Pharmaceutical Biology

ISSN: 1388-0209 (Print) 1744-5116 (Online) Journal homepage: https://www.tandfonline.com/loi/iphb20

Pharmaceutical properties and toxicology of

Dioclea grandiflora

Rita de Cássia da Silveira e Sá, Reinaldo Nóbrega de Almeida & Jnanabrata

Bhattacharyya

To cite this article: Rita de Cássia da Silveira e Sá, Reinaldo Nóbrega de Almeida & Jnanabrata
Bhattacharyya (2013) Pharmaceutical properties and toxicology of Dioclea�grandiflora,
Pharmaceutical Biology, 51:5, 659-667, DOI: 10.3109/13880209.2012.755208

To link to this article: https://doi.org/10.3109/13880209.2012.755208

Published online: 01 Feb 2013.

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ISSN 1388-0209 print/ISSN 1744-5116 online
Editor-in-Chief: John M. Pezzuto
Pharm Biol, 2013; 51(5): 659–667
! 2013 Informa Healthcare USA, Inc. DOI: 10.3109/13880209.2012.755208

REVIEW ARTICLE

Pharmaceutical properties and toxicology of Dioclea grandiflora

Rita de Cássia da Silveira e Sá1, Reinaldo Nóbrega de Almeida2, and Jnanabrata Bhattacharyyay
1
Department of Physiology and Pathology and 2Laboratory of Pharmaceutical Technology, Center of Health Sciences, Federal University
of Paraı́ba (UFPB), João Pessoa, Paraı́ba, Brazil

Abstract Keywords
Context: Since the beginning of civilization, herbal medicines have been an important source for Bull’s-eye, phytochemicals, therapeutic prop-
human beings to treat their ailments. Despite the large number of synthetic remedies available erties, toxicology
in the market, the use of plants is seen as a great challenge in the search for new substances
endowed with therapeutic properties. One example is Dioclea grandiflora Mart. ex Benth. History
(Leguminosae) employed in traditional medicine to treat prostate disorders and kidney stones.
Objectives: This work presents a brief overview of D. grandiflora, including a description of the Received 12 September 2012
plant, its chemical composition and pharmacological properties. Revised 28 November 2012
Methods: This review gathers information available in the scientific literature compiled from Accepted 29 November 2012
databases such as Science Direct, PubMed, Dr. Dukes Phytochemical and Ethnobotany, Missouri Published online 1 February 2013
Botanical Garden and The International Plant Names Index.
Results: The information found in the literature showed that flavonoids are the major
constituents of D. grandiflora that account for most of the pharmacological properties so far
disclosed. Several studies have revealed that D. grandiflora possesses antinociceptive,
cardiovascular, antioxidant and anti-inflammatory activities.
Conclusion: Research shows that D. grandiflora is a potential source of compounds pertaining
medicinal applications. It provides an interesting subject in the search for new drugs of natural
origin.

