Phytochem Rev (2015) 14:443–457
DOI 10.1007/s11101-015-9399-z
Edible freshwater macrophytes: a source of anticancer
and antioxidative natural products—a mini-review
Tsun-Thai Chai • Keng-Fei Ooh • Yixian Quah
Fai-Chu Wong
Received: 17 December 2014 / Accepted: 5 March 2015 / Published online: 12 March 2015
Ó Springer Science+Business Media Dordrecht 2015
Abstract Edible freshwater macrophytes (EFM) are
edible, macroscopic freshwater plants, many of which
are also used as traditional/folk medicine. This mini-
review highlights phytochemical and pharmacological
evidence pertaining to anticancer and antioxidative
natural products derived from EFM, with special
attention to Centella asiatica (Indian pennywort),
Nelumbo nucifera (sacred lotus), Nasturtium officinale
(watercress), Ipomoea aquatica (water spinach) and
Ludwigia adscendens (water primrose). Current
knowledge gaps and further research opportunities
are also discussed. EFM is a promising source of
anticancer and antioxidative natural products which
warrants more extensive exploration. More research is
needed before such natural products can be exploited
for application in food and medicine.
Keywords Centella asiatica
Ipomoea aquatica

Ludwigia adscendens
Nasturtium officinale

Nelumbo nucifera
T.-T. Chai (&)
F.-C. Wong
Center for Biodiversity Research, Universiti Tunku Abdul
Rahman, 31900 Kampar, Malaysia
e-mail: chaitt@utar.edu.my
T.-T. Chai
K.-F. Ooh
Y. Quah
F.-C. Wong
Department of Chemical Science, Faculty of Science,
Universiti Tunku Abdul Rahman, 31900 Kampar,
Malaysia
•
Introduction
Freshwater macrophytes (EFM) refer to macroscopic
freshwater plants that occur in aquatic and semi-
aquatic habitats, including freshwater bodies such as
lakes, ponds, streams, rivers and waterlogged soils.
Such plants may be further categorized as emergent,
submergent, and floating macrophytes, depending on
their growing patterns. Terrestrial plants and micro-
scopic freshwater photosynthetic organisms (e.g.
algae) have attracted substantial attention in bio-
prospecting research aimed at new drug discovery. By
contrast, the importance of EFM, especially the edible
and medicinal ones, has often been overlooked.
Table 1 presents some well-known examples of edible
EFM.
The fact that numerous EFM species have been
traditionally used as food and medicine in different
geographical regions implies that such plants are
valuable sources of natural products which can be
developed into therapeutic agents and functional/
health-promoting food ingredients. Hence, in this
review, we aimed to highlight pharmacological and
phytochemical findings that substantiate the bioactive
potential of selected EFM. Current knowledge of
anticancer and antioxidative natural products of Cen-
tella asiatica, Nelumbo nucifera, Nasturtium of-
ficinale, Ipomoea aquatica and Ludwigia adscendens
(Fig. 1) is the main focus of our discussion. We also
identify gaps in knowledge and propose future
research opportunities.
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Table 1 Examples of EFM and their uses as medicine and food

Species Use as food Use in traditional/folk medicine References

Alternanthera sessilis Leave eaten raw or cooked
(Red sessile
joyweed)
Centella asiatica Leaves eaten raw or cooked and
(Indian pennywort) used to prepare herbal tea
Colocasia esculenta Leaves, petioles and corm eaten
(Taro) cooked as vegetable
Corm also made into cakes,
chips and snacks
Ipomoea aquatica Leaves and stem consumed as
(water spinach) vegetable
Ludwigia adscendens Consumed as vegetable in China
(water primrose)
Nasturtium officinale Young shoots and leaves eaten
(watercress) raw or cooked, and made into
soups
Nelumbo nucifera Seeds, flower petals, young
(sacred lotus) leaves and rhizomes eaten raw
or cooked
Rhizome flour used for baking
Oryza sativa (rice) Grains used as staple food for
more than half of the global
populations
Wasabia japonica Rhizome used for preparation of
(Wasabi/Japanese sauces and condiments
horseradish)
Remedy for stomach disorders, diarrhea, dysentery, Jansen (2004)
fever, bronchitis, and asthma
Decoction used to treat asthma, bronchitis, cancer, Ong (2008)
and diarrhea
Leaves and corm pounded and applied on bites, Ong (2008)
burns, and wounds
Corm boiled in vinegar and used to treat skin
diseases
Leaves and stem pounded and applied on boils, skin Ong (2008)
diseases and swellings
Remedy for food poisoning, headache, hematuria,
hemorrhoids, and cough
Treatment of cough, gonorrhea, measles, erysipelas Huang et al.
and furuncle (2007)
Decoction or soup used as antiscorbutic, diuretic, Ong (2008)
laxative and as remedy for cough, hypertension and
mouth ulcers
Leaf decoction used to treat diarrhea, fever and Mukherjee et al.
intestinal inflammations (2009), Ong
Flower decoction used to treat cholera, dysentery, (2008)
and fever
Seed decoction used to improve circulation and treat
diarrhea
Rhizome decoction used to treat hemorrhage,
hematemesis, fever and lung ailments
Treatment of inflammation, gastrointestinal ailments, Burlando and
hypercholesterolemia, diabetes and skin diseases Cornara
(2014)
Treatment for stomach disorders Hashimoto et al.
(2010)

Overview of phytochemistry and pharmacology
of edible freshwater macrophytes
A wide range of bioactive phytochemicals have been
identified in or isolated from EFM. Such phyto-
chemicals belong to diverse chemical groups, which
include flavonoids, alkaloids, glycosides, phenolic
acids, triterpenoids and vitamins (Mukherjee et al.
