Phytochemical screening and antioxidant activity of methanolic extracts of 53 antimalarial plants from Bagira in Eastern DR Congo

A previous study inventoried 53 plants used in traditional medicine in Bagira in Eastern Democratic Republic of Congo (DRC) in the management of malaria. During malaria disease, oxidative stress is responsible for the worsening of the patient's condition. This study aims to identify phytochemical groups and to evaluate antioxidant activity of 53 plants used in traditional medicine in Bagira to treat malaria. The phytochemical screening was carried out by conventional reactions in solution and antioxidant activity used in vitro method with 1,1-diphenyl-2picrylhydrazyl radical (DPPH). Chemical screening has identified secondary metabolites with both antimalarial and antioxidant potential such as coumarins, steroids, saponins, tannins and terpenoids in more than 70% of plants. Antioxidant screening revealed for the first-time antioxidant activity of 18 plants, among which Dalbergia katangensis, Dialium angolense and Solanecio cydoniifolius with IC50 ≤ 1.6 μg / mL having the highest activities. This study shows that among plants used as antimalarial in Bagira several possess antioxidant power and contain many of groups presumed to be both antioxidant and antimalarial. This suggests that further studies continue to isolate compounds responsible for the proven activity.


Introduction
Oxidative stress results from a profound imbalance between oxidative systems and the body's antioxidant capacities in favor of the former [1]. Unbalanced, it leads to irreversible cell damage [2] responsible for aging and many conditions such as obesity [3], type 2 diabetes [4], atherosclerosis [5], cancer [6] or virus diseases [7] requiring the use of antioxidants. Several synthetic antioxidants used in the past have been abandoned because of their increased risk of toxicity in favor of natural antioxidants [8], which motivates the screening of plants with antioxidant potential.
Studies have shown that during a malarial disease oxidative stress occurs which can progress to cerebral malaria or anemia [9,10]. Thus, studies have been carried out with a view to seeking both plants with antioxidant and antimalarial potential. This is the case with the work of Saliq et al [11] or Sulistyaningsih et al [12] like so many others [13][14][15][16]. Another advantage of this approach is that it allows the discovery of new antimalarial molecules with new mechanisms of action likely to overcome the resistance problems facing current antimalarials [17,18]. This approach to screening plants with dual potential has seen some studies lead to the isolation of natural molecules that are both antimalarial and antioxidant. This is the case of mammea A / AA cyclo D, a coumarin isolated from the stem bark of Mesua borneensis (P. F. Stevens), a Calophyllaceae [19] or that of Lonchocarpol A, a flavonoid isolated from the stem bark of Erythrina crista-galli L., a Fabaceae [20]. Furthermore, the isolation of a bioactive molecule is conventionally preceded by the search for secondary metabolites with the desired potential. Beyond this interest, this screening also makes it possible to provide new knowledge on the chemical composition of the plant concerned on the major phytochemical groups of secondary metabolites of plants. In the case of malaria, bibliographical reviews [21][22][23] have highlighted alkaloids, flavonoids, coumarins, quinones, steroids, terpenoids as phytochemical groups with antimalarial potential. Among these phytochemical groups, flavonoids, coumarins, and terpenoids are particularly reported as groups with antioxidant potential [24,25].
This study focused on 53 plants used in traditional medicine in Bagira, in the treatment of malaria, to assess their antioxidant potential in vitro and to search for phytochemical groups with antiplasmodial potential. These plants come from an ethnobotanical study carried out on antimalarial plants from Bagira, such as the city of Bukavu in the eastern DRC.

Plant material
The plant material consisted of the leaves, stems, roots, flowers, fruits, and aerial parts of 53 plant species taken from a database of a survey we conducted in Bagira in 2013-2014. These plants have been collected in Bukavu in the company of traditional healers and the herbaria created for this occasion were deposited at the IRS Lwiro herbarium where the identity of the plants was determined ( Table 2). After drying at room temperature, the plant material was ground using a stainless-steel electric mill (Plymix PX-MFC 90 D, Belgium) and then kept cool before handling. The choice of organs to screen was related to availability at harvest. Thus, for the herbs, we screened the aerial parts consisting mainly of leaves and stems without discrimination.

