HPLC/DAD Analysis, Determination of Total Phenolic and Flavonoid Contents and Antioxidant Activity from the Leaves of Cariniana domestica (Mart) Miers
The species Cariniana domestica, popularly known in Brazil as jequitibá-roxo, belongs to Lecythidaceae and is very little studied, although it is used in folk Brazilian medicine. In this work, antioxidant potential, total phenolic and flavonoid contents from the leaves of the plant were investigated. Additionally, HPLC/DAD analysis of ethyl acetate fraction revealed five active phenolic compounds (gallic, chlorogenic and caffeic acids, rutin and kaempferol). Antioxidant potential was determined using DPPH assay. Total phenolics were measured using Folin Ciocalteau and total flavonoid, using aluminium chloride. HPLC/DAD analysis was performed using gradient system. The leaves of C. domestica exhibited high antioxidant activity, as well as a large content of phenolic compounds and flavonoids. IC50 obtained with DPPH assay varied from 5.12±0.34 to 14.17±0.32 μg mL-1. Total phenolic varied from 214.32±0.33 to 510±0.21 μg mL-1 and flavonoid content, from 15.26±0.63 to 39.92±0.03 μg mL-1. The species C. domestica exhibited high antioxidant potential, being ethyl acetate the most active fraction. Several bioactive phenolics were identified and quantified in this fraction. All compounds are described for the first time for the genus Cariniana.
to cite this article:
V. Janovik, A.A. Boligon, R.V. Bandeira and M.L. Athayde, 2011. HPLC/DAD Analysis, Determination of Total Phenolic and Flavonoid Contents and Antioxidant Activity from the Leaves of Cariniana domestica (Mart) Miers. Research Journal of Phytochemistry, 5: 209-215.
Received: July 30, 2011;
Accepted: October 10, 2011;
Published: October 29, 2011
The species Cariniana domestica, commonly known in Brazil as jequitibá-roxo
belongs to Lecythidaceae, a family consisting of 25 genera and 400 species that
occur in the form of large trees, with pantropical distribution (Tsou
and Mori, 2007). Several important bioactive compounds have been isolated
from species of this family and therefore, it is important for the conduction
of further studies on this genus (De Carvalho et al.,
1998; Lima et al., 2002). There are few reports
in the literature concerning to chemical constituents and ethnopharmacological
uses of the genus Cariniana, although the stem bark and the leaves infusion
of these species are commonly used as anti-inflammatory and antimicrobial remedies
(Silva et al., 2009).
Previously, Lima et al. (2002) has isolated
triterpenoidal saponins from the barks of Cariniana rubra Gardner and
Miers, whereas some constituents have been isolated from other species
of this family, including pentacyclic triterpenes, saponins, ellagic acid and
indol-quinazoline alkaloids (De Carvalho et al.,
1998). Further works related to Cariniana spp. describe the species
as skin-lightening cosmetic by inhibition of hyaluronidase and also shown anti-tyrosinase
activity (Baurin et al., 2002) and also as antifungal
(Silva et al., 2009). A previous work from our
group led to the isolation, identification and quantification of four bioactive
substances from the dichloromethane fraction obtained with the barks of C.
domestica (β-sitosterol, stigmasterol, lupeol and β-amyrin) (Janovik
et al., 2011).
Several authors have researched different extracts of plants in order to improve
knowledge about antioxidant capacity. According to Kintzios
et al. (2010), it is important to identify the compounds involved
in this capacity as well as to evaluate the free radical-scavenging from crude
extracts because it is the common form of usage in popular medicine. Polyphenolic
compounds have been largely studied as antioxidant compounds and its dietary
ingest have shown protective effect against diseases such as coronary heart
(Engler and Engler, 2006) and neurodegenerative (Lau
et al., 2005).
Preliminary phytochemical investigations of our group concerning the leaves of C. domestica indicated the presence of a large number of phenolic compounds. This feature allied to the importance of the oxidative stress in the pathogenesis of various diseases prompts us to better evaluate the antioxidant potential of this plant and to determine the phenolic and flavonoid contents. Simultaneously, HPLC/DAD was performed to identify which were the mainly phenolic compounds and to quantify them.
