffee grinder and added to H2O
(40 mL) at 22, 40, 60, 80, and 100 °C as triplicate samples. The mix-
tures were maintained at these temperatures for 30 min in hot
water baths. Following 30 min of incubation, each MC was pre-
pared as described above. Analysis for percent yield (weight of
MC as a percent of starting fresh weight of leaves) and ITC content
was determined.
Once the optimum temperature (22 °C) and dilution factor (1:5)
were established using micro preparations, larger batches of MC
were made using the Vitamix blender (200 g: 1000 mL). Triplicate
samples of MC prepared in this manner from 3 separate batches of
moringa leaves were compared for reproducibility tests.
and filtered through a 0.45 lm filter prior to purification by pre-
parative high-performance liquid chromatography (HPLC).
Replicate HPLC injections of an aliquot of the EtOAc fraction
(100
lL, 200 mg/mL) were eluted with CH3CN-H2O-TFA
(50:50:0.05) to yield 5 (Rt = 8.2 min) and 8 (Rt = 17.5 min). The elu-
ent of HPLC solvent at these Rt was collected and dried initially by
rotary-evaporation, followed by lyophilization. The compounds ap-
peared as white solids. Compound 8 was scraped and placed on a
petri dish for imaging at 10X magnification (Fig 4 insert). The
chemical purity of isolated ITCs was confirmed by liquid chroma-
tography mass spectrometry (LCMS) and 1H NMR spectroscopic
analyses. Reversed phase HPLC was carried out on a Waters System
consisting of a four channel Waters 616 pump with semi-prepara-
tive pump heads operated by a Waters 600 Controller; Waters
490E Programmable Multiwavelength Detector set to monitor at
222 nm; and Waters 717 Plus Autosampler. Compounds were sep-
arated on a Phenomenex semi-preparative Synergi Hydro column
Compound stability
Triplicate samples (100 mg) of optimized MC were placed at
room temperature (ca.25 °C) and in a 37 °C dark incubator and
subjected to LCMS analysis for quantification of ITCs at 0, 20 and
30 days for room temperature experiments and 0, 18 and 30 days
for 37 °C experiments.
(4
l
M, 250 ꢁ 20 mm, 80 Å) with a mobile phase flow rate of
10 mL/min.
Compound quantification
Characterization of extract
UV peak area of LCMS injections of 5 and 8 (>98% purity) at 3
concentrations (3x) were averaged and used to generate standard
curves to quantify ITC content in MC preparations. 1 lL injections
The optimized MC preparation, prepared at 22 °C H2O using a
ratio 1:5 (w/v) of fresh leaves to H2O was additionally character-
ized for TP content and ORAC. TP content was quantified by the Fo-
lin–Ciocalteu method (Singleton and Rossi, 1965) and sample
absorbance was read at 726 nm against a gallic acid standard
(3ꢁ) of 5 at 20, 100, and 200 ng/
lL dissolved in CH3CN-H2O (1:1)
generated a standard curve (y = 123xꢀ0.098, R2 = 1), and 8 at 10,
50, and 100 ng/lL generated a standard curve (y = 104.32xꢀ
curve. ORAC was determined as lM TE using fluorescein as the
0.098, R2 > 0.99). The content of compounds 6 and 7 were esti-
mated by using the standard curve of 8, but was not included when
total ITC content was calculated.
fluorescent probe and 2,2’-azobis(2-amidinopropane) dihydrochlo-
ride (AAPH) as a peroxyl radical generator in a procedure adapted
from previously published methods (Prior et al., 2003).
LCMS analysis was performed using the DionexÒ UltiMate 3000
RSLC ultra-high pressure liquid chromatography system, consisting
of a workstation with DionexÒ Chromeleon v. 6.8 software pack-
age, solvent rack/degasser SRD-3400, pulseless chromatography
pump HPG-3400RS, autosampler WPS-3000RS, column compart-
ment TCC-3000RS, and photodiode array detector DAD-3000RS.
