Anais da Academia Brasileira de Ciências (2017) 89(3): 1565-1578
(Annals of the Brazilian Academy of Sciences)
Printed version ISSN 0001-3765 / Online version ISSN 1678-2690
http://dx.doi.org/10.1590/0001-3765201720160286
www.scielo.br/aabc | www.fb.com/aabcjournal
Clusia criuva Cambess. (Clusiaceae): anatomical characterization,
chemical prospecting and antioxidant activity
KARLA M.M. DA SILVA1, ANDREA B. DA NÓBREGA2, BRUNO LESSA2, MARIA CAROLINA ANHOLETI3,
ADRIANA Q. LOBÃO3, ALESSANDRA L. VALVERDE4, SELMA R. DE PAIVA3 and ANA JOFFILY3
¹Instituto de Pesquisas do Jardim Botânico do Rio de Janeiro, Rua Pacheco Leão,
915, Jardim Botânico, 22460-030 Rio de Janeiro, RJ, Brazil
2
Fundação Oswaldo Cruz, Instituto de Tecnologia em Fármacos, Far-Manguinhos/FIOCRUZ,
Av. Comandante Guaranys, 447, Jacarepaguá, 22775-903 Rio de Janeiro, RJ, Brazil
3
Setor de Botânica, Departamento de Biologia Geral, Instituto de Biologia, Universidade Federal Fluminense,
Outeiro de São João Batista, s/n, Campus do Valonguinho, Centro, 24210-130 Niterói, RJ, Brazil
4
Lapromar, Departamento de Química Orgânica, Instituto de Química, Universidade Federal Fluminense,
Outeiro de São João Batista, s/n, Campus do Valonguinho, Centro, 24210-130 Niterói, RJ, Brazil
Manuscript received on June 2, 2016; accepted for publication on August 23, 2016
ABSTRACT
This study aims the anatomical description and chemical characterization of aerial parts of Clusia criuva
Cambess., Clusiaceae in addition to the evaluation of the antioxidant activity of crude extracts, correlated
to the flavonoid content. The morphological characterization was performed using traditional techniques of
plant anatomy. For phytochemical studies, crude extracts were obtained by static maceration and analyzed
by thin layer chromatography. The antioxidant activity and the flavonoids content were determined by
colorimetric methods involving, respectively, 2,2-diphenyl-1-picrylhydrazyl free radical and aluminum
chloride. C. criuva has uniseriate epidermis, paracytic stomata, hypostomatic leaves, cuticular flanges
and cordiform vascular cylinder with accessory bundles. Chemical prospecting confirmed the abundant
presence of terpenes and phenols in the extracts of leaves and of fruits. The methanolic extract of seeds
showed the lowest EC50 value, but the methanolic extract of pericarps exhibited the highest maximum
antioxidant activity. The results suggested a high percentage of flavonoids in the hexanic extract of
pericarps, however, this could represent, in fact, the presence of benzophenones. Secretory ducts and the
shape of the midrib are diagnostic for C. criuva. The antioxidant activity is not directly related to the
flavonoids. The results indicate the importance of future studies with C. criuva chemical constituents.
Key words: morphoanatomy, antioxidant activity, chemical prospecting, Clusia criuva, Clusiaceae,
flavonoids.
INTRODUCTION
The genus Clusia (Clusiaceae) is represented by
nearly 200 species (Judd et al. 2009), some of
Correspondence to: Karla Marins Mattos da Silva
E-mail: karlamariins@gmail.com
which have been used as purgatives as well as
germicides in the treatment of leprosy and skin
infections, helping to heal wounds and the new
borns navel. They have also been prescribed for
the relief of headaches and used in veterinary
medicine (Cavalcante and Frikel 1973, CoelhoAn Acad Bras Cienc (2017) 89 (3)
KARLA M.M. DA SILVA et al.
1566
Ferreira 2009, Valadeau et al. 2009, Odonne et al.
2013). Species of this genus are known as sources
MATERIALS AND METHODS
EQUIPMENT AND REAGENTS
of poliisoprenylated benzophenones, terpenoids,
1999, Compagnone et al. 2008, Silva and Paiva
All solvents used were of analytical grade and
obtained from VETEC, Brazil. The 2,2-diphenyl1-picrylhydrazyl (DPPH) and rutin were purchased
from Sigma. The recordings were made by a UVVIS Biospectro spectrometer SP -220.
2012).
PLANT MATERIAL
benzoquinones and flavonoids (Andrade et al.
