Evaluation of Antioxidant Activities and Estimation of Zinc Content of Aqueous and Methanolic Extracts of Three Medicinal Plants: Cochlospermum Planchonii, Pericopsis Laxiflora and Harungana Madagascariensis

Malaria disease is a pathology which occurs as a result of the oxidative stress. Three plants of the Ivorian pharmacopoeia are particularly used in the traditional medicine for the treatment of malaria in Côte d'Ivoire. They are Cochlospermum planchonii (Placo), Pericopsis laxiflora (Laper) and Harungana madagascariensis (Madhar).The objective of this study was to evaluate the in vitro antioxidant activity of the aqueous and methanolic extracts of three medicinal plants of Côte d’Ivoire. The quantitative measurement of total flavonoids gave the following results: Laper (560.80 mg QE/g ± 7.40), Madhar (352.50 mg QE/g ± 9.46) and Placo (314.20 mg QE/g ± 4.41). The total polyphenols were more concentrated in Laper (262.50 mg GAE/g ± 9.47) and Placo (203.20 mg GAE/g ± 2.21). Noticeable zinc content was found in Laper (13.56 ppm) and Placo (11.40 ppm) against 3.43 ppm in Madhar. The radical scavenging activity by 2,2-diphenyl-1-picryl hydrazyl (DPPH) and antiradical power measurement by ABTS (2,2' azino bis-3-ethylbenzothiazoline-6-sulphonic acid) assay have showed good activity for Laper (IC50 = 7.50 μg/mL) and placo (IC50 = 8.01 μg/mL). The standard antioxidant compound (quercetin) gave an IC50 = 4.00 μg/mL. This study suggested that the high content of total flavonoids, total phenols and minerals in these plants could partly justify the therapeutic use of at least two of the three plants.


Introduction
In recent time, phytomedicines has drawn special attention as therapeutics giving wide range of treatment options against diseases. Sometimes they are more useful than synthetic drugs due to their economic price, less adverse effects and efficacy in multidrug resistant incidences [1][2][3]. Today, natural antioxidants molecules from plants sources are required [4,5]. Indeed, the polyphenols compounds are widely found in plants which have proven beneficial effects on human health [6]. Their role as natural antioxidants arouses more and more interest for the prevention and the treatment of cancer, inflammatory and cardiovascular diseases [7]. They are also used as additives in agro-alimentary, pharmaceutical and cosmetic industry [4].
Scientific research was developed for extraction, identification and quantification of natural compounds starting from the various sources such as the agricultural and horticultural cultures or the medicinal plants [8]. It is also necessary to add total flavonoids which are natural compounds, almost universal as the vascular plants. They constitute pigments responsible for yellow colorings, orange and red of various plants organs [9] .The properties of the flavonoids best described are their antioxidant activity and their capacity to scavenge the free radicals like their role in anti-inflammatory drug [ 10,11 ] . Likewise, Zinc has been shown to have an antioxidant role in defined chemical systems. Two mechanisms have been elucidated; the protection of sulfhydryl groups against oxidation and the inhibition of the production of reactive oxygens by transition metals. Supraphysiological concentrations of Zinc have antioxidant-like effects in organelle-based systems and isolated cell-based systems in vitro [12]. The concept of which oxygen, molecule essential for life, can lead to significant cellular damage through the formation of active oxygenated derivatives is still badly perceived in the medical environment. However, many epidemiological studies suggested the role of these oxygenated free radicals 32 Evaluation of Antioxidant Activities and Estimation of Zinc Content of Aqueous and Methanolic Extracts of Three Medicinal Plants: Cochlospermum Planchonii, Pericopsis Laxiflora and Harungana Madagascariensis (OFR) in the development of many pathological processes associated with the atherosclerosis, cancerogenesis and malaria [13,14] .To protect itself from the toxic effects of oxygen, the body organism developed antioxidant systems of defense composed of enzymes (glutathion peroxidase), vitamins (A, C, E), trace elements (selenium, zinc) and proteins (ferritin). Lipoperoxidation is the most accessible aspect of oxidative stress [15]; It is also the most evoked part currently in physiopathology. Several in vitro studies [16,17], in vivo [18] and somewhat in humans [19] were done for the evaluation of the evolution of oxidative stress during malaria [20]. They showed that during the course of the disease, there was an increase in lipoperoxidation measured by malondialdehydes (MDA) and a decrease in the antioxidant system [21,22]. To overcome this physiological imbalance, exploration of new sources of natural antioxidants is necessary in view of the potential toxicological risks of synthetic antioxidants. This work is part of the research and development of natural bioactive substances, focused on three plants of the Ivorian pharmacopoeia. Cochlospermum planchonii, Pericopsis laxiflora and Harungana madagascariensis, traditionally used to treat malaria in Côte d'Ivoire and in the West African region [23,24]. The main objective of this study was to evaluate the in vitro antioxidant activity of three medicinal plants by the DPPH free radical scavenging effect and antiradical power by ABTS assay and determine the content of total polyphenols, total flavonoids and zinc, well known for their antioxidant activity.

