Evaluation of Antioxidant Activity, Total Flavonoids, Tannins and Phenolic Compounds in Psychotria Leaf Extracts

The antioxidant activity of Psychotria carthagenensis, P. leiocarpa, P. capillacea and P. deflexa (Rubiaceae) extracts were investigated, and the concentrations of total phenolics, flavonoids, condensed tannins and flavonols were determined. The chemical compositions of the extracts were investigated using the high performance liquid chromatography (HPLC/PAD) method. We used 1,1-diphenyl-1-picrylhydrazyl free radical (DPPH), β-Carotene bleaching and 2,2-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical cations to determine antioxidant activity. The ability to scavenge radical was measured in these experiments by the discoloration of the solution. Concentrations of constituents were measured spectrophotometrically. P. carthagenensis and P. capillacea exhibited the highest antioxidant activity, in the DPPH test, β-carotene bleaching and ABTS system. The highest phenolic, flavonoid, condensed tannin and flavonol concentration was found in P. carthagenensis and P. capillacea extracts. HPLC-PDA analysis of P. carthagenensis and P. capillacea revealed hydroxycinnamic acid (p-coumaric acid). This is the first report on the antioxidant properties and constituent analysis of these Psychotria extracts.

a different analytical methodology. We also determined the concentrations of phenolic compounds, condensed tannins, flavonoids, and flavonol.

β-Carotene/Linoleic Acid Method
The β-carotene solution was prepared by dissolving 2 mg β-carotene in 10 mL chloroform; 1 mL of this β-carotene-chloroform solution was mixed with 20 mg linoleic acid and 0.2 g Tween 40. Subsequently, the chloroform was removed by a rotary evaporator at 45 °C. Distilled water (50 mL) was slowly added with vigorous agitation to form an emulsion. Emulsion aliquots (5 mL) were transferred with 0.2 mL of the extracts different concentrations (10-200 μg/mL, sample stock 1.0 mg/mL). Control samples were prepared with 0.2 mL methanol devoid of extract [36][37][38]. As soon as the emulsion was added to each tube, absorbance was read at 470 nm against blank (zero time). Tubes were placed in a water bath at 50 °C, and oxidation was monitored by absorbance at 15 min intervals until the color of β-carotene in the control sample had disappeared (105 min). BHT was used as reference. The analyses were performed in triplicate. Antioxidant activity (AA) was calculated as percent inhibition relative to the control: Ai = absorbance of sample at zero time, At = absorbance of sample after incubation (105 min) at 50 °C, A'i = absorbance of control at zero time, and A't = absorbance of control after incubation (105 min) at 50 °C. , prepared daily. 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 reagents except the test samples [39]. BHT was used as the positive control. Assays were carried out in triplicate. The percentage of inhibition of DPPH (I%) was calculated using the following equation: A0 is the absorbance of the blank solution and A is the absorbance of the methanolic extract. The IC50, the concentration giving 50% inhibition of DPPH, was read off a graph of I% (percentage inhibition) versus extract concentration.

ABTS + Scavenging Activity
Total antioxidant activity was measured using an improved azinobis (ethylbenzothiazoline-6-sulphonic acid) (ABTS) radical scavenging method [40] with minor modifications. Sample stock solutions (1.0 mg/mL) were diluted to final concentrations of 250, 125, 50, 25, 10 and 5 μg/mL in methanol. Briefly, 7.0 mM ABTS and 140 mM potassium persulphate were mixed and kept in the dark for 16 h at ambient temperature. Before usage, the ABTS + solution was diluted to get an absorbance of 0.700 ± 0.05 at 734 nm with ethanol (P.A.). Then, 3 mL of ABTS + solution was added to 30 μL of different sample concentrations (5-250 μg/mL). After 30 min, the absorbance was taken at 734 nm using spectrophotometer. The ABTS + scavenging activity was calculated using the following equation: A0 is the absorbance of the blank solution and A is the absorbance of the methanolics extracts.