Introduction and other natural products are seen as promising sources of

new components displaying idealistic pharmaceutical profiles,
Natural products have long been employed to treat various
i.e., with no side effects and no addictive potential, to be
forms of human diseases. However, it was not until the
used in the treatment of human diseases (McCurdy & Scully,
nineteenth century that individual compounds were isolated
2005). In this context, D. grandiflora Mart. ex Benth.
from these natural sources and shown to exert the desired
(Leguminosae) and its constituents have been the subject of
effects. With the development of new technology by the
interest and research by many authors that have demonstrated
pharmaceutical industry, drug discovery became a complex
their activities on the CNS. For instance, it has been shown
subject that went beyond the use of only natural products, and
that D. grandiflora displays central antinociceptive effects
made the synthetic production of drugs a profitable
(Almeida et al., 2000, 2003; Batista et al., 1995; Mattei et al.,
enterprise. Today, there is resurgence in the study of natural
products with a special interest in the use of plants as a
1995; Sá et al., 2010), and cardiovascular, antioxidant and 13
20
anti-inflammatory properties. This provides evidence that
primary source in drug discovery efforts.
this species may represent a new effective alternative or
Increasing knowledge of organ systems, for example the
complement to synthetic compounds in the search for a
central nervous system (CNS) organization and function, has
potential therapeutic agent (Almeida et al., 2002; Botelho
become an important contributing factor to the uncovering of
et al., 2007; Guabiraba et al., 2010). Hence, this work provides
new compounds to treat human disorders. In particular, it led to
a brief overview of D. grandiflora with emphasis on its
a better understanding of mechanisms of pain transmission in
chemical composition and pharmacological properties with the
the nervous system, which combined with the discovery of new
attempt to develop a better understanding of D. grandiflora
natural substances, may contribute to an improved under-
biological actions.
standing of pharmacological mechanisms. Therefore, plants
The data used in this study were compiled from major
databases such as PubMed, Dr. Dukes Phytochemical and
yDr Jnanabrata Bhattacharyya is now deceased. Ethnobotany, Missouri Botanical Garden, The International
Correspondence: Rita de Cássia da Silveira e Sá, Department of Plant Names Index and Science Direct by searching terms that
Physiology and Pathology, Federal University of Paraı́ba (UFPB), Av
comprised the species name, phytochemicals and pharmaco-
Antonio Lira, 950, Cabo Branco, 58045-030, João Pessoa, Paraı́ba,
Brasil. Tel: þ55 83 3247 1810. Fax: 55 83 3216 7504. E-mail: logical application from the available scientific literature
ritacassia.sa@bol.com.br since 1980.
660 RCS Sá et al.
Plant description
Dioclea grandiflora is popularly known as ‘‘mucunã’’,
‘‘mucunã-de-caroço’’ and ‘‘olho-de-boi’’ or bull’s-eye. The
scientific name Dioclea was homage to Diocles Carystius, a
very well-known Greek physician who lived in the fourth
century B.C., not long after the time of Hippocrates, the father
of modern medicine (Smith, 1870).
This species is a liana unique to Brazil’s Caatinga, an
indigenous term meaning ‘‘white forest’’ found in an area of
approximately 800 000 km2 that extends continuously through
parts of the states of Piauı́, Ceará, Rio Grande do Norte,
Paraı́ba, Pernambuco, Alagoas, Sergipe, Bahia and Minas
Gerais (Castro, 2010). It is a high woody climber with erect
inflorescences covered with soft, short hair. The flowers
exhibit bright purple petals at the branch tips, and the leaves
are dark green above and grey-green beneath. The fruit is stiff
and the unripe pods are green. At maturity each pod holds 3–4
large and hard seeds (2.5 cm) commonly known as bull’s-eye
(Figure 1) (Nunes et al., 2012).
Chemical composition
The phytochemical investigation of D. grandiflora has shown
the presence of various substances, particularly flavonoids and
lectins. In the roots of D. grandiflora, dioclein (Figure 2a), a
flavanone, appears as its major constituent. The structure
of this compound has been determined on the basis of
spectral analysis as 5,20 ,50 -trihydroxy-6,7-dimethoxyflavanone
(Bhattacharyya et al., 1995).
The chromatographic fractions obtained during the isola-
tion of dioclein showed the presence of several other minor
constituents, such as dioclenol (Figure 2b), a flavanonol, with
the structure 3,5,7-trihydroxy-8-methoxy-6-prenylflavanone
(Bhattacharyya et al., 1997); dioflorin (Figure 2c), a
flavanone, with the structure 5,7,20 -trihydroxy-8-methoxy-6-
(3-methyl-2-butenyl)flavanone (Bhattacharyya et al., 1998);
agrandol (5,7-dihydroxy-6-methoxy-8-prenylflavanone;
Figure 2d), diosalol (3,5,7,20 ,50 -pentahydroxy-6-methoxy-8-
prenylflavanone; Figure 2e) and paraibanol (3,5,7,20 ,50 -
pentahydroxy-6-methoxyflavanone; Figure 2f), three flava-
