Journal of
Medicinal Plants Research

  • Abbreviation: J. Med. Plants Res.
  • Language: English
  • ISSN: 1996-0875
  • DOI: 10.5897/JMPR
  • Start Year: 2007
  • Published Articles: 3684

Full Length Research Paper

Application of a degreasing process and sequential ultrasound-assisted extraction to obtain phenolic compounds and elucidate of the potential antioxidant of Siparuna guianensis Aublet

Ilsamar Mendes Soares
  • Ilsamar Mendes Soares
  • Research Laboratory of Natural Products, Biodiversity and Biotechnology Post Graduate Program of the Amazon (Bionorte), Federal University of Tocantins, Palmas, Tocantins 77020-210, Brazil.
  • Google Scholar
Maria de Fátima Ribeiro2,
  • Maria de Fátima Ribeiro2,
  • Research Laboratory of Natural Products, Agroenergy Post Graduate Program,Federal University of Tocantins, Palmas, Tocantins 77020-210, Brazil.
  • Google Scholar
Odelio Joaquim da Costa1,
  • Odelio Joaquim da Costa1,
  • Research Laboratory of Natural Products, Biodiversity and Biotechnology Post Graduate Program of the Amazon (Bionorte), Federal University of Tocantins, Palmas, Tocantins 77020-210, Brazil.
  • Google Scholar
Éber Eurípedes de Souza1
  • Éber Eurípedes de Souza1
  • Research Laboratory of Natural Products, Biodiversity and Biotechnology Post Graduate Program of the Amazon (Bionorte), Federal University of Tocantins, Palmas, Tocantins 77020-210, Brazil.
  • Google Scholar
, Aline Aires Aguiar1,
  • , Aline Aires Aguiar1,
  • Research Laboratory of Natural Products, Biodiversity and Biotechnology Post Graduate Program of the Amazon (Bionorte), Federal University of Tocantins, Palmas, Tocantins 77020-210, Brazil.
  • Google Scholar
Robson dos Santos Barbosa3
  • Robson dos Santos Barbosa3
  • Agroenergy Post Graduate Program, Federal University of Tocantins, Palmas, Tocantins 77020-210, Brazil.
  • Google Scholar
, Tarso da Costa Alvim4,
  • , Tarso da Costa Alvim4,
  • Faculty of Food Engineering, Agroenergy Post Graduate Program, Federal University of Tocantins, Av. NS 15, Block Number III, 26 Room, Palmas, Tocantins 77020-210, Brazil.
  • Google Scholar
Sérgio Donizeti Ascêncio5*
  • Sérgio Donizeti Ascêncio5*
  • Faculty of Medicine, Research Laboratory of Natural Products, Biodiversity and Biotechnology Post Graduate Program
  • Google Scholar
Raimundo Wagner de Souza Aguiar6
  • Raimundo Wagner de Souza Aguiar6
  • Department of Biotechnology, Biodiversity and Biotechnology Post Graduate Program of the Amazon (Bionorte) Gurupi, Federal University of Tocantins, Tocantins 77404-970, Brazil.
  • Google Scholar


  •  Received: 07 April 2017
  •  Accepted: 23 May 2017
  •  Published: 03 June 2017

 ABSTRACT

This study investigated appropriate means to determine the total phenolic content and antioxidant activity of the leaves of Siparuna guianensis. Evaluation by thin layer chromatography (TLC) revealed phenolic compounds in all extracts. The results were confirmed by high-performance liquid chromatography (HPLC), with the identification and quantification of catechin, epigallocatechin gallate and rutin. The methanolic extract had the highest content of phenolic compounds, total tannins and condensed tannins (221.16 ± 3.39 mg GAE/g, 183.72 ± 1.85 mg GAE/g and 135.92 ± 1.00 μg/mL CE/g, respectively) and the strongest radical scavenging activity (IC50 = 37.54 ± 0.41 and 73.13 ± 2.09 μg/mL for DPPH and ABTS, respectively). The ethanolic and hydroethanolic extracts had the highest flavonoid (111.28 ± 4.59 and 103.27 ± 3.00 mg RE/g, respectively) and flavonol content (99.73 ± 0.64 and 77.31 ± 0.33 mg RE/g, respectively). In the correlation analysis, it was verified that tannins are the main phenolic compounds and antioxidants of S. guianensis leaves, and their flavonoid content consists mainly of flavonols. The data obtained establish S. guianensis as a new alternative source of antioxidant compounds, implying its potential application as a food preservative or in the production of nutraceutical and pharmacological products.

