Iron Reducing and Radical Scavenging Activities of 13 Medicinal Plants From Côte d ’ Ivoire

Objective: Oxidative stress has been involved in the development of varied human diseases. The aim of this study was to evaluate the iron reducing power and the antiradical activity of 13 plants traditionally used as medicinal plants in Côte d’Ivoire. Materials and Methods: FRAP (ferric reducing antioxidant power) and ABTS (2,2’-azino-bis(3-ethylbenzothiazoline6-sulfonic acid)) assays were used to assess the antioxidant property of 80% methanol extracts prepared from the 13 plants. Results: A high iron reducing activity was exhibited by extracts from leaves of Leea guineensis (42.76 ± 28.54 mg of TE/gdry extract) and Bersama abyssinica (39.77 ± 31.29 mg of TE/g dry extract). Smeathmannia pubescens (% ABTS = 92.44 ± 12.93%), L. guineensis (%ABTS = 89.73 ± 15.10%), Keetia venosa (% ABTS = 88.78 ± 17.36 %) and Sapium ellipticum (%ABTS = 85.86 ± 25.10%), showed promising antiradical activity with IC50 values of 4.50, 5.00, 5.40 and 5.70 μg/mL respectively. These values are (p < 0.05) close to those of Trolox (CI50 = 4.10 μg/mL) and ascorbic acid (CI50 = 4.90 μg/mL). Conclusion: Our findings confirm the traditional use of the studied plants in treatment of various ailments. The results obtained provide promising baseline information for using these medicinal plants for improving the health status of the population.


INTRODUCTION
Iron is a useful mineral for the metabolism of living organism.In the blood, it contributes to numerous physiological functions such as hematite maturation, oxygen transportation for cellular respiration, DNA and proteins synthesis. 1 However, the accumulation of iron in some essential organs such as liver, heart and spleen leads to free radical production via oxidative stress. 2his iron overload is called hemochromatosis and is implicated in the genesis or the complication of diseases like cancer, Alzheimer, malaria and diabetes. 3,4Iron overload may contribute to the development of hepatocellular carcinoma 5 and about 85 % of the hepatocellular carcinoma occurs in developing countries. 6According to WHO, cancers figure among the leading causes of death worldwide, accounting for 8.2 million of death.In Africa, cancer is a real issue for people due to the lack of facilities and the relative expensive cost of treatments. 7In addition, iron overload can be cause by some heath conditions such as malaria which is endemic in Africa.This disease is a major cause of death especially for children under five years old, and more than 90% of malaria deaths occur in Sub-Saharan Africa. 8One of the defense strategy of the host is to restrict iron availability to pathogens in order to reduce their virulence. 9 major ''trade-off '' of this host defense strategy is accumulation of toxic iron in tissues and organs, which can act in a pro-oxidant and cytotoxic manner.This can lead to tissue damage, enhancing rather than preventing disease severity.Synthetic iron chelators are used to facilitate iron overload elimination and are good antioxidants.However they are reported to be toxic 10 and naturally occurring compounds are needed as alternative treatments.Plants are good source of natural antioxidants and largely used as food and medicines.Phytocompounds such as flavonoids are well known for their antioxidant properties.As such they can strongly contribute to the treatment of iron and free radical-related diseases 11 This study investigated 13 medicinal plants for their iron reducing antioxidant power and antiradical activity.

Plant material
The studied plants were selected on the basis of ethnobotanical surveys carried out in Côte d'Ivoire and elsewhere in Africa.These plant species are used in traditional medicine in the treatment of cancer, malaria, diabetes and gastric ulcer.These plants were collected in August 2008 in the Bouaflé savannah (Western-central Côte d'Ivoire) (Figure 1).Botanical identification of each species was performed at the herbarium of Centre Suisse de Recherches Scientifiques of Côte d'Ivoire and authenticated by Professor Aké-Assi Laurent † at the Centre National de Floristique of the University Félix Houphouët Boigny (Abidjan, Côte d'Ivoire).

Preparation of plant extracts
Parts of the selected plants were harvested, cleaned with tap water, dried in air-conditioned room at 18°C for two to three weeks, and then powdered.Fifteen grams of each powder was macerated in 150 mL of 80% methanol under mechanical stirring (200 rd/min) for 24 hours at room temperature.The filtrates were dried to obtained extracts.The yield for each extract was calculated using the formula: (weight of dry extract/ weight of powder plant material) x 100%.