vones (Jenkins et al., 1999); b-amyrin (Figure 2g); 5,7,20 ,50 -
tetrahydroxy-6-methoxy-8-prenylflavanone (Figure 2h)
(Jenkins et al., 1999); and floranol (Figure 2i), a flavanone,
Figure 1. Dioclea grandiflora seed pod and seeds.
Pharm Biol, 2013; 51(5): 659–667
0
with the structure 3,5,7,2 -tetrahydroxy-6-methoxy-8-prenyl-
flavanone (Lemos et al., 2002).
Pharmacological properties
Antinociceptive effect
In folk medicine, the seed and root bark of D. grandiflora
have been widely used to treat prostate disorders and kidney
stones (Lima, 1989). Previous studies reported a significant
CNS activity of the chloroform (CHCl3) and ethanol extracts
obtained from the dried root bark (Batista et al., 1995), the
hydroalcoholic extract from the seeds (Almeida et al., 2003),
and the ethanol extract from the seed pod of this plant
(Sá et al., 2010).
In contrast to pain defined as a subjective sensation,
nociception is defined as the mechanism through which
harmful peripheral stimuli are transmitted to the CNS without
showing the emotional component. The search for more
effective pain relievers has become a major target for modern
medicine, particularly with respect to the discovery of
substances that exert a central action (known as opioids)
with a lower incidence of side effects. Another group of drugs
also involved in the relief of pain comprises the peripheral
analgesics, known as non-steroid anti-inflammatory sub-
stances, which decrease the sensibilization of primary afferent
fibers.
Generally, opiates are considered to act on the CNS
exerting their effects through m,  and d opioid receptors
(Almeida et al., 2001). However, accumulating evidence has
shown that opioid antinociception can be initiated by
activation of opioid receptors found outside of the CNS
(Stein, 1993). Several aspects should be considered though
when evaluating the opioid antinociception in the periphery.
For instance, the criteria for opioid receptor-mediated effects
that can be met through antagonism by naloxone, a m-opioid
receptor competitive antagonist used to block the pharmaco-
logical effects of chemical mediators such as morphine
(Santos et al., 1998).
Extracts from D. grandiflora are reported to have central
antinociception effects (Almeida et al., 2003; Batista et al.,
1995; Sá et al., 2010). The acute treatment of rats and mice
with a hydroalcoholic extract from the seeds of D. grandiflora
at doses of 250 and 500 mg/kg, by intraperitoneal (i.p.) or oral
administration, produced a significant antinociceptive effect
in the tail-flick and hot-plate tests. This effect was reversed by
naloxone indicating a potential mode of interaction with
opioid receptors (Almeida et al., 2003). It is postulated that
the antinociceptive effect could be related to the reduction in
calcium influx at the afferent nerve fiber ending, which
induced a decrease in adenylyl cyclase activity, resulting in
lower levels of cyclic AMP and potassium efflux. The latter
led to hyperpolarization of the nerve fiber and an apparent
antinociceptive effect (Yaksh, 1999).
The administration of the aqueous fractions (12.5, 25 and
50 mg/kg, i.p.) and dioclein (25, 50 and 100 mg/kg, i.p.)
obtained from the ethanol extract of D. grandiflora root bark
produced an antinociceptive effect in mice and rats submitted
to the acetic acid–induced writhing and tail-flick tests,
respectively (Batista et al., 1995). In the tail-flick assay,
after the application of the thermal stimulus, both dioclein
DOI: 10.3109/13880209.2012.755208
Figure 2. Dioclea grandiflora major constituents. (a) dioclein, (b) dioclenol, (c) dioflorin, (d) agrandol, (e) diosalol, (f) paraibanol, (g) b-amyrin,
(h) 5,7,20 ,50 -tetrahydroxy-6-methoxy-8-prenylflavanone, and (i) floranol.
and the aqueous fractions exhibited a significant increase in
the reaction time in rats, a feature considered an important
parameter of the central antinociceptive activity. When
compared with morphine, dioclein displayed a prolonged
action by showing a significant analgesic activity 2 h after its
administration in the rats (18.9  7.5 and 57.2  13.8 mg/kg,
respectively). The attenuation of the antinociceptive action of
the aqueous fractions and dioclein after treatment with
naloxone suggests that both substances acted centrally,
possibly involving an opioid-like mechanism.
The intraperitoneal administration of the ethanol extract
from the seed pod of D. grandiflora (EDgP) at doses of 75,
150 and 300 mg/kg also exerted significant antinociceptive
effects in mice subjected to different algesiometric tests, such
as the acetic acid–induced writhing and formalin tests (Sá
et al., 2010). The acetic acid-induced writhing test in mice is
an efficient nociceptive model for the screening of analgesic
and anti-inflammatory drugs and is also regarded as a useful
procedure to assess visceral inflammatory pain (Koster et al.,
1959; Tjølsen & Hole, 1997). The formalin test comprises
two excitatory phases and one inhibitory interphase of the
nociceptive response by employment of tonic stimulus
Overview of D. grandiflora 661
(Abbott et al., 1995). The first phase (neurogenic pain)