Obtaining extracts
The extracts were obtained by maceration of 350 g of powder in 1.5 L of methanol (Sigma Aldrich, USA) for 72 hours at room temperature then filtered through paper (Whatman, USA) and concentrated on a rotary evaporator (Büchi R -210, Switzerland) at a pressure of 180 mbar and a temperature of 40 ° C.

Substrate and positive control
DPPH (Sigma Aldrich, United Kingdom) was used as a substrate for the evaluation of antioxidant activity. It was prepared at 0.002% (w / v) in methanol. L-ascorbic acid (Sigma Aldrich, China) used as a reference antioxidant substance made it possible to prepare a standard curve with 5 successive dilutions of order 2 carried out from a solution of ascorbic acid at 40 µg/mL ( y = 0.0298X +0.0071; r 2 = 0.9997).

Identification of secondary metabolites
The phytochemical screening was carried out using conventional reactions in solution in tubes, based on staining, precipitation, or the formation of foams. It consisted in looking for alkaloids, anthocyanins, coumarins, flavonoids, quinones, saponins, steroids, tannins and terpenoids for their antiplasmodial or antioxidant potential and cyanogenic heterosides for their toxic potential, following the protocols previously described [26][27][28].

Alkaloids
The detection of alkaloids consisted in precipitating them using six precipitation reagents. Briefly, 1 g of powder of dry plant material was macerated in 10 mL of methanol at room temperature for 24 hours and then in an oven at 50 ° C for 4 hours. The solution obtained was filtered then the marc washed three times with portions of hot methanol. The filtrate was evaporated to dryness in an oven at 50 ° C and the residue was collected twice with 2 mL of hot 1% hydrochloric acid solution (Sigma-Aldrich, USA). The acid solution obtained was basified with 1 mL of concentrated ammonia (Sigma-Aldrich, UK), placed in a separating funnel (VWR, Belgium) and then mixed with 5 mL of chloroform (Sigma-Aldrich, USA). After stirring, the two phases were separated, and the operation was repeated three times. The organic phase was evaporated to dryness in the open air, the residue obtained was taken up in 0.5 mL of chloroform and the solution, transferred to a test tube, was mixed with 0.5 mL of 1% HCl thus forming two phases. The aqueous phase, which is above, was removed using a Pasteur pipette. Six drops were placed on a microscope slide. Each of these drops was treated with one drop of one of six precipitation reagents namely Dragendorff, Mayer, Hager, Wagner, Bertrand, and Sonnenschein reagent. The presence of alkaloids was only considered certain if each of the six reagents gave a precipitate.

Coumarins
Coumarins were identified by the alkaline reaction. Briefly 0.5 g of the moistened various extracts was taken in a test tube. The mouth of the tube was covered with filter paper treated with 1 N NaOH solution. Test tube was placed for 5 minutes in boiling water and then the filter paper was removed and examined under the UV light for yellow fluorescence indicated the presence of coumarins.

Flavonoids and anthocyanins
The flavonoids have been demonstrated by the Shinoda test. Briefly, 5 g of plant material placed in an Erlenmeyer flask was infused in 50 mL of distilled water for 30 minutes. 5 mL of filtrate were then treated successively with 5 mL of concentrated HCl, 5 drops of isoamyl alcohol and 1 mg of magnesium shavings. The red-orange (flavone), red or redviolet (flavonones), cherry red (flavonol) coloration appeared in the supernatant layer if the solution contained the flavonoids. Likewise, the reaction carried out for two minutes in a water bath in the absence of magnesium chips allowed the characterization of anthocyanins with the appearance of a red color.

Cyanogenic heterosides
Cyanogenic heterosides were identified by the reaction with picric acid. Briefly, 5 g of vegetable powder was placed in an Erlenmeyer flask with 10 mL of distilled water. The container was closed with a stopper to which was attached a strip of picrosodium paper lightly moistened with water and the contents were slightly heated (to 60 ° C). The yellow picrosodium paper turned orange or red if the plant extract had released hydrocyanic acid.