 

Key words: Flavonoid, polyphenol, condensed tannins, flavonol, plant extract, free radical scavenging


 INTRODUCTION

The current century is marked by the incidence of a number of lifestyle-related diseases, aggravated mainly by excess free radicals in the body. Interestingly, free radicals are byproducts of aerobic metabolism, and the human body’s defenses against these harmful agents are limited, involving only the enzymes superoxide dismutase, catalase and glutathione peroxidase (Peterson, 2001). Several factors, such as environmental pollution, increased exposure to ultraviolet radiation, smoking, stress, etc., have led to an increase in the rate of these chemical species in the human body (Dal and Sigrist, 2016), contributing significantly to the appearance of various diseases associated with the oxidation of biomolecules such as cancer, diabetes, hepatic disorders, hypertension, atherosclerosis, Alzheimer’s disease and arthritis among others (Kala et al., 2016). It is evident, then, that the search for products capable of neutralizing free radicals constitutes an emergency for humanity.
 
With regard to the development of products capable of combating free radicals, plants are currently the most promising sources, since this ability is intrinsic to them, being one of the factors that guarantee them survival in the environment (Agati et al., 2013). Moreover, many plants already have a long history of use by traditional populations; this contributes to the development of safe products. Research with plant bioactive compounds aimed at this purpose demonstrates that there is potential for both the development of antioxidant drugs and functional foods and natural preservatives (Rashid et al., 2016).
 
Siparuna guianensis Aublet, popularly known as negramina, is a plant that shows potential for obtaining drugs because it has a broad ethnobotanical indication, including the treatment of stomach disorders, malaria, hypertension, migraines, rheumatism, snake bites, as a postpartum bath and an insect repellent (Valentini et al., 2010; Aguiar et al., 2015). It is an important medicinal species in several countries of Latin America, distributed from southern Mexico to northern Paraguay, and from the Pacific to southeastern Brazil (Renner and Hausner, 2005).
 
The leaves of S. guianensis have already been evaluated for the potential elimination of free radicals (Andrade et al., 2013). However, because it is an aromatic plant, the evaluation was restricted to its essential oil and provided unsatisfactory results regarding this biological role. On the other hand, studies of S. guianensis chemical composition reported the presence of phenolic compounds in its leaves (Leitao et al., 2005; Negri et al., 2012). This class of compounds is nowadays the most promising for natural antioxidant products (Kala et al., 2016). As no work that determined the total phenolic content of this plant was found, it was inferred that research aimed at extracting this class of substances could evidence the antioxidant potential of S. guianensis leaves and further validate the use of the herb by popular medicine.
 
Thus, ultrasound-assisted extractions were performed, this being considered one of the most efficient and economical means of recovering polyphenols and other valuable compounds (Zhu et al., 2016). These extractions were preceded by a degreasing process, which is a fundamental step in the success of the extraction of phenolic compounds since the presence of interfering substances such as waxes, fats, terpenes and chlorophylls is an aspect that directly affects the efficiency of the extractive process of this class of compounds (Naczk and Shahidi, 2004).
 
This work aimed to establish adequate means to evaluate and quantify antioxidant compounds in leaves of S. guianensis. In view of this, the objective of this work was to determine the phenolic composition and in vitro antioxidant activity of leaf extracts of S. guianensis.


 MATERIALS AND METHODS

Plant material
 
The leaves of S. guianensis were harvested in Gurupi City, Tocantins, Brazil (GPS coordinates: 11° 43' 45" latitude S, 49° 04' 07" longitude W) in August 2014. Access to the genetic patrimony for the purposes of research was authorized by the National Council for Scientific and Technological Development (CNPq) under registry no. 010580/2013–1. The plant was identified in the Herbarium of the Federal University of Tocantins and a voucher specimen was deposited (no. 10.496). The leaves were dried in a drying oven at 50°C and powdered using a knife mill. The powder obtained was stored in glass bottles and kept at room temperature sheltered from light and moisture.
 
