Antioxidant and Antiplasmodial Activities of Extracts from Gardenia erubescens Stapf et Hutch. and Fadogia agrestis Schweinf. ex Hiern. (Rubiaceae)

Two medicinal plants, Gardenia erubescens and Fadogia agrestis were selected to evaluate their biological activities. Their total phenolic and flavonoid contents were assessed using folin-ciocalteu and aluminum chloride regents’ methods. The antioxidant activity was estimated using DPPH (1,1-diphényl-2-picrylhydrazyl), ABTS (2,2’-azinobis-[3-ethylenzothiazoline-6-sulfonic acid]) and FRAP (ferric reducing antioxidant power). The antiplasmodial activity of the extracts was determined in vivo on 42 NMRI mice. The results indicate that all the extracts from both two plants contain some polyphenols. The ethanolic extract of the leaves of Gardenia erubescens showed the best antioxidant activity by the method of DPPH. The aqueous extracts of the leaves of Gardenia erubescens and the whole plant of Fadogia agrestis have a reducing power similar to control (quercetin). All the extracts have a low capacity to scavenge the ABTS radical cation compared to the controls (trolox and quercetin). Concerning the antiplasmodial activity, all the extracts presented moderate antiplamodial activities. This result could justify the traditional uses of Gardenia erubescens and Fadogia agrestis to treat of malaria in Burkina Faso.


Introduction
Malaria is a public health problem around the world [1] [2]. In 2018, there are estimated 228 million cases and 405,000 malaria deaths worldwide, and 94% of these deaths occur in Sub-Saharan Africa. In South Asia and Africa, malaria continues to affect pregnant women and children more strongly with around 80% of deaths in pregnant women and children under 5 years [3]. In Burkina Faso, malaria remains the leading cause of morbidity and mortality with 3,501,245 cases and 1002 deaths [4]. It remains the primary reason for consultation in health facilities. For the treatment of this disease, WHO recommends artemisinin-based combination therapy (CTA) for uncomplicated malaria caused by P. falciparum. For severe malaria, it is preferable to use injectable artesunate (intramuscularly or intravenously) for 24 hours followed by a complete CTA of 3 days once the patient can tolerate oral medications [5]. However, the treatment of this erythrocytopathy leads more and more problems with the resistance of the parasites to the usual antimalarial drugs, such as artemisinin and its derivatives [6] and anopheles to insecticides [7]. This resistance problem has sparked a renewed interest in traditional medicine [8]. The medicinal plants constitute a source of molecules of therapeutic interest [9] which can contribute to the development of new molecules which are both effective and accessible to the poorest populations, the main and vulnerable target of malaria. Many molecules namely quinin and artemisinin used in therapeutics are from plant origin [10]. A previous study has shown that the aqueous extract of Fadogia agrestis (F. agrestis) has an antiplasmodial activity in vitro and an IC50 of 4 < IC50 < 10 g/ml [11]. Regarding Gardenia erubescens (G. erubescens), the ethanolic extract of the barks has an antioxidant activity [12]. These results prove that studies have been done on these 2 species but concerning their in vivo antiplasmodial activity. This is how the present work, although preliminary, was initiated in order to determine the antiplasmodial and antioxidant activities of the ethanolic and aqueous extracts of Gardenia erubescens and Fadogia agrestis.

Plants materials
The leaves, barks (trunk) of G. erubescens and the whole plant of F. agrestis  Plasmodium berghei Plasmodium berghei (strain ANKA) is continuously maintained in the entomology laboratory of Health Sciences Research Institut at Bobo-Dioulasso by an acyclic passage from infected mice to healthy mice through an injection of parasitized blood.

Extracts Preparation
Preparation of aqueous extracts Decoctions were performed by adding to 500 mL of distilled water to the sample (50 g), heated and boiled under reflux for 15 min. The decoctions were frozen and lyophilized.
Preparation of ethanolic extracts The plant samples (20 g) were sequentially extracted with 200 ml of petroleum ether and ethanol using a soxhlet apparatus. The extracts were first concentrated to maximum using soxhlet dispositive and then to dryness at room temperature in the Petri dishes.