1998). Among the phenolic substances in Clusia,
flavonoids, especially biflavonoids, may have
particular importance in medicine (Chedier et al.
Studies involving plants have contributed
very significantly in the search for new treatments
for various diseases, showing effectiveness with
several biological activities (Montanari and Bolzani
2001). The potential medicinal use of a species has
a direct relationship to the metabolites that the plant
produces for their own defence, that are usually
characteristic of a species or a group of species.
Therefore, detailed morphological, anatomical
and biochemical studies are needed to identify the
species and the chemical substances responsible for
their potential biological activity. Such chemical
and anatomical research is important to prevent the
dangerous and inaccurate use of medicinal plants
(Coelho et al. 2012).
Evidence that free radicals are involved in
many degenerative diseases have increased interest
in antioxidants, mainly of natural origin, as the
current synthetic forms are restricted mainly due
to their carcinogenic potential (Degáspari and
Waszczynskyj 2004, Sousa et al. 2007).
The aim of this study was to perform the
anatomic description of C. criuva leaves, the
chemical prospecting of the crude extracts of the
leaves and fruits, the evaluation of the antioxidant
activity and determination of total flavonoids of
these extracts. This will enhance the knowledge
about this species adding to the potential for its use
in medicine.
An Acad Bras Cienc (2017) 89 (3)
Vegetative and reproductive parts of three pistillate
individuals (collected numbers: SP08, SP09, SP10)
of C. criuva were collected at Floresta Nacional da
Tijuca. One voucher was deposited at the Jardim
Botânico Herbarium, registered under the number
RB 603158.
ANALYSIS OF CELLULAR ORGANIZATION
The middle of the petiole and leaf blade fragments
were removed from the midrib, intercostal and
edge regions of C. criuva leaves. The fragments
were embedded in polyethylene glycol MW 1500
(PEG) proposed by Burger and Richter (1991). The
sectioning was performed on a rotary microtome
(RMC Products MT990) and sections stained with
Astra Blue and Basic Fuchsin (Johansen 1940).
The dissociation of the epidermis fragments was
carried out by immersion in a solution of acetic
acid and hydrogen peroxide (1:1) and subsequently
the epidermal surfaces were stained with safranin.
The already stained sections were made into semipermanent slides (50% glycerin) for viewing
and photography in a Zeiss Primo Star optical
microscope. To characterize the leaf surface
micromorphology, the samples were processed
according to the usual technique for observation
under scanning electron microscopy (SEM) (Klein
et al. 2004). The epicuticular wax was classified
according to Barthlott et al. (1998).
Clusia criuva: ANATOMY AND CHEMISTRY
HISTOCHEMICAL ANALYSIS
Freehand cut sections of freshly collected leaves
were embedded in specific reagents for the
identification of the following chemical classes:
alkaloids (Dragendorff Solution), starch (Lugol),
cellulose (Astra Blue), phenols (2% Ferric
Chloride), lipids (Sudam III and IV), lignin
(Phloroglucinol), calcium oxalate (Sulfuric acid)
and tannins (Chloridric vanillin), accordingly to
SantAnna-Santos et al. (2006) with modifications.
PROCESSING AND EXTRACTION OF PLANT
MATERIAL
Leaves and fruits (separated into pericarps and
seeds) of dioecious individuals with pistillate
flowers of C. criuva were dried at 40°C, fragmented
and submitted to static maceration using hexane and
1567
methanol as solvents. Three successive exchanges
of solvent were made, followed by filtration of the
extract and evaporation under reduced pressure.
ANALYSIS OF CHEMICAL PROSPECTING
Chemical analysis of the extracts were performed
by Thin Layer Chromatography, adapted from
Farnsworth (1966), Marini-Bettòlo et al. (1981)
and Wagner et al. (1984). Dry extracts (50 mg) were
solubilized in 1 mL of the respective solvent used
in the extraction. To 300 µL of each solubilized
extract was added 1.2 mL of methanol. The results
of applications to the chromatographic silica gel 60
G plates (Merck) were taken by a ATS4 and TLC
Visualizer, controlled by WinCATS 1.4.4 software
(Camag - Muttenz). The list of reagents used in
chemical tests is presented in Table I.
TABLE I
List of reagents used in chemical prospecting tests by TLC (thin layer chromatography) (Farnsworth 1966, MariniBettòlo et al. 1981, Wagner et al. 1984).