Plant extract preparation
 
The plant material (45 g of leaf powder) was initially defatted with petroleum ether using Soxhlet apparatus (50°C for 24 h). Then, it was extracted successively by ultrasound with methanol, ethanol, 70% aqueous ethanol and distilled water. In sonication extraction, the sample was mixed with 350 mL of extracting solvent in a beaker (1000 mL). The sample flask was immersed in an ultrasound cleaning bath (USC1600, ULTRASONIC CLEANER, UNIQUE, São Paulo, Brazil) which has a frequency of 40 kHz (135 W), for a 1 h cycle at room temperature. For each solvent extraction, the process was repeated five times combining the supernatants. Each time, before extracting with the next solvent, the powdered material was air-dried at room temperature. After extraction, the resultant slurries were filtered under vacuum. The respective extracts had the solvents removed on a rotary evaporator at -600 mm Hg and 45°C, and were then lyophilized and stored in an amber bottle and kept in a desiccator until analysis.
 
Content of phenolic compounds
 
Total phenolic and total tannin content
 
Total phenolic and total tannin content was quantified using the Folin-Ciocalteu method coupled with the casein precipitation method as described by Amorim et al. (2008), with slight modification. To quantify the total phenolic content, methanolic solutions (0.2 mL) of S. guianensis extracts (0.25 mg/mL, w/v) or the standard used (gallic acid, 3-100 μg/mL, w/v) were mixed with Folin-Ciocalteu reagent (0.5 mL of 10%, v/v), sodium carbonate (1 mL of 75%, w/v) and purified and deionized water in a Millipore® Milli-Q purifier gradient system (Milli-Q water) (8.3 mL). The mixture was gently agitated and kept for 30 min in the dark. The absorbance was measured at 760 nm using a UV-visible spectrophotometer (Biospectro® SP-220, SP, Brazil). Total phenolic content was determined by interpolation of the absorbance of the samples against a calibration curve constructed with different concentrations of gallic acid in methanol (y = 1.3631x + 0.0213, adjusted r2 = 0.962). The result was expressed as mg gallic acid equivalents (GAE) per gram of S. guianensis extract (mg GAE/g). To quantify the tannin content, the extract (1 mL, 1 mg/mL) was mixed with casein (0.1 g) and distilled water (5 mL) followed by vigorous shaking until homogenized. This solution was allowed to stand for 3 h and then centrifuged at 1358 × g for 10 min at 10°C. In the supernatant, the non-tannin phenolic constituents were determined in a way similar to the total phenolic content. The amount of tannins was calculated as the difference between total phenolic and non-tannin phenolic content in the extract. The total tannin content was expressed as milligrams of GAE per gram of S. guianensis extract (mg GAE/g).
 
Total condensed tannins
 
Condensed tannins were determined according to the method of Burns (1971) with slight modification, by using catechin as a reference compound. A volume of 400 µL of diluted extract in methanol (1 mg/mL) was added to 0.5 mL of a solution of hydrochloric acid (10% in methanol). The mixture was allowed to stand for 10 min. After that, 0.5 mL of vanillin (1% in methanol) was added and the mixture incubated for 10 min in a water bath at 60°C. Afterward, the absorbance was measured at 500 nm against methanol as a blank. Total condensed tannins were determined by interpolation of the absorbance of the samples against a calibration curve constructed with different concentrations of catechin in methanol (y = 2.5218x + 0.0072, adjusted r2 = 0.9878). The amount of total condensed tannins is expressed as mg catechin equivalents per gram of S. guianensis extract (mg CE/g).
 
Total flavonoids
 
Total flavonoid content was determined by using a method described by Soares et al. (2014) with some modifications. Briefly, reactions were performed in triplicate by mixing 0.5 mL of methanolic solutions of the extract (0.25 mg/mL w/v) or standard (8–400 μg/mL w/v) with an aqueous solution of 0.5 mL of 60% acetic acid, 2 mL methanolic solution of 20% pyridine (v/v), 1 mL of 5% aluminum chloride (w/v) and 6 mL of Milli-Q water. The blank was made up of all of the reaction components with the extract or standard and replacing the aluminum chloride with methanol. This reaction mixture was gently stirred, kept for 30 min in  the  dark  and then its absorbance was measured at 420 nm in a spectro-photometer. Total flavonoid content was determined by interpolating the absorbance of the samples against a calibration curve (y = 1.3324x + 0.0074, adjusted r2 = 0.999) constructed with different concentrations of rutin standard and expressed as milligrams of rutin equivalents (RE) per gram of dry extract (mg RE/g). All experiments were performed in triplicate.
 
