Phenolic compounds of Hibiscus sabdariffa and influence of organic residues on its antioxidant and antitumoral properties

The aim of this study was to evaluate the phenolic and flavonoids contents and the antioxidant and antitumoral activity of leaf and calyx methanolic extracts from Hibiscus sabdariffa (roselle) cultivated with poultry litter and organosuper under three modes of application. The total phenolic content in the each extract was determined using the Folin-Ciocalteu reagent and for aluminium chloride flavonoids. The antioxidant parameters were analyzed using a 2, 2-diphenyl-1picrylhydrazyl (DPPH.) free radical scavenging assay. An antitumor colorimetric assay using sulforhodamine B. The highest contents of phenolic and flavonoids were observed in leaf extracts (389.98 and 104.52 mg g–1, respectively) and calyx extracts (474.09 and 148.35 mg g–1, respectively) from plants cultivated with organosuper, although these values did not differ significantly from those observed for the other treatments. The average IC50 of leaves (43.48 μg mL –1) and calyces (37.15 μg mL–1) demonstrated that both have substances that may contribute to free radical scavenging action. The methanol extract from calyces showed significant selective activity against a leukemia line (K-562), with IC50 values of 0.12 mg mL –1 (organosuper) and 1.16 mg mL–1 (poultry litter), with concentration-dependent, cytotoxic and cytocidal effects.


Introduction
Hibiscus sabdariffa L., (Malvaceae), commonly known as "roselle", is an important medicinal plant native to India and Malaysia, although it grows widely in the tropics and subtropics of both hemispheres and has become naturalized in many areas in Central America (Morton, 1987). The plant is an annual, erect, herbaceous sub-shrub with a deep root system. The plant has fibrous stems, small branches, as well as bright red and acidic-tasting calyces.
In folk medicine, an infusion from the calyces is used as a diuretic and to treat gastrointestinal disorders, liver diseases, fever, hypercholesterolemia, and hypertension (Monroy-Ortiz and Castillo-Espana, 2007). Extracts from the calyces are reported to have a variety of therapeutic effects in vivo and in vitro, including anticancer (Olvera-Garcia et al., 2008;Lin et al., 2005) and antioxidant (Farombi and Fakoya, 2005;Ramakrishna et al., 2008) properties.
Many studies also demonstrated an additional cardioprotective effect of anthocyanins, flavonoids and polyphenols, either through antioxidant-related mechanisms or by other means. H. sabdariffa extracts inhibited lowdensity lipoprotein (LDL) oxidation in vitro and decreased serum cholesterol levels in cholesterol-fed rats and rabbits (Chen et al., 2003(Chen et al., , 2004. Antioxidants are considered to be effective inhibitors of carcinogenesis as well as other conditions that are pathogenically associated with oxidative mechanisms. Many studies have supported the hypothesis that antioxidant nutrients and/or medicines play a protective role in human health (Ames et al., 1993). Therefore, as calyces are consumed throughout the world with culinary and medicinal objectives, and as calyces show potential for therapeutic use, it is necessary to investigate the potential production of H. sabdariffa and evaluate its constituents.
The amount of bioactive components in plants is influenced by agricultural management in which they are grown (fertilization, cultivation practices, etc.), and the efficacy of these phytochemicals depends on the concentration in the plants. Thus, the incorporation of organic fertilization is a logical step towards increasing production of medicinal plants. Furthermore, poultry litter represents a rich feedstock of carbon, nitrogen, phosphorous, potassium, and other nutrients (Mullins and Bendfeldt, 2001). However, studies with different extracts of H. sabdariffa poultry litter and different spacing cultivated were evaluated in antioxidant activity and content of phenols and flavonoids. The spacing did not influence on content of constituents, but the addition of poultry litter has enhanced the responses both in aqueous and ethanolic extract. The ethanolic extract has higher antioxidant activity (Ramos et al., 2011). The data indicate the importance of the continuity of phytochemical and cultivation studies and especially in the evaluation of other biological activities.
Thus, the aim of this study was to evaluate the phenolic and flavonoid contents, as well as the antioxidant and anticancer activities, of leaf and calyx extracts from H. sabdariffa that were cultivated with poultry litter and organosuper  under three modes of application. Brazil. This area is located in the southern part of Mato Grosso do Sul, at an average altitude of 452 m (22°14'16" S, 54°49'2" W). The climate is Cwa, by the Köppen classification system. The soil, originally from Cerrado vegetation, is classified as distroferric red latosol, with a sandy texture and a flat topography.