Determination of Total Phenolics and Total Flavooides
Total phenolics: Total phenolics were evaluated according to the colometric method described by Meda et al. in [13]. To 0.125 ml of the sample solution was added 0.625 ml of Folin-ciocalteu reagent (FCR 0.2N) and the whole was incubated in the dark for 5 minutes. 0.5 ml of sodium carbonate (75 g/L) was then added to the mixture and incubated again for 2 hours in the dark. The absorbances were read at 760 nm against a standard gallic acid curve (y = 4668e-3 * x-0.034, r 2 = 0.9991). A series of 3 tests were carried out for each extract, then the average of 3 measurements was calculated. The results are expressed in mg of gallic acid equivalent per 100 milligrams of the extract (mg EAG/100mg).
Total flavonoids: Total flavonoids were assessed by the aluminum chloride method [13]. 0.625 ml of the sample solution was mixed with 0.625 ml of 2% AlCl 3 . After waiting 10 minutes in the dark, the absorbances were read at 415 nm using a quercetin calibration curve (Y = 1.259e-02 * x; r 2 = 0.9990). A series of 3 tests were carried out for each extract; then the average of the 3 measurements was calculated. The results were expressed in mg of quercetin equivalent (QE)/100mg of the extract.

Evaluation of Antioxidant Activity in Vitro
The antioxidant power of the ethanolic and aqueous extracts of plants was achieved by 3 methods: the scavenging of the free radical (DPPH), the scavenging of the cation radical (ABTS) and the reduction of ferric chloride (FRAP). DPPH (2,2-diphenyl-1-picrylhydrazyl): The antioxidant activity was evaluated by the 2,2-diphenyl-1-picrylhydrazyl (DPPH) method as described by [14]. A series of cascade dilutions of the initial solution (10 mg/ml) was carried out for each extract. In each tube a volume of 0.375 ml of the sample solution was mixed with 0.75 ml of the DPPH solution (20 mg/l) and the whole is incu-bated for 15 minutes in the dark. The absorbances are read at 517 nm and methanol is used as the blank sample. The antioxidant activity is expressed as a percentage of inhibition according to the following formula: % inhibition = (White absorbance − Sample absorbance)/white absorbance × 100%. Three readings are taken per extract and the average of the IC 50 (concentration causing 50% inhibition of the DPPH radical) determined graphically, is calculated. ABTS (2,2'-azinobis-[3-ethylenzothiazoline-6-sulfonic acid]): The ABTS method was carried out according to the method described by [13]. The cation radical ABTS •+ was regenerated by mixing an aqueous solution of ABTS (7 mM) with 2.5 mM potassium persulfate (final concentration) and the mixture is kept in the dark at room temperature for 12 hours before use. The mixture was then diluted with ethanol to give an absorbance of 0.70 ± 0.02 at 734 nm using the spectrophotometer. 10 µl of the sample solution was added to 990 µl of the ABTS reagent and the whole is incubated for 15 minutes, protected from light. The absorbances were read in a spectrophotometer at 734 nm against a standard curve of ascorbic acid. A series of 3 tests were carried out for each extract, then the average of the 3 measurements was calculated. The results were expressed in µmol equivalent ascorbic acid per 1 gram of extract (µmol EAA/g of extract).
FRAP (Ferric Reducing Antioxidant Power): The reducing power by the FRAP method was determined according to the method described by [13]. The sample solution (0.250 ml) was mixed with 0.625 ml of the phosphate buffer solution (0.2 M; pH 6.6) and 0.625 ml of potassium hexacyanoferrate (1%). The whole was incubated at 50˚C for 30 minutes in a sonicator. After incubation, 0.625 ml of trichloroacetic acid (10%) was added and the mixture was centrifuged at 3000 rpm for 10 minutes. After centrifugation was completed, 0.3125 ml of the supernatant was added to 0.3125 ml of distilled water and 0.0625 ml of freshly prepared iron chloride (0.1%). The absorbances were read at 700 nm against a standard curve of ascorbic acid (y = 3,270e-3 * x, r 2 = 0.9990). A series of 3 readings was carried out and the reducing power was expressed in μmol ascorbic acid equivalent for 1 gram of extract (μmol EAA/g of extract).