Total flavonols
 
The total flavonol content was measured based on the reported method of Miliauskas et al. (2004). Briefly, 0.5 mL of extracts (1 mg/mL) was mixed with 0.5 mL of aluminum chloride (20 mg/mL), followed by 1.5 mL of sodium acetate (50 mg/mL). The mixture was incubated at room temperature for 2.5 h. The absorbance was measured by a spectrophotometer at 440 nm. Total flavonol content was determined by interpolation of the absorbance of the samples against a calibration curve (y = 6.2744x + 0.0016, adjusted r2 = 0.999) constructed with different concentrations of rutin in methanol (2-400 μg/mL, w/v). The result was expressed as mg RE per gram of S. guianensis extract (mg RE/g).
 
Evaluation of antioxidant activity
 
DPPH radical scavenging assay
 
The quantification of antioxidant activity performed by the 1,1-diphenyl-2-picrylhydrazyl (DPPH) (Sigma-Aldrich) assay followed the description from Peixoto Sobrinho et al. (2011).  Briefly, six dilutions (varying between 10 and 250 µg/mL in methanol) of samples or positive control substances (rutin and ascorbic acid (Sigma-Aldrich)) were prepared. An aliquot (0.5 mL) of each dilution was mixed with DPPH (3 mL of 40 μg/mL in methanol). For contrast of the extractive solution color with the assay reaction, a blank was prepared, replacing the DPPH solution with methanol. After 30 min the absorbance was measured at 517 nm. The percentage scavenging values were calculated from the absorbance using the following equation:
 
AA (%) = [(A0 - (AS - Ablank))/A0] × 100
 
Where AA is the antioxidant activity, AS is the absorbance of the sample, Ablank is the absorbance of the blank and A0 is the absorbance of the 40 μg/mL DPPH (without sample). The IC50 value was also calculated, denoting the concentration of a sample required to decrease the absorbance at 517 nm by 50%. The IC50 was expressed in μg/mL.
 
ABTS radical scavenging assay
 
The ABTS assay was performed according to the method described by Re et al. (1999), modified by Liu et al. (2014). Initially, a radical cation (ABTS+•) was prepared with potassium persulfate solution (5 mL of 2.45 mM) and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) aqueous solution (2.5 mL of 7 mM). This mixture was kept in the dark at room temperature for 16 h before use. The final solution was diluted with ethanol to an absorbance of 0.70 ± 0.05 at 734 nm. A 200 μL sample in methanol at different concentrations (1.25-500.00 μg/mL) was mixed with 3.0 mL ABTS+• solution. After 6 min the absorbance was measured at 734 nm. Rutin and ascorbic acid served as positive controls. The percentage inhibition was calculated by the following formula:
 
ABTS radical scavenging activity (%) = [1 - (As/Ac)] × 100
 
Where Ac is the absorbance of the blank (without sample) and As is the absorbance of the sample. The IC50 value was also calculated to measure the concentration of a sample which was required to decrease the absorbance at 734 nm by 50%.
 
Profiling of phenolic compounds
 
TLC analysis
 
Qualitative analysis of investigated extracts was evaluated by using the TLC method described by Wagner and Bladt (1996). The test was performed on a 0.20 mm Silica gel 60 Alugram® Sil G plate (Macherey-Nagel GmbH & Co., Düren, Germany) using benzene-ethyl acetate-formic acid-methanol (60/30/10/5, v/v/v/v) as the mobile phase. The developed plates were dried with a hairdryer, then sprayed with NEU/PEG reagent (spray with a 1% solution of 2-aminoethyl diphenylborinate in methanol and spray with a 0.5% ethanolic solution of polyethylene glycol). After a new drying, the plates were visualized under UV light at 365 nm using a darkroom viewing cabinet (SOLAB®, model SL 204) equipped with 365 and 254 nm lamps and then photographed using a Samsung WB100 digital camera. The retention factor (Rf) values were calculated and compared with reference standards rutin, quercetin, catechin and gallic acid.
 