After the photodiode array detector, the eluent flow was guided
to a Varian 1200L (Varian Inc., Palo Alto, CA) triple quadrupole
mass detector with electrospray ionization interface, operating in
the negative ionization mode. The voltage was adjusted to
ꢀ4.5 kV, heated capillary temperature is 280 °C, and sheath gas
(zero grade compressed air) for the negative ionization mode.
The mass detector was used in scanning mode from 65 to 1500
atomic mass units. Data from the Varian 1200L mass detector
was collected, compiled and analyzed using Varian’s MS Worksta-
tion, v. 6.9, SP2. Compounds were separated on a Phenomenex™
Cell culture
All reagents were supplied from Sigma–Aldrich Co. (St. Louis,
MO) unless otherwise noted. RAW 264.7 macrophages (ATCC TIB-
71) were maintained in Dulbecco’s modified Eagle’s medium
(DMEM) (Caisson, North Logan, UT) supplemented with 100 U/mL
penicillin, 100 lg/mL streptomycin, and 10% fetal bovine serum.
Cells were incubated at 37 °C with 5% CO2 humidified atmosphere
and subcultured by cell scraping. For experiments, RAW cells were
plated at a density of 4 ꢁ 105 cells/mL in 24-well plates. Cells were
incubated overnight (18 h), washed with warm PBS, and replaced
with fresh DMEM media. Cells were pretreated with designated
doses of MC, 5, 8 or vehicle (EtOH–H2O, 1:1, v/v). LPS (1 lg/mL)
was added after 2 h incubation with treatments to elicit inflamma-
tory responses. Cells were treated in duplicate or triplicate. After an
additional 6 h incubation period, media were collected and then
cells were washed with PBS prior to collection in TRIzolÒ Reagent
(Life Technologies, Carlsbad, CA). Samples were stored at ꢀ80 °C
prior to processing.
C8 reversed phase column, size 150 ꢁ 2 mm, particle size 3
lm,
pore size 100 Å. The mobile phase consisted of 2 components:
Solvent A (0.5% ACS grade AcOH in double distilled de-ionized
H2O, pH 3–3.5), and Solvent B (CH3CN). The initial conditions of

C. Waterman et al. / Phytochemistry 103 (2014) 114–122
121
Gene expression analyses
TNFa levels were quantified using a reference standard curve pro-
vided with the kit. Absorbance was read at 450 nm and corrected
Total RNA was extracted from RAW macrophage cells according
at 570 nm.
to manufacturer’s specifications. Briefly, CHCl3 (200
lL) was added
to TRIzol harvested samples (600 L). Samples were vigorously
l
Nitric oxide production analysis
mixed for 30 s, incubated at room temperature for 5 min, centri-
fuged at 12,400g and i-PrOH was added to the aqueous phase to
obtain a ratio of 0.7 supernatant to i-PrOH. Samples were mixed
by inverting, vortexed briefly and incubated for 10 min at ꢀ20 °C.
Samples were centrifuged at 12,400g for 15 min at 4 °C. Next,
supernatant was removed and the sample was washed twice with
EtOH–H2O (75:25, v/v) and centrifuged at 6000g for 10 min. Sam-
ples were allowed to dry and resuspended in diethylpyrocarbonate
(DEPC) treated-H2O. RNA integrity was evaluated by running RNA
Collected media, as described above, was assayed in duplicate
following the Griess Reagent System provided by Promega (Prome-
ga Corporation; Madison, WI). The nitrite standard (0.1 M NaNO2)
reference curve was built performing a serial dilution (0 to
100
Cell viability
Effect of treatments on cell viability was measured using MTT
lM). Absorbance was read at 540 nm.
(ca. 1 lg) on a 1% agarose gel.
RNA was then treated with Deoxyribonuclease I Amplification
grade (Life Technologies), following the manufacturer’s guidelines.
RNA quality was checked on the NanoDrop 1000 system (Nano-
Drop Technologies, Wilmington, DE). A ratio of OD 260/280 P 2.0
and OD 260/230 P 1.8 was considered to be good quality RNA.