FRAP assay
The ability of the plant extracts to reduce iron was assessed using the Ferric reducing antioxidant power (FRAP) assay. 12In the presence of reductive compounds, iron ferric form (Fe 3+ ) non colored is transformed into iron ferrous form (Fe 2+ ) which is blue.The fresh FRAP reagent (10: 1: 1) was prepared from acetate buffer (300 mM, pH 3.6), TPTZ (10 mM in HCl 400 mM) and ferric chloride (10 mM) and kept at 37°C.Plant extracts were serially diluted to obtain a range of concentrations (100, 50, 25, 12.5 and 6.25 μg/mL).Trolox (2.4, 1.2 and 0.6 μg/mL) was used as reference for standard curve Y = 0.065X + 0.043 where X is the value of absorbance and Y the Trolox equivalent value.Subsequently, the FRAP reagent (2850 μL) was added to 150 μL of each concentration of plant extract.The mixture was maintained for 30 min at room temperature, and then absorbance was measured at 593 nm against methanol as blank.The results were expressed in mg of TE/g of dry extract according to the formula. 13

FRAP value (mg of TE/g of dry extract) = [(A e -A 0 ) / (slope)]x[V/v)] / [w][1000]
When: A e = absorbance of sample; A 0 = absorbance of blank; w=weight of the dry extract; V=total volume of extract; v=used volume of the extract and 1000=conversion factor.

ABTS assay
The ABTS radical scavenging activity of plant extracts was determined using the method described. 14with slight modifications.Briefly, 2850 μL of ABTS reagent were added to 150 μL of the tested extracts, and mixed thoroughly at room temperature, for 2 hours in dark conditions.This reagent was obtained from 7 mM of ABTS solution and 2.42 mM potassium persulfate (1: 1 v/v), incubated at 23°C for 6 hours to 3 days prior use and absorbance was adjusted to 1.1 ± 0.2 at 734 nm by adding methanol.Then the absorbance was measured at 734 nm using a UV spectrophotometer, methanol was used as blank.The percentage inhibition was calculated using the formula: % ABTS = [(A 0 -Ae)/(A 0 )] x 100 with A 0 =absorbance of control; A e = absorbance of sample.The different extracts were grouped according to their inhibition percentage: high activity, moderate activity and weak activity.The IC 50 was graphically determined using Trolox (100, 50 and 25 μM) as the reference compound for calibration.A low IC 50 value indicates strong antioxidant activity in a sample. 15

Statistical analysis
The software STATISTICA 7.1 was used for data analysis.Results obtained were reported as means ± SD of duplicate experiments.One-way analysis of variance (ANOVA 1) was performed to test the influence of concentrations on the percentage of inhibition and iron reducing power of extracts.The difference was significant at p<0.05.The least significant difference (LSD) test was used to determine the difference in the inhibition percentage of ABTS and the iron reducing power among the extracts.The relationship between concentrations of extract and percentage of inhibition was determined, R 2 ≥ 0.90 was considered as strong correlation. 16

Ferric reducing antioxidant activity
The FRAP values ranged from 42.76 ± 28.54 to18.06 ± 23.90 mg of TE/g of dry extract.There was a significant difference between the plant extracts tested (α=0.05,p<0.001 and F=6.04).The leaves extract of Leea guineensis showed the highest ferric reducing power (42.76 ± 28.54 mg of TE/g of dry extract).Moderate effect was obtained for leaves of Bersama abyssinica (39.77 ± 31.29 mg of TE/g of dry extract), roots of L. guineensis (37.60 ± 28.36 mg of TE/g of dry extract), stem bark of Sapium ellipticum (32.67 ± 27.44 mg of TE/g of dry extract) and root bark of Flacourtia indica (31.92 ± 23.69 mg of TE/g of dry extract).The leaves of Sapium ellipticum (29.19 ± 23.88 mg of TE/g of dry extract) showed weak iron reducing power (Table 1).

ABTS radical scavenging activity
The results of ABTS assay are presented in Table 2.The tested extracts showed antiradical activity ranging from 92.44 ± 12.93 to 52.08 ± 32.25%.There was a very high significant difference (α=0.05,p< 0,001, F=7.20) between ABTS inhibition of the extracts.The multiple comparison with the LSD test showed that the stem bark extract of Smeathmania pubescens (92.44 ± 1.97%) possessed the highest scavenging activity.The extract of Leea guineensis (roots), Keetia venosa (leaves) and Sapium ellipticum (stem bark) showed moderate scavenging activity with inhibi-tory percentage of 89.73 ± 15.10, 88.78 ± 17.36 and 85.86 ± 25.10% respectively.The remaining extracts showed low activity (Figures 2 and 3).Of the studied plant extracts, Smeathmania pubescens showed the lowest CI 50 value of 4.50 μg/mL which was lower than that of Trolox (CI 50 =4.90μg/mL), used as reference.The CI 50 of Leea guineensis and Keetia venosa were slight low, with values of 5.00 and 5.40 μg/mL respectively (Table 2).