2009; Nagendra Prasad et al. 2008; Orhan 2012; Shilpi
et al. 2010). N. nucifera (sacred lotus) is an excellent
example of EFM in which bioactive phytochemicals
have been identified or isolated from different plant
organs. The seed of N. nucifera is a good source of
alkaloids (e.g. nuciferine and dauricine), in addition to
containing gallic acid, a phenolic acid. The leaves of
the species are rich in alkaloids (e.g. nuciferine) but
also contain glycoside (e.g. nelumboside) and flavo-
noids (e.g. quercetin). The flower of N. nucifera
contains flavonoids and their derivatives (e.g.
kaempferol, kaempferol 3-O-b-D-galactopyranoside,
and quercetin 3-O-b-D-glucopyranoside. The rhizome
of the plant contains betulinic acid, which is a steroidal
triterpenoid, as well as vitamins (thiamine, riboflavin,
niacin and ascorbic acid) (Mukherjee et al. 2009). In
the example of N. nucifera, the discovery of bioactive
constituents in the aforementioned plant organs is
notable as they are also the plant parts used for food
and medicine. Such findings suggest that the different
organs of an EFM species may be exploited as sources
of bioactive compounds.

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Phytochem Rev (2015) 14:443–457 445

Fig. 1 Examples of EFM species known to produce anticancer and antioxidative natural products. a Centella asiatica, b Nelumbo
nucifera, c Nasturtium officinale, d Ipomoea aquatica, e Ludwigia adscendens

Research to-date has revealed a diverse range of
bioactive or pharmacological effects in extracts and
compounds derived from EFM. These include an-
tioxidant, anticancer, antimicrobial, antiviral, antiaging,
antiinflammatory, antidiabetic, antimalarial, hepatopro-
tective, and neuroprotective activities. Extracts and
compounds derived from a single EFM species often
exhibit multiple bioactive or pharmacological effects.
The seed extract of N. nucifera, for example, exerted
antioxidant, hepatoprotective, antiproliferative and an-
tiinflammatory effects when tested on cellular and/or
animal models (Mukherjee et al. 2009). Asiaticoside, a
key active compound of C. asiatica, is also multifunc-
tional, exhibiting wound healing, antiinflammatory and
antitumor effects (Al-Saeedi 2014). Among the numer-
ous bioactivities associated with natural products
derived from EFM, anticancer and antioxidant activities
have been the subjects of extensive studies.
Anticancer natural products of edible freshwater
macrophytes
Need for natural anticancer agents
Cancer chemotherapy is one of the common therapeu-
tic approaches for fighting cancers (Kakde et al. 2011).
Chemotherapy often causes side effects, such as

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fatigue, nausea, vomiting, decreased blood cell counts,
hair loss, mouth sores, and pain (Reddy et al. 2003).
Therefore, the search for alternative or novel anti-
cancer agents with minimal or no side effects has
always been a research priority. Previous success of
cancer drug discovery from plants is a key driving
force behind current interests on edible and medicinal
plants among researchers (Prakash et al. 2013). A
recent review has comprehensively discussed the
diverse array of phytochemicals of edible plants which
exhibit anticancer potential through their effects on the
mitochondrial functionality of targeted cells (Forbes-
Hernandez et al. 2014). Notably, although underex-
plored compared with terrestrial plants, EFM species
may also be an equally valuable source of potent
anticancer natural products as indicated by the grow-
ing body of evidence in the literature.
Common experimental approaches
Solvents such as methanol, ethanol, chloroform and
water are widely used to extract anticancer com-
pounds from EFM (Babykutty et al. 2008; Dantas-
Santos et al. 2012; Mukherjee et al. 2009; Pittella et al.
2009). Sequential fractionation of active extracts with
organic solvents, for example, petroleum ether, ace-
tone, dichloromethane, ethyl acetate, butanol and
methanol (Khlifi et al. 2013; Tagne et al. 2014) has
also been reported. The cytotoxicity assay most
widely used for estimating the anticancer potential
of EFM is the 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) assay (Babykut-
ty et al. 2008; Fan et al. 2014; Liu et al. 2014; Park
et al. 2005), but other assays such as the sulphorho-
damine B (SRB), neutral red, and lactate dehydroge-
nase (LDH) leakage assays have also been used
(Aboul-Enein et al. 2014; Prasad et al. 2005; Rose
et al. 2005). The MTT assay gives an indication of
whole cell cytotoxicity. The assay can be readily
performed on a wide range of cell lines, with high
reliability and versatility (McCauley et al. 2013).
Cancer cell lines used to test the cytotoxicity of EFM
included human breast cancer (MDA-MB-231; MCF-
7), human hepatocellular carcinoma (HepG2), human
lung carcinoma (A549), human colon adenocarcino-
ma (HT29; SW480), human cervix adenocarcinoma
(HeLa), rat glioma (C6), and mouse melanoma (B16F1)
cell lines (Babykutty et al. 2008; Boyd et al. 2006;
Pittella et al. 2009; Rose et al. 2005).
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Phytochem Rev (2015) 14:443–457
The mechanisms of action of anticancer extracts
and compounds obtained from EFM have been studied
at the cellular and molecular levels. Flow cytometric
analysis, apoptosis-specific assays (i.e., acridine or-
ange/ethidium bromide staining; annexin/propidium
iodide staining), mitochondrial membrane potential
visualization assay, DNA fragmentation assay, gene
expression analysis, reverse transcription-polymerase
chain reaction (RT-PCR) assay, and Western blot
analysis have been used to study the induction of
apoptosis by extracts and compounds isolated from
EFM (Aboul-Enein et al. 2014; Babykutty et al. 2008;
Chen et al. 2014; Dwarka 2012; Engel et al. 2014;
Hussin et al. 2014; Zhang et al. 2013b). Instrumental
analyses such as reversed-phase high performance
liquid chromatography (RP-HPLC), gas chromatog-
raphy (GC), mass spectrometry (MS), infrared (IR)
and nuclear magnetic resonance (1D and 2D NMR)
(Boyd et al. 2006; Fan et al. 2014; Ooh et al. 2014)
were used to detect, characterize, and identify poten-
tial anticancer compounds from EFM.
