Evaluation of Antioxidant Properties of Phaulopsis fascisepala C.B.Cl. (Acanthaceae).

The antioxidant activities of crude extract of Phaulopsis fascisepala leaf were evaluated and compared with alpha-tocopherol and BHT as synthetic antioxidants and ascorbic acid as natural-based antioxidant. In vitro, we studied its antioxidative activities, radical-scavenging effects, Fe(2+)-chelating ability and reducing power. The total phenolic content was determined and expressed in gallic acid equivalent. The extract showed variable activities in all of these in vitro tests. The antioxidant effect of P. fascisepala was strongly dose dependent, increased with increasing leaf extract dose and then leveled off with further increase in extract dose. Compared to other antioxidants used in the study, alpha-Tocopherol, ascorbic acid and BHT, P. fascisepala leaf extract showed less scavenging effect on alpha,alpha,-diphenyl-beta-picrylhydrazyl (DPPH) radical and less reducing power on Fe(3+)/ferricyanide complex but better Fe(2+)-chelating ability. These results revealed the in vitro antioxidant activity of P. fascisepala. Further investigations are necessary to verify these activities in vivo.

Introduction

Cancer is a leading cause of death and may result from chronic injury to the epithelium by oxidants and other carcinogens (1–3). Epidemiological and experimental studies also offer strong evidence that implicates oxidative damage in the etiology of brain, heart and nervous system diseases (4–6). Although the body has effective defence systems that protect it against oxidative stress, the capacity of these protective systems decreases with aging (7) creating a need to provide the body with a constant supply of phytochemicals through dietary supplements.

Phaulopsis fascisepala C.B.Cl. (Acanthaceae) is an erect or decumbent herb or shrub found throughout the forest zones from Senegal to S. Nigeria and has been used by local indigenous groups for a wide range of medicinal purposes. Dried and powdered, it is applied to staunch wounds in Nigeria. In Ivory Coast, it is used to treat sores caused by skin-parasites e.g. ringworm and fungal infections and is sometimes put into baths or steam baths to treat fever stiffness and rheumatic pain (8). Since the relationship between oxygen free radicals and damage to skin is well documented (6,9) the ethnopharmacological use of P. fascisepala for skin diseases inspired us to investigate its antioxidant properties.

Methods

Chemicals and Reagents

Deionized water, 2.0 M Folin-Ciocalteu phenol reagent, gallic acid, anhydrous sodium carbonate, 2,6-Di-tert-buytl-4-methylphenol (BHT), α-Tocopherol (Vitamin E), 90% 1,1-diphenyl-2-picrylhydrazyl (DPPH), FeCl2 tetrahydrate, anhydrous ferric chloride, potassium ferricyanide, [4,4′- [3-(2-pyridinyl-1,2,4-triazine-5,6-diyl]bisbenzenesulfonic acid] (ferrozine) and trichloroacetic acid were purchased from Sigma Chemical Company (St Louis, MO). Ascorbic acid and all other chemicals were of analytical grade BDH Chemical Laboratory (England, UK).

Plant Material

The aerial part of P. fascisepala collected in Ibadan, Nigerias was identified by Mr Felix Usanga of Forest Research Institute of Nigeria (FRIN). Voucher specimen with FHI number 107231 was deposited at the herbarium. The leaves were plucked from the aerial portion, dried separately at 30°C, crushed into fine particles (∼250 μm) and then extracted.

Extraction

About 500 g of powdered leaves of the plant was extracted with methanol (3l) using Soxhlet apparatus for 48 h. The extract was concentrated under reduced pressure using rotatory evaporator until a semi-solid sticky mass was obtained. The yield was 13.0%w/w.

Evaluation of Antioxidant Activities

Total Phenolic Content

The total phenolic content of the leaf extract was determined separately using the method of Macdonald et al. (10) with modifications. Calibration curve was prepared by mixing ethanolic solution of gallic acid (1 ml; 0.025–0.400 mg ml−1) with 5 ml Folin-Ciocalteu reagent (diluted ten fold) and sodium carbonate (4 ml, 0.7 M). We measured absorbance at 765 nm and drew the calibration curve. One millimeter of ethanolic plant extract (5 g l−1) was also mixed with the reagents above and after 2 h the absorbance was measured to determine total plant phenolic contents. All determinations were carried out in triplicate. The total content of phenolic compounds in the extract in gallic acid equivalents (GAE) was calculated by the following formula: where T = total content of phenolic compounds, milligram per gram plant extract, in GAE; C = the concentration of gallic acid established from the calibration curve, milligram per milliliter; V = the volume of extract, milliliter; M = the weight of ethanolic plant extract, gram.

