In Vitro and In Silico Potential Inhibitory Effects of New Biflavonoids from Ochna rhizomatosa on HIV-1 Integrase and Plasmodium falciparum

The aim of this study was to identify bioactive secondary metabolites from Ochna rhizomatosa with potential inhibitory effects against HIV and Plasmodium falciparum. A phytochemical study of O. rhizomatosa root barks resulted in the identification of three new biflavonoids (1–3), along with four known ones (4–7). Compound 7 (Gerontoisoflavone A) was a single flavonoid present in the rootbark of the plant and was used as a reference. Compound 1 (IC50 = 0.047 µM) was the only one with a noteworthy inhibitory effect against HIV-1 integrase in vitro. Chicoric acid (IC50 = 0.006 µM), a pure competitive inhibitor of HIV-1 integrase, was used as control. Compound 2 exhibited the highest antiplasmodial activity (IC50 = 4.60 µM) against the chloroquine-sensitive strain of Plasmodium falciparum NF54. Computational molecular docking revealed that compounds 1 and 2 had the highest binding score (−121.8 and −131.88 Kcal/mol, respectively) in comparison to chicoric acid and Dolutegravir (−116 and −100 Kcal/mol, respectively), towards integrase receptor (PDB:3LPT). As far as Plasmodium-6 cysteine s48/45 domain inhibition is concerned, compounds 1 and 2 showed the highest binding scores in comparison to chloroquine, urging the analysis of these compounds in vivo for disease treatment. These results confirm the potential inhibitory effect of compounds 1 and 2 for HIV and malaria treatment. Therefore, our future investigation to find inhibitors of these receptors in vivo could be an effective strategy for developing new drugs.


Introduction
Malaria and HIV/AIDS are among the main illnesses Sub-Saharan Africa is facing. In many countries in this area, such as Cameroon, both diseases are endemic, and the HIV/AIDS infections may increase the burden of malaria. This may occur by increasing the susceptibility to malaria infection, hence, making these diseases deadlier [1].

Extraction of Plant Materials and Isolation of Compounds
The root barks of Ochna rhizomatosa (500 g) were dried and crushed before extraction using CH2Cl2/MeOH 1:1 (3 × 3 L) at ambient temperature to afford 87 g of crude extract after evaporation under reduced pressure. Part of the crude extract (80 g) was chromatographed on silica gel using a stepwise gradient condition and a mixture of CH2Cl2/MeOH (100:0 to 5:1) as solvents, resulting in 185 fractions A-H. Fraction D (CH2Cl2/MeOH; 30:1) was further purified by column chromatography using Sephadex LH-20 in acetone to yield compounds 2 (16 mg) and 7 (30 mg). The purification of fraction E (CH2Cl2/MeOH; 20:1) by column chromatography was also performed using Sephadex LH-20 in the same conditions and afforded compounds 4 (15 mg) and 5 (20 mg). For fraction F (CH2Cl2/MeOH; 20:1) a purification via a conventional RP-C18 column eluting with an MeOH/H2O system resulted in the isolation of compound 3 (100 mg). The fraction G was chromatographed by a RP-C18 column using the same conditions to yield compound 1 (150 mg). Lastly, fraction H [CH2Cl2/MeOH (5:1)] was subjected to repeated Sephadex LH-20 column chromatography eluted with an isocratic elution of CH2Cl2/MeOH (10:1), and this resulted in the isolation of compounds 1 (11 mg) and 6 (40 mg). 20 D ) were measured in methanol on a JASCO 810 polarimeter in a 1 cm tube. The FTIR spectra were recorded on a Perkin Elmer FT-IR spectrometer (Thermo Scientific, Madison, WI, USA). The NMR spectral data were recorded using a Bruker Avance DPX-400 spectrometer operating at the frequencies of 400 MHz ( 1 H) and 100 MHz ( 13 C). The samples were dissolved in MeOH-d 4 prior to recording. Chemical shift values were given in parts per million (ppm) from the internal standard. HRESIMS spectral data resulted from a MSQ Thermo Finnigan. Column chromatography (CC) was carried out on a medium preparative liquid chromatography (MPLC) system (RP-18, 0.040-0.063 mm, Merck MGaA, Darmstadt, Germany), Sephadex LH-20 (0.025-0.100 mm; GE Healthcare, Danderyd, Sweden) using silica gel 60 (Merck, 0.040-0.063 mm Qingdao, China). Thin layer chromatography (TLC) experiments were performed on pre-coated Merck Kieselgel 60 F 254 plates (20 × 20 cm 2 , 0.25 mm, Qingdao, China).