Selected examples
Centella asiatica
The antiproliferative effects of C. asiatica extracts
have been demonstrated in several cancer cell lines,
such as MDA-MB-231, MCF-7, B16F1, and C6 cell
lines (Babykutty et al. 2008; Pittella et al. 2009). The
hydromethanolic extract of C. asiatica induced apop-
tosis in MCF-7 cells, as evidenced by nuclear conden-
sation, increased annexin staining, pertubation of
mitochondrial membrane potential, and induction of
DNA breakage (Babykutty et al. 2008). Notably, the
aqueous extract of C. asiatica was not cytotoxic to
normal hamster kidney (BHK-21) cell lines (Pittella
et al. 2009). Acetone extract of C. asiatica was non-
toxic to normal human lymphocytes; more important-
ly, it protected the cells from the damaging effects of
cyproterone acetate, a genotoxic and tumor-initiating
agent (Siddique et al. 2008).
The anticancer effects of C. asiatica in animal
models have also been reported. Aqueous extract of C.
asiatica protected against the formation of azoxy-
methane-induced aberrant crypt foci (ACF) in rats;
ACF are precursor lesions of colon cancer (Bunpo et al.
2004). The extract suppressed ACF development by
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inhibiting cell proliferation and inducing apoptosis in
the colonic crypts. Chemopreventive effect against
colon tumorigenesis was substantiated by reduced
incidences and sizes of neoplasms as well as reduced
numbers of intestinal adenocarcinomas in rats fed with
C. asiatica extract (Bunpo et al. 2004). Protective effects
of topically applied aqueous extract of C. asiatica
against 7,12-dimethylbenz[a]-anthracene (DMBA)-in-
duced skin tumorigenesis in mice were evidenced by
significant reduction in the incidence and size of tumor,
as well as the number of papillomas (Rai et al. 2011b).
Furthermore, oral administration of aqueous extract of
C. asiatica was found to increase survival time and
reduce tumor volume in mice implanted with melanoma
cells (B6F10) (Rai et al. 2011a).
Nine triterpenes (e.g. ursolic acid, asiatic acid,
corosolic acid, and b-sitosterol 3-O-b-glucopyra-
noside) and rosmarinic acid were isolated from the
methanolic extract of C. asiatica (Yoshida et al. 2005).
The 10 compounds were cytotoxic to human gastric
adenocarcinoma (MK-1), human uterine carcinoma
(HeLa), and murine melanoma (B16F10) cells to
different degrees. Ursolic acid, corosolic acid, and b-
sitosterol 3-O-b-glucopyranoside exhibited the stron-
gest cytotoxicity against MK-1, HeLa, and B16F10
cells, respectively (Yoshida et al. 2005). Another
study has isolated cadiyenol, a polyacetylene com-
pound which was cytotoxic to mouse lymphoma cells
(P388D1), from C. asiatica. Cadiyenol did not disrupt
the cell cycle regimen of the P388D1 cells; it induced
cell death mainly by apoptosis (Govindan et al. 2007).
The cellular and molecular mechanisms underlying
the antiproliferative effects of asiatic acid have been
examined in several cancer cell types. In MCF-7 and
MDA-MB-231 cells, apoptosis and cell cycle arrest
induced by asiatic acid were attributed to the activa-
tion of mitogen-activated protein kinases (MAPKs)
pathways (extracellular signal-regulated kinase
(ERK1/2) and p38 kinase) (Hsu et al. 2005). In human
liver cancer cells (HepG2), asiatic acid induced
apoptosis by elevating intracellular Ca2? concentra-
tion, which in turn, enhanced the expression of p53
protein (Lee et al. 2002). A recent study reported that
the antiproliferative effect of asiatic acid on HepG2
cells was mediated by enhanced stability and up-
regulated expression of the p21WAF1/CIP1 protein, a
regulator of cell cycle progression at G1 phase (Chen
et al. 2014). In myeloma (RPMI 8226) cells, cell cycle
arrest induced by asiatic acid was associated with
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reduced expression of the focal adhesion kinase (FAK)
protein. The antiproliferative effect of asiatic acid in
RPMI 8226 cells was suggested to be mediated by the
inhibition of signal transduction mediated by FAK
(Zhang et al. 2013b). In human melanoma cells (SK-
MEL-2), the induction of apoptosis by asiatic acid was
mediated by increased generation of reactive oxygen
species (ROS), alteration of Bax/Bcl-2 ratio, and
enhanced of caspase-3 activity (Park et al. 2005). In
SW480 cells, asiatic acid-induced apoptosis was
mediated by loss of mitochondrial membrane poten-
tial, enhanced cytochrome c release, as well as
activation of caspase signaling pathways and poly
(ADP-ribose) polymerase cleavage (Tang et al. 2009).
Current lines of evidence suggest that the anticancer
mechanisms of asiatic acid are different, depending on
the cancer cell types examined.
The anticancer activity of asiaticoside, another
bioactive compound of C. asiatica, has also been
demonstrated in in vitro and in vivo cancer models.
Asiaticoside induced apoptosis in MCF-7 cell line. In
rats that developed DMBA-induced multiple mam-
mary tumors, tumor regression and reduction were
observed upon asiaticoside treatment (Al-Saeedi
2014). Asiaticoside also showed synergistic effect
with vincristine in triggering apoptosis in human
mouth epidermoid carcinoma (KB), multidrug-resis-
tant KB (KBv200), MCF-7, and adriamycin-resistant
MCF-7 (MCF-7/ADM) cell lines (Huang et al. 2004).
Nelumbo nucifera
The mechanism of anticancer effects of neferine, a
major bisbenzylisoquinoline alkaloid isolated from the
embryos of N. nucifera, has been investigated in three
cancer cell lines. In hepatocellular carcinoma (Hep3B)
cells, neferine downregulated the dephosphorlyation of
cdc2 and expression of c-Myc, cyclin D1, D3, CDK4,
and E2F-1 proteins; this likely forms the molecular
basis of the cell cycle arrest in Hep3B cells (Yoon et al.