HPLC analysis
 
The S. guianensis extracts were analyzed by high-performance liquid chromatography (HPLC) using a Shimadzu® chromatograph (LC-10 Avp series, Kyoto, Japan) equipped with an LC-10AD pump, DGU-14A degasser, UV-vis SPD-10A detector, CTO-10A column oven, Rheodyne manual injector (loop 20 μL) and a CLASS LC-10A integrator. The separation was carried out using a 250 × 4.6 mm C18 reverse-phase Phenomenex Luna column of 5 µm particle size. The mobile phase consisted of water/acetic acid (100 : 1, v/v) (A) and acetonitrile (B). The linear gradient program was: 10-24% B from 0-10 min, 24-25% B from 10-12 min, 25% B from 12-23 min, 25-28% B from 23-25 min, 28-30% B from 25-26 min, 30% B from 26-35 min, 30-100% B from 35-40 min and 100% B from 40-50 min. The flow rate was 0.87 mL/min, column oven temperature 30°C and UV detection at 280 nm. Identification was based on retention time by comparison with commercial standards, such as catechin, epigallocatechin gallate and rutin (Sigma®). To confirm the identification of the compounds in the extracts, co-chromatography was performed. The quantities of the compounds were determined by correlating the area of the analyte with the calibration curves of the standards. The calibration curves for each substance were: catechin, y = 24246x + 20852 (r2 = 1.0000); epigallocatechin gallate, y = 35453x - 72831 (r2 = 0.9879) and rutin, y = 62021x + 8132.5 (r2 = 0.9942). The results were then expressed in micrograms per milligram of extract (μg/mg). Before being injected into the equipment, the samples (1 mg/mL) and reference substances (5-300 μg/mL) were dissolved in methanol-acetonitrile solution (80 : 20 v/v) and filtered through a 0.45 μm membrane filter (Millipore).
 
Statistical analysis
 
All tests were performed in triplicate. Results were expressed as the means ± SD (n = 3). Results showing a probability of occurrence of the nullity hypothesis of less than 5% (p < 0.05) were considered statistically different applying analysis of variance (ANOVA), followed by multiple comparisons by the Tukey test in ASSISTAT 7.0 software. Pearson’s correlation coefficient was determined between results of the different assays: Total phenolic content, total tannin   content,  total   condensed   tannins,  total  flavonoids,  total flavonol, IC50 DPPH and IC50 ABTS.


 RESULTS AND DISCUSSION

Several studies report that S. guianensis leaves constitute a material rich in terpenes, which are extracted for various purposes (Valentini et al., 2010; Portella et al., 2014). However, terpene presence is not a feature favorable to phenolic compound extraction in plants because these two classes of metabolites act in a competitive way for some solvents in extraction (Naczk and Shahidi, 2004), this perhaps justifies the scarcity of quantitative data regarding the total phenolic content of the S. guianensis species. In the present work, the degreasing process seems to have eliminated this interference, favoring phenolic compound extraction. This aspect has also been observed by Zhu et al. (2010) under study with Portulaca oleracea L.
 
The sequential extraction carried out on the defatted powder of S. guianensis leaves provided 9.584 ± 0.294 g (21.299 ± 0.007%) of methanolic extract, 1.947 ± 0.173 g (4.327 ± 0.004%) of ethanolic extract, 2.262 ± 0.182 g (5.028 ± 0.004%) of hydroethanolic extract and 1.123 ± 0.107 g (2.495 ± 0.002%) of aqueous extract. This result suggests that absolute methanol is a better solvent for the extraction of the degreased powder of S. guianensis leaves.
 
The sequence in which the solvents were used in the extraction proved to be adequate, mainly because it showed some selectivity for specific groups of polyphenols in each extract. Based on the results presented in Table 1, flavonoids and flavonols predominated in the ethanol and hydroethanolic extracts. The lowest content of phenolic compounds was found in the aqueous extract, which was constituted mainly by flavonoids. The solvent selectivity was also evident in the observation that in the hydroethanolic extract, obtained at intermediate polarity (70% ethanol), the tannin content was equivalent to that of flavonoids. Alothman et al. (2009), in a study of tropical fruits from Malaysia, also found that the recovery of phenolic compounds is dependent on the solvent used and its polarity.
 
