Antiplasmodial Activities of Flavonoids from Leaves of Securidaca longepedunculata Fresen (Polygalaceae)

The discovery of new molecules for fighting against malaria is still relevant to overcome Plasmodium sp resistance. Phenolic compounds from medicinal plants have shown antiplasmodial properties. In addition, the targets of flavonoids on P. falciparum are multiple. This work aimed to identify the antiplasmodial compounds from methanol extract of Securidaca longepedunculata leaves. The inhibition of β-hematin formation was used to detect antiplasmodial compounds through a bio-guided chromatographic fractionation procedures. W2 strain was inhibited by flavonoids fractions Fc1 and Fb4 with 6.98 and 10.39 μg/mL as IC50 respectively. Also, fractions of phenol acids have shown good activities on the inhibition of β-hematin formation. The HPLC analyze showed that S. longepedunculata leaves extract contained quercetin, 3-β-quercetin, luteolin, chrysin, isorhamnetin, hyperoside, rutin, gallic acid, ellagic acid, chlorogenic acid, tannic acid and ferulic acid. Among these compounds identified, some had shown antiplasmodial and inhibitory activities on the formation of β-hematin. The antimalarial activity of the leaves of S. longepedunculata would be due in part to phenolic acids and flavonoids. The antiplasmodial activity observed in this work would be due in part to the ability of flavonoids from S. longepedunculata leaves to inhibit the formation of β-hematin. This finding could justify partially the S. longepedunculata uses in malaria treatment in Burkina Faso.


Introduction
The number of malaria cases recorded in 2016 was 216 million, an increase of 5 million compared to the previous year. Effective treatment of Malaria remains a challenge for modern medicine as part of its eradication. The complexity of development cycle and the ability of P. falciparum to resist against new antiplasmodial molecules posed a threat to the global health system, especially in sub-Saharan Africa [1]. Despite the progress made in the search for a vaccine against malaria, the observation is that none has passed the last experimental phase [2]. One of alternative to conventional medicine, is to promote heavily the use of medicinal plants for the malaria treatment [3,4]. These plants have always constituted an investigation source to research new interest compound [5]. Indeed, molecules belonging to different chemical groups with very interesting antiplasmodial activities have been isolated from antimalarial plants [6,7].
Securidaca longepedunculata Fresen (Polygalaceae) is a plant used to treat malaria in African traditional medicine [8]. The dichloromethane leaves extracts were inhibited the sensitive P. falciparum 3D7 strain grow with 6.69µg/mL as IC 50 [9,10]. The roots of this plant are very used [8]. In previous studies it was shown that after leaves extract defatted, the methanol extract had a very high antiplasmodial activity on the chloroquine-resistant strain K1 (IC 50 =2.33 µg/mL) [11]. This work consisted in a bio-guided fractionation of leaves methanolic extracts by using β-hematin formation inhibition assay. The results could help on the knowledge of the active compounds and one of the possible mechanisms of action.

Chromatographic Analysis
The Fractions with interesting inhibition activity were selected for the flash chromatography (Puriflash 215; column 50 STD -25.0 g, 15 bars). The sub-fractions resulting from flash chromatography were grouped according to their thin layer chromatography profile followed by the assessment of their inhibitory activity. The active sub-fractions were refractionated by using preparative thin layer chromatography for isolating the compounds fractions with β-hematin inhibitory activity.
Main bioactive fraction was analyzed using an HPLC-UV system (Ultimate 3000, C18 column (150mm×4.6mm), λ=254 or 365 nm). Data from HPLC was integrated by Ultimate 3000 software and results were obtained by comparison with external standards retention time (t R ) and UV spectral. Mobile phase consisting of solvent A (Water +Formic acid 0.2%), solvent B (methanol+ Formic acid 0.2%) and at a flow rate of 15mL/min. The isocratic elution program was used for chromatograms development.

Inhibition of β-hematin Formation
The inhibition of β-hematin formation was assessed by according to methods from the literature [12,13]. Freshly prepared hemin chloride solution (100 µL, 0.5mg/ mL in DMSO) was mixed with sodium acetate buffer (200 µL, 0.5M in water, pH 4.4) and test sample at 100µg/mL in mixture. Test mixture was incubated (37°C for 18 to 24h) and centrifuged (4000 rpm for 10 min) for discarding supernatant. Total pellet was suspended in DMSO (400 µL) and centrifuged (4000 rpm for 10 min) for dissolving the remaining hemin chloride. β-hematin pellet within tubes is solubilized with NaOH (400 µL, 0.1M). The negative control contained water. The absorbance (Abs) of β-hematin solution was read at 405nm. Data were expressed as the percentage of inhibition of β-hematin formation and calculated by using the equation (1).

Inhibition of Plasmodium Falciparum Parasite Growth
The antiplasmodial activity was determined according to Makler et al. method [14]. Test sample) was mixed within culture medium (O + blood: 2% hematocrit, parasitaemia: 1.5%). After incubation (72 hours, 37°; 5% CO 2 , 2% O 2 , 93% N 2 and 95% of humidity) parasite growth was measured with Malstat reagent and NBT/PES solution. The absorbance (Abs) was recorded at 650nm against a blank (culture medium without test sample). Data were expressed as inhibition percentage of parasite growth and calculated by the equation (2). The concentration inhibiting 50% of the parasite growth (IC 50 ) was determined.