CHEMICAL CLASS
REAGENT
ELUENT
Tannins and poliphenols
1% Potassium ferrocyanide and 2%
ferric chloride
Ethyl acetate: formic acid: acetic acid: water
(100:11:11:27)
Flavonoid glycosides
NP/PEG
Ethyl acetate: formic acid: acetic acid: water
(80:9:9:22).
Flavonoid aglycones
NP/PEG
Toluene: acetone: chloroform:
acetic acid (40:25:35:25)
Anthraquinone glycosides
5% Methanolic solution of 5%
potassium hydroxide
Ethyl acetate: formic acid: acetic acid: water
(80:9:9:22)
Anthraquinone aglycones
5% Methanolic solution of 5%
potassium hydroxide
Toluene: acetone: chloroform (25:10:10).
Triterpenes and Steroids
Liebermann-Burchard
Hexane: ethyl acetate (80:20).
Coumarins
5% Methanolic solution of 5%
potassium hydroxide
Toluene: ethyl ether: acetic acid (30:30:30).
Saponins
Anisaldehyde sulfuric
Chloroform: methanol: water (64:50:10)
Cardioactive glycosides
Keddes Reagent
Ethyl acetate: methanol: water (84:11:8)
Alkaloids
Dragendorff Reagent
Ethyl acetate: methanol: water (40:10:20).
Proanthocyanidins
50% Sulfuric acid
Ethyl acetate: formic acid: acetic acid: water
(100:11:11:27)
An Acad Bras Cienc (2017) 89 (3)
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KARLA M.M. DA SILVA et al.
TOTAL ANTIOXIDANT ACTIVITY TEST
Total antioxidant activity (TAA) of the extracts
of C. criuva was evaluated by the scavenging of
2,2-diphenyl-1-picrylhydrazyl (DPPH) radical
according to the methodologies described by
Mensor et al. (2001) and Rufino et al. (2007), with
modifications proposed by Silva and Paiva (2012).
Solutions of the extracts with concentrations of
250, 125, 50, 25, 10 and 5 μg/ml were prepared
using methanol as solvent. To aliquots of 2.5
mL of each solution was added 1 ml of DPPH
solution (0.3 mM in methanol) and the mixture was
homogenized on a vortex agitator. It was used 2.5
mL of methanol with 1 mL of DPPH solution as
negative control and 2.5 ml of each extract solution
with 1 mL of methanol as blank. The absorbance
was read at 518 nm at every 5 minutes for 30
minutes. The final absorbance was used to calculate
the concentration of sample able to reduce in 50%
the initial concentration of DPPH (EC50), expressed
as grams of sample / grams of DPPH (Rufino et al.
2007). It was used rutin as positive control and the
procedures were performed in triplicate.
DETERMINATION OF TOTAL FLAVONOIDS
The content of total flavonoids, expressed as
flavonols and flavones, was determined in extracts
of C. criuva using a colorimetric method involving
reaction with aluminum chloride, using rutin as a
standard, as described by Chang et al. (2002) with
modifications.
STATISTICAL PROCESSING OF DATA
The results were expressed as the mean ± standard
deviation of three independent experiments
performed in triplicate. Statistical significance of
the differences observed between different assays
for the same sample and among the different samples
were assessed using ANOVA (simple analysis of
variance). In case of rejection of the null hypothesis
by ANOVA, the Tukey-Kramer test was used.
An Acad Bras Cienc (2017) 89 (3)
The correlation between EC50 values of the crude
extracts of C. criuva, the maximum antioxidant
activity and the percentage corresponding to the
flavonoid content (expressed as flavones and
flavonols) were analysed and classified using the
Pearson correlation coefficient (Cohen 1988, Filho
and Junior 2009).
RESULTS
ANALYSIS OF CELLULAR ORGANIZATION
Petiole
In cross section, the middle region of the petiole
of C. criuva has a plan-convex shape (Fig. 1a).
The epidermis is uniseriate, glabrous and thick,
has cuticle with flanges and internal periclinal,
cutinized cell walls. Underlying the adaxial
epidermis, 2-3 chlorophyll parenchyma layers are
present, followed by fundamental parenchyma.
On the abaxial surface, there are 10-12 layers of
ring collenchyma (Fig. 1b) and the cells near the
epidermis contain chloroplasts. Toward the vascular
system fundamental parenchyma and idioblasts
are present. The vascular system is composed of
30-35 cordiform bundles fused together, and are
surrounded by perivascular fiber groups. In the
phloem, the transport elements and companion
cells occur in clusters, separated by parenchyma.