Total Phenol Concentration
The total phenol concentration of the samples was determined using folin reagent [41]. Briefly, 100 μL of extract in methanol (1 g/L) were mixed with 1.0 mL of distilled water and 0.5 mL of folin-ciocaleu's reagent (1:10 v/v). After mixing, 1.5 mL of 2% aqueous sodium bicarbonate were added, and the mixture was allowed to stand for 30 min with intermittent shaking. The absorbance was measured at 765 nm using a spectrophotometer. Total phenolic concentration is expressed as gallic acid equivalent in mg per gram of extract. The methanol solution was used as a blank. All assays were carried out in triplicate.

Total Flavonoid Concentration
The amount of total flavonoids in the extracts was measured spectrophotometrically as previously reported [42]. Briefly, 500 μL of each extract was mixed with 1.50 mL of 95% ethanol, 0.10 mL of 10% aluminium chloride (AlCl3.6H2O), 0.10 mL of sodium acetate (NaC2H3O2.3H2O) (1 M) and 2.80 mL of distilled water. After incubation for 40 min, absorbance was measured at 415 nm using a spectrophotometer. To calculate the concentration of flavonoids, we prepared a calibration curve using quercetin as standard. The flavonoid concentration is expressed as quercetin equivalents in mg per gram of extract. All assays were carried out in triplicate.

Condensed Tannin Concentration (CT)
CT concentrations were determined by a modified version of a method reported previously [41,42]. Samples were mixed with 5 mL vanillin-HCl (8% conc. aq. HCl and 4% vanillin in methanol). Absorbance at 500 nm was read after 20 min. Catechin was used as the standard. The condensed tannin concentration is expressed as catechin equivalents in mg per gram of extract.

Flavonol Concentration
Total flavonols in the plant extracts were estimated using the method reported previously [41,43]. To 2 mL of sample, 2 mL AlCl3 (2%)/ethanol and 3 mL (50 g/L) sodium acetate were added. The mixture was shaken and incubated for 2.5 h at 20 °C. Absorbance was read at 440 nm. Total flavonols are expressed as mg of quercetin equivalents per gram of dry weight (mg QE/g extract) using the calibration curve with quercetin.

HPLC/PDA Analysis
The Psychotria extracts and standards were analyzed using an analytical HPLC (Varian 210, Varian, Sugar Land, TX, USA) system, with a ternary solvent delivery system and an auto-sampler. A photodiode array detector (PAD) was monitored at λ = 200-800 nm. The HPLC column was C-18 (25 cm × 4.6 mm; particle size, 5 μm; Luna, Phenomenex, Torrance, CA, USA), with a small pre-column (2.5 cm × 3 mm) containing the same packing to protect the analytical column. The flow rate and injected volume were 1.0 mL min −1 and 10 μL, respectively. All chromatographic analyses were performed at 22 °C. The elution of P. leicocarpa and P. deflexa extracts and standards was carried out using acetonitrile with formic acid (pH 2.64) (solvent A) and water (solvent B). The solvent gradient program was as follows: 0 min, 70% B; 30 min, 37% B; in 45 min returning to the initial condition. The P. carthagenensis and P. capillacea extracts and standards were eluted using acetonitrile with formic acid (pH 2.64) (solvent A) and water (solvent B). The solvent gradient program was as follows: 0 min, 84% B; 15 min, 59% B; 20 min, 79% B; 40 min, 0% B, in 50 min returning to the initial condition.
Stock solutions of standards (caffeic acid, p-coumaric acid, luteolin, quercetin and apigenin), created by dissolving individual solutions in acetonitrile at 10 μg/mL and were employed in the identification of compounds using a PDA detector (200-800 nm). This did not reveal coeluting substances.