Material and Methods
The treatments were composed of combinations of two organic waste sources, poultry litter (10 t ha -1 ) and organosuper  (10 t ha -1 ), and three application modes (coverage, incorporated and coverage + incorporated) plus a control. The treatments were arranged in a 2 × 4 factorial, randomized block design with four replicates. The coverage + incorporated mode was applied at 5 t ha -1 + 5 t ha -1 . Leaves were harvested when the plants began flowering at 95 days after transplant (DAT). Two harvests of calyces were made from the remaining plants from each plot at 139 and 185 DAT, using the size of calyces (larger than 3.5 cm) as an indication of the harvest point.

Preparation of methanol extracts
Leaves and calyces from H. sabdariffa cultivated with two organic waste sources, poultry litter (10 t ha -1 ) and organosuper  (10 t ha -1 ), and three application modes (coverage, incorporated and coverage + incorporated), separately were dried in an oven (AC-035/81) of circulating air at 45°C for four days and subsequently crushed in a grinder of knives (MA340/A). The material obtained (50 g) was subjected to maceration for a period of 24 h in 300 mL of methanol (Dynamics  ) at room temperature, followed by filtration by filter paper. The process was repeated five times and the filtrates were pooled and concentrated under reduced pressure by rotary evaporator (MA-120) and lyophilized.

Determination of total phenolic content
Total phenolic compounds in the methanolic extract from leaves and calyces of H. sabdariffa were determined using the Folin-Ciocalteu method (Meda et al., 2005).
Specifically, to each 100 μL of extract in methanol (1 g L −1 ) was added 1.0 mL of distilled water and 0.5 mL of Folin-Ciocalteu ' s (1:10 v/v) reagent. After 3 min, a 1.5 mL saturated solution of Na 2 CO 3 (2%) was added. After 30 min the optical density was measured by the absorbance at 765 nm using a spectrophotometer. The quantification was performed on the basis of a standard curve of gallic acid prepared in 80% methanol, and the results were expressed in milligrams of gallic acid equivalent per gram of extract. A methanol solution was used as a blank. The assays were performed in triplicate.

Determination of total flavonoid content
To determine the level of flavonoids, each 500 μL of sample was combined with 1.50 mL of 95% ethanol, 0.10 mL of 10% aluminum chloride (AlCl 3 ·6H 2 O), 0.10 mL of sodium acetate (NaC 2 H 3 O 2 ·3H 2 O) (1 mol L -1 ) and 2.80 mL of distilled water. The tubes were kept at room temperature for 40 min. The optical density was measured by absorbance at 415 nm using a spectrophotometer. The same procedure was used in the analysis of a blank (Lin and Tang, 2007). To calculate the concentration of flavonoids, a calibration curve was prepared (2.5, 5.0, 10.0, 20.0, 25.0, 50.0, 100.0 and 125.0 μg) using quercetin as a standard. A linear regression was calculated from these data, and a line equation was obtained for subsequent use in the quantification of actual samples. The results were expressed in milligrams of quercetin equivalents per gram of extract. The assays were performed in triplicate.

Qualitative analysis of antioxidant activity
The extracts were analyzed by TLC using quercetin as a positive comparison. The plates were eluted in chloroform/ methanol 10% and, after drying, were nebulized with a solution of 0.4 mmol L -1 DPPH in methanol. The cells were observed until the appearance of yellow spots on a background of purple coloration, indicating possible antioxidant activity .