2013). In the same study, neferine also induced
endoplasmic reticulum stress and apoptosis in the
Hep3B cells. In A549 cells, neferine induced apopto-
sis, activation of MAPKs, lipid oxidation, depletion of
cellular antioxidants, loss of mitochondrial membrane
potential, and accumulation of intracellular calcium
(Poornima et al. 2014). In human osteosarcoma cells,
the inhibitory effect of neferine was largely attributed
to cell cycle arrest at G1 (Zhang et al. 2012).
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7-Hydroxydehydronuciferine isolated from the leaf
of N. nucifera inhibited the proliferation of melanoma,
prostate and gastric cancer cells; but the compound
also killed normal cells as revealed by MTT assays.
The cytotoxicity of 13 other compounds isolated from
N. nucifera, i.e., lysicamine, (-)-anonaine, (-)-asim-
ilobine, (-)-caaverine, (-)-N-methylasimilobine,
(-)-nuciferine, (-)-nornuciferine, (-)-roemerine,
cepharadione B, b-sitostenone, stigmasta-4,22-dien-
3-one, pheophytin-a and aristophyll-C, showed lower
cytotoxicity compared to 7-hydroxydehydronucifer-
ine (Liu et al. 2014).
Nasturtium officinale
Nasturtium officinale, besides broccoli, is the richest
natural source of phenethyl isothiocyanate (PEITC), a
natural product with remarkable anticancer potential.
PEITC can be released from its precursor gluconas-
turtiin by the action of myrosinase; the enzyme can be
activated by cutting or chewing N. officinale. The
chemopreventive and chemotherapeutic effects of
PEITC, along with references to different cruciferous
vegetables (including N. officinale) as examples, have
been recently reviewed (Gupta et al. 2014). Earlier
studies have also associated the anticancer potential of
N. officinale with their phenolic constituents and other
isothiocyanates. The chemoprotective potential of N.
officinale extract has been demonstrated by using
in vitro models for the three important stages of
carcinogenesis, namely initiation, proliferation, and
metastasis (Boyd et al. 2006). In the study, N.
officinale extract exhibited anti-genotoxic effects in
HT29 cells challenged with H2O2 and fecal water. The
extract also induced cell cycle delay in the HT29 cells.
Moreover, the extract inhibited the invasion of HT115
colon cancer cells through matrigel. The authors
proposed that the observed effects can be at least partly
attributed to quercetin glycosides (including rutin) and
hydroxycinnamic acid derivatives detected in the
extract by HPLC–ESI–MS analysis (Boyd et al.
2006). In another study, hydromethanolic extract of
N. officinale inhibited 12-O-tetradecanoylphorbol-13-
acetate-induced matrix metalloproteinase-9 (MMP-9)
activity and the invasive potential of MDA-MB-231
cells. LC–MS analysis found such effects to be
associated with the presence of 4-methylsulfinylbutyl
and 7-methylsulfinylheptyl isothiocyanates in the
extract (Rose et al. 2005).
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Ipomoea aquatica
A number of natural products isolated from I.
aquatica have been shown to be cytotoxic against
human cancer cell lines. The isolation of 11 resin
glycosides (aquaterins I–XI) from I. aquatica was
recently reported (Fan et al. 2014). Based on MTT
assays, most of the resin glycosides exhibited low
cytotoxicity against the human cancer cell lines
tested. In the HepG2 cell line, cytotoxicity of such
compounds was associated with their ability to
elevate intracellular Ca2? concentration. Aquaterin
IV was the most cytotoxic resin glycoside against the
HepG2 cells, which had an IC50 value (2.4 lM) lower
than that of cis-platinum (IC50 6.4 lM). Cell cycle
analysis found that aquaterin IV inhibited HepG2 cell
proliferation by G0/G1 arrest and apoptosis induction
(Fan et al. 2014). 7-O-b-D-glucopyranosyl-dihydro-
quercetin-3-O-a-D-glucopyranoside (DHQG) isolat-
ed from I. aquatica crude methanolic extract was
investigated for cytotoxicity by using MTT and SRB
assays. DHQG concentrations required to kill 50 %
of normal African green monkey kidney (Vero),
A549, and human larynx epithelial carcinoma (Hep-
2) cells were 387, 394 and 156 mg/mL, respectively
(Prasad et al. 2005). Whether aquaterin IV and
DHQG have any in vivo anticancer effects remains to
be confirmed.
Quercetin and chlorogenic acid, two potential
anticancer agents, have also been isolated from I.
aquatica leaves (Sivaraman et al. 2014). Quercetin
inhibited the growth of several human cancer cell
lines at different phases of the cell cycle, without
compromising the growth of normal, non-trans-
formed cells (Gibellini et al. 2011). Quercetin also
induced apoptosis in A549 cells (Zheng et al. 2012)
and KB and KBv200 cells (Zhang et al. 2013a). The
cellular and molecular mechanisms underlying the
pro-apoptotic effects of quercetin in different cancer
cell lines have been detailed in a recent review
(Forbes-Hernandez et al. 2014). The ability of
chlorogenic acid to promote tumor cell apoptosis
and its molecular basis have also been reported (Xu
et al. 2013b).
Ludwigia adscendens
RP-HPLC analysis revealed the presence of gallic
acid in L. adscendens leaves (Ooh et al. 2014). Gallic
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acid is known to induce apoptosis in various cancer
cell lines, including esophageal cancer (TE-2) and
human melanoma (A375.S2) cells (Lo et al. 2010) as
well as several human lung cancer cell lines (Ohno
et al. 1999). Gallic acid was nontoxic to non-
cancerous cell line (CHEK-1, immortalized human
esophageal cell) (Faried et al. 2007). Another study
reported that rosmarinic acid was a major constituent
in this plant (Huang et al. 2011). Supplementation of
rosmarinic acid to 1,2-dimethylhydrazine-treated
mouse decreased the incidence and distribution of
tumors along the colon (Venkatachalam et al. 2013).