Quinones
Quinones were identified by the Borntrager test. Briefly, 5g of powdered plant material was macerated for 24 hours in 50mL of petroleum ether. After filtration, 10 mL of ethereal filtrate was treated with 5 mL of 10% NH3. The appearance of a purplish red color in the aqueous phase indicated the presence of free quinones and that of yellow or orange colors, the bound quinones.

Saponins
Saponins were identified by the foaming reaction. Briefly, 10 g of coarsely ground plant material was treated with 100 mL of distilled water to make a decoction for 30 minutes and the mixture was filtered through filter paper after cooling. 15 mL of the decocts were then introduced into a test tube 16 mm in diameter and 160 mm in height. The contents of the tube were shaken tightly for one minute and then allowed to stand for 10 minutes. The appearance of a persistent foam greater than 10 mm in height indicates the presence of saponins.

Steroids and terpenoids
Both steroids and terpenoids have been defied by the reaction with sulfuric acid. Briefly, 5 g of plant material was macerated for 24 hours in 100mL of petroleum ether. After filtration, the solvent was evaporated to dryness. In the residue obtained, were added successively and with stirring, 2 mL of chloroform and three drops of concentrated sulfuric acid. The appearance of purple or green colorings indicated the presence of steroids. The identification of terpenoids followed the same pattern as that of steroids. In addition to the reagents used for steroid testing, a few drops of Hirschson reagent (concentrated acetic anhydride) were added to 4 mL of the acidified solution. Yellow staining turning red indicated the presence of terpenoids.

Tannins
The tannins were identified according to the protocol below: 5 g of plant material were infused in 50 mL of water contained in an Erlenmeyer flask for 30 minutes. 5 mL of the infused was taken and mixed with 1 mL of 1% ferric chloride. The test was considered positive when either a precipitate appeared or a blue-green, dark blue or green color. 15 mL of Stiasny reagent (10 ml 40% formalin and 5 mL concentrated HCl) was mixed with 30 mL of the infused and the mixture was brought to a water bath at 90 ° C. The appearance of a precipitate indicated the presence of catechetical tannins. The solution was then filtered, and the filtrate was saturated with sodium acetate before adding a few drops of ferric chloride thereto. The formation of a precipitate in this case revealed the presence of gallic tannins.

Antioxidant activity test with DPPH
Antioxidant activity was assessed using the DPPH assay [29]. Briefly, 50 µL of extract or positive control prepared at different dilutions of order 2 in methanol from a 100 µg / mL solution were interacted with 1950 µL of 0.002% DPPH in test tubes. (Nunc WVR, Germany). After mixing and incubating in the dark for 30 minutes, the absorbance of the solution was read at 492 nm (Thermo Fisher Scientific Inc. spectrophotometer, Waltham, USA). The tests were carried out in triplicate and the percentage of antioxidant activity was calculated by the formula: with Ab = absorbance measured in the presence of the negative control, Ae = absorbance measured in the presence of the extract and % AAO = Percent inhibition and expresses antioxidant activity. This percentage of activity made it possible to generate the IC50 or concentration at which the extract has 50%, to categorize the extracts.

Statistical analysis of data
GraphPad Prisme version 6 software (GraphPad Software, La Jolla, USA) was used to perform statistical analysis of the data and generate the IC50s. The analysis of the variables was carried out by one-way ANOVA with the significance level set at 95%.

Classification of species according to the number of phytochemical groups identified
Depending on the number of phytochemical groups identified within each plant, all the organs together, the 53 plant species can be grouped into 5 classes (Classes A to E). Although almost 70% of plant species contain 7 phytochemical groups, only 6% of plants contain the nine phytochemical groups with therapeutic potential (Figure 1).
Class A: species with 5 phytochemical groups; Class B: species with 6 phytochemical groups; Class C: species with 7 phytochemical groups; Class D: species with 8 phytochemical groups; class E: species with 9 phytochemical groups.