Statistical Analyses
The results were expressed as mean value of three (n=3) independent experiments ± standard deviation. Table curve 2D v.2 was used to determine IC 50 . Statistical analysis of data was performed with the XLSTAT pro 7.5. ANOVA for p<0.05.

Inhibition Activity of ß-hematin Formation
The ability of crude extract and 12 fractions to inhibit the formation of β-hematin was summarized in the Figure 1. The percentages of inhibition were ranged from about 5.97 to 82.96%. The extract inhibited 53.24% of β-hematin formation in vitro. The Fractions with ameliorated inhibiting effect were F4 (73.79%), F5 (81.96%), F6 (71.66%), F7 (82.96%), F8 (72.71%) and F11 (53.57%). All activities from fraction were less than chloroquine inhibitory activity (91.51%). This increased inhibition activity in some fractions suggested that the increasing of bioactive compounds concentration in these fractions. The fractionation by flash chromatography of one gram of the mixture of F7 and F8 gave twenty-seven (27) sub-fractions (F1'-F27'). According to their TLC chromatographic profile, these sub-fractions were grouped into 4 groups which were Fa (F1'-F'7), Fb (F'8), Fc (F9'-F'25) and Fd (F26'-F27') which were submitted to the β-hematin inhibition assay and the result was indicated by the Figure 2. The high activity was recorded with the sub-fractions Fb and Fc that showed 76.52% and 75.06% as inhibition percent respectively. Again, the best sub-fraction inhibition activity was less than Chloroquine inhibition activity. The observed activities of fractions could be due the presence of flavonoids and phenolic acids detected by TLC analysis. Particularly, flavonoid can form complex with iron (iii) ions and induced the non-formation of βhematin [15,16]. The data were expressed as the percentage of inhibition. Data followed by same letter are not significantly different at p>0.05.

Figure 2. Inhibition of β-hematin formation by sub-fractions (Fa-Fd).
The data were expressed as the percentage of inhibition. Data followed by same letter are not significantly different at p>0.05.

Sub-fractions Antiplasmodial Activities
The figures 3 and 4 showed the inhibition of β-hematin formation and W2 growth of fractions from sub-fractions Fb and Fc. The best sub-fractions were Fb4 (78.14%), Fc1 (72.09%), Fb3 (66.68%), Fc4 (64.82%) and Fb2 (61.94%) concerning the inhibition of β-hematin formation. But their activity was less than chloroquine inhibition effect. Fc1 and Fb4 were able to inhibit significantly the W2 development according to the figure 4 with IC 50 as 6.98 and 10.39 µg/mL respectively. Fc1 was twice active than fraction F8 and 8 times active than the methanol extract according to this data. These indications confirmed the presence of antiplasmodial compound in the extract and its fractions. In TLC, Fb4 and Fc1 are flavonoid fractions having the same frontal references as quercetin (Rf = 0.91) and 3-βquercetin (Rf = 0.56) respectively.  IC 50 : The concentration inhibiting 50% of the parasite growth. Data followed by same superscript letter are not significantly different at p>0.05. DHA: Dihydroartemisinin (ng/mL). Fb4 and Fc1 are flavonoid fraction from subfractions Fb and Fc respectively. F8: fraction 8 from crud extract.

Compounds Identification by HPLC
In-depth phytochemistry study of the F8 fraction by HPLC analysis allowed to have the data in the table 1. The leaves extract from S. longepedunculata contained phenolic acids such as gallic, ellagic, chlorogenic, tannic, ferulic acids and flavonoids such as luteolin, rutin, chrysin, isorhamnetin, hyperoside, quercetin and 3-β-quercetin. The identification of these compounds in the leaves extract of S. longepedunculata was reported in the first time. The flavonoids fractions Fb4 and Fc1 would be or would content quercetin and 3-βquercetin respectively according TLC data and their identification by HPLC analysis in the bioactive fraction. The richest in flavonoids and phenolic acids confirmed the TLC profile and could supported the biological data. So, the ability of quercetin to inhibit β-hematin formation was reported by Manu et al. [17,18] who found 0.15 µg/mL as IC 50 and 3-β-quercetin can prevent red blood cell membrane damage [17,18]. This preventive property would be involved in cell membrane protection against lipid peroxidation induced by free radical during P. falciparum infection [19]. The inhibitory activity of 3-β-quercetin on the formation of β-hematin and P. falciparum resistant grown is the first to be reported according to our best bibliographic knowledge. Quercetin and luteolin were able to inhibit falciparum sp. like 3D7, 7G8 and NF54 [17,20,21]. Again, rutin inhibited RIO-2, the P. falciparum enzyme involved in cell cycle progression, chromosome stability [22]. The gallic acid were 3D7 antiproliferative compound and tannic acid was inhibited β-hematin formation [23,24]. The mechanism action of flavonoids and phenolic acids were attributed to their hydroxyl groups [25].

Conclusion
The methanol extract and its fractions from leaves of S. longepedunculata showed some antiplasmodial potential and this activity was explained partially by the 5 phenolic acids and 7 flavonoid compounds. These compounds could contribute to the leaves antimalarial uses by various mechanisms in synergic action that allow resistance disturbing. The present work results could be a preliminary basis for further studies such as evaluating the effect of S. longepedunculta leaves extracts on P. falciparum gametogenesis or combinatory activity with some standard antiplasmodial compounds.