Plant Material
The plant material consisted of barks of Pericopsis laxiflora (Laper) and Harungana madagascariensis (Madhar) and leaves of Cochlospermum planchonii, (Placo) ,collected in the locality of Moronou about 20 km from Toumodi central region of Côte.d'ivoire, between July and September 2014 [24].They were washed and dried under the shade, the fine powder of drug was obtained after crushing using a mechanical crusher (Retsh, M26951).

Aqueous Extraction
100 g of drug was homogenized in 1L of water. After two cycles of homogenization for 5 mn, the homogenized was collected in a square clean fabric and was pressed with the hand by application of strong pressures. The solution collected was filtered twice on absorbent cotton then on Whatmann 3 paper then the solution obtained was dried in a drying oven of the type Heraeus® at the temperature of 50 °C for three days [25].

Methanolic Extraction
50 g of drug powder were homogenized in 1.5 L of pure methanol in a mixer; the following steps were identical to those of the aqueous extraction above [ [27] was used for the measurement of total polyphenols. A volume of 2.5 mL of diluted Folin-Ciocalteu reagent FCR (1/10) was added to 30 µL of extract. The mixture was maintained for 2 minutes in the dark at room temperature and 2 ml of calcium carbonate solution (75 g/L) was added. Then the mixture was placed for 15 minutes in a water bath at 50°C, and then cooled quickly. The absorbance was measured at 760 nm using a spectrophotometer and the polyphenol concentration expressed in milligram of gallic acid equivalent per gram of extract (mg GAE /g).

Determination of total Flavonoids by Spectrophotometrically
Method of Marinova and al., (2005) [28] was used for the measurement of the total flavonoïdes. In a flask of 25 ml, 0.75 ml of sodium nitrite (NaNO 2 ) of 5 % (m/v) was added to 2.5 ml of extract. The mixture was added to 0.75 ml of aluminium chloride (AlCl 3 ) of 10% (m/v), and then incubated in the dark for 6 minutes. After incubation, 5 mL of sodium hydroxide solution (NaOH 1N) were added then volume was supplemented to 25 mL. The absorbance was read at 510 nm and the flavonoid concentration expressed in milligrams of quercetin equivalent per grams of extract (mg QE/g) was determined using a calibration line established in the same conditions.

Evaluation of DPPH Radical Scavenging Activity
The evaluation of the anti-oxidant activity of the plant extract was performed using the 2.2-diphényl-1-picryl-hydrazyl (DPPH) radical scavenging assay according to the method of Parejo et al., (2000) [29] with some modifications) range of concentrations (0-200 µg/mL of extract and Quercetin (antioxidant molecule of reference) was prepared in the ethanol / eau (70/30) (v/v). A volume of 100 µL of this solution, was mixed with 3.9 ml of DPPH (70 µM) prepared in methanol. After homogenization, the mixture was incubated at the room temperature (25 °C) in the dark. After 15 minutes of incubation the absorbance was read at 517nm and the lC 50 which is the Quercetin concentration responsible for 50 % of inhibition of DPPH radicals, was determined by projection from 50 % on the graph representing the percentage of inhibition of the DPPH according to the concentrations of the extracts and Quercetin.