involves the direct stimulation of sensorial afferent C-fibers
by formalin, while the second phase (inflammatory pain)
involves a peripheral inflammatory process and is believed to
arise from nociceptive spinal neuron hyperactivity (Tjolsen
et al., 1992).
EDgP produced a higher antinociceptive response on the
second phase than that displayed by morphine, particularly at
the dose of 150 mg/kg. Thus, the antinociceptive effects of
D. grandiflora extract in the writhing test and in both phases
of the formalin test not only suggest a central (neurogenic)
and peripheral (inflammatory) action, but also imply that this
extract exerts anti-inflammatory activity. These results
corroborate the findings of Batista and collaborators (1995)
that showed the antinociceptive action of the aqueous fraction
obtained from the ethanolic extract of D. grandiflora, at a
lower dose level (50 mg/kg, i.p.), in mice that underwent
acetic acid-induced writhings. However, contrary to the data
reported by these authors, the administration of naloxone
(6 mg/kg, i.p.) did not reverse the antinociceptive effect of
EDgP; instead, it increased this effect at the dose level of
300 mg/kg while considerably reducing the morphine
662 RCS Sá et al.
antinociceptive effect. These data indicate that central or
peripheral active endogenous opioids do not seem to be
mediating the antinociceptive effect of EDgP in the formalin
test (Sá et al., 2010).
The treatment of mice with dioflorin (10 mg/kg) and
dioclenol (10 mg/kg), administered intraperitoneally, showed
that both substances have acute central analgesic activity
(Almeida et al., 2000). In the tail-immersion, a test reported to
be specific for agents producing central antinociception, both
substances were effective, whereas in the acetic acid-induced
writhing test, dioflorin produced a near maximal inhibition of
the writhing response (4.8  2.1 writhings) similar to
morphine (5.2  2.7 writhings; dioclenol 11.2  6.0 wri-
things; vehicle/control 31.8  5.8 writhings).
Despite the reported antinociceptive effects of D. grand-
ifora, no precise receptor binding data has appeared in the
literature to date; therefore it is unclear if this plant is
producing antinociception selectively through direct interac-
tion with opioid receptors.
Cardiovascular action
Flavonoids consist of a large group of compounds derived
from plants that are largely consumed in the daily diet (Yao
et al., 2004). Its consumption is reported to prevent
cardiovascular diseases (Riemersma et al., 2001) which
could be related to their pharmacological properties such as
vasodilators, anti-inflammatory, inhibitors of platelet aggre-
gation and antioxidants (Rezende et al., 2009). The latter is
believed to delay the onset of atherogenesis by reducing
peroxidative reactions and decreasing thrombotic tendency
(Aviram & Fuhrman, 1998; Rice-Evans et al., 1997).
The vasorelaxant effect of the flavonoid dioclein was
investigated in the rat aorta. In phenylephrine pre-contracted
vessels, dioclein induced an endothelium-dependent relaxa-
tion, which probably involved increased production of nitric
oxide (NO) by endothelial cells. This effect was reduced after
NO synthase activity inhibition by L-NAME (NG nitro-L-
arginine-methyl-ester) and absent after endothelium removal
(Lemos et al., 1999). The data available in the literature
indicate that most flavonoids show a relaxant effect regardless
of the presence of a functional endothelium, and a less potent
action than dioclein (Herrera et al., 1996). The stimulation of
NO production by dioclein represents an important factor in
vascular homeostasis by exerting modulatory effects on the
vascular tone and growth of vascular smooth muscle cells,
and decreasing platelet adhesion and aggregation (Scott-
Burden & Vanhoutte, 1993). NO deficiency in the vascular
endothelial cells milieu can be related to endothelium
dysfunction, a condition that may contribute to the develop-
ment of severe pathologies, such as atherosclerosis and
hypertension (Busse & Fleming, 1996). Hence, the discovery
of substances that enhance the production of NO by
endothelium cells can become a useful tool in the treatment
of cardiovascular diseases.
Trigueiro and collaborators (2000) demonstrated that
dioclein induced relaxation of aortic rings in endothelium-
denuded vessels precontracted with noradrenaline and KCl. In
the absence of extracellular calcium, dioclein reduced the
contraction induced by noradrenaline but not that induced
Pharm Biol, 2013; 51(5): 659–667
by caffeine. It also inhibited the sustained contractions
induced by the phorbol ester 12-O-tetradecanoyl phorbol-
13-acetate in normal and calcium-free solution. According to
these authors, the endothelium-independent vasorelaxant
effect of dioclein could be related to the inhibition of
contractions dependent on activation of protein kinase C,
voltage-dependent calcium influx and on the release of
intracellular calcium stores sensitive to noradrenaline.
The effect of dioclein on coronary flow was investigated in
isolated rat heart elsewhere. It was observed that dioclein