Rosmarinic acid also lowered the incidence of oral
tumor induced in hamsters by topical application of
DMBA (Baldasquin-Caceres et al. 2014). Hence,
current evidence, although limited, points to the
presence of anticancer natural products in L.
adscendens.
Antioxidative natural products of edible
freshwater macrophytes
Need for natural antioxidants
Synthetic antioxidants such as butylated hy-
droxytoluene and butylated hydroxylanisole were
introduced to the food industry in 1940s (Berdahl
et al. 2010). They are cheap and efficient antioxidants,
but were linked to potential carcinogenesis (Chen
et al. 1992; Sun and Fukuhara 1997). Such risk,
together with consumer preference for natural food
ingredients, has led to a growing demand for natural
antioxidants (Berdahl et al. 2010). An advantage of
natural antioxidants is that they may not require
extensive safety testing prior to use (Huang et al.
2011). Besides the food industry, there is also
increasing demand for natural antioxidants in the
cosmetic industry and for applications in therapy
against oxidative stress-related diseases (Lobo et al.
2010). Food and medicinal plants have been long
regarded as potential sources of natural antioxidants
by researchers. Natural products from non-aquatic
food plants such as oregano, sage (Fasseas et al.
2007), rosemary (Sebranek et al. 2005), and black
pepper (Martı́nez et al. 2007) have been shown to
possess potent antioxidant activities. By contrast,
the antioxidant potential of many EFM is still
underexplored.
449
Common experimental approaches
Polar solvents, such as water, methanol, ethanol, and
ethyl acetate, are commonly used to extract an-
tioxidants from EFM. Hexane has been used as
extractant; however, the antioxidant activity of hexane
extract was inferior to those of polar solvent extracts
(Wijeweera et al. 2006). Soxhlet extraction, heat-
reflux extraction (Ma et al. 2010), sequential solvent
extraction (Anand et al. 2010; Wijeweera et al. 2006)
and ionic liquid-based microwave-assisted extraction
(Ma et al. 2010) have been used to extract antioxidants
from EFM. The common range of extraction tem-
perature for antioxidant extraction from EFM varied
from cold maceration (4 °C) to 65 °C and the duration
of extraction ranged from 1 to 72 h.
Chromatographic techniques such as RP-HPLC
have been used to detect phytochemicals in EFM
extracts exhibiting antioxidant activities (Ooh et al.
2014; Orhan 2012). More advanced technologies
employed to identify and characterize antioxidants
from EFM included HPLC–electrospray ionization–
mass spectrometry (HPLC–ESI–MS) (Fu et al. 2011),
photodiode array detection-electrospray ionization-
mass spectrometry (HPLC–DAD–ESI–MS/MS) (Chen
et al. 2012b), and NMR analysis (Marzouk et al. 2007).
Multiple in vitro assays have been used to
characterize the antioxidant potential of EFM ex-
tracts and compounds isolated from them. Such
in vitro antioxidant assays included 2,2-diphenyl-1-
picrylhydrazyl (DPPH) radical scavenging assay
(Anand et al. 2010; Ooh et al. 2014; Orhan 2012),
nitric oxide (NO) scavenging assay (Ooh et al. 2014;
Rai et al. 2006), metal chelating assay (Bahramikia
and Yazdanparast 2010; Ooh et al. 2014; Orhan
2012), Ferric Reducing Antioxidant Power (FRAP)
assay (Anand et al. 2010; Orhan 2012), and hydroxyl
radical scavenging assay (Anand et al. 2010; Das-
gupta and De 2007).
Studies on animal models have also been carried
out to investigate the antioxidant effects of EFM
extracts and their compounds on oxidative markers,
such as the activities of superoxide dismutase (SOD),
catalase (CAT), glutathione peroxidase (GSH-Px)
(Gnanapragasam et al. 2004; Hussin et al. 2007;
Subathra et al. 2005), as well as the level of lipid
peroxidation (Fu et al. 2011; Yazdanparast et al.
2008). Oxidative stress markers were studied in
different organs/tissues of the animal models, most
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frequently in brain tissues, followed by serum, red
blood cells, liver, and heart tissues.
Selected examples
Centella asiatica
Several studies found that the whole plant extracts of
C. asiatica increased SOD, CAT, and GSH-Px
activities, as well as reducing malondialdehyde
(MDA) content, an index of lipid peroxidation, in
rat brain (Orhan 2012; Subathra et al. 2005;
Veerendra Kumar and Gupta 2002). Such evidence
suggests that the neuroprotective effects of C.
asiatica extract is attributable to its potent an-
tioxidant activities (Orhan 2012; Orhan et al. 2013;
Subathra et al. 2005).
Asiatic and madecassic acids are the two major
triterpene saponosides, whereas asiaticoside and
madecassoside are the two major heterosides in C.
asiatica. These phytochemicals are believed to have
protective effects against brain aging (Orhan 2012).
Madecassoside has been shown to be a more potent
antioxidant than nimodipine, an antioxidant with
neuroprotective effects. Madecassoside ameliorated
oxidative stress by reducing oxygen free radicals in
rat brain and reducing the levels of NO in rat serum.
Thus, madecassoside may protect against cerebral
ischemia–reperfusion injury in rats (Luo et al. 2014).
Madecassoside also decreased MDA contents and
increased GSH contents in rat brain tissue. The
balanced redox reaction in tissue microenvironment
was proposed to be the most possible mechanism
whereby madecassoside exerts its neuroprotective
effect (Xu et al. 2013a). Asiaticode was shown to
significantly reverse the neurotoxic effects of
1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine
(MPTP) in the rat model of Parkinsonism. MDA level
was lowered and GSH level increased in the substan-
tia nigra of the rat brain. The reversal balance of redox
in tissue microenvironment is likely the mechanism
by which asiaticode exerts its neuroprotective effect
(Xu et al. 2012). In a nutshell, phytochemicals found
in C. asiatica may be potential therapeutic agents or
lead compounds for discovery of drugs for the
management of neurodegenerative diseases, such as
Parkinson’s disease, and other oxidative stress-related
diseases.