Extraction of Plant Materials and Isolation of Compounds
The root barks of Ochna rhizomatosa (500 g) were dried and crushed before extraction using CH 2 Cl 2 /MeOH 1:1 (3 × 3 L) at ambient temperature to afford 87 g of crude extract after evaporation under reduced pressure. Part of the crude extract (80 g) was chromatographed on silica gel using a stepwise gradient condition and a mixture of CH 2 Cl 2 /MeOH (100:0 to 5:1) as solvents, resulting in 185 fractions A-H. Fraction D (CH 2 Cl 2 /MeOH; 30:1) was further purified by column chromatography using Sephadex LH-20 in acetone to yield compounds 2 (16 mg) and 7 (30 mg). The purification of fraction E (CH 2 Cl 2 /MeOH; 20:1) by column chromatography was also performed using Sephadex LH-20 in the same conditions and afforded compounds 4 (15 mg) and 5 (20 mg). For fraction F (CH 2 Cl 2 /MeOH; 20:1) a purification via a conventional RP-C 18 column eluting with an MeOH/H 2 O system resulted in the isolation of compound 3 (100 mg). The fraction G was chromatographed by a RP-C 18 column using the same conditions to yield compound 1 (150 mg). Lastly, fraction H [CH 2 Cl 2 /MeOH (5:1)] was subjected to repeated Sephadex LH-20 column chromatography eluted with an isocratic elution of CH 2 Cl 2 /MeOH (10:1), and this resulted in the isolation of compounds 1 (11 mg) and 6 (40 mg).
(R)-rhizomatobiflavonoid A (1): Yellow powder; Pharmaceutics 2022, 14, x FOR PEER REVIEW Some of the isolates were evaluated for their antiplasmodial and an [12]. In this study, the chemical constituents of the CH2Cl2/MeOH; (1:1 bark of O. rhizomatosa were investigated in order to find a potential integrase and antiplasmodial lead compounds. As a result, three ne type biflavonoids formed through rare types of Ca1-Ca2 and C1″-C described here for the first time along with four known compounds (4were elucidated by means of their UV, IR, NMR, HRESIMS, and CD analyses. The absolute configuration at (Ca2) of the new biflavonoids the first time. Structure-based-drug design has long been recognized to discover drug inhibitors and has become a famous strategy for dis compounds for drug development. Taking advantage of molecular binding potential of the isolates was investigated on integrase and Pla which are key enzymes involved in HIV and Plasmodium invasion of h

General Experimental Procedures
Optical rotations ([ᾳ] 20 D) were measured in methanol on a JASCO a 1 cm tube. The FTIR spectra were recorded on a Perkin Elmer F (Thermo Scientific, Madison, WI, USA). The NMR spectral data wer Bruker Avance DPX-400 spectrometer operating at the frequencies of 100 MHz ( 13 C). The samples were dissolved in MeOH-d4 prior to record values were given in parts per million (ppm) from the internal st spectral data resulted from a MSQ Thermo Finnigan. Column chroma carried out on a medium preparative liquid chromatography (MPL 0.040-0.063 mm, Merck MGaA, Darmstadt, Germany), Sephadex LH-2 GE Healthcare, Danderyd, Sweden) using silica gel 60 (Merck, 0.040-0 China). Thin layer chromatography (TLC) experiments were perform Merck Kieselgel 60 F254 plates (20 × 20 cm 2 , 0.25 mm, Qingdao, China).