Antioxidant Activity
Due to the complexity of some plant extracts, the use of several different methods is recommended for the evaluation of antioxidant activity [43]. Currently used methods include the DPPH (1,1-diphenyl-2-picrylhydrazyl) assay [35], which measures the ability of a substance to scavenge the DPPH radical, reducing it to hydrazine. When a substance that acts as a donor of hydrogen atoms is added to a solution of DPPH, hydrazine is obtained, with a change in color from violet to pale yellow. 2,2-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS + ) is a decolorization technique, in that the radical is generated directly in a stable form prior to reaction with putative antioxidants, which involves the production of the blue/green ABTS + chromophore through the reaction of ABTS with potassium persulfate. The β-carotene bleaching method [44] evaluates the ability of a substance to prevent the oxidation of β-carotene, protecting it from the free radicals generated during the peroxidation of linoleic acid.
The effects of methanolic extracts of Psychotria species in the DPPH, linoleic acid peroxidation and ABTS assays are shown in Table 1. P. carthagenensis and P. capillacea exhibited the highest scavenging activity with an IC50 of 16.92 ± 4.58 and 30.05 ± 6.22 μg/mL, respectively (Table 1), which was comparable to that of the standard antioxidant butylated hydroxytoluene (BHT) (IC50 = 16.72 ± 1.34 μg/mL) and ascorbic acid (IC50 = 22.28 ± 0.53 μg/mL). The comparison of the obtained free-radical scavenging data indicated potent activity for the P. carthagenensis and P. capillacea at 100 μg/mL, with %FRS values of 93.52% ± 8.41% and 91.78% ± 4.23%, respectively. These results show that the DPPH radical scavenging activity of these extracts was similar to BHT and ascorbic acid (Table 1). Gallic acid (6.56%, standard), P. carthagenensis (6.47%) and P. capillacea (7.32%) also resulted in a remarkable reduction of the DPPH remaining, compared with P. leicocarpa and P. deflexa (56.26% and 63.51%), respectively, when recorded after 60 min (Figure 1). It is clear that the more DPPH that remains, the lower the radical-scavenging activity of the tested samples is. All these data clearly indicate that P. carthagenensis and P. capillacea extracts are effective electrons or hydrogen atoms donor to DPPH.
Higher antioxidant activity was also found for P. carthagenensis (79.1% ± 3.70%) with the β-carotene bleaching method compared to quercetin and BHT (Table 1). This higher activity was not observed for ascorbic acid, which has relatively high polarity. We propose that this extract contains lipophilic compounds, which act by inhibiting or retarding oxidation of β-carotene.
Phenolic compounds are known as high-level antioxidants because of their ability to scavenge free radicals and active oxygen species, such as singlet oxygen, superoxide free radicals and hydroxyl radicals [47]. The radical-scavenging activity is attributed to replacement of hydroxyl groups in the aromatic ring systems of the phenolic compounds as a result of their hydrogen donating ability [44].

HPLC/PAD Analysis from Psychotria Extracts
After determination of the antioxidant potential and levels of constituents, we analyzed the methanolic extracts obtained from Psychotria in an analytical LC (Figure 3). The standards were easily identified based on their UV absorption spectra and retention times. The substances found in the extracts were unambiguously identified by performing co-injection experiments in which aliquots of extract and standard were mixed and diluted to a known volume, and analyzed by HPLC. Only p-coumaric acid (tr = 24.91 min) was found in P. carthagenensis and P. capillacea ( Figure 3A,B). The antioxidant activity of the pure p-coumaric acid at 40 μg/mL concentration was determined. DPPH radical scavenging activity of p-coumaric acid was found to be 55.6% ± 3.4%; the β-carotene bleaching with 78.6% ± 2%; and 87.14% ± 5.7% in ABTS radical scavenging activity. Comparison of the obtained of P. carthagenensis and P. capillacea data indicated the potent activity for the pure p-coumaric acid.
p-Coumaric acid (4-hydroxycinnamic acid), a phenolic acid, hydroxyl derivative of cinnamic acid, is widely used in the chemical, food, health, cosmetic, and pharmaceutical industries. Studies reported that antioxidants, such as p-coumaric acid and others hydroxycinnamic acids, function as chemoprotective agents by quenching carcinogenic nitrosating agents in several biological compartments, including salivary and gastric fluids [48]. However, minor attention has also been directed to the activity of simple phenolic acids, such as benzoic or cinnamic acids, and their derivatives.

Conclusions
According to data obtained from the present study, P. carthagenensis and P. capillacea were found to be effectives antioxidants in different in vitro assay including DPPH radical, ABTS radical and β-carotene bleaching activities when they are compared to standard antioxidant compounds, such as BHT, gallic acid, quercetin and ascorbic acid. The results indicate that the antioxidant activity to these plants can be attributed the presence the p-coumaric acid. Further, more detailed, studies on the chemical composition of those extracts, as well as studies with other models, such in vivo assays, are essential to characterize them as biological antioxidants.