First strand cDNA synthesis was performed using ABI High-Capac-
ity cDNA Reverse Transcription kit (Applied Biosystems, Foster
City, CA) with RNAse I inhibitor, according to the manufacturer’s
[3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide]
(TCI, Portland, OR) (Mosmann, 1983). MTT (5 mg/mL) was dis-
solved in PBS (Cayman Chemical, Ann Arbor, MI), filtered through
a 0.22 lm membrane and added to treated cells during the last
3–4 h of treatment. Media were carefully aspirated; cells were dis-
solved in DMSO and the absorbance was read at 570 nm.
Statistical analysis
instructions using RNA (1 lg). The thermal cycle program was
set as follows: 10 min, 25 °C; 60 min, 37 °C; 60 min, 37 °C; 5 s,
85 °C, and final hold at 4 °C.
Data were expressed as mean SEM. Statistical comparisons for
optimization experiments were made by use of 1-way analysis of
variance (ANOVA) followed Tukey’s post hoc test in the MC optimi-
zation and stability experiments. Statistical comparisons for anti-
inflammatory experiments were made by use of ANOVA followed
by a Dunnett’s or Wilcoxon test as indicated and p < 0.05 were
considered significant. ⁄⁄⁄ = p < 0.001, ⁄⁄ = p < 0.01, ⁄ = p < 0.05. For
statistical analysis GraphPad Prism version 6.02 for Windows
(GraphPad Software, Inc.) was employed.
Synthesized cDNAs were diluted 25-fold and the diluted sample
(5
(12.5
nology Performance Certified (BPC) grade H2O (Sigma) to a final
reaction volume (25 L). Exon-spanning primer sequences were
l
L) was used for qPCR with Power SYBR Green PCR master mix
l
L, Applied Biosystems), primers (0.5 L, 6 M) and Biotech-
l
l
l
previously designed (Dey et al., 2006) on Primer ExpressÒ (Life
Technologies) and are as follows: b-actin forward 50-AAC CGT
GAA AAG ATG ACC CAG AT-30, reverse: 50-CAC AGC CTG GAT GGC
TAC GT-30, IL-1b forward 50-CAA CCA ACA AGT GAT ATT CTC CAT-
30, reverse 50-GAT CCA CAC TCT CCA GCT GCA-30, iNOS forward
50-CCC TCC TGA TCT TGT GTT GGA-30, reverse 50-TCA ACC CGA
GCT CCT GGA A-30, COX-2 forward 50-TGG TGC CTG GTC TGA
Acknowledgements
This project was supported by P50AT002776-01 from the
National Center for Complementary and Alternative Medicine
(NCCAM) and the Office of Dietary Supplements (ODS), which
funds the Botanical Research Center and Botanical Research Center
Pilot Program Sub award 5P50AT002776-08 S12-50318. CW and
DC were also supported by NIH training Grant T32:
5T32AT004094-04. PRS was supported by the SENESCYT 2011 Fel-
lowship. We would like to thank Rodney Perdew from Moringa
Farms, CA for supplying high-quality moringa leaves. The photo
of moringa leaves in the graphical abstracts was provided courtesy
of ilovemoringa.com. IR and MAL have equity in Nutrasorb LLC,
which licensed moringa-related intellectual property from Rutgers
University.
TGA TG-30, reverse 50-GTG GTA ACC GCT CAG GTG TTG-30, TNF
a
forward 50-TGG GAG TAG ACA AGG TAC AAC CC-30, reverse 50-
CAT CTT CTC AAA ATT CGA GTG AGA A-30, IL-6 forward 50-TCG
GAG GCT TAA TTA CAC ATG TTC-30, reverse 50 TGC CAT TGC ACA
ACT CTT TTC T-30. All primers were validated by analyzing amplifi-
cation efficiencies and melt curve profiles.
Quantitative PCR amplifications were performed on an ABI 7300
Real-Time PCR System (Applied Biosystems) with the following
thermal cycler profile: 2 min, 50 °C; 10 min, 95 °C; 15 s, 95 °C;
1 min, 60 °C for the dissociation stage; 15 s, 95 °C; 1 min, 60 °C;
15 s, 95 °C. Inflammatory marker mRNA expressions were
analyzed by the comparative DDCt method and normalized with
respect to the average Ct value of b-actin. Vehicle with LPS treat-
ment served as the calibrator for DDCt analysis and was assigned
a value of 1.0. Lower values indicate inhibition of gene expression
relative to vehicle treated with LPS control. All experimental
samples were run in triplicate and each reaction plate included
no template controls.