Radical Scavenging Activity

We estimated the effect of extract, α-Tocopherol, ascorbic acid and BHT on the DPPH radical according to the method of Koleva et al. (11) with modification. The plant extract (0.1–2.0 mg), BHT, ascorbic acid or α-Tocopherol (0.02–1.00 mg), in 4 ml of distilled water was added to a methanolic solution of DPPH (1 mM, 1 ml). The mixture was shaken and allowed to stand at 20°C for 30 min; the absorbance of the resulting solution was measured spectrophotometrically at 517 nm. We then calculated the ability to scavenge DPPH radical using the following equation: where Ao was the absorbance of the control and A1 was the absorbance in the presence of the sample of extract and standard.

Reducing Power

Using the modified method of Yen and Chen (12), we determined the reducing power of leaf extract, α-Tocopherol, ascorbic acid and BHT. The extract (0.1–1.5 mg), α-Tocopherol, ascorbic acid or BHT (0.1–1.0 mg) in 1 ml of methanol was mixed with phosphate buffer (2.5 ml, 0.2 M, PH 6.6) and potassium ferricyanide [K3Fe(CN)6] (2.5 ml, 1%); the mixture was incubated at 50°C for 20 min. A portion (2.5 ml) of trichloroacetic acid (10%) was added to the mixture, which was then centrifuged at 1000 g for 10 min. The upper layer of the solution (2.5 ml) was mixed with distilled water (2.5 ml) and FeCl3 (0.5 ml, 0.1%) and the absorbance was measured at 700 nm. Increased absorbance of the reaction mixture indicated increased reducing power.

Determination of Fe2+-Chelating Ability

Fe2+ chelating ability was determined according to the method of Gulcin et al. (13). The Fe2+ was monitored by measuring the formation of ferrous iron-ferrozine complex at 562 nm. The P. fascisepala extract (0.1–2.0 mg) in 5 ml of methanol was mixed with FeCl2 (0.5 ml, 2 mM) and ferrozine (1.0 ml, 5 mM). The resulting mixture was shaken and left to stand for 10 min at room temperature. The absorbance of the resulting solution was measured at 562 nm. The lower the absorbance of the reaction mixture, the higher the Fe2+-chelating ability. The capability to chelate the ferrous iron was calculated using the following equation: where Ao was the absorbance of the control and A1 was the absorbance in the presence of the sample of extract and standard.

Statistical Analysis

All data were expressed as mean ± SD. Analysis of variance was performed by ANOVA procedures and P < 0.05 was considered to be statistically significant.

Results

Total Phenolic Content

The total phenolic content of the P. fascisepala leaf extract measured by Folin Cicalteu reagents in terms of gallic acid equivalent (GAE) was 29.27 ± 1.46 mg g−1.

Radical Scavenging Increased with Antioxidant Concentration

Figure 1 shows the dose response curve for the radical scavenging effect of P. fascisepala extract, α-Tocopherol, ascorbic acid and BHT by the DPPH coloring method. The radical scavenging effects of extract and synthetic antioxidants used as positive control on DPPH radical were concentration dependent. The scavenging of DPPH radicals increased with increasing antioxidant concentration. The radical scavenging activity of the extract and positive controls decreased in the following order: ascorbic acid > BHT > α-Tocopherol > P. fascisepala The scavenging percentage on the DPPH radical was 53.4% for P. fascisepala extract at the dose level of 0.3 mg and 61.7, 53.2 and 67.4% for α-Tocopherol, ascorbic acid and BHT at the dose 0.2, 0.05 and 0.1 mg.

Figure 1.

Scavenging effects of Phaulopsis fascisepala, α-Tocopherol, ascorbic acid and BHT on DPPH radical. Each value represents mean ± SD (n = 3).

Reducing Powers Increased with Increasing Concentrations

Figure 2 shows the reducing powers of the extract, α-Tocopherol, ascorbic acid and BHT. We discovered that the reducing powers of the extract, BHT and natural-based antioxidants like α-Tocopherol and ascorbic acid on Fe3+ were concentration dependent. The reducing powers increased with increasing concentration but the reducing powers of α-Tocopherol, ascorbic acid and BHT were significantly (P < 0.05) higher than that of P. fascisepala extract. For instance, the absorbances at 700 nm were found to be 0.76, 0.86, 0.95 and 0.74 for P. fascisepala, α-Tocopherol, ascorbic acid and BHT at the doses of 1.0, 0.1, 0.1 and 0.1 mg. This means to reach a similar reducing power, the dose required for P. fascisepala was ∼10-fold than required for α-Tocopherol, ascorbic acid and BHT.