The xylem is composed of proto- and meta-xylem
elements, arranged in a radial series, separated by
parenchyma cells. In the region corresponding to
the medulla, isodiametrical parenchyma cells of
varying sizes occur (Fig.1a).
Leaf blade
In front view, the epidermis is glabrous on both
sides. The cells are polygonal shaped, slender
with straight anticlinal walls on the upper side
and slightly sinuous walls on the abaxial surface
(Fig. 1c). Epicuticular wax “smooth layer” type,
registered under electron microscopy, forms
Clusia criuva: ANATOMY AND CHEMISTRY
1569
Figure 1 - Leaf anatomy of C. criuva. a: Overview of the petiole cross-sectional; b: Abaxial face of the epidermis, showing
the cuticular flanges and the collenchyma ring; c: Adaxial and abaxial face of the epidermis, showing paracytic stomata; d:
Detail of cuticle in adaxial surface of the epidermis in scanning electron microscopy; e: Detail of cuticle on the abaxial surface
of the epidermis in scanning electron microscopy; f: Overview of the mesophyll; g: Intermediate layer showing elongated cells
and intercellular spaces; h: Detail of edge of leaf, showing the cuticular flanges and impregnation of internal periclinal wall; i:
Overview of the midrib; j: Details of vascular bundles of the midrib. mp: Medullary parenchyma; cp: Cortical parenchyma; ch:
Collenchyma; cut: Cuticle; ec: Epidermal cells; st: Stomata; pp: Palisade parenchyma; sp: Spongy parenchyma; fp: Fundamental
parenchyma; vb: Vascular bundles; sf: Sclerenchyma fibers. Scale bar = 100 µm, except in Fig. 1d (Scale bar = 5 µm).
An Acad Bras Cienc (2017) 89 (3)
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KARLA M.M. DA SILVA et al.
a continuous coat on the leaf surface without
prominent protrusions (Fig. 1d and 1e). The
leaves are hypostomatic with paracytic stomata.
The subsidiary cells, in general, are far larger than
the guard cells and stomata are on the same level
as the other epidermal cells. Transverse sections
show that the epidermis is uniseriate, covered with
smooth cuticle and less thickened when compared
to other parts of the leaf, and penetrates between
the anticlinal walls. The mesophyll of C. criuva
is dorsiventral (Fig. 1f). The palisade parenchyma
has 2-3 layers of elongated cells of similar size.
Between the palisade and spongy parenchyma
are 1-2 layers of loose tissue formed by rounded
or slightly elongated cells (Fig. 1g). The spongy
parenchyma is loose with large intercellular spaces,
with 10-12 layers of cells with isodiametric format
and with little variation in size. The vascular tissue
is composed of bundles, with xylem towards the
upper side and phloem at the abaxial surface. The
bundles are surrounded by perivascular fibers.
At the end of the leaf margin the epidermis is
uniseriate with elongated cells, with smooth cuticle
and more thickened when compared to other parts
of the leaf, with flanges and internal periclinal
cutinized cell walls. The layers underlying the
epidermis are occupied by cells of the fundamental
parenchyma (Fig. 1h).
In the transverse plane of the leaf blade,
at the midrib level, the epidermis is uniseriate
and glabrous. In this region, the cuticle has
characteristics similar to those observed in the
petiole (Fig. 1i). Underlying the epidermis at the
upper side, 2-3 chlorophyll parenchyma layers are
presents, followed by fundamental parenchyma
cells that increase in size towards the vascular
cylinder. Along the abaxial surface, 3-5 layers
of collenchyma of the annular type are observed
which, in turn, are followed by 7-15 layers of
parenchyma cells. The vascular system is formed
by 30-37 fused bundles that are arc-shaped with
invaginated ends. Along the cord parenchyma, there
An Acad Bras Cienc (2017) 89 (3)
are 5-7 accessory bundles with the region facing the
phloem at the abaxial surface. The vascular tissue
has the same organization described for the petiole.
Groups of perivascular phloem fibers occur along
the entire length of tissue of the vascular system
forming a sclerenchymatic sheath (Fig. 1j).