 
The methanolic extract had the highest concentration of phenolic compounds and tannins in comparison with those obtained by other solvents. There is no information in the literature investigating quantification of this class of compounds in S. guianensis leaves. However, methanolic extraction in the aerial parts of other plants has been described (Wijekoon et al., 2011). The results of the S. guianensis leaf tannin quantification are in accordance with those obtained by Naima et al. (2015), who compared the impact of different extraction methods of tannins from Acacia mollissima bark and reported that the highest polyphenol content was obtained using methanol.
 
In light of the expressive concentrations of phenolic compounds obtained in this study, it can be said that the broad ethnobotanical indication of S. guianensis may have a strong relationship with this class of compounds, since they have diverse biological roles. Valentini et al. (2010) reported that this plant is used for therapeutic purposes by different populations of Latin America.
 
Among the non-enzymatic molecules produced by plants, phenolic compounds, especially flavonoids and phenolic acids, are the most effective in free radical neutralization; as a consequence of this they are also the most investigated to obtain natural antioxidants (Gülçin, 2012; Shebis et al., 2013). The data for total phenolic content quantification and evaluation of the antioxidant activity in S. guianensis leaves allowed us to infer that this plant is efficient in this biological role, and could be used in the development of pharmacological products capable of acting on disease-related oxidative damage.
 
The total antioxidant activity evaluated by the DPPH method in the methanolic extract, ethanolic extract, hydroethanolic extract, aqueous extract and of reference substances, rutin and ascorbic acid, at concentrations of 30, 60 and 120 μg mL are shown in Figure 1. These data make it clear that free radicals were eliminated by the
different extracts and the positive controls in a concentration-dependent manner.
 
 
The methanolic and hydroethanolic extracts were the most active. When comparing the efficiency of the extracts with reference substances, the antioxidant potential of S. guianensis became more evident since, at concentrations of 30 and 120 μg/mL, the percentage of total antioxidant activity of the methanolic extract was statistically higher that the rutin positive control.
 
The minimum amount of sample or reference substance (rutin and ascorbic acid) which was able to inhibit oxidation by 50% (IC50), evaluated by the DPPH and ABTS methods, also showed significant effects of the extracts of S. guianensis leaves on the elimination of free radicals. As shown in Table 2, the effect of eliminating ABTS radicals from the different extracts showed a similar trend to the DPPH radical scavenging activity. The IC50 value revealed that the extract obtained with methanol had a higher free radical scavenging activity in comparison to other extracts and was more efficient than the rutin positive control. These results are consistent with previous research reporting this biological role for tannins (Zhao et al., 2011), especially condensed tannins (Ujwala et al., 2012). According to Sandhar et al. (2011), the free radical scavenging ability of phenolic compounds is attributed mainly to the high reactivity of the substituent hydroxyls that readily interact with free radicals.
 
 
Results of correlation analysis between different pairs of assays are shown in Table 3. A strong correlation was observed between the DPPH and ABTS methods (p < 0.0001). The DPPH assay of antioxidant activity correlated significantly (p = 0.0522) with the total tannins. Moreover, significant correlations were observed between total phenolics and total tannins (p = 0.0131), indicating that tannins are the main constituents of this plant’s leaves.
 
 
Another important observation led by the correlation analysis is the fact that S. guianensis leaf flavonoids are constituted mainly by flavonols (p = 0.0286). That suggests S. guianensis leaves are a good source of bioactive compounds since flavonoids have a wide pharmacological spectrum, such as antibacterial, antiviral, anti-inflammatory and antioxidant properties (Zhou et al., 2015), among these, flavonols are effective compounds against human pathogenic microorganisms (Sandhar et al., 2011).
 
There were no significant correlations (p > 0.05) between antioxidant activity and total phenolic (p = 0.0815 with DPPH and p = 0.1103 with ABTS), total flavonoid (p = 0.1747 with DPPH and p = 0.1751 with ABTS), total flavonol (p = 0.6286 with DPPH and p = 0.6896 with ABTS) and total condensed tannin content (p = 0.1747 with DPPH and p = 0.1751 with ABTS). This may be related to the presence of other compounds, especially sugars and ascorbic acid, which, according to Nur Syukriah et al. (2014), are able to interfere with quantification and antioxidant activity assays. The lack of significant correlation between total tannin content and total condensed tannins (p = 0.1018) indicates that other groups of the class may be present in S. guianensis leaves.
 