Extraction of Plant Materials and Isolation of Compounds
The root barks of Ochna rhizomatosa (500 g) were dried and crushe using CH2Cl2/ Some of the isolates were evaluated for their antiplasmodial and [12]. In this study, the chemical constituents of the CH2Cl2/MeOH; bark of O. rhizomatosa were investigated in order to find a potent integrase and antiplasmodial lead compounds. As a result, three type biflavonoids formed through rare types of Ca1-Ca2 and C described here for the first time along with four known compounds were elucidated by means of their UV, IR, NMR, HRESIMS, and analyses. The absolute configuration at (Ca2) of the new biflavono the first time. Structure-based-drug design has long been recogniz to discover drug inhibitors and has become a famous strategy for compounds for drug development. Taking advantage of molecu binding potential of the isolates was investigated on integrase and which are key enzymes involved in HIV and Plasmodium invasion

General Experimental Procedures
Optical rotations ([ᾳ] 20 D) were measured in methanol on a JAS a 1 cm tube. The FTIR spectra were recorded on a Perkin Elm (Thermo Scientific, Madison, WI, USA). The NMR spectral data Bruker Avance DPX-400 spectrometer operating at the frequencie 100 MHz ( 13 C). The samples were dissolved in MeOH-d4 prior to rec values were given in parts per million (ppm) from the interna spectral data resulted from a MSQ Thermo Finnigan. Column chro carried out on a medium preparative liquid chromatography ( Some of the isolates were evaluated for their an [12]. In this study, the chemical constituents of the bark of O. rhizomatosa were investigated in orde integrase and antiplasmodial lead compounds. A type biflavonoids formed through rare types o described here for the first time along with four kn were elucidated by means of their UV, IR, NMR analyses. The absolute configuration at (Ca2) of th the first time. Structure-based-drug design has lo to discover drug inhibitors and has become a fam compounds for drug development. Taking adva binding potential of the isolates was investigated which are key enzymes involved in HIV and Plas

Plant Materials Collection
The root barks of Ochna rhizomatosa F. Hoff Dang district, Adamaoua region, Cameroon (GPS December 2019. The plant was identified at the N a voucher specimen (No. 39120HNC) was deposi

General Experimental Procedures
Optical rotations ([ᾳ] 20 D) were measured in m a 1 cm tube. The FTIR spectra were recorded (Thermo Scientific, Madison, WI, USA). The NM Bruker Avance DPX-400 spectrometer operating 100 MHz ( 13 C). The samples were dissolved in Me values were given in parts per million (ppm) spectral data resulted from a MSQ Thermo Finnig carried out on a medium preparative liquid ch 0.040-0.063 mm, Merck MGaA, Darmstadt, Germ GE Healthcare, Danderyd, Sweden) using silica g  Table 1. The HIV-1 Integrase strand transfer inhibition assay was adapted from previously described methods [20,21]. Briefly, 20 nM double-stranded biotinylated donor DNA (5 -5-BiotinTEG/ACCCTTTTAGTCAGTGTGGAAAATCTCTAGCA-3 ) annealed to (5 -ACTGCTA-GAGATTTTCCACACTGACTAAAAG-3 ) was immobilized in wells of streptavidin-coated 96 well microtiter plates (R&D Systems, Minneapolis, MN, USA). Following incubation at room temperature for 40 min and a stringent wash step, 5 µg/mL purified recombinant HIV-1 subtype CIN in integrase buffer 1 (50 µM NaCl, 25 µM Hepes, 25 µM MnCl 2 , 5 µM β-mercap to ethanol, 50 µg/mL BSA, pH 7.5) was added to individual wells. Test compounds and chicoric acid were added to individual wells to a final concentration of 20 µM. Recombinant HIV-1 subtype CIN was assembled on to the pre-processed donor DNA through incubation for 45 min at room temperature. Strand transfer reaction was initiated through the addition of 10 µM (final concentration) double-stranded FITC-labelled target DNA (5 -TGACCAAGGGCTAATTCACT/36-FAM/-3 ) annealed to (5 -AGTGAATTAGCCCTTGGTCA-/36-FAM/-3 ) in integrase buffer 2 (same as buffer 1, except 25 µM MnCl 2 was replaced with 2.5 µM MgCl 2 ). After an incubation period of 60 min at 37 • C, the plates were washed using PBS containing 0.05% Tween 20 and 0.01% BSA, followed by the addition of peroxidase-conjugated sheep anti-FITC antibody (Thermo Scientific, Waltham, MA, USA) diluted 1:1000 in the same PBS buffer. Finally, the plates were washed and peroxidase substrate (Sure Blue Reserve™, KPL, Gaithersburg, MD, USA) was added to allow for detection at 620 nm using a Synergy MX (BioTek ® ) plate reader. Absorbance values were converted to % enzyme activity relative to the readings obtained from the control wells (enzyme without inhibitor).