induced a sustained increase on the coronary flow without
modification of inotropic and chronotropic contractions of the
heart and electric activity, through a mechanism independent
on the production of NO and cyclooxygenase-derived factors.
These effects suggest that dioclein could aid the treatment of
vasospasm associated with myocardial ischemic complica-
tions (Almeida et al., 2002).
A combined in vivo and in vitro study showed that dioclein
lowered arterial pressure in normotensive rats by decreasing
the peripheral vascular resistance. Pharmacological evidence
suggested that the mechanism involved in the hypotensive
vasorelaxant effect of dioclein in the mesenteric arteries of
rats was mediated by activation of potassium (Kþ) channels,
namely the opening of Kca and Kv channels, and subsequent
membrane hyperpolarization (Côrtes et al., 2001). Vascular
tone of small arteries and arterioles is the basis for the
maintenance of peripheral resistance in the circulation and is
the major contributor to the control of blood pressure (White
et al., 1996). As Kþ channels play an important role in the
control of vascular tone, it is believed that they are involved in
controlling the cellular membrane potential. Various sub-
stances that act on Kþ channels by increasing their open
probability are shown to dilate arteries by causing hyperpo-
larization in vascular smooth muscle cells (Quast, 1993).
Therefore, the effects of Kþ channels opener could be useful
in the treatment of hypertension (Kuriyama et al., 1995;
Lawson, 2000).
Despite the therapeutic benefits obtained from flavonoids,
the expected outcome is still dependent on the improvement
of their pharmacokinetic profile after oral administration
(Rezende et al., 2009). As the use of pharmaceutics can be
limited due to the lack of oral activity or poor water solubility,
in their natural form, flavonoids are poorly absorbed in the
intestine. They undergo extensive degradation by intestinal
microorganisms or enzymes, producing different metabolites,
which, if absorbed, may undergo further degradation by
hepatic enzymes producing new metabolites of varying
bioactivity (Stahl et al., 2002).
Like many flavonoids, dioclein has been reported to have
poor oral absorption (Rezende et al., 2009; Sildeberg et al.,
2005) probably due to reduced dissolution rate (Liu et al.,
2006) or degradation by enzymes in the gastrointestinal tract
and liver (Stahl et al., 2002). In order to improve its intestinal
absorption, dioclein has been associated with cyclodextrins,
natural macrocyclic oligosaccharides that are used as tools to
generate aqueous drug solutions without the use of organic
co-solvents, surfactants or lipids, as formulation adjuncts
(Polyakov et al., 2004). Cyclodextrins form inclusion com-
plexes that increase the guest’s in vivo stability against
hydrolysis, oxidation, decomposition and dehydration,
DOI: 10.3109/13880209.2012.755208
consequently increasing bioavailability (Côrtes et al., 2001).
The inclusion of dioclein in b-cyclodextrins has been shown to
improve the hypotensive effect of this substance by increasing
its bioavailability and to enable it to be effective after oral
administration, probably as consequence of a protective effect
of b-cyclodextrins against in vivo biodegradation and possibly
increased water solubility (Rezende et al., 2009).
In addition to dioclein, floranol has also been reported to
exert vasorelaxant activity in the rat aorta smooth muscle. In
phenylephrine-precontracted aortic rings, floranol induced a
vasodilator effect (IC50 ¼ 19.9  2.4 mM) probably by exerting
a direct action in the smooth muscle cells as even after the
removal of endothelium or pretreatment of vessels with
L-NAME, the IC50 value for floranol-induced vasorelaxation
did no change (Lemos et al., 2006).
Ansiolitic effect
In recent years, anxiety disorders have been shown to have a
relatively high prevalence in modern society. The most
prescribed medications to treat this disorder are benzodiaze-
pines; however, their clinical use is limited by their side effects,
such as psychomotor impairment, potentiation of other central
depression drugs, and dependence liability (Lader, 2008). The
search for new remedies to treat anxiety is focused on the use of
medicinal plants because recent studies have suggested that
many substances present in plants affect the mind of mammals.
For instance, the administration of caffeine (Tang et al., 1989)
and dietary soy phytoestrogens (Lund & Lephart, 2001) was
reported to decrease anxiety in rats.
The ansiolitic-like effect of D. grandifora was observed in
mice after the administration of the alcoholic fraction of the
stem bark of D. grandiflora (doses of 15, 30 and 60 mg/kg)
and dioclenol (dose of 10 mg/kg) using several behavioral
assays (marble-burying, hole-board and elevated plus maze
tests). In addition, the same treatment increased the duration
of the sleeping time induced by sodium pentobarbital and
showed a significant increase on protection against pentyle-
netetrazole-induced convulsion (De Almeida et al., 2010).
Since many flavonoids and many flavones derivatives were