DPPH free radical scavenging assay
Free radical scavenging activity of the methanolic extract was determined using a 1,1-diphenyl-1-picrylhydrazyl (DPPH) free radical method (Blois, 1958). Various concentrations of the samples were added to 2 mL of daily prepared methanol solutions of DPPH (0.1 mM). The mixture was shaken and left to stand at room temperature in the dark. After 30 min, absorbance was measured at 517 nm against a blank containing all of the reagents except the test samples. Butylated hydroxytoluene (BHT) was used as the standard. The assays were performed in triplicate.

The evaluation of anticancer activity
The evaluation of anticancer activity of methanol extracts of leaves and calyces grown with organic waste (poultry litter and organosuper  ) was performed by a colorimetric method employing sulforhodamine B (Skehan et al., 1990), with doxorubicin as a positive control. One hundred microliters of a cell suspension were added to RPMI-1640/SFB (10% gentamicin, 50 μg mL -1 ) medium, in 96-well plates and incubated for 24 h at 37°C in 5% CO 2 . After this period, a control plate was fixed by adding trichloroacetic acid to determine the number of cells at the time of drug addiction. The extracts were tested at concentrations of 0.25, 2.5, 25 and 250 μg mL -1 and incubated for 48 h. The plates were then stained by adding 50 μL of the protein dye sulforhodamine B (SRB) 0.4% (w/v), dissolved in 1% acetic acid, and incubated at 4°C for 30 min. The dye was resuspended in a solution of Trizma base (10 μM, pH 10.5), and the absorbance at 540 nm was measured on a microplate reader.

Statistical Analysis
Statistical data were analyzed using variance analysis (ANOVA) followed by the Tukey test (HSD, honestly significant differences), with the level of significance set at (p < 0.05) (Ribeiro Júnior and De Melo, 2008).

Results
The results for the determination of total phenols by Folin-Ciocalteu method, expressed as gallic acid equivalents per g of methanol extract of leaves and calyces of H. sabdariffa grown under different organic waste and application conditions are presented in Table 1. The methanolic extract from leaves and calyces of H. sabdariffa cultivated with incorporated organosuper  showed the highest phenol contents (389.98 mg g -1 and 474.09 mg g -1 , respectively); however, no significant difference was observed between treatments. The highest flavonoid contents were also observed in leaf extract (104.52 mg g -1 ) and calyces (148.35 mg g -1 ) in plants grown with incorporated organosuper  , and these values did not differ from the other treatments (Table 1).
Preliminary qualitative evaluation of methanol extracts of leaves and calyces from different treatments (by TLC) suggested the existence of substances with antioxidant activity, as indicated by the chromatographic spots of yellow on purple background that resulted from the reduction of DPPH radical. The test data against the free radical are presented in Table 2. The average IC 50 values for leaves (43.48 μg mL -1 ) and calyces (37.15 μg mL -1 ) (Table 2) demonstrate that both have substances that may contribute to free radical scavenging action compared to the positive control BHT (16.7 μg mL -1 ). Figure 1 shows the concentration-response curves of the control condition and for the methanol extract from the leaves and calyces of plants cultivated with incorporated poultry litter and organosuper  for the tested cell lines.
These curves compare the percentage of cell growth and the concentration of the sample used. The curves A and B show that the methanol extracts of leaves exhibited low activity against the leukemia cells, with IC 50 values of 43.20 mg mL -1 , when compared with the calyces (Table 3). The curves D and E show that the methanol extract of calyces showed significant selective activity for leukemia cells (K-562), with IC 50 values of 0.12 mg mL -1 and 1.16 mg mL -1 , respectively. This effect was concentration dependent, and the extracts had cytotoxic and cytocidal effects. However, when the curves C and F were evaluated, no difference was observed in the activity of extracts from the leaves and calyces compared to the control (Table 3).