Figure 2.

Reducing power of Phaulopsis fascisepala, α-Tocopherol, ascorbic acid and BHT. Each value represents mean ± SD (n = 3).

Iron (II) Chelating Ability of the Extract

The extract of P. fascisepala leaf showed a moderate ferrous ion chelating ability. This ability increased with increasing P. fascisepala leaf extract concentration and reached 34.4% at a concentration of 2.0 mg ml−1. However, α-Tocopherol, ascorbic acid and BHT showed no such ability.

Discussion

The phenolic content of P. fascisepala leaf extract was 29.27 ± 1.46 mg g−1 in terms of GAE. Phytochemicals, especially plant phenolics constitute a major group of compounds that act as primary antioxidants (14). They can react with active oxygen radicals, such as hydroxyl radicals (15), superoxide anion radicals (16) and lipid peroxy radicals (17), and inhibit lipid oxidation at an early stage. They also inhibit cyclooxygenase and lipoxygenase of platelets and macrophages, thus reducing thrombotic tendencies in vivo (18).

Proton radical-scavenging action is one mechanism for oxidation. DPPH has a proton free radical and shows characteristic absorption at 517 nm (purple). When it encounters proton radical scavenger, its purple color fades rapidly (19–21), suggesting that antioxidant activity of P. fascisepala leaf extract is due to its proton donating ability. Phaulopsis fascisepala leaf extract was less able to scavenge radicals than α-Tocopherol and BHT and these differences were statistically significant (P < 0.05). The dose of P. fascisepala leaf extract required to reduce the absorbance of DPPH control solution by 50% (EC50) was calculated at 0.50 mg and those of α-Tocopherol, BHT and ascorbic acid were 0.35, 0.24 and 0.1 mg (Table 1). In other words, P. fascisepala leaf extract required a 1.5–5.0-fold dose to scavenge DPPH as well as α-Tocopherol, BHT and ascorbic acid. Also by simple regression analysis, we found that the total phenolic content of the extract positively correlated (r = 0.991) with its DPPH radical scavenging effect, suggesting that phenolic constituents of the plant contributed to the scavenging. Further studies such as ability of the extract to suppress 2,2′- azobis(2-amidinopropane) dihydrochloride- induced plasma oxidation (22) may be necessary to verify these activities.

Table 1.

Antioxidant activities of extracts, α-Tocopherol and BHT as expressed by half–effective doses (ED50)

Antioxidant reaction	Antioxidant	ED50 (mg)
DPPH radical	P. fascisepala	0.50
	α-Toc	0.35
	BHT	0.24
	Ascorb	0.10

In this assay, the presence of antioxidants in the sample reduced Fe3+/ferricyanide complex to the ferrous form. Fe2+ can be monitored by measuring the formation of Perl's Prussian blue at 700 nm. This reducing capacity of compounds could serve as an indicator of potential antioxidant properties (23) and increasing absorbance could indicate an increase in reducing power. Although the greatest reducing power was obtained from ascorbic acid, α-Tocopherol and BHT compared to P. fascisepala, our results showed that P. fascisepala leaf extract is an electron donor and could react with free radicals, convert them to more stable products and terminate radical chain reaction (24). Also, a strong positive correlation (r = 0.980) between total phenolic content and the reducing power of the extract suggests that phenolic compounds in the plant may determine the ferric ion reducing antioxidant power (FRAP) of the extract. Further investigations such as evaluation of blood total antioxidant capacity (TAC) (25) and antioxidant status in streptozotocin–nicotinamide-induced type 2 diabetic rats (26) may be necessary to verify our findings in vivo.

As a transitional metal, the capability of iron to generate free radicals from peroxides by Fenton reactions has been implicated in cardiovascular disease (27). Since Fe2+ has been shown to induce oxyradical production and lipid peroxidation, reduction of Fe2+ concentrations in the Fenton reaction protects against oxidative damage (28,29). Thus, P. fascisepala leaf extract may offer protection against oxidative damage through its chelating properties. Further work such as deoxyribose degradation assay in the absence of EDTA (22) could also be carried out to further establish these iron chelating properties.

Conclusion

Phaulopsis fascisepala leaf extract demonstrated moderate antioxidant activity, reducing power, as well as scavenging and chelating effects. Purification of the extract may lead to increased activity in its bioactive compounds. The antioxidant activities of P. fascisepala leaf extract may be due to its proton donating capability as shown in DPPH radical scavenging results. Acting as an electron donor that can react with free radicals, it converts them to more stable products and terminates radical chain reactions. This mechanism may explain its use for the treatment of wounds and sores.