found to be ligands for the g-aminobutyric acid type A
(GABAA) receptors in the CNS, it has been hypothesized that
these substances act as benzodiazepine-like molecules and
bind to the benzodiazepine binding site with resulting
depressant actions in mice (Marder & Paladini, 2002).
Therefore, the ansiolitic effect of the alcoholic fraction of
the stem bark of D. grandiflora and dioclenol was suggested
to occur in the benzodiazepine site of GABA receptors
(De Almeida et al., 2010). In this same study, an antic-
onvulsant effect has also been attributed to D. grandiflora and
dioclenol; however, an earlier study reported that the
intraperitoneal administration of the aqueous extract from
the seeds of D. grandiflora to male Swiss mice (at doses of
150, 342, 520 and 685 mg/kg, representing approximately 20,
50, 75 and 100 times the doses used by human beings,
respectively) did not produce anticonvulsant action, although
a significant protection with regard to the latency time of the
occurrence of convulsion was observed. This particular factor,
however, was not considered sufficient to justify the use of
this plant as an anticonvulsant (Mattei et al., 1995).
Overview of D. grandiflora 663
Anti-inflammatory activity
Inflammation is a protective mechanism used by the organism
to eliminate the injurious stimuli and is caused by release of
chemicals, such as cytokines and chemokines, from tissues
and migrating cells. The assessment of the effects of dioclein
on the production of mediators of inflammation in vitro using
murine macrophages showed that dioclein efficiently
decreased the production of cytokines, chemokines and NO
more effectively than butylated hydroxytoluene (BHT), an
antioxidant, and rolipram, a PDE4 (cyclic nucleotide
phosphosdiesterase type 4) inhibitor. Diocelin inhibited
PDE4 activity (IC50 of 16.8  1.4 mM) and decreased the
concentration of reactive oxygen species more effectively
than BHT in cell and cell-free systems. These effects indicate
that dioclein exerted anti-inflammatory effects by reducing
the production of mediators of inflammation, such as
cytokines, chemokines and reactive oxygen species by
macrophages (Guabiraba et al., 2010).
Antioxidant activity
Several studies have shown that the oxidative modification of
low-density lipoprotein (LDL) plays an important role on the
mechanism responsible for atherosclerosis. According to the
oxidation hypothesis of atherosclerosis, the initiating event in
the development of this condition is an oxidative modification
of LDL that significantly increases its uptake into the arterial
intima (Ylä-Herttuala et al., 1989). Moreover, studies of
atherosclerotic lesions led to the proposition that transition
metal ions are involved on the LDL oxidation in vivo.
The use of external antioxidants is of therapeutic
importance to maintain a proper internal redox environment.
As indicated by several reports in the literature, the most
relevant biological property of flavonoids is the prevention of
cardiovascular diseases, an ability associated with the
decrease of LDL oxidation, macrophage foam cell formation
and atherosclerosis (Botelho et al., 2007). The antioxidant
activity could be explained by two possible factors: the
scavenging of free radical or chelation of metal ions, such as
copper (Cu) and iron (Fe).
The antioxidant activity of floranol was assessed through
the evaluation of its capacity to inhibit Cu(II)- and Fe(III)-
mediated oxidation of LDL. It is known that Cu and Fe in their
reduced oxidation state can promote the formation of free
radicals, which can oxidize LDL. The high values of the
stability constants observed in the experiment indicated that
floranol efficiently bound Cu(II) and Fe(III) ions, thus
preventing their effect on LDL oxidation. Therefore, the
antioxidant activity of floranol, which probably involves
the sequestering of metals, may have an important role on
the inhibition of lipid peroxidation in vitro, a property
that could be beneficial in reducing atherosclerosis (Botelho
et al., 2007).
Lectin
Lectins comprise a heterogeneous group of proteins posses-
sing at least one non-catalytic domain that binds reversibly to
a specific mono- or oligosaccharide (Van Damme et al.,
1998). Cells are coated with carbohydrate that can
664 RCS Sá et al.
specifically bind to lectins and, because of this association,
several roles have been attributed to this class of molecules in
biological research (Silva Lima et al., 1993). For instance,
lectins were shown to act as inflammatory agents, such as
concanavalin A (Con A) – a lectin from Canavalia ensiformes
(L.) DC. (Leguminosae), a species from the Diocleinae tribe
that has been reported to induce histamine release from
basophils and mast cells (Ennis et al., 1981). In addition,
lectins have been used as probes to differentiate diagnosis of
tumor from inflammation (Kojima & Jay, 1987) or to detect
microheterogeneity of a-1-antichymotrypsin in sera from
patients with various inflammatory syndromes (Pos et al.,
1990).
The seed lectin from D. grandiflora is a D-mannose-(D-