Measurement of Antioxidant Activity by Reduction of ABTS +
This method was based on the ability of the extracts to scavenge the ABTS + radical cation (2, 2' azino bis-3-ethylbenzothiazoline-6-sulphonic acid). The test was carried out according to the method described by Choong et al., (2007) [30]. The reduction of ABTS + was produced by reaction of 8 mM ABTS (87.7 mg in 20 mL of distilled water) and 3 mM of potassium peroxodisulphate (K 2 S 2 O 8 ) (0.0162 g in 20 ml distilled water) in a ratio 1:1 (v/v). The mixture was then incubated in the dark at room temperature for 12-16 hours. This solution of ABTS + was diluted with methanol to obtain a solution whose absorbance value was 0.7 ±0.02 which was read at 734 nm. Thus, a test specimen of 3.9 ml of this diluted solution of ABTS + was added to 100 µL of the compound to test. After Agitation, the mixture was incubated for 6 minutes in the dark (T = 30 ±2°C). The addition of antioxidant reduces the radical and causes the discoloration of the mixture. This discoloration of the radical measured by spectrophotometry at 734 nm is proportional to the concentration of antioxidants expressed in µmol of Trolox equivalent of antioxidants activity per liter of extract (µmol TE/L).

Measurement of Zinc Content
The zinc content was measured by atomic absorption spectrometry [31].

Statistical Analysis
The data were analyzed using Graph Pad PRISM 5 software (Microsoft, USA). The average value accompanied by the standard error (mean ±SD). The difference between two values was considered to be significant when P < 0.05. The statistical analysis of the results was carried out using ANOVA followed by the test of Tukey for the multiple comparisons and the determination of the degree of significance. Figure 1 showed the contents of total phenols that measured by Folin-Ciocalteu reagent in terms of gallic acid equivalent (mg GAE/g). Total phenols varied from 109.20 ±2.21 mg GAE/g to 262.50 ±9.47 mg GAE/g in the extracts. Laper with total phenol contents 262.50 ±9.47 mg GAE/g had the highest amount in this study followed Placo (203 ±2.21 mg GAE/g) and madhar (109.20 ±2.21 mg GAE/g). The results obtained in Placo and Laper are not statistically different (p>0.05). Madhar has a relatively lower value compared to the other two plants.

Total Flavonoids Content
Flavonoids contents of the extracts in term of quercetin equivalent (mg QE/g) were between 314.20 ±4.41 mg QE/g to 560.80 ±7.40 mg QE/g (Figure 2). The flavonoid content in extract of Laper (560.80 ±7.40 mg QE/g) was higher than extracts of Madhar (352.50 ±9.46 mg QE /g) and Placo (314.20 ±4.41 mg QE/g). A peak value in flavonoids content was obtained with Laper but the results obtained in Placo and Madhar are also important and are not statistically different (p>0.05).

Measurement of Antioxidant Activity
The different extract showed dose dependent DPPH radical scavenging activity. These results show amount of each extract needed for 50% inhibition (IC 50

Inhibiting Capacity of ABTS++ Radical
Reduction in ABTS radical was also used for measuring the antioxidant activity of the extracts. This method was based on the capacity of the compounds to reduce ABTS radical. The antioxidant activity was measured by inhibition of ABTS ++ radical obtained starting from the ABTS (ammonium salt of the 2, 2' azino-bis -3-ethylbenzothiazoline -6-sulphonic acid) compared to a reference antioxidant. Trolox (acid 6 hydroxy-2, 5, 7,8tetramethylchroman -2-carboxylic), of which the cyclic molecular structure is similar to that of the vitamin E. So the antioxidant capacity in Trolox equivalent corresponds to the concentration of Trolox (µmol T E/L) having the same activity as the same unit concentration of the studied extract. Thus, two plants had an antioxidant activity: Laper (44.26±1.63 µmol Trolox E /L) and Placo (43.00±2.01 µmol Trolox E /L) (Figure 4). On the other hand, Madhar had a weak antioxidant activity (10.29± 0.14 µmol Trolox E /L).