Evaluation of Antiplasmodial Assay
The assessment of antiplasmodial activity in vitro was evaluated against the erythrocytic stages of chloroquine-sensitive Plasmodium falciparum strain NF54 using a 3 Hhypoxanthine incorporation assay previously described [22], of which the chloroquine and pyrimethamine-resistant NF54 strain originated from South Africa, and the standard drug chloroquine was obtained from Sigma-Aldrich (St. Louis, MO, USA). Compounds were dissolved in DMSO at 10 µg/mL and added to parasite cultures incubated in RPMI 1640 medium without hypoxanthine, supplemented with HEPES (5.94 g/L), NaHCO 3 (2.1 g/L), neomycin (100 U/mL), Albumax, and washed human red cells A + at 2.5% hematocrit (0.3% parasitemia). Serial sample dilutions of 113-fold dilution steps covering a range from 100 to 0.002 µg/mL were prepared. The 96-well plates were incubated in a humidified atmosphere at 37 • C; 4% CO 2 ; 3% O 2 and 93% N 2 . After 48 h, 50 µL of 3 H-hypoxanthine (0.5 µCi) was added to each well.

Molecular Docking
Ligand preparation: The 3D structures of compounds 1, 2 and 7 were drawn using Molview software. Energy minimization was performed using the MM2 force field and saved as a MOL format. The missing charges and hybridization states of compound structures were assigned with the help of the MVD software.
Protein preparation: The 3D structures of different receptors were retrieved from the Protein Data Bank (PDB) (http://www.rcsb.org accessed on 22 July 2022). Integrase (PDB ID: 3LPT), and plasmodium falciparum key protein (PDB ID: 2LOE) were chosen respectively as targets. The proteins had one or two polypeptides and were co-crystallized with ligands. The targets were visually inspected, and reference ligands were identified for each receptor. Receptors were prepared for docking by removal of water molecules, ligands, cofactors and assigning bonds, bond order, hybridization and charges using the MVD software.
Docking search algorithm and scoring functions: MVD uses a Piecewise Linear Potential (PLP) algorithm as a scoring function for computational screening. In this study, the MolDock simplex evolution search algorithm was used for docking. Docking of compounds 1, 2 and 7 in different receptors was performed, and the best poses generated were used based on the docking scores (expressed in Kcal/mol). Docking of compounds 3, 4, 5 and 6 were not pursued because no binding poses were obtained (data not shown). MVD software uses two scoring functions: the MolDock score and the ReRank score, where MolDock score is an E score (docking scoring function) defined as: E score = E inter + E intra. E inter: Sum of the ligand-protein interaction energy, ligand-water interaction energy and ligand-cofactor interaction energy [23]). E intra: internal energy of the ligand. ReRank score provides an estimation of the ligand-receptor interaction strength [24].