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Phytochem Rev (2015) 14:443–457
Nelumbo nucifera
Quercetin 3-O-b-D-glucopyranoside and quercetin
3-O-b-D-glucuronopyranoside were two major flavo-
noids detected using HPLC and isolated from N.
nucifera leaf extract. The two flavonoids showed
superior inhibition against rat lens aldose reductase
(AR) activity and the formation of advanced glycation
endproducts (AGE) (Jung et al. 2008). AR-related
polyol pathway and AGE formation contribute to
oxidative stress particularly in eye lenses and nerves
tissues; these can lead to oxidative stress-related
diabetic complications (Chung et al. 2003). Quercetin
was another potent phytochemical isolated from the
ethanolic fraction of N. nucifera. Notably, quercetin
was a better inhibitor of AR than quercetin 3-O-b-D-
glucopyranoside and quercetin 3-O-b-D-glucuronopy-
ranoside (Jung et al. 2008). In another study, quercetin
reduced the levels of erythrocyte and pancreatic tissue
MDA as well as serum NO in streptozotocin-induced
diabetic rats; increased SOD, CAT and GSH-Px
activities in pancreatic tissues were also observed
(Coskun et al. 2005). Thus, quercetin, quercetin 3-O-
b-D-glucopyranoside, and quercetin 3-O-b-D-glu-
curonopyranoside are N. nucifera natural products
with potential applications in the management of
diabetes and other oxidative stress-related patho-
logical conditions.
A number of other flavonoids have also been
isolated from N. nucifera, such as N-nornuciferine, O-
nornuciferine, nuciferine (Luo et al. 2005), hyperoside
(Deng et al. 2009), isohamnetin, and diosmetin
derivatives (Chen et al. 2012a). The in vivo and
in vitro antioxidative effects of these flavonoids
remain to be confirmed in future.
Nasturtium officinale
Ethanolic extract of N. officinale exhibited in vitro
scavenging activity against DPPH, 2,2-azinobis-3-
ethylbenzothiazoline-6-sulfonate (ABTS) and NO, as
well as metal chelating activity and ferric reducing
activity. The extract also inhibited lipid peroxidation
in rat liver homogenate, likely attributable to its ability
to chelate Fe2? ions (Bahramikia and Yazdanparast
2010). In vivo antioxidant effect of hydroethanolic
extract of N. officinale was demonstrated by the its
ability to increase SOD and CAT activities, reduce
lipid peroxidation, and increase GSH levels in the liver
Phytochem Rev (2015) 14:443–457
of hypercholesterolemic rats (Yazdanparast et al.
2008). Hypercholesterolemia is known to weaken
antioxidant defense in vivo, resulting in increased
levels of lipid peroxidation (Anila and Vijayalakshmi
2003; Daniel et al. 1998). Another study found that
daily supplementation of diet with 85 g of raw N.
officinale for 8 weeks decreased lymphocyte DNA
damage in human subjects, with a concomitant
increase in plasma antioxidant concentrations of b-
carotene and lutein (Gill et al. 2007). N. officinale
supplementation had no effects on the plasma con-
centrations of a-tocopherol, retinol, and ascorbic acid
as well as the ferric reducing ability of plasma. Neither
were erythrocyte GSH-Px and SOD activities altered
by N. officinale supplementation in the study (Gill
et al. 2007).
In a phytochemical characterization and an-
tioxidant study of N. officinale, HPLC–MS and
HPLC–DAD analyses revealed two major classes of
phytochemicals which were phenolics and glucosino-
lates. The major phenolics were chlorogenic acid,
quercetin-3-O-rutinoside (rutin), dicaffeoyltartaric
acid, and isorhamnetin; the major glucosinolate was
gluconasturtiin (Aires et al. 2013). In the same study,
the hydromethanolic extract exhibited good an-
tioxidant activity when tested with the DPPH scav-
enging assay and reducing power antioxidant assay.
Pearson correlation analysis and primary component
analysis confirmed that the antioxidant activities of N.
officinale can be accounted for by caffeic acid, rutin,
and gluconasturtiin (Aires et al. 2013). Hydroethanolic
extract of N. officinale and its dichloromethane, ethyl
acetate, and butanol fractions exhibited good DPPH
scavenging activity and inhibited Fe2?-induced lipid
peroxidation in rat brain homogenates. HPLC–DAD
analysis detected rutin, chlorogenic acid, and caffeic
acid in the hydroethanolic extract of N. officinale
(Boligon et al. 2013).
A GC–MS study of N. officinale reported nine,
eight, and 15 essential oils from the leaves, stems and
flowers of the species, respectively (Amiri 2012).
Myristicin, a-terpinolene and limonene are the major
constituents in the essential oil of the leaves. The
major compounds of the oils of stems and flowers
were caryophyllene oxide, p-cymene-8-ol, a-ter-
pinolene and limonene. DPPH scavenging and b-
carotene bleaching assays found that the essential oils
of the leaves, stems and flowers all exhibited
451
antioxidant activity, but the leaves’ oil was the most
active. a-terpinolene and limonene were key compo-
nents in the essential oils of all three plant organs
(Amiri 2012), suggesting that the two compounds may
possess antioxidant activity. Myristicin was the most
abundant component (57.6 %) of the leaves’ oil,
which had the strongest antioxidant activity; thus
myristicin is also worthy of further investigations.
Ipomoea aquatica
The major phytochemicals in I. aquatica are carote-
noids, namely b-carotene and lutein (Chen and Chen
1992; Tee and Lim 1990) with smaller fractions of
violaxanthin, neoxanthin and lutein epoxide (Chen
et al. 1991; Wills and Rangga 1995). b-carotene
purified from I. aquatica leaf had stronger DPPH
radical scavenging activity compared with lutein and
violaxanthin (Fu et al. 2011). The strong scavenging
activity of b-carotene was possibly due to the allylic
position of hydrogen at C-4 of two ring b-ionone
found in b-carotene (Fu et al. 2011; Woodall et al.
1997).