Classification of phytochemical groups identified in the 53 plants
The

Antioxidant activity of methanolic extracts from 53 selected plants
Regarding the IC50 values, the 147 extracts obtained from the 53 plants can be grouped into 4 classes. Class 1 is that of very active extracts (IC50 ≤ 1.6 µg / mL), class 2 is that of active extracts (1.6 <IC50 ≤ 50 µg / mL), class 3 contains weakly  (Table 2).

Discussion
During this study, 53 plants selected from an ethnobotanical survey carried out on plants known to be antimalarial in Bagira in eastern DRC were studied. This study focused on the search for secondary metabolites in various organs of these plants, and the demonstration of the antioxidant potential of methanolic extracts from their organs used as antimalarial drugs in Bagira. The interest in evaluating the antioxidant potential of antimalarial substances comes from the fact that plants with antioxidant potential could prevent the oxidative stress which occurs in malaria disease. As for the phytochemical screening of secondary metabolites, it constitutes the first step towards the isolation and characterization of the active compounds. The results of this study show that 30 of these 53 plants are already known from a phytochemical point of view although no antimalarial molecules have been reported (Table 1). This study confirmed previous results for some of these species. This is the case with flavonoids and terpenoids in Chenopodium opulifolium [45] or flavonoids and terpenoids in Ekebergia benguellensis [51]. Furthermore, some previous phytochemical knowledge has not been confirmed by this study. This is the case of the species Ekebergia benguelensis where we did not find coumarins in the root bark and yet previously 4-methoxy-5-hydroxymethylcoumarin had been isolated there [50]. The fact that the plants were not harvested in the same environment is a likely explanation for these observed disparities given that the phytochemical composition of the plant in secondary metabolites depends on several factors such as climate, age of the plant or the place of harvest [115]. It could also be varieties different from those studied previously. It would therefore be interesting to carry out a simultaneous study between these different specimens to have a clear point of view.
Terpenoids and flavonoids were the most frequent alongside several metabolites with antiplasmodial and antioxidant potential (Table 1 and Figure 3). Note that several studies have reported the preponderance of these metabolites among phytochemical groups with antimalarial potential [21,22]. The identification of these phytochemical groups within these 53 plants could constitute a first orientation for a more in-depth screening possible mainly on the 11 plants which were until then little known from a phytochemical point of view.
Only 18 plants (32%) have never been assessed for prior antioxidant activity ( Among the 18 plants so far unrecognized from the point of view of antioxidant activity, 11 are also unknown from the phytochemical and antimalarial point of view [116]. These species are Aframomum laurentii, Clematis villosa, Crassocephalum montuosum, Crassocephalum picridifolium, Dalbergia katangensis, Dialium angolense, Isoberlinia angolensis, Isoberlinia tomentosa, Julbernardia paniculata, Rothmannia engleriana and Solanecio cydoniifolius. Over 80% of these plants contain coumarins (100% of plants) and terpenoids (81.2% of plants). However, it has been previously reported that coumarins [114,117] and terpenoids [118,119] constitute true groups of natural antioxidants. Their frequent presence in the plants could therefore constitute an explanation of the antioxidant activity demonstrated in the extracts examined. On the other hand, the fact that 68% of plants have already been investigated for antioxidant activity suggests that there is a high probability of encountering in the 32% of plants not studied, interesting antioxidant compounds.

Conclusion
This study highlights for the first time the antioxidant activity of 18 plants in which several phytochemical groups with both antioxidant and antimalarial activity such as flavonoids and terpenoids are frequently found. It shows that among these plants, 11 are also unrecognized from the phytochemical antioxidant and antimalarial point of view. She suggests that these plants, such as Dalbergia katangensis, Dialium angolense and Solanecio cydoniifolius, whose antioxidant activity has just turned out to be interesting, be further investigated in the hope of discovering compounds that are both anti-free radicals and antimalarial.