Idioblasts are also found in all parts, filled with
organic compounds or containing calcium oxalate
crystals in the form of druse, the latter being more
common (Fig. 2a and 2b). Secretory structures
formed by the schizogenous process result in
secretory cavities of varying sizes, with 9-14
rectangular epithelial cells. The secretory cavities
are abundant in the leaf of C. criuva, mainly in
collenchymatic tissue, fundamental parenchyma
of the petiole and midrib, parenchyma underlying
the edge of the epidermis on edge, and intercostal
region (Fig. 2b).
ANALYTICAL HISTOCHEMISTRY
Positive results for the possible presence of alkaloids
were obtained in the parenchyma of the cortex and
medulla of the petiole (Fig. 2c), in the midrib and in
the palisade parenchyma of the intercostal region.
Starch grains were seen in all parts of the leaf,
especially in the cortical and medullar parenchyma
of the petiole, the midrib near the vascular cylinder,
the parenchyma near the epidermis of the petiole
and midrib, and in the palisade parenchyma of
the intercostal region (Fig. 2d). The presence of
cellulose was confirmed, except in the cuticle and in
the conducting tissue fibers. Phenols were detected
in the parenchyma of the petiole, the midrib (Fig.
2e), and in the intercostal region (mainly the
palisade parenchyma). In the cuticle, the more
conspicuous edge because this structure is thicker
in relation to other parts of the sheet (Fig. 2f),
lipophilic substances were detected in the walls of
the underlying epidermal cells in certain parts of the
sheet, in many idioblasts present in the cortical and
medullary parenchyma of the petiole and midrib,
Clusia criuva: ANATOMY AND CHEMISTRY
and in the phloem vessels and parenchyma present
in all parts of the sheet. Lignin was detected in the
sclerenchyma fibers forming a sheath around the
vascular bundles in the leaf blade (Fig. 2g), and in
the vessel elements of all parts of the plant. Tannins
were observed in the parenchyma of the petiole and
midrib (Fig. 2f) and conductive elements of these
same parts of the plant. The presence of numerous
calcium oxalate crystals in the form of druse was
confirmed throughout the leaf lamina and petiole.
Analysis of chemical prospecting
Table II summarizes the results for the chemical
prospecting tests by thin layer chromatography
(TLC).
1571
EVALUATION OF ANTIOXIDANT ACTIVITY
The EC50 values obtained for extracts of C. criuva
and the standard rutin are shown in Table III. Results
are expressed as mean ± standard deviation of three
independent experiments. The curves obtained
by linear regression showed a good coefficient of
determination, according to Mensor et al. (2001)
(R2> 0.80), for all extracts. The statistical treatment
of the data by ANOVA showed that for each extract
(except for the hexanic extract of pericarps), there
was no significant difference (p> 0.05) between the
three independent assays. For the hexanic extract
of pericarps only two independent assays were
considered. Table III shows that all of C. criuva
crude extracts exhibited EC50 values greater than
TABLE II
Detection of different chemical classes of extracts of C. criuva by TLC.
EXTRACT
CCLH
CCPH
CCSH
CCLM
CCPM
CCSM
Tannins and poliphenols
-
-
-
-
+
+
Flavonoid glycosides
-
-
-
+
+
-
Flavonoid aglycones
-
-
-
-
+
+
Anthraquinone glycosides
-
-
-
-
-
-
Anthraquinone aglycones
-
-
-
-
+
-
Triterpenes and Steroids
+
+
+
+
+
+
Coumarins
-
-
-
-
-
+
Saponins
-
-
-
+
+
+
Cardioactive glycosides
-
-
-
+
+
-
Alkaloids
-
-
-
+
+
+
Proanthocyanidins
-
-
-
-
+
+
CHEMICAL
CLASSES
CCLH: crude hexanic extract of leaves; CCPH: crude hexanic extract of pericarps; CCSH: crude hexanic extract of seeds; CCLM:
crude methanolic extract of leaves; CCPM: crude methanolic extract of pericarps; CCSM: crude methanolic extract of seeds.
- absence of the substance;
+ presence of the substance.
An Acad Bras Cienc (2017) 89 (3)
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KARLA M.M. DA SILVA et al.
that found for the standard rutin. However, the
Tukey-Kramer statistical test suggests that there
was no significant difference between the EC 50
values of rutin and the extracts analyzed, except
when compared with the hexanic extract of leaves.