Zinc Content
Measurement of zinc content by atomic absorption spectrometry made it possible to note a good concentration level in Laper with 13.56±0.97 ppm and Placo 11.40±0.53 ppm against 3.43±0.32 ppm in Madhar ( Figure 5).
It arises from the analysis of these data that Laper has at the same time a good polyphenol content, total flavonoids and zinc. It is followed in the order by Placo and Madhar regarding content of the same compounds.

Discussion
DPPH radical scavenging method is standard procedure applied to evaluation of antiradical activity. This method is easy, rapid and sensitive way to survey the antioxidant activity of a specific compound or plant extracts [32]. DPPH free radicals, which are stable in methanol shows maximum a proton donating substances such as antioxidant. Radicals would be scavenged and absorbed. The different extracts showed dose dependent DPPH radical scavenging activity. IC 50 of the standard compound (Quercetin) was 4.00µg /mL, which is the highest radical scavenging activity. It is similar to the one found by Bidié et al. (2010) [33] with quercetin (IC 50 = 2 µg/mL). The difference between quercetin and the others is significant. But the radical scavenging activity in the plant extracts is also important and decreased in the following order: Laper (IC 50 = 7.5 µg/mL) > Placo (IC 50 = 8.01 µg/mL) > Madhar (IC 50 = 147 µg/mL). Results in Laper and Placo did not differ significantly (P>0.05). These results are similar to those obtained by Bidie et al. (2010) [33]. Indeed, these authors found an IC 50 of 7.5 μg / mL and 10.5 μg / mL in the extracts of Trichilia prieuriana and Mitragyna ciliata respectively. Their study showed that both plants had very good antioxidant activity due to their high polyphenols and flavonoids content.An antioxidant is defined like a substance which, added at a lower dose with a naturally oxidable product with the air, is able to slow down or to inhibit the phenomenon of oxidation.
This definition can be extended to all the substances which protect the body systems against the potential noxious effects of the processes or reactions which generate an excessive oxidation [34]. Thus, several substances acting as antioxidants in vivo were selected. They include β-carotene, albumin, uric acid, oestrogens, polyamines, flavonoids, ascorbic acid, vitamin E, phenolic compounds, the flavonoïds, zinc and selenium [6]. In this study we studied the correlation between the flavonoïds, total polyphenols and zinc content and the antiradical capacity of three extracts of plant used in traditional medicine to treat malaria [35,36]. Therefore, it was observed a strong correlation between the antiradical activity and the simultaneous presence of the three compounds which are the polyphenols, total flavonoïds and zinc. On the other hand, Madhar which contains a relatively low concentration of polyphenols and zinc has a relatively very poor free radical scavenging activity (IC 50 > 100 µg/mL). The higher polyphenols concentration observed in laper and placo could be very interesting because the polyphenolic compounds are used more and more in therapeutic [37]. In addition, N'guessan et al. (2007) [38] showed the existence of a correlation between the total phenols content and the free radical scavenging activity. Zinc is essential for human development and function. It provides conformational stability to numerous metalloenzymes. Zinc deficiency is associated with oxidative damage in numerous diseases, including cancer, cirrhosis, coronary artery disease, diabetes and some skin disorders. Abundant evidence demonstrates the antioxidant role for zinc [39]. These investigations provide a comprehensive profile of the antioxidant activity of plant extracts with respect to their phenols and flavonoids content. Many reports of natural antioxidants of plants have been published and their importance in health, food and preventive medicine has been well documented [40,41]. The abundance of polyphenol and flavonoids compounds would confirm the therapeutic properties that there assigned in ethnotherapy.

Conclusions
The plants studied have a good total polyphenols, total flavonoids and zinc contents. Laper and Placo are sure to have antioxidant activity, unlike Madhar whose antioxidant activity has not been demonstrated. At the end of this work, we noted that medicinal plants concentrate in their tissues chemical substances, such as those mentioned above, which formed the basis of their pharmacological properties. In line of this, Laper and Placo could be potential sources of natural antioxidants.