References
Alberti, K.G., Eckel, R.H., Grundy, S.M., Zimmet, P.Z., Cleeman, J.I., Donato, K.A.,
Fruchart, J.C., James, W.P.T., Loria, C.M., Smith, S.C., 2009. Harmonizing the
metabolic syndrome: A joint interim statement of the International Diabetes
Federation Task Force on Epidemiology and Prevention; National Heart, Lung,
and Blood Institute; American Heart Association; World Heart Federation;
International Atherosclerosis Society; and International Association for the
Study of Obesity. Circulation 120, 1640–1645.
Amaglo, N.K., Bennett, R.N., Lo Curto, R.B., Rosa, E.A.S., Lo Turco, V., Giuffrida, A.,
Curto, A.L., Crea, F., Timpo, G.M., 2010. Profiling selected phytochemicals and
nutrients in different tissues of the multipurpose tree Moringa oleifera L., grown
in Ghana. Food Chem. 122, 1047–1054.
Bao, B., Wang, Z., Li, Y., Kong, D., Ali, S., Banerjee, S., Ahmad, A., Sarkar, F.H., 2011.
The complexities of obesity and diabetes with the development and progression
of pancreatic cancer. Biochim. Biophys. Acta – Rev. Cancer 1815, 135–146.
Bennett, R.N., Mellon, F.A., Foidl, N., Pratt, J.H., Dupont, M.S., Perkins, L., Kroon, P.A.,
2003. Profiling glucosinolates and phenolics in vegetative and reproductive
tissues of the multi-purpose trees Moringa oleifera L. (Horseradish Tree) and
Moringa stenopetala L. J. Agric. Food Chem. 51, 3546–3553.
TNFa secretion analysis
RAW 264.7 macrophages were cultured, treated with MC or
ITCs, and subjected to LPS-induced inflammation as stated above.
After treatments, media (1 mL) was collected and immediately
centrifuged at 13,500g and 4 °C for 10 min. The supernatant was
preserved at ꢀ80 °C until further processing with the BD OptEIA™
Mouse TNF ELISA kit (BD Bioscience, San Jose, CA) following the
manufacturer’s protocol. All samples were assayed in duplicate.

122
C. Waterman et al. / Phytochemistry 103 (2014) 114–122
Bhargava, P., Lee, C., 2012. Role and function of macrophages in the metabolic
syndrome. Biochem. J. 442, 253–262.
Brunelli, D., Tavecchio, M., Falcioni, C., Frapolli, R., Erba, E., Iori, R., Rollin, P., Barillari,
J., Manzotti, C., Morazzoni, P., D’Incalci, M., 2010. The isothiocyanate produced
from glucomoringin inhibits NF-kB and reduces myeloma growth in nude mice
in vivo. Biochem. Pharmacol. 79, 1141–1148.
Pandey, A., Pandey, R.V., Tripathi, P., Gupta, P.P., Haider, J., Bhatt, S., Singh, A.V.,
2012. Moringa oleifera Lam. (Sahijan) – a plant with a plethora of diverse
therapeutic benefits: an updated retrospection. Med. Aromatic Plants 1, 1–8.
Park, E.J., Cheenpracha, S., Chang, L.C., Kondratyuk, T.P., Pezzuto, J.M., 2011.
Inhibition of lipopolysaccharide-induced cyclooxygenase-2 and inducible
nitric
oxide
synthase
expression
by
4-[(20-O-acetyl-
a-L-
Cheenpracha, S., Park, E.-J., Yoshida, W.Y., Barit, C., Wall, M., Pezzuto, J.M., Chang,
L.C., 2010. Potential anti-inflammatory phenolic glycosides from the medicinal
plant Moringa oleifera fruits. Bioorgan. Med. Chem. 18, 6598–6602.
Dey, M., Ribnicky, D., Kurmukovl, A.G., Raskin, I., 2006. In vitro and in vivo anti-
rhamnosyloxy)benzyl]isothiocyanate from Moringa oleifera. Nutr. Cancer 63,
971–982.