The kinetic profiles of reactions between DPPH
and extracts of C. criuva and also with the standard
rutin are shown in Figure 3. The analyses show
that the reaction kinetics of all extracts had almost
instantaneous responses for every concentration
tested. In Fig. 3a, the standard rutin reached its
maximum antioxidant activity at a concentration of
25 µg / ml, at 10 to 15 minutes, resulting in about
98% of the maximum antioxidant activity. Among
the extracts, the methanolic extract of seeds showed
the lowest EC50 value (Fig. 3g), but the extract
that showed the largest percentage of maximum
antioxidant activity was the methanolic extract of
pericarps, which at a concentration of 250 µg /
ml, resulting in approximately 4.5% of remaining
DPPH (Fig. 3f). Leaf extracts showed the lowest
antioxidant activities in terms of both EC50 values
and maximum antioxidant activity especially the
hexanic extract which showed an EC50 value higher
than the standard and the other extracts analyzed.
DETERMINATION OF TOTAL FLAVONOIDS
Figure 2 - Anatomy and histochemistry of C. criuva. a:
Secretory duct and druse in the collenchyma ring of the midrib;
b: Secretory cavity in the cortical parenchyma of the petiole;
c: Overview of the petiole stained with the Dragendorff
reagent; d: Starch grains in chlorophyllian parenchyma of the
intercostal space; e: Overview of the midrib stained with ferric
chloride; f: Overview of the leaf edge stained with Sudan
III; g: Overview of the midrib stained with phloroglucinol,
showing sclerenchyma fibers around the vascular cylinder; h:
Midrib stained with hydrochloric vanillin, structures containing
tannins are indicated by arrows. mp: Medullary parenchyma;
cp: Cortical parenchyma; cut: Cuticle; sd: Secretory ducts; id:
Idioblasts; dr: Druse. Scale bar = 100 µm, except in Fig. 1D
(Scale bar = 5 µm).
An Acad Bras Cienc (2017) 89 (3)
Table III presents the values of total flavonoid
content obtained for extracts of C. criuva. Results
are expressed as mean ± standard deviation of
three independent experiments. The statistical
treatment of the data by ANOVA showed that
three independent experiments for each extract
were statistically equivalent (p ≥ 0.05). The results
suggest the highest percentages of flavones and
flavonols in the hexanic extracts. The hexanic
extract of pericarps had the highest percentage
of flavonoids among the tested extracts (14.62 ±
0.76%), followed by the hexanic extract of leaves
(4.78 ± 0.17%). These results were not expected,
since flavonoids usually exhibits polar character
Clusia criuva: ANATOMY AND CHEMISTRY
1573
Figure 3 - Reaction kinetics with DPPH (2,2-diphenyl-1-picrylhydrazyl). (a) Rutin. (b) CCLH: crude hexanic extract of leaves.
(c) CCPH: crude hexanic extract of pericarps. (d) CCSH: crude hexanic extract of seeds. (e) CCLM: crude methanolic extract of
leaves. (f) CCPM: crude methanolic extract of pericarps. (g) CCSM: crude methanolic extract of seeds.
An Acad Bras Cienc (2017) 89 (3)
KARLA M.M. DA SILVA et al.
1574
TABLE III
Content of total flavonoids (flavones and flavonols), EC50
and maximum antioxidant activity (MAA) of C. criuva
extracts.
Samples
Flavonoids
%
Mean ± SD
EC50 (Mean ± SD)
(g extract/ g DPPH)
%
MAA
CCLH
CCPH
CCSH
CCLM
CCPM
4.78 ± 0.17
14.62 ± 0.76
3.14 ± 0.16
1.57 ± 0.14
0.63 ± 0.12
34.66 ± 19.42
10.94 ± 0.36
8.32 ± 0.30
12.54 ± 1.43
5.01 ± 0.16
22.60
71.43
72.75
64.52
95.49
CCSM
1.40 ± 0.19
4.06 ± 1.12
77.23
Rutin
-
0.61 ± 0.11
98.91
CCLH: crude hexanic extract of leaves. CCPH: crude hexanic
extract of pericarps. CCSH: crude hexanic extract of seeds.
CCLM: crude methanolic extract of leaves. CCPM: crude
methanolic extract of pericarps. CCSM: crude methanolic
extract of seeds.
and are not present in hexanic extracts, as observed
in TLC analysis. In order to understand what might
be happening, possible interactions between the
reagent used in this assay and the other chemical
classes occurring in these extracts were studied.
CORRELATION BETWEEN ANTIOXIDANT
ACTIVITY AND FLAVONOID PERCENTAGE
The correlation between EC50 values of the crude
extracts and the percentage of flavonoid content
showed a coefficient of +0.1728, indicating a
weak positive correlation. It means that there is a
tendency that high values of EC50 are associated
with high percentages of flavonoids in the extracts.
