Phytochemical, biological and computational investigations of Erythrina fusca Lour. to assess antimalarial property against Plasmodium falciparum

For centuries medicinal plants have been traditionally used for prophylaxis and ailment of diseases. Nowadays it’s easy to isolate, purify, and characterize bioactive compounds with high efficacy. To investigate the medicinal especially antimalarial property of traditionally used plants, a number of Erythrina spp have been reviewed systematically where Erythrina fusca has been selected for further analysis. Phytochemical investigation included chromatographic separation and purification of compounds followed by characterization using NMR. In-vitro antimalarial drug sensitivity ELISA was carried out against chloroquine (CQ) sensitive 3D7 and resistant Dd2 strains. Additional biological tests such as central and peripheral analgesic, antioxidant, anti-diarrheal, hypoglycemic, thrombolytic, and membrane stabilization activities were also investigated. Molecular docking was performed using the isolated compounds against clinically important 14 Plasmodium falciparum proteins. For the first time, Phaseolin, Phytol, β-amyrin, Lupeol, and Stigmasterol are reported here and extracts showed significant antimalarial activity against 3D7 and Dd2 strains (IC50 4.94-22 μg/mL). Potent central analgesic, antioxidant and anti-diarrheal activities (p<0.05) and mild thrombolytic and membrane stabilization properties were also observed. Molecular docking of Phaseolin bolsters its potential as a new antimalarial drug candidate. This study projects significant medicinal values and necessitates further investigations to reveal its potential as a novel source of therapeutics.

isolation, characterization using chromatographic and spectroscopic techniques. The purpose includes biological investigations of the different fractions through bio-guided assays [4]. Depending the characterization of the isolated compounds, in-silico ligand-protein interaction was also considered. On ethno-pharmacological point of view, Erythrina spp. a flowering plant in the family of Fabaceae, had been systemically reviewed. There are about 130 species in this family which are mainly distributed in tropical and subtropical regions all over the world. Erythrina fusca is one of the less investigated plants comparing other species of this genus and locally known as "Harikakra" in Bangladesh. E. fusca is a medium to large multi-branched, soft-wooded, spreading, deciduous tree usually ranging from 10-15m (max 30m). It is widely grown in tropical and Central America as a shade tree for cocoa and coffee as well as for ornamental purpose. Currently plants of this genus have been used for its antibacterial, anxiolytic, antinociceptive, antioxidant, antiplasmodial, etc. activities [5]. Therefore, it requires searching other potential properties as well as screening of medicinal values of unsearched species of this genus.

Antimalarial activity:
The crude as well as the partitionates have high to moderate antimalarial activity against CQsensitive 3D7 and resistant Dd2 strains (IC50 4.94-22 µg/mL) as summarized in Table 1. The aqueous fractions exerted the most potent activity with IC50 4.94µg/mL against 3D7 strain. For the Dd2 strain, the n-hexane partionates proved to be the most potent one with IC50 05µg/ml.

TPC test:
The total phenolic content of the partitionates were found between 73.87 to 164.16mg of gallic acid equivalent (GAE)/gm of extractives which is depicted in Table 2. The highest TPC was demonstrated by n-hexane partitionates with 164.84mg of GAE/gm of extractives.

DPPH Assay:
In the DPPH assay, the IC50 values ranged from 14.02 to 57.25 (µg/ml) with the highest free radical scavenging activity found in the chloroform fraction (IC50 57.25 µg/ml) as shown in Table 2.

Thrombolytic and anti-inflammatory response:
Moderate thrombolytic activity was observed among the partitionates with chloroform fraction possessing the highest one (42.06 ± 0.33%). Streptokinase was used as standard with lysis of 69.99 ± 0.55% at 37°C and a minimal lysis was observed in the blank.
For anti-inflammatory response investigations, chloroform soluble fraction showed highest inhibition in hypotonic solution induced hemolysis with 86.19 ± 0.27% inhibition compared to 79.30 ± 0.93% inhibition in standard acetyl salicylic acid (ASA). In heat induced hemolysis method, aqueous fraction exerted 38.32 ± 0.92% inhibition compared to 43.70 ± 0.97% inhibition found in ASA. These results are found in Table 3.

CNS activity:
Statistical analysis indicated that both doses of crude extracts of E. fusca at 200mg and 400mg/kg body weight exerted extremely central analgesic effect after 30mins, 60mins and 90mins of administration as shown in Table 4.

PNS Activity:
The crude extract of E. fusca showed significant peripheral analgesic activity with high percentage inhibition of writhing both at 200mg/kg and 400mg/kg body weight. Thus it exposes significant peripheral analgesic activity which is depicted in Table 5.

Antidiarrheal Activity:
The methanolic crude extract of bark of E. fusca at both doses of 200mg/kg and 400mg/kg body weight showed moderate activity at the first and second hour of administration of the test compound.
However, the activity decreased significantly at the third and fourth hours as shown in Table 6.