MATERIALS AND METHODS
Reagents, standards and apparatus: Silica Gel 60, Silica Gel 60 F254
coated plates, solvents for the extractions, dichloromethane, ethyl acetate,
ethanol, methanol and n-butanol, Folin-Ciocalteau and aluminum chloride were
purchased from Merck (Darmstadt, Germany). Ascorbic acid, 2,2-diphenyl,1-picrylhydrasyl
(DPPH), quercetin, rutin, kaempferol, gallic, chlorogenic and caffeic acids
standards were acquired from Sigma Chemical Co. (St. Louis, MO, USA). 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. Spectrophotometer
Plant collection and extraction: The leaves 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 in a knife mill. The powder was macerated with ethanol 70% 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 after ethanol evaporation was partitioned with solvents in increasing polarity to obtain the following fractions: dichloromethane, ethyl acetate and n-butanol.
Radical-scavenging activity-DPPH assay: The antioxidant activity of
the fractions and crude extract was evaluated by monitoring its ability in quenching
the stable free radical DPPH, according to a slightly modified method previously
described (Choi et al., 2002). 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. Ethanol was used to calibrate the spectrophotometer. The test
was performed in triplicate and the calculation of the antioxidant activity
followed the equation:
where, AbsS is the absorbance of each fraction; AbsB is the absorbance of fractions without adding the DPPH; AbsC is absorbance the ethanolic solution of 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
contents was performed by the Folin-Ciocalteu method (Chandra
and Mejia, 2004). The total phenolic content was expressed in milligrams
equivalents of pyrogallic acid (PGA) 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 (r = 0.9984).
Determination of total flavonoids: The determination of flavonoid content
was performed using the method described by Woisky and Salatino
(1998). Total flavonoid content was expressed in milligrams equivalents
of rutin (RT) per gram of each fraction. The equation obtained for the calibration
curve of rutin was Y = 20.394 x-0.2033 (r = 0.9997).
HPLC-DAD qualitative and quantitative analysis of polyphenols: Reverse
phase chromatography analyses were carried out with a Phenomenex C-18 column
(4.6 mm x 250 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 slightly modified. 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.
Statistical analysis: Correlation coefficient (r) was used to determine the relationship between two variables and the standard deviations in the DPPH, total phenolic and total flavonoid assays were calculated from the data obtained from three separate experiments using MS Excel for Windows.
RESULTS AND DISCUSSION
The phenolic substances such as tocopherols, flavonoids and phenolic acids
are specially mentioned as antioxidants due to their efficient scavenging capacity
of reactive oxygen species which may cause a large number of disorders by reacting
with cellular lipids, proteins, carbohydrates and nucleic acids (Chanwitheesuk
et al., 2005). Besides, natural antioxidants pursuits better benefits
in comparison to synthetic ones (Stankovic et al.,
In this study, the leaves of C. domestica exhibited very good antioxidant activity, especially ethyl acetate fraction. The results are shown in Table 1.
Ethyl acetate was the most active fraction. The IC50 (the amount of extract of the plant tested necessary to decrease the concentration of initial DPPH absorbance by 50%) value obtained was 5.12±0.34 μg mL-1 (Table 1). Butanolic fraction showed slightly lower IC50 value (5.73±0.81 μg mL-1) than ethyl acetate fraction, being also very active and the crude extract IC50 was 6.95±0.62 μg mL-1. The lowest antioxidant activity (IC50 = 14.17±0.32 μg mL-1) was observed for dichloromethane fraction. Ethyl acetate, butanolic and crude extract antioxidant activities were found to be higher than the well known antioxidant ascorbic acid, commonly used as a standard (IC50 = 9.02±0.21 μg mL-1).