Statistical Analysis
Data were represented as mean ± SD. Statistical analysis of the data was carried out using analysis of variance (ANOVA). Differences with p-value < 0.05 were considered statistically significant.
Data were represented as mean ± SD. Statistical analysis of the data was carried out using analysis of variance (ANOVA). Differences with p-value < 0.05 were considered statistically significant.
The 13 C-NMR spectrum (  Compound 1 was isolated as a yellow powder and gave a positive reaction with Neu's reagent, indicative of biflavonoids [12]. The HR-ESIMS spectrum showed a sodium adduct ion peak at m/z 533.1196 [M + Na] + , which corresponds to the molecular mass of C 30 H 22 O 8 Na (calc. 533.1046 [M + Na] + ). The spectral data from the UV analysis displayed absorptions at λ max 243, 247 and 277 nm, suggestive of an isoflavonoid nucleus [27]. The IR spectrum disclosed vibration bands at υ max 3287 (hydroxyl groups), 1652 (conjugated carbonyls) and between 1564 and 1506 cm −1 , indicative of aromatic ring and conjugated double bonds, respectively.
The 1 H-1 H COSY spectrum allowed the establishment of the following spin-system sequences: H-2 to H-3 , H-5 to H-6 , H-2 to H-3 , H-5 to H-6 , H-5 to H-6, H-5 to H-6 and H-a 2 to H-b 2 , which confirmed the presence of two aromatic AA BB spin systems, two aromatic ABX spin systems, and also an aliphatic AB system at δ H 6.01 and 4.67 (d, J = 11.0 Hz, H-a 2 /b 2 ).
The 13 C-NMR spectrum (  8, suggesting the presence of a dihydrochalcone backbone, indicating that compound 1 is a biflavonoid composed of one unusual isoflavone together with a dihydrochalcone monomer. The connection between Ca 1 and Ca 2 and also between C-1 and C b2 was unambiguously assigned by the HMBC correlations ( Figure 2) from H-a 2 (δ H 6.01) to C-a 1 (δ C 122.7); C-c 1 (δ C 177.2) and C-b 1 (δ C 157.5) and H-b 2 (δ H 4.67) to C-1 (δ C 134.9) and C-2 (δ C 130.5). The presence of five oxygenated aromatic carbons displayed in the 13 C-NMR spectrum confirmed the existence of five hydroxyl groups located on benzene rings. These findings supported a Lophirone A skeleton for compound 1 [12,17,25,26,29]. unusual isoflavone together with a dihydrochalcone monomer. The connection between Ca1 and Ca2 and also between C-1″ and Cb2 was unambiguously assigned by the HMBC correlations ( Figure 2) from H-a2 (δH 6.01) to C-a1 (δC 122.7); C-c1 (δC 177.2) and C-b1 (δC 157.5) and H-b2 (δH 4.67) to C-1″ (δC 134.9) and C-2″ (δC 130.5). The presence of five oxygenated aromatic carbons displayed in the 13 C-NMR spectrum confirmed the existence of five hydroxyl groups located on benzene rings. These findings supported a Lophirone A skeleton for compound 1 [12,17,25,26,29]. with the assigned absolute configuration [16]. Moreover, a high-amplitude positive Cotton effect is consistent with the β-orientation of proton H-a2. Based on the above comprehensive spectroscopic data analysis of the compound, the planar structure of compound 1 was established as depicted in Figure 1 as (R)-3-(1-(2′,4′-dihydroxyphenyl)-3,3-bis(4″,4‴-hydroxyphenyl)-1-oxopropan-2-yl)-4-hydroxy-chromenone and named (R)rhizomatobiflavonoid A (Figure 1). Compound 2 was isolated as a yellow powder eluted with CH2Cl2/MeOH (20:1) and it gave a positive reaction with Neu's reagent. Its HR-ESIMS spectral data exhibited a protonated molecular ion peak at m/z 567.1274 [M+H] + , which corresponds to the molecular mass of C34H30O8 (calc. 567. 3501 [M+H] + ). The UV and FTIR spectral data for characteristic functional groups of compound 2 were similar to those obtained for compound 1. When compared to the 1 H and 13 C NMR spectral data of compound 1 (Table  1), compound 2 exhibited a unique difference as four of the five hydroxy groups were substituted by methoxy groups, which resonated at δH 3.63, 3.75, 3.78 and 3.63 with the corresponding carbon signals at δc 55.7, 55.6, 56.2 and 54.9. This was further confirmed by the molecular formula of C34H30O8, bearing only one hydroxy group and four methoxy groups compared to compound 1.
As for compound 1, the CD spectral data of 2 ( Figure S22) exhibited a successive highamplitude positive Cotton effect for the n→* electronic transition at 305 nm and high- The CD spectral data of compound 1 ( Figure S21) exhibited the cotton effects of the electronic transition n→π* and π→π* transitions of the two flavonoid moieties. Successive high-amplitude positive Cotton effects for the n→π* electronic transition at 309 nm and high-amplitude positive Cotton effect for the π→π* transition at 248 nm, respectively, suggest (R) absolute configuration of the stereogenic center (Ca 2 ). The spectrum also displayed a low-amplitude positive Cotton effect for the π→π* at 208 nm, in agreement with the assigned absolute configuration [16]. Moreover, a high-amplitude positive Cotton effect is consistent with the β-orientation of proton H-a 2 . Based on the above comprehensive spectroscopic data analysis of the compound, the planar structure of compound 1 was established as depicted in Figure 1 as (R)-3-(1-(2 ,4 -dihydroxyphenyl)-3,3-bis(4 ,4 -hydroxyphenyl)-1oxopropan-2-yl)-4-hydroxy-chromenone and named (R)-rhizomatobiflavonoid A (Figure 1).
Compound 2 was isolated as a yellow powder eluted with CH 2 Cl 2 /MeOH (20:1) and it gave a positive reaction with Neu's reagent. Its HR-ESIMS spectral data exhibited a protonated molecular ion peak at m/z 567.1274 [M + H] + , which corresponds to the molecular mass of C 34 H 30 O 8 (calc. 567. 3501 [M + H] + ). The UV and FTIR spectral data for characteristic functional groups of compound 2 were similar to those obtained for compound 1. When compared to the 1 H and 13 C NMR spectral data of compound 1 (Table 1), compound 2 exhibited a unique difference as four of the five hydroxy groups were substituted by methoxy groups, which resonated at δ H 3.63, 3.75, 3.78 and 3.63 with the corresponding carbon signals at δc 55.7, 55.6, 56.2 and 54.9. This was further confirmed by the molecular formula of C 34 H 30 O 8 , bearing only one hydroxy group and four methoxy groups compared to compound 1.
Compound 3 was isolated as a yellowish amorphous powder, eluted with CH 2 Cl 2 /MeOH (20:1), and showed UV and FTIR spectral data similar to those of compounds 1 and 2. The 1D and 2D NMR spectral data revealed a high degree of similarity with compound 1 in chemical shifts as well as the coupling patterns and substituent positions except for both the carbon and proton signals at C-4 and C-4 . When compared with the NMR spectral data of compound 1, compound 3 displayed one proton signal at δ H 3.65 (6H, s) in the 1 H NMR (Table 1)  , and also indicated the presence of two additional methoxy groups. However, the position of these two methoxy groups was endorsed by the correlations found in the HMBC spectral data of the first methoxy proton at δ H 3.65 with the carbon atom at δ C 159.6 (C-4 ) and the second at δ H 3.65 with the carbon atom at δ C 159.7 (C-4 ), which proved that these two methoxy groups were positioned at C-4 and C-4 , respectively (Figure 2).