Determination of Antiplasmodial Activity in Vivo
This activity was carried out according to the protocol described by [15]. Seven groups of six mice including six test groups and one control group were used to carry out this test.
Mouse infestation: On D 0 , 10 7 parasitized erythrocytes, obtained by dilution of parasitized blood from mice infested with P. berghei ANKA were administered intraperitoneally to the groups of mice. The volume of parasitized blood to be injected into each mouse was 200 µL. Mouse treatment: 2 hours after the infestation, the mice except the control group receive orally, the dose of extract (250 mg/kg) intended for the group and the control group receives the dilution solution of the extract (distilled water). The dose is administered once a day for 4 successive days from D 0 to D 3 . The mice were observed during these 4 days to note any changes in behavior that Open Journal of Applied Sciences

Determination of Total Phenolics and Total Flavonoids
Total phenolic contents: The results of the phenolic contents were as shown in

Evaluation of Antioxidant Activity in Vitro
DPPH(2,2-diphenyl-1-picrylhydrazyl) In Figure 1  Comparison between the total flavonoids of aqueous and ethanolic extracts from leaves, barks and whole plant of Gardenia erubescens and Fadogia agrestis G. P-value *: difference was significant, P-value **: difference was very significant.  Regarding the ability to reduce ferric ions, the antioxidant activities of ethanolic and aqueous extracts of two plants varied from 11.52 to 39.24 µmol EAA/g extract. The aqueous extracts from leaves of G. erubescens and whole plant of F. agrestis had the best antioxidant activities compared to those of the other extracts. However, the ability of aqueous extracts from leaves of G. erubescens and whole plant of F. agrestis to reduce iron III to iron II was very low compared to that of trolox but similar to that of quercetin ( Figure 3). In view of these results, the aqueous extracts of the leaves of G. erubescens and of the whole plant of F. agrestis had interesting antioxidant activities by FRAP method.

Determination of Antiplasmodial Activity in Vivo
Impact The parasitaemia reduction percentages of ethanolic and aqueous extracts of leaves and bark of G. erubescens, as well as those of whole plant extracts of F. agrestis were between 10% and 50%. According to the scale of [16], these percentages of reduction were moderate ( Figure 5).

Discussion
In this study, the secondary metabolites, more precisely the total phenolics and total flavonoids, were determined by the method described in [13]. All the extracts were rich in total phenolics and flavonoids but their contents varied from one extract to another. This variation could be explained by the fact that the total phenolic and flavonoid contents varied according to the season and the geographical location [17] of the different plants. These results could also be explained by the extraction conditions (sample/solvent sample, extraction time, Open Journal of Applied Sciences temperature) [18]. As for the antioxidant activity, all the extracts by the three methods namely DPPH, ABTS and FRAP had exhibited antioxidant activities.
The presence of these activities within these various extracts could be due to the phenolics and total flavonoids previously measured in this study. As a reminder, total phenolics and total flavonoids are considered to be very powerful natural     during the injection of various plant extracts, considered to be foreign bodies for the body. In fact, inflammation is the first reaction to an attack on the body [24].
This inflammation will lead to an increase in the number of white blood cells, particularly neutrophils which are the first cells to arrive in large numbers at the site of inflammation [25]. During malaria there is an increased rate of lipoperoxidation which leads to an increase in parasitaemia [26]. In addition, the electrons produced during the oxidation of Fe II to Fe III following the degradation of hemoglobin by the parasite react with molecular oxygen to form free radicals [27]. These different phenomena lead to a decrease in the antioxidant system and to an increase in parasitaemia. Thus, the total phenolics and flavonoids which represent antioxidants dosed in our extracts could be at the origin of the inhibition of the production of reactive species leading to moderate reduction percentages of the parasitaemia of all the aqueous and ethanolic extracts of the two plants.

Conclusion
In present work, efficacy of ethanolic and aqueous extracts from leaves and bark of G. erubescens and whole plant of F. agrestis was investigated. For this, quanti-