Previous work has found similar good results for ethyl acetate and butanolic
fractions and have documented that these fractions are sources of antioxidant
substances (Tung et al., 2007). Antioxidant compounds
of different chemical structure can be achieved using solvents of different
polarity (Stankovic et al., 2010) and some investigations
of plants compositions have demonstrated that polar fractions usually concentrates
high amounts of phenolic compounds (Canadanovic-Brunet et
It is important to compare data obtained on C. domestica with other
plants under the same assay conditions (Schubert et al.,
2007). If compared with green tea (Camellia sinensis), the results
obtained with C. domestica were better than those obtained for this plant,
IC50 = 11.8 μg mL-1 (Almeida
et al., 2008). Ilex paraguariensis, a popular species from
South Brazil which achieves antioxidant properties, have exhibited superior
values for ethyl acetate fraction (IC50 = 13.26 μg mL-1)
when compared with C. domestica (Schubert et al.,
For the determination of phenolic content, the ethyl acetate fraction also exhibited the highest value (510±0.21) (Table 1). Butanolic fraction expressed the second highest value (486.22±0.87). On the other hand, dichloromethane fraction showed the lowest phenolic content (214.32±0.33). When comparing the results obtained with phenolic and flavonoid contents assays, we may observe a correlation between both dosages for ethyl acetate fraction. Differently, butanolic fraction, despite presenting high levels of phenolic compounds, ranged a low value in the flavonoid determination. Dichloromethane fraction exhibited a high content of flavonoids (38.01±0.1 mg g-1), being the second best value. On the other hand, the value obtained for total phenolic content was the lowest (214.32±0.33 mg g-1).
Ethyl acetate fraction furnished high concentrations of polyphenols and flavonoids, as well as the best IC50 value in DPPH assay. Therefore, we have performed an HPLC/DAD analysis with the purpose of identify and quantify its mainly phenolic compounds.
|| Total phenolics and flavonoid contents and antioxidant activities
(IC50/DPPH) from the leaves of C. domestica
|Values are as Mean±SE. PGA: Pyrogallic acid, RT: Rutin,
SE: Standard error, IC50 = The amount of extract necessary to
decrease the concentration of initial DPPH absorbance in 50%
||HPLC/DAD chromatograms obtained with ethyl acetate fraction.
a = ethyl acetate, 254 nm;
|ethyl acetate, 327 nm; c = ethyl acetate, 365 nm. Peaks:
|gallic acid (RT = 12.8 min); 2 = chlorogenic acid (RT = 24.6 min);
|caffeic acid (25.8 min); 4 = rutin (RT = 38.2 min); 5 = kaempferol (RT
= 55.1 min)
|| HPLC/DAD of identified and quantified phenolic compounds
in ethyl acetate fraction obtained from the leaves of C. domestica
|aResults are expressed as Mean (n = 3) ±SD.
G: Gallic acid; CFA: Caffeic acid; CLA: Chlorogenic acid; RT: Rutin; QT:
Quercetin; KF: Kaempferol
Ethyl acetate fraction was investigated for the presence of the following compounds: gallic, chlorogenic and caffeic acids, quercetin, rutin and kaempferol. Identification was performed by comparison of their retentions time and UV absorption spectrum with those of the standards.
The results and chromatograms are shown in Table 2 and Fig.
1. All tested substances are well-known antioxidants. Gallic acid is a derivative
from benzoic acid widely distributed in various plants and foods and its various
biological effects have been reported, such as anti-proliferative, pro-apoptotic
and anti-tumorigenic effects against prostate (Kaur et
al., 2009). In this work, gallic acid was found in minor quantities
in ethyl acetate fraction (2.5±0.82%).
Caffeic and chlorogenic acids have been reported as good free radical scavengers
(Arrua et al., 2010). Our analysis revealed that
caffeic acid constitutes 12.82±0.93% of whole ethyl acetate fraction.
In the HPLC/DAD analysis, chlorogenic acid was found in major quantities (15.27±1.21%).
Rutin (quercetin 3-O-rhamnoglucoside) which is commonly found in plants, upon
the hydrolysis of the glycoside bond produces quercetin, a highly antioxidative
aglycon. This flavonoid is an active and natural antioxidant, involved in protection
and prevention of pathologies like diabetes mellitus, as an example (Fernandes
et al., 2010). In this analysis, rutin constitutes 11.45±0.32%
of whole fraction.
Finally, kaempferol, a flavonol with a free hydroxyl group at the C-3 position, represented 0.85±1.11% from ethyl acetate fraction.
Values for antioxidant activity and concentrations of phenolic and flavonoid contents obtained for crude extract and fractions from C. domestica exhibited positive correlations. Ethyl acetate fraction ranged the highest flavonoid and phenolic content and this significant correlation indicates that these compounds contribute to the antioxidant capacity. All results obtained are reported for the first time for C. domestica.
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