Discussion
Generally, the highest concentrations of phenols and flavonoids were observed in the calyces, with values ranging from 454.66 to 474.09 mg g -1 and 140.29 to 148.35 mg g -1 , respectively, regardless of the form of plant cultivation. According to Juliani et al. (2009), the total content of anthocyanins (by HPLC) in calyces of H. sabdariffa varies Table 2. Antioxidant activity of methanol extract from leaves and calyces of H. sabdariffa cultivated with two organic wastes and three modes of application plus a control a .  from 0.3 to 2.4%, suggesting that Hibiscus calyces can be a rich source of these compounds compared to other natural plant sources. However, when the methanol extracts of leaves and calyces were evaluated individually, the total phenols and flavonoids were higher with poultry litter and incorporated organosuper  . The content of phenolic compounds and other phytochemicals present in medicinal plants, as well as in fruits and vegetables, is largely influenced by the type of cultivation, genetic factors, environmental conditions, in addition to the degree of maturation and the variety of the plant (Koleva et al., 2002;Melo et al., 2006). The fact that the highest phenol content was obtained using soil with poultry litter and incorporated organosuper  is due to an improvement in soil physical properties, such as an increased soil water retention (Karamanos et al., 2004); this is in addition to an improvement in the balance of soil nutrients (Blaise, 2006).

Organic waste
Of the various chemical methods used to determine the antioxidant capacity of a compound in capturing free radicals, DPPH is one of the most used because it is considered to be practical, fast and stable (Espin et al., 2000). This method is based on the ability of compounds to donate a proton to DPPH and form stable resonance structures. The test data against the free radical are presented in Table 2. IC 50 values (concentration for 50% inhibition of DPPH remaining) were calculated via the graph of % I (inhibition percentage) versus the extract concentration in mg mL -1 . The percentage of DPPH inhibition (% I) was calculated using the equation % I: where A 0 is the absorbance of DPPH (control) and A is the absorbance of sample plus DPPH. The amount of extract required to inhibit 50% of DPPH was not influenced by organic waste or the application mode studied.
The antioxidant effects of extracts were most likely due to a subgroup of flavonoids, anthocyanins, which have been reported in the literature as the main constituents of H. sabdariffa (Strack and Wray, 1986;Mazza and Miniati, 1993;Tsai et al., 2002). These substances display conjugation to form stable resonance structures during the formation of free radicals, highlighting the high response to the test with the free radical DPPH.
Studies evaluating cell proliferation suggest that the inhibition of HeLa cell proliferation observed for a hot water extract of H. sabdariffa flowers may be correlated with the acidic condition induced by the presence of phenolic compounds. These compounds include protocatechuic acid, anthocyanins, quercetin, as well as organic acids, such as citric, malic and tartaric acids (Olvera-Garcia et al., 2008). Studies with human leukemia cells incubated for 24 and 48 h with protocatechuic acid (3,4-dihidroxibenzóico, PCA) at concentrations of 0.2, 0.5, 1.0 and 2.0 mM showed a concentration-dependent inhibitory effect on cell growth (Ali et al., 2005;Falade et al., 2005). This compound induced internucleosomal DNA fragmentation and cellular morphological changes characteristic of apoptosis .
All of the extracts of H. sabdariffa that were studied as a function of cultivation with organic wastes and application modes exhibited varied phenolic content and antioxidant and anticancer effects. However, the intensity of these effects did not differ significantly with the crop system employed. Furthermore, the anticancer potential was observed in the extract of calyces, independently of the cultivation conditions. Thus, the results of this study should stimulate further studies of H. sabdariffa using other forms of cultivation in an attempt of increase the concentration of potentially active substances, ensuring the effectiveness and subsequent commercialization of the species. Further studies to elucidate the possible compounds involved in anticancer activity will be carried out.