The in vitro antioxidative activity of I. aquatica
extracts has been reported. Water extract showed
excellent DPPH, superoxide, and hydroxyl radical
scavenging activities compared with other leafy
vegetables; the extract also inhibited lipid peroxida-
tion in an assay using egg yolk homogenates as lipid-
rich medium (Dasgupta and De 2007). Another study
has demonstrated the ability of ethanolic extract of I.
aquatica stem to scavenge DPPH radicals and reduce
Fe3? ions, as well as dampening linoleic acid oxida-
tion (Huang et al. 2005).
Several studies have demonstrated the hepatopro-
tective activity of I. aquatica extracts, which are
associated with their ability to enhance in vivo
antioxidative defense of animal models. Administra-
tion of the hydromethanolic extract of I. aquatica to
carbofuran-treated rats restored the CAT activity in
the liver to normal levels. SOD activity was increased
in the liver, plasma and brain tissue; GSH level was
increased in the liver and plasma (Datta et al. 2013).
In the aforementioned study, RP-HPLC analysis
detected rutin, quercetin, and apigenin as major
phenolic components in the extract. Oral administra-
tion of freeze-dried red-stemmed I. aquatica to carbon
tetrachloride-treated mice also suppressed lipid
123
452
peroxidation in the liver (Hirai et al. 2011). In another
study, ethanolic extract of I. aquatica restored the
activities of SOD and CAT and reduced MDA levels
in the liver of thioacetamide-treated rats (Alkiyumi
et al. 2012).
A study on streptozotocin-induced diabetic rats
found that administration of methanolic extract of the
plant had antihyperglycemic effects (Saha et al. 2008).
Notably, the extract attenuated oxidative stress in vivo
by enhancing CAT activity and GSH content in the
liver, kidney and pancreas tissues (Saha et al. 2008).
Although preliminary in nature, the finding suggests
that I. aquatica extract may be useful for preventing
oxidative stress-related complications associated with
diabetes.
Ludwigia adscendens
Rosmarinic acid, quercetin 3-O-b-D-glucopyranoside
and kaempferol 3-O-b-D-glucopyranoside were found
to be the major antioxidants in L. adscendens,
following bioactivity-guided chromatographic frac-
tionation of a methanol/chloroform extract as well as
NMR, mass spectral and RP-HPLC analyses. The
order of in vitro antioxidant potency of the three
compounds were rosmarinic acid [ quercetin 3-O-b-
D-glucopyranoside [ kaempferol 3-O-b-D-glucopyra-
noside, based on results of DPPH and hydroxyl
scavenging assays as well as reducing power assay
(Huang et al. 2007). In a more recent study, RP-HPLC
analysis has detected gallic acid, p-coumaric acid,
chlorogenic acid, myricetin and rutin in the hot water
extracts of L. adscendens leaf and stem (Ooh et al.
2014). The same study also found strong DPPH and
NO scavenging activity as well as iron chelating
activity in the L. adscendens leaf extract, which
correlated to its high contents of gallic acid, p-
coumaric acid and myricetin (Ooh et al. 2014). This
link between myricetin content and high antioxidant
activity in L. adscendens corroborates an earlier
animal study which suggested a link between the
bioactivity of L. adscendens and its flavonoid contents.
An ethyl acetate extract of L. adscendens showed
in vivo antioxidant effects when orally administered
into animal models, leading to decline in serum MDA
levels and increase in hepatocellular GSH levels.
Thirteen flavonoids were isolated from the extract,
which included quercetrin, hyperin, rutin, kaempferol
and quercetin (Marzouk et al. 2007).
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Phytochem Rev (2015) 14:443–457
Gaps in knowledge and further research
opportunities
From the examples of EFM highlighted in this
review, it is clear that EFM species, in general, are
a valuable source of anticancer and antioxidative
natural products (Table 2). Notably, some of such
natural products have been shown to have concurrent
anticancer and antioxidant effects. Notwithstanding,
knowledge gaps exist and further research is neces-
sary before the potential of such natural products can
be more effectively harnessed for food and biome-
dical applications.
Current knowledge of the bioactive phytochemicals
occurring in EFM species is still limited. For example,
I. aquatica, and L. adscendens are still not well-
explored with regards to their anticancer and an-
tioxidant constituents. The anticancer and antioxida-
tive properties of not only these two but many other
EFM species were confirmed primarily in crude and/or
partially purified extracts, not in isolated compounds.
Thus more studies are needed to isolate and identify
active compounds from such active EFM extracts. One
knowledge gap is that concerning the potential toxic
effects of antioxidative and anticancer natural prod-
ucts of EFM in vivo. A previous study has shown that
natural products from EFM (e.g. 7-hydroxydehy-
dronuciferine) may be simultaneously toxic to both
cancer and non-cancer cells (Liu et al. 2014). Thus,
despite the edibility of EFM, the safety of bioactive
natural products purified or isolated from the plants
cannot be simply assumed. In general, potent cyto-
toxic agents which preferentially or specifically target
cancer cells, with minimal or no detectable toxicity on
normal cells will be highly worthy for further inves-
tigations on animal models.