Correlation
Strong and positive correlation (R 2 ≥0.90) was established between the concentrations of extracts and inhibitory percentage of ABTS radical for leaves of Cissus doeringii, Vernonia guineensis and Cuviera macroura, stems of Leea guineensis, leaves of Smeathmannia pubescens and stem bark of S. pubescens (Table 2).For the extracts of Sapium ellipticum, L.

CONCULSION
Iron reducing and radical scavenging activities of these plants reveal that methanol extract of all the studied plants have either iron reduction or scavenging activities.Some of the plants such as Leea guineensis present the both activities.These results suggest that the active plants could be candidates to prevent oxidative stress related diseases.We plan to undergo further investiga tions such as study of the influence of season and location on antioxidant activity, elucidation of compounds responsible for the activity and toxic ity in order to develop antioxidant nutraceutics.

DISCUSSION
This study investigated the antioxidant activity of 13 plants used in traditional medicine in Côte d'Ivoire.The most active plants were S. pubescens, L. guineensis, Bersama abyssinica, K. venosa and C. doeringii.S. pubescens showed the highest inhibitory percentage (% ABTS=92.44 ± 1.97 %).This strong antioxidant activity may explain some traditional uses of this plant, such as the treatment of toothache. 17Free radicals are implicated in the genesis of pain and fatigue. 18All the 13 plants studied were found to have good antioxidant power.The leaves of L. guineensis and B. abyssinica showed high iron reducing power.The stem bark of S. pubescens (4.50 μg/mL), root of L. guineensis (5.00 μg/mL), leaves of K. venosa (5.40 μg/mL) and C. doeringii (5.70 μg/mL) showed high antiradical activities close to that of Trolox and vitamin C.There is a good association between the traditional uses against chronic fatigue, malaria, cancers and diabetes of studied plants and their antioxidant properties.
In traditional medicine, L. guineensis is used to treat malaria, ulcer, cancers and epilepsy. 19This plant also is used as analgesic.From what we discovered, it's worth mentioning that this plant possesses good iron reducing antioxidant power.Oxidative stress is aggravating factor of malaria, ulcer and cancer. 20Iron overload can be observed during malaria due to host defense strategy. 9The plant showed iron chelating activity revealing it may act by reducing the accumulation of iron during the treatment.B. abyssinica is traditionally used for the treatment of malaria, 21 cancer, ulcer, rheumatism, wounds 22 and diabetes. 23Free radicals are involved in the genesis of most of these diseases.Interestingly, the plant showed high activity in the current study.To the best of our knowledge, this is the first report of the iron reducing activity of B. abyssinica.Antioxidants are known to protect organisms against oxidative stress due to malaria. 24lso iron overload can generate an oxidative stress responsible for inflammation in rheumatism. 25The antioxidant properties of this studied plant may justify its traditional use for treating diseases and improving the health status of people.Our study revealed high antioxidant power for Keetia venosa.This plant is traditionally used to treat intercostals pains, rheumatism and is also used as tonic. 26Muscular fatigue is strongly linked to the production of free radicals in the body. 27The antiradical properties of K. venosa support its use in traditional medicine as a tonic.
Cissus doeringii showed a strong antiradical activity with IC 50 =5.70µg/ mL.This plant has shown analgesic activity and antirheumatism property. 28,29oxidative stress, due to iron overload may exacerbate rheumatism.Antioxidant compounds possess good anti-inflammatory properties useful in the treatment of rheumatism. 30

Figure 1 :
Figure 1: Map of the Department of Bouaflé showing the studying site, the Tibéita village (BNEDT/CCT; 2008)

Figure 2 :
Figure 2: Mean of percentage of inhibition of extract for 5 different concentrations For all the concentration of extracts, the difference was significant; bands having the same letters are not statistically different according to LSD Fisher post hoc test. 1 F: leaves of Cissus doeringii, 3 F: leaves of Vernonia guineensis, 5 ET: stem bark of Sapium ellipticum, 6 F: leaves of Cuviera macroura, 7 R: root of Leea guineensis, 7 F: leaves of Leea guineensis, 7 T: stem of Leea guineensis, 10 F: leaves of Smeathmannia pubescens, 10 ET: stem bark of Smeathmannia pubescens, 12 F: leaves of Keetia venosa, 12 ER: root bark of Keetia venosa, 13 F: leaves of Pouteria alnifolia, 18 ET: stem bark of Anthocleista nobilis, T: Trolox, AA: ascorbic acid.

Table 1 : Iron chelating potential (mg de TE/g of dry extract) of plant species tested
TE: Trolox equivalence, F: Fisher statistical, FRAP: Ferric Reducing Antioxyant Power, SD: Standard deviation for 5 different concentrations.Values having the same letters are not statistically different according to LSD Fisher post hoc test.

Table 2 : ABTS radical scavenging power of tested plants and standards
Fisher statistical, IC 50: concentration of extract required to obtain 50% inhibition of ABTS radical.