By definition the EC50 represents the concentration
of sample able to reduce in 50% the initial
concentration of DPPH, so, the best samples are
those with the lowest values of EC50. Based on
that, the results show that the extracts with best
antioxidant activities were not the ones with the
highest levels of flavonoids.
This data is corroborated by the correlation
between the maximum antioxidant activity of
extracts and the flavonoid content, which showed
a coefficient of -0.1729, indicating a weak negative
An Acad Bras Cienc (2017) 89 (3)
correlation. It suggests that for Clusia criuva
extracts there is not a direct relationship between
antioxidant activity and the presence of flavonoids.
DISCUSSION
Clusia criuva anatomical presents characteristics
considered common to Clusiaceae as uniseriate
epidermis, paracytic stomata, hypostomatic
leaves, presence of cuticular flanges, dorsiventral
mesophyll, among others (Metcalfe and Chalk
1950, Stevens 2007).
Some authors also consider the presence of
hypodermis in Clusiaceae a hallmark for the family.
The hypodermis is a subepidermal layer, which
originates in the meristem, that works as a storer
tissue of water, and it is mostly found in xerophytes
(Esau 1974). The presence of subepidermal layers
has been mentioned in several studies about Clusia
(Paula 1976, Schneider 1985, Silva et al. 2005,
Fernandes 2007). However, in this study it was
not observed the presence of these layers, possibly
because the individuals were collected in Floresta
Atlântica, where the climate is more humid than
the habitats of C. criuva from studies cited earlier.
This indicates a plasticity regarding the presence
of subepidermal layers, showing that this feature is
not essential to species. Fernandes (2007) reports
that the presence of a storer tissue of water, in
fact, is not essential. Since plants of some specific
areas are not exposed to long droughts, and when
it occurs, it can be an adaptation to periods of lowrainfall.
Fernandes (2007), Guimarães et al. (2013),
Rocha et al. (2014) and Silva et al. (2014) also
cite secretory ducts and cavities for species
of Clusia. Paula (1976) notes the presence of
schizogenous and schizolisigenous ducts in Clusia
aff. macropoda. Although there are not ontogenesis
studies for secretory structures found in Clusia,
Fernandes (2007) indicates the absence of traces
of lysis of epithelial cells and suggests that the
Clusia criuva: ANATOMY AND CHEMISTRY
observed structures have schizogenous origin.
The development of sheath sclerenchyma fibers
involving the vascular bundles was also mentioned
in Clusia (Paula 1976, Boeger and Wisniewski
2003). According to Esau (1974), sclereids are
cells sclerenchyma constituents presenting variety
in shape and having thick lignified secondary wall
with numerous pits. Edwards et al. (2000) point out
the possibility for the appearance of sclerophylly in
forest plants: adaptation to seasonal water deficits,
the adaptation or consequence of living soils with
low nutrients and emphasis longevity leaf, assisting
in their leaf protection (defense against herbivores)
or increasing leaf carbon gain per unit of investment.
Such assumptions are not deleted and contribute to
clarify the presence of sclerophyllous vegetation in
tropical forest (Boeger and Wisniewski 2003).
Fernandes (2007) shows the format of the main
vein as a distinctive anatomical character among
the species of the genus Clusia. This structure, in
C. criuva has a plan-convex format with rounded
abaxial face. The vascular bundles present
cordiform disposition with accessory bundles,
which can differentiate it from other species.
The presence of alkaloids was detected by
histochemical tests and confirmed in chemical
prospecting of leaves. These substances can act
as factors against herbivory - as well as calciumoxalate and control of pathogens (Wittstock and
Gershenzon 2002).
Starch grains and lignin are widely present in
plants. Starch is the main reserve substance of plant
and lignin is the mainly heteropolymer present
in cell walls of vascular plant cells (Opsahl and
Benner 1995, Amaral et al. 2007).
Lipids detected in leaves of C. criuva are also
substances widely present in plants. They have
structural functions, constituting membranes and
cuticular waxes (Barthlott et al. 1998, Moreau et
al. 1998). Essential oils and resins are considered
lipids that have been indicated in Clusia (Nogueira
et al. 2001, Ferreira et al. 2014). In the chemical
1575
prospecting, were detected triterpenes and steroids.