In silico studies:
High binding affinity were observed between the proteins and the ligand. Among the 14 proteins, pdb id 5jaz (-9.3kacl/mole), 5k8s (-8.8 kcal/mole) and 6ee4 (-8.7 kcal/mole) expressed excellent binding affinities which are described in Table 7 and       Table 4: Tail immersion effect of the crude extracts of bark of E. fusca   Among these, to the best of our knowledge, all compounds appear to be the first report from E. fusca.
Phaseolin (1)  Hz) was assigned to the proton at C-12 suggesting a skeleton of the olean-12-ene-form. Hence, 001 was characterized as β-amyrin by comparing its spectral data to reported values in addition to co-TLC with genuine sample [6]. Additionally, the doublet (J = 6.4 Hz) at δ 0.91 integrated for three proton indicated the primary methyl attached to C-28 (H3-29). The above spectral features have resemblance for stigmasterol that was further confirmed by Co-TLC with original sample [8].
Phytol (5) A doublet observed at δ 0.89 was assigned to the six methyl protons positioned at C-7 and C-11.
Moreover, doublet at δ 1.08 (J = 7.6 Hz) was ascribed to H3-16 and H3-17 (C-15) for another six protons of methyl groups. These features were similar to those of phytol and therefore, 003 was characterized as a diterpene alcohol Phytol [9].
There are many species in the Erythrina genus that had been extensively studied for different biological activity and diversified medicinal values. The species investigated in this studies is also found throughout the world [10]. . Bactericidal effects had been reported in the leaves aquadest extract of the E. fusca [11]. Other than antibacterial effect, it is also effective against malaria parasites [12]. Subal et. al experimented on the antiepileptic and antioxidant property of the hydro-alcoholic extract of bark of E. fusca [13,14]. The antiepileptic effect noticed might be due to the enhancing of the gamma amino butyric acid (GABA) [13] and they also found an increase in the superoxide dismutase activity and reduced glutathione levels as well as reduced level of malondialdehyde (MDA) that may be due to its antioxidant property [14]. In our study, we performed DPPH assay and fusca against multi-drug resistant Pf strain K1 [12]. However, in that study the stem bark of E. fusca had not been used for medicinal resources and only three of the isolated compounds expressed activity at concentration <12.5µg/ml [12]. In comparison, we performed antimalarial assay of the crude extract of the bark of E. fusca and its different Kupchan partitionates against CQ sensitive 3D7 and resistant Dd2 strain and observed significant antimalarial effect. Here, IC50 4.94µg/mL and 05µg/ml were obtained in aqueous fractions and n-hexanes fractions against 3D7 and Dd2 strains respectively. Their antimalarial activity was categorized as: high (IC50<5 µg/mL), promising (5< IC50<15 µg/mL), moderate (15< IC50<50 µg/mL) and inactive (IC50>50 µg/mL) based on previous reports. [15][16][17] Other than the aforementioned biological activities, significant central analgesic activity has been observed through high percentage of elongation time in mice (71. 16

Preparation of Plant Extract and Fractions:
The bark was shade dried at rooftop for a week and ground to a coarse powder. Subsequently, 500gm dried coarse powder was soaked in 2L of methanol for 2 weeks with regular frequent stirring for the extraction of compound(s) through cold maceration. The filtrate was then collected through multiple filtrations with cotton plug, Whatman no. 1 filter paper as well as decantation and evaporated to dryness using rotary evaporator (Büchi ® rotary evaporator Model R-200). The fractionation was done using slightly modified version of Kupchan method of solvent-solvent partitioning [18] and yielded three partitionates; n-hexane, chloroform, and left over mother methanolic aqueous fraction which were stored in 4°C in aliquots of different concentration range (10-100µg/mL) for further biological activity analysis.

Size Exclusion Chromatography:
Three gm of n-hexane and chloroform extracts were subjected to size exclusion chromatography using lipophilic Silica 60G and later 600mg of similar fractions were subjected to Sephadex (LH-20).
Gradient solvent system of n-hexane, ethyl acetate, and methanol were used resulting in 48 different fractions. For the Sephadex column, the solvent system was n-hexane: chloroform: methanol (2:5:1) initially and then 10% methanol in chloroform with gradual increase by 10% up to 100% methanol.
The preparative thin layer chromatography (PTLC) was performed on several fractions based on their TLC behavior. Eight of the purified compounds, four from n-hexane and four from chloroform partition, were spectroscopically analyzed. and CQ-resistant Dd2 (MRA-156) strains were used from BEI-resources (MR4/ATCC® Manassas, VA, USA). The continuous in-vitro culture of asexual Pf blood stage was maintained as described by Trager and Jensen with slight modification [19].
(ii) Evaluation of in-vitro antimalarial activity: Antimalarial assay was performed using HRPII based enzyme-linked immunosorbent assay (ELISA) method described by Noedl et al. and WWARN [20][21][22]. The data is analyzed using GraphPad  [30]. Crystal structures were obtained from the Protein Data Bank (PDB) and prior to docking ligand, water molecule removal and necessary energy minimization was performed. The centre grid box was positioned to cover all the active sites in x,y and z directions and torsional rotation was allowed for optimal flexible docking (see Fig 1).

Conclusions
Repeated chromatographic separation and purification of the barks of Erythrina fusca Lour.
(family: Fabaceae) afforded eight compounds; among which five were identified. The extractives of E. fusca revealed significant antimalarial, central analgesic, and antioxidant activities and moderate anti-diarrheal, thrombolytic and membrane stabilization properties. Moreover, molecular docking of the 3D simulated compound phaseolin in silico expressed excellent binding affinity towards clinical antimalarial drug target in virtual screening via AutoDock Vina that indicates its potential as future antimalarial drug. Therefore, the plant materials can be further investigated extensively to find out its unexplored efficacy that may be helpful in the discovery of a new lead compound.