In recent years, the interest in finding natural antioxidants from plant materials
has been increased, as seen that crude extracts and pure natural compounds from
plants were reported to have antioxidant activities (Barla
et al., 2007).
Oxidative stress can be defined as the imbalance between the production of
Reactive Oxygen Species (ROS) and the ability of enzymatic and non-enzymatic
biological systems to protect against their injury. Different therapeutic strategies
have been proposed for the prevention and treatment of ROS-mediated diseases,
with special emphasis on antioxidant therapy (Paz et
Researches suggest that a wide variety of phytochemicals, such as phenolics
and carotenoids may prevent oxidative stress-induced damage leading to cancer
(Alesiani et al., 2010). Reports have confirmed
that lower incidence of age-related chronic diseases is associated to the consumption
of carotene-rich vegetables (Sangheeta et al., 2009).
Lycopene exerts anti-inflammatory effects through its action as free radical
scavenger which may reduce cellular damage (Pennathur et
al., 2010). β-carotene, besides it is a known quencher of ROS,
is also applied in photoprotective compounds (Eicker et
Cariniana domestica belongs to Lecythidaceae and is known in Brazil
as jequitibá-roxo. Lecythidaceae is composed of 10 genera and 230 species,
being a dominant group in neotropical forests (Tsou and
Mori, 2007). The bark infusion of these species is commonly used as antioxidant,
anti-inflammatory and antimicrobial remedies (Neto and Morais,
2003; Silva et al., 2009). Preliminary phytochemical
investigations of our group concerning the barks of C. domestica demonstrated
the presence of triterpenoids and phenolic compounds (Janovik
et al., 2011). This feature allied to the importance of the oxidative
stress in the pathogenesis of various diseases led us to better evaluate the
antioxidant potential of this plant. Simultaneously, phytochemical analysis
led identification of several phenolic compounds, besides lycopene and β-carotene
and its HPLC/DAD analysis.
MATERIALS AND METHODS
Chemicals, apparatus and general procedures: Folin-Ciocalteu and solvents for the extractions, dichloromethane, ethyl acetate, ethanol, methanol and n-butanol were purchased from Merck (Darmstadt, Germany). Ascorbic acid, 2,2-diphenyl-1-picrylhydrazyl (DPPH), quercetin, rutin, kaempferol, gallic, chlorogenic and caffeic acids standards were acquired from Sigma Chemical Co. (St. Louis, MO, USA). β-carotene (>95%) and lycopene were acquired from Sigma-Aldrich. High performance liquid chromatography (HPLC-DAD) was performed with the HPLC system (Shimadzu, Kyoto, Japan), Prominence Auto-Sampler (SIL-20A), equipped with Shimadzu LC-20AT reciprocating pumps connected to the degasser DGU-20A5 with integrator CBM-20A, UV-VIS detector DAD SPD-M20A and software LC Solution 1.22 SP1. Absorbances were measured in a Shimadzu-UV-1201.
Plant collection and extractions: The barks of C. domestica were collected in Tangará da Serra-Mato Grosso do Sul, Brazil, in March of 2008. Exsiccate was archived as voucher specimen in the herbarium of Department of Pharmacology at UFSM (SMDB 11818).
Plant material was dried at room temperature and powdered. The powder was macerated with ethanol 70% (v/v) during ten days. After filtration, the extract was evaporated under reduced pressure. This procedure was repeated twice and the extracts were combined. The remained aqueous extract obtained was partitioned with solvents in increasing polarity: dichloromethane, ethyl acetate and n-butanol. At the end of all extractions procedures, each extract was evaporated under reduced pressure to obtain the dried fractions.
Radical-scavenging activity: DPPH assay: Antioxidant activity was evaluated
by monitoring its ability in quenching the stable free radical DPPH, according
to a slightly modified method previously described (Kussar
et al., 2006). The DPPH quenching ability was expressed as IC50.
Six different ethanol dilutions of each fraction and crude extracts at 250,
125, 62.5, 31.25, 15.62 and 7.81 μg mL-1 were mixed with 1.0
mL of DPPH 0.3 mM in ethanol solution. After 30 min, the readings were made
at 518 nm. A solution of DPPH (1 mL, 0.3 mM) in ethanol (2.5 mL) was used as
a negative control and ascorbic acid in the same concentrations used for the
fractions and the crude extracts provide the positive control. The test was
performed in triplicate and the calculation of the antioxidant activity followed
where, Abssample is absorbance of each fraction, Absblank is absorbance of fractions without adding the DPPH and Abscontrol is absorbance the solution of ethanol in DPPH.
The percentage of inhibition was calculated and a graphic of percentage of
inhibition versus concentration was constructed. Correlation coefficients were
optimised (Tsimogiannis and Oreopoulou, 2006).
Determination of total phenolics: The determination of total phenolic
content was performed by the Folin-Ciocalteu method (Chandra
and Mejia, 2004). Briefly, 0.5 mL of 2 N Folin-Ciocalteu reagent was added
to a 1 mL of each sample (0.15 mg mL-1) and this mixture was allowed
to stand for 5 min before the addition of 2 mL of 20% Na2CO3.