Figure 2 shows TLC chromatographic separation after chemical derivation with the NEU/PEG reagent. Following the criteria of Wagner and Bladt (1996), flavonoids were detected as zones of orange fluorescence in UV light at 365 nm and phenolic acids with cyan-blue fluorescence under the same conditions. Compounds were detected with Rf characteristics for rutin (Rf = 0.06), quercetin (Rf = 0.82), catechin (Rf = 0.43) and gallic acid reference substances (Rf = 0.62). The four extracts showed distinctive TLC chromatogram profiles, demonstrating selectivity by the solvents in the conditions used. This analysis was consistent with the HPLC analysis.
 
 
 
As noted in the HPLC analysis (Figure 3), most of the major peaks in the aqueous extract appeared at retention times < 18 min while in the methanolic, ethanolic and hydroethanolic extracts, peaks with retention times up to 26 min were observed. This analysis also allowed the identification of representative constituents of phenolic compounds (tannins and flavonoids), making spectrophotometric quantification for tannins, flavonoids and flavonols consistent. Moreover, it confirmed the evaluation performed by the DPPH and ABTS methods, because the identified compounds, catechin, epigallocatechin gallate and rutin, are reported in several studies as good antioxidants (Liu et al., 2015; Lins et al., 2017; Shi et al., 2017).
 
 
A comparison of the HPLC profiles of S. guianensis extracts and co-chromatography with reference substances showed that, among the identified compounds, epigallocatechin gallate (retention time of 12.0 min) was present in all extracts, although at different concentrations. The amounts found for this compound showed a tendency similar to the spectrophotometer measurements both for in vitro biologic activity and for the total phenolic content. Such an observation makes it evident that condensed tannins, besides being the predominant phenolic compounds in S. guianensis leaves, are also the most influential in the antioxidant activity of this organ of the plant.
 
The epigallocatechin gallate level was quantified to be 9.051 µg/mg in the methanolic extract, 4.753 µg/mg in the ethanolic extract, 7.254 µg/mg in the hydroethanolic extract and 4.860 µg/mg in the aqueous extract. Catechin
 
(retention time of 10.5 min) was detected only in the methanol (11.863 µg/g) and aqueous extracts (3.910 µg/mg). Rutin (retention time of 15.3 min) was present only in the methanolic (4.928 µg/mg), ethanolic (5.037 µg/mg) and hydroethanolic extracts (12.300 µg/mg), not in the aqueous extract.
 
The HPLC identifications are consistent with other studies of the S. guianensis species. Leitao et al. (2005) and Negri et al. (2012) have also reported the presence of phenolic compounds in leaves of S. guianensis.
 
The HPLC data (Figure 3) make it evident that the obtained extracts are rich in phenolic compounds of great pharmacological importance because, beyond their antioxidant properties, catechin and epigallocatechin gallate are also regarded as antifungal (Anand and Rai, 2016), anti-inflammatory, chemopreventive and lung protective (Shahid et al., 2016). Rutin presents significant therapeutic potential against cancer, being able to decrease the adhesion and migration of cancer cells (Sghaier et al., 2016) besides having antidiabetic activity (Aitken et al., 2017).
 
The unidentified peaks shown in the chromatograms (Figure 3) may represent phytocomponents with biological activity different from reported potential. Like this, additional research into S. guianensis leaves could expand the pharmacological spectrum of that plant.


 CONCLUSIONS

This study showed that degreasing with petroleum ether followed by ultrasound-assisted sequential extraction with different solvents is an efficient way to obtain antioxidants from S. guianensis leaves. Antioxidant activity evaluation allowed observation that S. guianensis leaves are rich in substances capable of scavenging free radicals, especially condensed tannins. Moreover, spectro-photometric quantification and HPLC analysis revealed that S. guianensis leaves are a potential source of compounds with pharmacological properties. Thus, this information set establishes S. guianensis as a new alternative source of antioxidant compounds, implicating its potential application as a food preservative or in nutraceutical and pharmacological products.


 CONFLICT OF INTERESTS

The authors have not declared any conflict of interests.



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