glucose)-binding protein with a molecular mass of 100 kDa
(Moreira et al., 1983) that shows a high degree of sequence
similarity with C. ensiformes Con A, differing only in 53 out
of 237 residues (Gupta et al., 1996; Richardson et al., 1984).
The carbohydrate-binding specificity of D. grandiflora lectin
has been addressed in several studies through various
experimental protocols, including calorimetric analysis and
fluorescence anisotropy titration, among others (Chervenak &
Toone, 1995, 1996; Dam et al., 2000, 2002, 2005; Lee et al.,
2000; Ramos et al., 1996; Weatherman et al., 1996).
Studies have shown that D. grandiflora lectin exhibits high
affinity for 3,6-di-O-(a-D-mannopyranosyl)-a-D-mannopyra-
nose, the core trimannoside of asparagine-linked carbohy-
drates, but lower affinity for biantennary complex
carbohydrates. The thermodynamics of D. grandiflora lectin
binding to deoxy analogs of the core trimannoside and to a
biantennary complex carbohydrate determined by isothermal
titration microcalorimetry showed that it recognizes specific
hydroxyl groups of the trimannoside similar to that of Con A
(Dam et al., 1998a; Rozwarski et al., 1998). In addition, the
thermodynamics data of binding of D. grandiflora lectin to a
complete set of monodeoxy analogs of the core trimannoside
as well as a tetradeoxy analog also determined by isothermal
titration microcalorimetry showed that D. grandiflora lectin
recognizes the 2-, 3-, 4- and 6-hydroxyl groups of the a(1,6)
Man residue, the 3- and 4-hydroxyl group of the a(1, 3) Man
residue, and the 2- and 4-hydroxyl groups of the central Man
residue of the trimannoside. Despite the similarity in the
overall pattern of data for D. grandiflora lectin binding to the
deoxy analogs and Con A, there are differences in the data for
certain monodeoxy analogs binding to the two lectins and also
in the thermodynamics of binding of D. grandiflora lectin and
Con A to a biantennary complex carbohydrate (Dam et al.,
1998b).
Selectins are a family of cell adhesion molecules important
for leukocyte recruitment in inflammation. Because they
possess a lectin domain present in their structure, it has been
postulated that plant lectin anti-inflammatory activity is
probably the result of a competitive blockage of a common
leukocyte and/or endothelial selectin carbohydrate ligand.
The investigation of the anti-inflammatory activity of
mannose-glucose binding lectins from species belonging to
the Diocleinae tribe (e.g., D. guianensis Benth., D. violacea
Mart. ex Benth., D. virgata Amsh. and Canavalia brasiliensis
Mart. ex Benth.) revealed that D. grandiflora lectin did not
exert any inhibitory effect on neutrophil migration, whereas
Pharm Biol, 2013; 51(5): 659–667
all the other lectins tested were able to reduce cell migration
into rat peritoneal cavities induced by N-formyl-methionine-
leucine-phenylalanine (fMLP) or carrageenan, and rat hind
paw edema induced by carrageenan (Assreuy et al., 1997).
The assessment of the in vivo stimulatory actions of
Diocleinae lectins, including that of D. grandiflora, on Balb/c
mice popliteal draining lymph nodes showed that these lectins
presented high stimulatory capacity on lymph node T cells.
Moreover, lectin stimulation was associated to apoptosis
induction in addition to causing other side effects such as
inflammation and high endothelial venule necrosis (Barbosa
et al., 2001).
The toxicity of lectins of the Diocleinae tribe, including D.
grandiflora, to Biomphalaria glabata Say and Artemia salina
Leach has been used as a promising tool to the search for
natural compounds with molluscicidal properties for the
vector control of schistosomiasis. The results indicated that all
lectins tested were toxic to A. salina and B. glabata, killing
90% at a concentration of 10 mg/mL, and 100% at 50 mg/mL,
respectively (Dos Santos et al., 2010). In a different context,
the lectins were tested for their potential to inhibit the
adherence of five streptococci species to acquired pellicle
in vitro by the use of fluorescence micrography. Adherence
inhibition was carried out on saliva-coated microtiter plates at
which lectins solutions were previously incubated followed by
incubation with the oral streptococci. The lectin from
D. grandiflora as well as the lectins from the other species
of the Diocleinae tribe effectively inhibited the adherence of
the microorganisms, suggesting the lectins could be useful in
anti-adhesion therapeutics (Teixeira et al., 2006).
Lectin extracted from D. grandiflora was compared to Con
A on the stimulation of lymphocyte proliferation and
interferon gamma production by peripheral blood mono-
nuclear cells from normal volunteers. D. grandiflora lectin
was able to induce intermediate levels of lymphocyte
proliferation and stimulation of interferon gamma production
when compared to that of Con A (Barral-Netto et al., 1992).
In another comparative study using Con A and D. grandiflora
lectin, the evaluation of peritoneal macrophage stimulation
(rapid spreading on glass surface and hydrogen peroxide
production) and inflammatory reaction (leukocyte accumula-