The solution was then allowed to stand for 10 min before reading at 730 nm.
Total phenolic content was expressed in milligrams equivalents of Pyrogallic
acid (PGAE) per gram of each fraction. The equation obtained for the calibration
curve of pyrogallic acid in the range of 0.005 to 0.030 mg mL-1 was
y = 34.443x-0.0942 (r2 = 0.9984).
In vitro Fe(II)-induced lipid peroxidation in rats brain: Male Wistar rats weighing 270-320 g and with age from 3 to 3.5 months, from our own breeding colony were kept in cages of 3 or 4 animals each, with continuous access to food and water in a room with controlled temperature (22±3°C) and on a 12 h light/dark cycle with lights on at 7:00 a.m. The animals were maintained and in accordance to the guidelines of the Brazilian Association for Laboratory Animal Science (COBEA).
Rats were decapitated under mild ether anesthesia and the encephalic tissue
was rapidly dissected and placed on ice. Tissues were immediately homogenized
in cold 10 mM Tris-HCl, pH 7.5 (1/10, w/v). The homogenate was centrifuged for
10 min at 4000 xg to yield a pellet that was discarded and a low-speed Supernatant
(SI) was used for the TBARS assay (Puntel et al.,
After centrifugation, an aliquot of 100 μL of S1 was incubated for 1 h
at 37°C with pro-oxidants agent (10 μM of Iron Sulfate) in presence
or absence of plant extracts and then used for TBARS determination. TBARS production
was determined as described by Puntel et al. (2005).
Data were analyzed statistically by one-way ANOVA, followed by Duncans
multiple range tests.
HPLC-DAD qualitative and quantitative analysis of polyphenols: Reverse
phase chromatography analysis were carried out with a Phenomenex C18
column (4.6x250 mm) packed with 5 μm diameter particles, volume injection
was 40 μL and the gradient elution was conducted according to the Evaristo
and Leitao (2001) method. The UV absorption spectra of the standards as
well as the samples were recorded in the range of 230-400 nm.
Stock solutions of standards were prepared in methanol in the range of 0.0025-0.045 mg mL-1. Quantification was carried out by the integration of the peaks using external standard method, considering the 254 nm for gallic acid, 327 nm for chlorogenic and caffeic acids and 365 nm for the three flavonoids (quercetin, rutin and kaempferol). All the samples and standards solutions as well as the mobile phase were degassed and filtered through 0.45 μm membrane filter (Millipore). The chromatographic operations were carried out at ambient temperature and in triplicate.
HPLC-DAD analysis of carotenoids: Standard solutions of β-carotene were prepared in mobile phase, at concentrations from 0.2 to 0.6 mg mL-1. The dichloromethane fraction was dissolved in mobile phase. Each concentration was injected tree times and the calibration curve was obtained by linear regression.
The β-carotene analysis was carried out under isocratic conditions using a C8 column (4.6x250 mm), packed with 5 μm diameter particles. The mobile phase was acetonitrile: methanol:ethyl acetate with 1% of acetic acid (88:8:4, v/v/v) and the flow rate, 0.8 mL min-1. Detections were done at 453 nm and the injection volume was 60 μL. Identification of β-carotene was achieved by comparing retention time and UV/DAD spectra with those of standard. All chromatographic operations were performed at room temperature and in triplicate.
Statistical analysis: Data from the TBARS assay were analyzed statistically
by one-way Analysis of Variance (ANOVA), followed by Duncans multiple
range tests when appropriated using the statistical software SPSS 10.0 for Windows.
TBARS graphics were constructed using the Slide Write 4.032 Bit Edition program.
Statistical p-values were calculated to quantify levels of significance for
each treatment type. A significant p-value (p<0.05, p<0.01, or p<0.001
when appropriate) means that there exists significant difference between the
two sets of data being analyzed. Correlation coefficient (r) to determine the
relationship between two variables and the standard deviations in the DPPH and
total phenolics assays were calculated from the data obtained from three separate
experiments using MS Excel for Windows.
RESULTS AND DISCUSSION
Total Phenolic contents (TP) assayed by the method of Folin-Ciocalteu expressed in milligrams equivalent of pyrogallic acid (PGAE) per gram of each fraction and IC50 determinations by the DPPH method are given in Table 1.
The quantitative determination of the polyphenolic content of C. domestica
used Folin-Ciocalteu as the main reagent and pyrogallic acid as reference. In
order, the phenolic contents obtained were ethyl acetate fraction> crude
extract>butanolic fraction>dichloromethane fraction. Investigations of
qualitative composition of plant extracts revealed the presence of high concentrations
of phenolics in the extracts obtained using polar solvents (Canadanovic-Brunet
et al., 2008).
Comparing the content of polyphenolic and antioxidant activity (IC50),
we can correlate the phenolic content of ethyl acetate fraction and its lower
IC50. This relationship of phenolic content and polarity is well
documented (Chandra and Mejia, 2004).