One further research opportunity would be to
identify the mechanisms of action of the anticancer
and antioxidative constituents isolated from EFM. For
such compounds, their bioactive effects should be
corroborated by detailed studies at the molecular,
cellular and physiological levels. In this respect,
transcriptome and proteome analyses on susceptible
cancer cell types will shed light on the molecular and
cellular events targeted by the active compounds. To
better characterize the physiological basis of the
anticancer effects of an EFM-derived anticancer
compound, studies on ex vivo and in vivo animal
models should be considered. Notably, the use of
Phytochem Rev (2015) 14:443–457 453

Table 2 Summary of anticancer and antioxidative natural products of Centella asiatica, Nelumbo nucifera, Nasturtium officinale,
Ipomoea aquatica, and Ludwigia adscendens
EFM Anticancer compounds Antioxidant compounds References

Centella Asiatic acid, asiaticoside, cadiyenol,
asiatica corosolic acid, rosmarinic acid,
ursolic acid, b-sitosterol 3-O-b-
glucopyranoside
Nelumbo 7-Hydroxydehydronuciferine,
nucifera neferine
Nasturtium 4-Methylsulfinylbutyl and
officinale 7-methylsulfinylheptyl
isothiocyanates, hydroxycinnamic
acid derivatives, phenethyl
isothiocyanate, quercetin glycosides
Ipomoea 7-O-b-D-glucopyranosyl-
aquatica dihydroquercetin-3-O-a-D-
glucopyranoside, aquaterins I–XI,
chlorogenic acid, quercetin
Ludwigia Gallic acid, rosmarinic acid
adscendens
Asiaticoside, madecassoside
Quercetin, quercetin 3-O-b-
D-glucuronopyranoside, quercetin
3-O-b-D-glucopyranoside
a-Terpinolene, caffeic acid,
caryophyllene oxide, chlorogenic
acid, gluconasturtiin, limonene,
myristicin, p-cymene-8-ol, rutin
Apigenin, lutein, quercetin, rutin,
violaxanthin, b-carotene
Chlorogenic acid, gallic acid, hyperin,
kaempferol, kaempferol 3-O-b-D-
glucopyranoside, myricetin, p-
coumaric acid, quercetin, quercetin
3-O-b-D-glucopyranoside,
quercetrin, rosmarinic acid, rutin
Al-Saeedi (2014), Govindan et al.
(2007), Huang et al. (2004),
Orhan (2012), Xu et al. (2013a),
Yoshida et al. (2005)
Jung et al. (2008), Liu et al. (2014),
Yoon et al. (2013)
Aires et al. (2013), Amiri (2012),
Boyd et al. (2006), Gupta et al.
(2014), Rose et al. (2005)
Fan et al. (2014), Fu et al. (2011),
Prasad et al. (2005), Sivaraman
et al. (2014)
Huang et al. (2011), 2007, Marzouk
et al. (2007), Ooh et al. (2014)

xenograft and genetically engineered mouse models
will be a valuable approach for gleaning information
on the in vivo therapeutic effects of the anticancer
compounds of EFM.
Current evidence for the antioxidative effects of
EFM-derived natural products is mainly obtained from
in vitro chemical assays. As pointed out in a recent
review, the commonly used in vitro antioxidant assays
often produce inconsistent results among themselves
and may not correlate with in vivo antioxidative
capacity of the natural products under investigation
(Niki 2011). In vitro assays are convenient for early
screening and can shed light on in vitro antioxidant
mechanisms. Notwithstanding, in the light of the long-
term goal of applying EFM-derived antioxidants in
food for human consumption, establishing the in vivo
efficacy of such compounds in animal and, eventually,
human studies is imperative. To this end, the effects of
antioxidant supplementation on various lipid-, pro-
tein- and DNA-related biomarkers of oxidative stress
in biological fluids and tissues may be assessed (Niki
2011). Bioavailability is one of the key determinants
of in vivo antioxidant capacity which cannot be readily
predicted from in vitro antioxidant capacity (Niki
2011). Thus, oral bioavailability studies of promising
EFM-derived antioxidants on animal models should
also be a priority in future. Prior to proceeding to
animal studies, future studies may also consider
testing the EFM-derived antioxidants with the cellular
antioxidant activity assay (Wolfe and Liu 2007),
which is more accessible than animal studies. When
used in conjunction with suitable microscopic and
molecular biology tools, the cellular antioxidant
activity assay may also facilitate the elucidation of
the molecular and cellular basis of the antioxidative
effects of natural products. Although the cellular assay
is relatively in vitro in nature in comparison with
animal studies, such an assay will provide more
biologically relevant information when compared
with in vitro chemical assays.
In cases where certain anticancer and/or antioxida-
tive natural products of EFM have been identified as
lead compounds for drug development, research
opportunities also arise for structural modification of
such compounds. Structural optimization may aid
manipulating the molecular size and complexity of

123
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the active compounds, in addition to improving their
potency, selectivity, pharmacokinetic properties as
well as eliminating side effect (Guo 2012). On the
other hand, many EFM species may not be easily
accessible or readily harvested in adequate quantities
owing to their aquatic or semi-aquatic habitats. More-
over, the extraction of some EFM natural products may
not be cost-effective due to suboptimal or low yield.
Hence, in the long run, cell and tissue culture of a
promising species for in vitro production and harvest of
desired anticancer and antioxidative natural products is
also worthwhile of future investigations.
Yet another promising area for future research is the
application of anticancer and antioxidant phyto-
chemicals derived from EFM, in the form of crude or
partially purified extracts, as health supplements,
ingredients of personal care products, and food ingre-
dients/preservatives. Products such as herbal supple-
ments, cosmetics, shampoos, and mouthwash liquid
based on leaf extracts of C. asiatica, N. officinale and
N. nucifera have been marketed. More studies to
discover the commercial applications of EFM extracts
whether as health-promoting agents or in personal care
products may help to boost the market value of EFM-
derived natural products. On the other hand, water
extracts of rhizome knots and leaves of N. nucifera
have been shown to protect porcine and bovine ground
meat from lipid oxidation, thus potentially extending
their shelf life (Huang et al. 2011). Therefore, the
potential use of antioxidants and other phytochemicals
derived from EFM as natural food preservatives/
additives should be addressed in future research.
Conclusions
EFM, as exemplified by C. asiatica, N. nucifera, N.
officinale, I. aquatica and L. adscendens, are a
promising source of anticancer and antioxidant phy-
tochemicals. Phytochemical and pharmacological re-
search to-date has revealed EFM-derived natural
products which exhibited anticancer and antioxidative
effects in in vitro, cellular and in vivo models.
However, knowledge gaps exist and further research
opportunities abound as highlighted above. More
thorough knowledge of EFM-derived anticancer and
antioxidative natural products is necessary to pave the
way for their future applications in food and therapy.
123
Phytochem Rev (2015) 14:443–457
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