Pereira, F.M.V., Rosa, E., Fahey, J.W., Stephenson, K.K., Carvalho, R., Aires, A., 2002.
Influence of temperature and ontogeny on the levels of glucosinolates in
broccoli (Brassica oleracea Var. italica) sprouts and their effect on the induction
of mammalian phase 2 enzymes. J. Agric. Food Chem. 50, 6239–6244.
Prior, R.L., Hoang, H., Gu, L., Wu, X., Bacchiocca, M., Howard, L., Hampsch-Woodill,
M., Huang, D., Ou, B., Jacob, R., 2003. Assays for hydrophilic and lipophilic
antioxidant capacity (oxygen radical absorbance capacity (ORAC(FL))) of plasma
and other biological and food samples. J. Agric. Food Chem. 51, 3273–3279.
Siddhuraju, P., Becker, K., 2003. Antioxidant properties of various solvent extracts of
total phenolic constituents from three different agroclimatic origins of
drumstick tree (Moringa oleifera Lam.) leaves. J. Agric. Food Chem. 51, 2144–
2155.
Singleton, V., Rossi, J.A., 1965. Colorimetry of total phenolics with
phosphomolybdic-phosphotungstic acid reagents. Am. J. Enol. Viticult. 16,
144–158.
Song, L., Thornalley, P.J., 2007. Effect of storage, processing and cooking on
glucosinolate content of Brassica vegetables. Food Chem. Toxicol. 45, 216–224.
Sreelatha, S., Padma, P.R., 2009. Antioxidant activity and total phenolic content of
Moringa oleifera leaves in two stages of maturity. Plant Foods Hum. Nutr. 64,
303–311.
inflammatory
activity
of
a
seed
preparation
containing
phenethylisothiocyanate. J. Pharm. Exp. Ther. 317, 326–333.
Dillard, C.J., German, J.B., 2000. Phytochemicals: nutraceuticals and human health. J.
Sci. Food Agric. 80, 1744–1756.
Dinkova-Kostova, A.T., Talalay, P., 2008. Direct and indirect antioxidant
properties of inducers of cytoprotective proteins. Mol. Nutr. Food Res. 52,
S128–S138.
Eylen, D.V., Oey, I., Hendrickx, M., Loey, A.V., 2008. Effects of pressure/
temperature treatments on stability and activity of endogenous broccoli
(Brassica oleracea L. cv. Italica) myrosinase and on cell permeability. J. Food
Eng. 89, 178–186.
Fahey, J.W., 2005. Moringa oleifera:
a review of the medical evidence for its
nutritional, therapeutic, and prophylactic properties. Trees Life J. 1, 5.
Ferrante, A.W., 2007. Obesity-induced inflammation: a metabolic dialogue in the
language of inflammation. J. Intern. Med. 262, 408–414.
Force, L.E., O’Hare, T.J., Wong, L.S., Irving, D.E., 2007. Impact of cold storage on
glucosinolate levels in seed-sprouts of broccoli, rocket, white radish and kohl-
rabi. Postharvest Bio. Tech. 44, 175–178.
Franklin, S.J., Dickinson, S.E., Karlage, K.L., Bowden, G.T., Myrdal, P.B., 2013. Stability
of sulforaphane for topical formulation. Drug Dev. Ind. Pharm., 1–9.
Gallaher, C.M., Gallaher, D.D., Peterson, S., 2012. Development and validation of a
spectrophotometric method for quantification of total glucosinolates in
cruciferous vegetables. J. Agric. Food Chem. 60, 1358–1362.
Giusti, M.M., Wrolstad, R.E., 2003. Acylated anthocyanins from edible sources and
their applications in food systems. Biochem. Eng. J. 14, 217–225.
Hobbs, A.J., Higgs, A., Moncada, S., 1999. Inhibition of nitric oxide synthase as a
potential therapeutic target. Annu. Rev. Pharmacol. Toxicol. 39, 191–220.
Hotamisligil, G.S., Murray, D.L., Choy, L.N., Spiegelman, B.M., 1994. Tumor necrosis
factor alpha inhibits signaling from the insulin receptor. Proc. Natl. Acad. Sci. 91,
4854–4858.