Butanolic fraction exhibited larger phenolic content than dichloromethane fraction
but its IC50 was higher. The structure of the phenolic groups and
possible changes in hydroxyl, by glycosylation as an example, cause a decrease
in antioxidant activity, due to the reduction in the number of hydroxyls and
the steric hindrance that sugar gives, difficulting the connection to the free
radical (Tsimogiannis and Oreopoulou, 2006). Dichloromethane
fraction expressed good IC50 value (16.4±0.35 μg mL-1)
and the lowest phenolic content.
|| Total phenolic content and antioxidant activity (IC50/DPPH)
for crude extract and fractions of C. domestica
|TP: Total phenolics expressed as pyrogallic acid equivalents
(mg g-1 fraction±SE), SE: Standard error, aIC50:
Values for inhibition by fractions and crude extract of DPPH oxidation capacity,
bIC50: Values for inhibition by fractions and crude
extract of TBARS production induced by Fe(II) in brain preparations
||Effects of different concentrations of crude extract, ethyl
acetate, dichloromethane and butanolic fractions from the barks of C.
domestica on Fe(II) (10 μM)-induced TBARS production in brain homogenates.
The samples were incubated for 1 h or without (basal) with Fe(II) and the
plant extracts. Data show means±SEM values average from 3 to 6 independent
experiments performed in duplicate
In TBARS assay, free Fe (II) can induce neurotoxicity via stimulation of the
Fenton reaction and its levels are increased in some degenerative disease (Bostanci
and Bagirici, 2008). In this study, ethyl acetate fraction presented the
best value of IC50 (11.11±0.85 μg mL-1), followed
by butanol fraction (15.2± 1.14 μg mL-1), crude extract
(18.1±2.02 μg mL-1) (Fig. 1). Dichloromethane
expressed the lowest value (44.5±2.31 μg mL-1). The results
have shown a positive correlation between HPLC/DAD analysis, in which all tested
phenolic compounds were identified and quantified in ethyl acetate fraction.
Previous study developed by Pereira et al. (2009)
evaluated the activity of flavonoids. Rutin (IC50 = 28.8 μg
mL-1), quercitin (12.2 μg mL-1) and quercetin (1.4
μg mL-1) have shown good results. We can assume that phenolic
compounds are closely related to inhibition of lipid peroxidation.
Within only a few years, there has been exponential growth in research to localize
carotenoid in plants and numerous chromatographic methods to assay carotenoids
in plants have been published (Serino et al., 2009).
In this study, both compounds were separated using mixture of acetonitrile:
methanol: ethyl acetate with 1% of acetic acid (88:8:4, v/v/v). For quantification,
a calibration curve was constructed with reference standard β-carotene
(Y = 44368x3707.2 with r = 0.9995). β-carotene (Peak 2, RT = 12.5
min) was found to represent 3.73±1.02% from whole fraction. Peak 1 was
identified as lycopene.
The bark extracts C. domestica were investigated for the presence of
the following polyphenolic compounds: gallic, chlorogenic and caffeic acids,
quercetin, rutin and kaempferol. Identification of the compounds was done by
comparison of their retentions time and UV/DAD absorption spectrum with
those of the standards. Results obtained for this HPLC screening (Fig.
2, Table 2) indicates that ethyl acetate fraction englobes
the larger number of phenolic compounds. All searched standards were found to
be represented in this fraction constitution. Caffeic acid was found in major
quantities, followed by chlorogenic acid and kaempferol.
||HPLC/DAD chromatograms obtained for polyphenols screening.
(a) Ethyl acetate at 254 nm, (b) Ethyl acetate at 327 nm, (c) Ethyl acetate
at 365 nm, (d) Dichloromethane at 254 nm, (e) Dichloromethane at 365 nm,
(f) Crude extract at 254 nm, (g) Crude extract at 365 nm and (h) n-butanol
at 365 nm, Peaks; 1: Gallic acid, 2: Caffeic acid, 3: Chlorogenic acid,
4: Rutin, 5: Quercetin, 6: Kaempferol
|| HPLC/DAD of identified and quantified polyphenols in crude
extract and fractions of C. domestica
|Values are Mean±standard deviation (n = 3), GA: Gallic
acid, CFA: Caffeic acid, CLA: Chlorogenic acid, RT: Rutin, QT: Quercetin,
In this study, we could establish a positive correlation between phenolic compounds and antioxidant activity with ethyl acetate fraction which shed a light on this positive relation, as seen that it presented the highest phenolic content and the lowest IC50-DPPH and TBARS assays. HPLC/DAD analysis performed with C. domestica revealed the presence of important bioactive phenolic acids, flavonoids and carotenoids.
The results furnished support to assume that C. domestica achieves antioxidant properties, which may be related to its popular uses. Considering the absence of scientific literature related to this species, this study is a contribution to the knowledge concerning the genus Cariniana.