Possibly, terpenoids detected in the methanolic
extracts have intermediate polarity, such as the
terpenes acids, as described in Clusiaceae family
(Tavares et al. 2001, Noldin et al. 2006, Guimarães
et al. 2008). The terpenoids are substances in
essential oils (mono- and sesquiterpenes) and latex
- characteristic of Clusia according to Metcalfe and
Chalk (1950). In some species of Clusia, triterpenes
have been isolated from epicuticular wax (Medina
et al. 2004, 2006).
Although the result for polyphenols had
been negative to leaves in chemical prospecting
test, phenols stood out in histochemical tests and
is widely present. The group of phenols include
various substances such as simple phenols,
phenolic acids, coumarins, flavonoids and tannins,
among others (Sousa et al. 2007). Several studies
indicate the presence of phenols in Clusia (Seo
et al. 1999, Compagnone et al. 2008, Silva and
Paiva 2012, Ferreira et al. 2014). The presence
of tannins, polyphenols and flavonoids were also
confirmed to extracts from pericarp and seeds
of C. criuva. Oliveira et al. (2012) highlights
biflavonoids in fruits of C. paralicola. in flowers of
Clusia, benzophenones have stood out, especially
poliisoprenylated benzophenones (Oliveira et al.
1996, 1999, Lokvam et al. 2000, Porto et al. 2000,
Compagnone et al. 2008, Silva et al. 2012).
The antioxidant potential of plant extracts
is shown in some studies related to the flavonoid
content (Peng et al. 2003, Proestos et al. 2006,
Oliveira et al. 2012, Silva and Paiva 2012). This
relationship is based on the fact that the chemical
structure of flavonoids favours the reduction of
free radicals by proton donation (Van Acker et al.
1996, Pietta 2000, Amić et al. 2003, Seyoum et al.
2006). In Silva and Paiva (2012) and Oliveira et al.
(2012), species of Clusia show potent antioxidant
activity in crude extracts, especially for fruit
extracts as observed in this work. Silva and Paiva
(2012) indicates a potential directly proportional to
An Acad Bras Cienc (2017) 89 (3)
1576
KARLA M.M. DA SILVA et al.
the flavonoid content described for crude extracts
of C. fluminensis, concluding the possibility of a
positive correlation between the two parameters.
Oliveira et al. (2012), emphasizes that crude
extracts of Clusia paralicola may have a better
potential antioxidant activity compared to isolated
flavonoids, biflavonoids in this case, which could
be explained if the substances present in crude
extracts are acting in synergy, or the activity of the
crude extracts are related to another substance that
has a controlling action relative to the other.
It was observed, according to the method
used, a higher content of flavonoids, expressed
as flavones and flavonols, in the hexanic extracts
of C. criuva. This result led us to speculate the
possible interaction between the aluminum
chloride and benzophenones, which could be
influencing the results obtained (Sancho et al.
2003). Thus, that high values of the flavonoid
content in hexanic extracts may represent; in fact,
the presence of benzophenones. Virginio (2015),
supports a flavonoid content of 6.22 ± 0.91% for
the ethanolic extract of the female flower of Clusia
lanceolata, however the literature does not indicate
the presence of flavonoids in Clusia flowers,
which are composed mainly by poliisoprenylated
benzophenones, fatty acids and terpenes (Nogueira
et al. 2001, Guimarães et al. 2013).
The result of anatomical characterization,
chemical prospecting and analysis of the potential
antioxidant activity involving leaves and fruits
of C. criuva described in this paper are related
to biological activities suitable for the species,
expanding knowledge about its pharmacological,
morphological and chemical potential. The results
highlight the importance of conducting isolation
studies, purification and identification of its
chemical constituents. In conclusion, the data can
assist taxonomic studies within the genus Clusia
and/or the Clusiaceae family, since they indicate
important anatomical characteristics of the species
C. criuva.
An Acad Bras Cienc (2017) 89 (3)
ACKNOWLEDGMENTS
We thank Pós-Graduação em Ciência e
Biotecnologia of the Universidade Federal
Fluminense, the funding agencies Coordenação
de Aperfeiçoamento de Pessoal de Nível Superior
(CAPES), Conselho Nacional de Desenvolvimento
Científico e Tecnológico (CNPq) and Programa
de Fomento à Pesquisa da Universidade Federal
Fluminense (FOPESQ-UFF) and the Laboratório
de Produtos Naturais Marinhos - UFF, the use of
facilities. We thank also Dr. Norman Rattclife for
the English review.
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