Srivastava, S.K., Singh, S.V., 2004. Cell cycle arrest, apoptosis induction and
inhibition of nuclear factor kappa B activation in anti-proliferative activity of
benzyl isothiocyanate against human pancreatic cancer cells. Carcinogenesis
25, 1701–1709.
Traka, M., Mithen, R., 2009. Glucosinolates, isothiocyanates and human health.
Phytochem. Rev. 8, 269–282.
Tsuji, P.A., Katherine, K., Stephenson, K.K., Wade, K.L., Liu, H., Fahey, J.W., 2013.
Structure-activity analysis of flavonoids: direct and indirect antioxidant, and
antiinflammatory potencies and toxicities. Nutr. Cancer 65, 1014–1025.
Vongsak, B., Sithisarn, P., Mangmool, S., Thongpraditchote, S., Wongkrajang, Y.,
Gritsanapan, W., 2012. Maximizing total phenolics, total flavonoids contents
and antioxidant activity of Moringa oleifera leaf extract by the appropriate
extraction method. Ind. Crop. Prod. 44, 566–571.
Wadsworth, T.L., Koop, D.R., 1999. Effects of the wine polyphenolics quercetin and
resveratrol on pro-inflammatory cytokine expression in RAW 264.7
macrophages. Biochem. Pharmacol. 57, 941–949.
Liu, H., Dinkova-Kostova, A.T., Talalay, P., 2008. Coordinate regulation of enzyme
markers for inflammation and for protection against oxidants and electrophiles.
Proc. Natl. Acad. Sci. 105, 15926–15931.
Mariappan, N., Elks, C.M., Sriramula, S., Guggilam, A., Liu, Z., Borkhsenious, O.,
Francis, J., 2010. NF-
j
B-induced oxidative stress contributes to mitochondrial
Wang, Y., Beydoun, M.A., 2007. The obesity epidemic in the United States—gender,
age, socioeconomic, racial/ethnic, and geographic characteristics: a systematic
review and meta-regression analysis. Epidemiol. Rev. 29, 6–28.
Wu, X., Beecher, G.R., Holden, J.M., Haytowitz, D.B., Gebhardt, S.E., Prior, R.L., 2004.
Lipophilic and hydrophilic antioxidant capacities of common foods in the
United States. J. Agric. Food Chem. 52, 4026–4037.
Xu, C.S.G., Chen, C., Gelinas, C., Kong, A.N., 2005. Suppression of NF-kappaB and
NFkappaB-regulated gene expression by sulforaphane and PEITC through
IkappaBalpha, IKK pathway in human prostate cancer PC-3 cells. Oncogene
24, 4486–4495.
and cardiac dysfunction in type II diabetes. Cardiovasc. Res. 85, 473–483.
Mbikay, M., 2012. Therapeutic potential of Moringa oleifera leaves in chronic
hyperglycemia and dyslipidemia: a review. Front. Pharmacol. 3, 1–12.
Mirza, S., Hossain, M., Mathews, C., Martinez, P., Pino, P., Gay, J.L., Rentfro, A.,
McCormick, J.B., Fisher-Hoch, S.P., 2012. Type 2-diabetes is associated with
elevated levels of TNF-alpha, IL-6 and adiponectin and low levels of leptin in a
population of Mexican Americans: a cross-sectional study. Cytokine 57, 136–
142.
Mocellin, S., Nitti, D., 2008. TNF and cancer: the two sides of the coin. Front. Biosci.
13, 2774–2783.
Mosmann, T., 1983. Rapid colorimetric assay for cellular growth and survival:
application to proliferation and cytotoxicity assays. J. Immunol. Methods 65,
55–63.
Xu, H., Barnes, G.T., Yang, Q., Tan, G., Yang, D., Chou, C.J., Sole, J., Nichols, A., Ross, J.S.,
Tartaglia, L.A., Chen, H., 2003. Chronic inflammation in fat plays a crucial role in
the development of obesity-related insulin resistance. J. Clin. Invest. 112, 1821–
1830.