tion) in C3H/HeJ mice revealed that the lectins improved
macrophage spreading three- to four-fold at 24–72 h and
stimulation of hydrogen peroxide release by Con A and
D. grandiflora lectin that lasted 1 and 3 d, respectively. Con A
induced a moderate increase in the total number of peritoneal
mononuclear cells, whereas administration of D. grandiflora
lectin increased the number of peritoneal cells two- to
three-fold (Rodriguez et al., 1992).
Lectin from D. grandiflora was also compared with Con A
for their ability to induce paw edema and peritoneal cell
immigration in rats. With a peak at 1 h after injection, Con A
produced a slight edema, while D. grandiflora lectin induced
a stronger and long-lasting edema with peak at about 6 h. The
use of different anti-inflammatory drugs revealed that a-D-
glucose partially blocked the edema caused by Con A but did
not reduce the edema induced by D. grandiflora lectin, while
a-methyl mannoside blocked the edema caused by
D. grandiflora lectin and Con A. At doses much lower than
those used to induce paw edema, the lectins promoted an
DOI: 10.3109/13880209.2012.755208
intense accumulation of neutrophil and mononuclear cells in
the rat peritoneal cavity and D. grandiflora lectin was more
potent than Con A. Despite the physicochemical similarities
between the lectins, D. grandiflora lectin exhibited stronger
pro-inflammatory effects than Con A. Such difference seems
to be related to their sugar-binding properties (Bento et al.,
1993). Further studies showed that D. grandiflora lectin,
when subcutaneously injected in mouse, induced inflamma-
tory cutaneous reaction whose histological analysis revealed
hemorrhagic ulceration with exudative reaction accompanied
by an influx of polymorphonuclear leukocytes and giant cells.
It was suggested that D. grandiflora lectin acted as an
inflammatory agent probably promoting exocytosis and
release of mediators (Silva Lima et al., 1993).
Toxicology
Various studies have investigated the side effects of
D. grandiflora after acute treatments, whereas information
on long-term side effects is still lacking.
A possible toxicity of the aqueous extract of D. grandiflora
(at doses of 150, 342, 520 and 685 mg/kg) was suggested due
to a probable induction of hepatotoxic effect after acute
treatment and by the total suppression of responses in a
conflict test (Mattei et al., 1995). Contrary to these findings,
the acute treatment of rats and mice with the hydroalcoholic
extract from the seeds of D. grandiflora (at doses of 250 and
500 mg/kg, i.p. or orally) did not produce any acute toxicity
(Almeida et al., 2003).
The administration of the aqueous extract from the stem
bark of D. grandiflora to mice produced low acute toxicity
(LD50 615 mg/kg) when compared with the root bark extract
(LD50 196.8 mg/kg) from this same species (Silva et al.,
2003). The contrasting values of the LD50 are probably due to
the presence of different components and chemical concen-
trations found in the different parts of this plant.
The locomotor activity of mice was significantly decreased
30 min after the injection of the alcoholic fraction obtained
from the stem bark of D. grandiflora (at doses of 30 and
60 mg/kg) and dioclenol (at the dose of 10 mg/kg) when
compared with the control group (saline 0.85%). The
reduction in locomotor activity was evident within 10 min
after treatment (De Almeida et al., 2010). Moreover, the
administration of the ethanol extract from the seed pod of
D. grandiflora (at the dose 300 mg/kg) caused a significant
reduction in motor coordination of mice at 30 min after
treatment, but not at two other time points (60 and 120 min),
pointing to a transient impairment of motility in mice (Sá
et al., 2010). In this study, the LD50 for the extract was
determined to be 753 mg/kg i.p. (95% confidence limits: 595–
929 mg/kg).
Conclusions
The scientific data available have not given sufficient
evidence to corroborate the therapeutic properties of
D. grandiflora. This matter needs to be addressed since this
species is currently used as folk medicine in Brazil. The
pharmacological data provided an interesting material for
further studies, especially in regard to the actions of its
chemical constituents such as dioclein. Other flavonoids
Overview of D. grandiflora 665
present in D. grandiflora represent a rich investigative field as
this class of polyphenolic compounds have been reported to
exert many biological actions such as anti-allergic, anti-
inflammatory, anti-microbial and antioxidant activities.
Extracts obtained from different parts of D. grandiflora
have shown that this plant is a potential pharmaceutical and
therapeutic source, particularly with respect to its antinoci-
ceptive activity. Despite the information available in the
literature regarding the biological activities of D. grandiflora,
further toxicological assessments need to be carried out,
namely long-term toxicological studies, in order to ensure a
safe use of this medicinal plant.
Declaration of interest
The authors report no declarations of interest.
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