Biological Activities of Isolated Biflavonoids
The anti-HIV-1 replication and antiplasmodial activities of the biflavonoids bearing hydroxy and methoxy groups were studied. Compound 7 (4 ,7-dihydroxy-3 ,5-dimethoxyisoflavone), is not a biflavonoid, but was included in the bioactivity studies since it represents the single isoflavone present in the rootbark and can be used as a reference and as a starting point to analyze structure-activity relationships of the compounds that were isolated.

Anti-HIV-1 Integrase Activity
As part of the protease and reverse transcriptase (RT), integrase is the only enzyme that is encoded by HIV-1. This enzyme first cleaves the last two nucleotides from each 3 -end of the linear viral DNA. Chicoric acid, a well-known active inhibitor of HIV-1 integrase, was used as a reference compound during the assay. The results for anti-HIV-1 replication inhibitory activity showed that compound 1 (IC 50 = 0.047 µM) is the only compound that presents prominent inhibition of the integrase enzyme. This activity is less pronounced than the inhibition obtained for the control compound chicoric acid (IC 50 = 0.006 µM), a pure competitive inhibitor of HIV-1 integrase used clinically in the early stages of viral replication. The results suggest that a higher number of free hydroxy groups seems to increase the inhibitory activity of HIV-1 integrase of the compound, since compound 1, which is a biflavonoid, is more active than its respective methoxy derivatives (compounds 2, 3, 4 and 5) ( Table 2). It can, therefore, be suggested from the structure-activity relationships that unusual biflavonoids consisting of an isoflavone unit fused with a dihydrochalcone skeleton with a higher number of free hydroxy groups increase the inhibition of HIV-1 replication, while the presence of methoxy groups as substituents decreases the inhibition ( Figure 3). However, this affirmation has been further supported by molecular docking data obtained in this study. Table 2. Anti-HIV-1 integrase activity of the compounds (1-7) of Ochna rhizomatosa.

Compounds
Assembly IC 50 (µM) decreases the inhibition (Figure 3). However, this affirmation has been further supported by molecular docking data obtained in this study.

Antiplasmodial Activity
The antiplasmodial activity of the studied compounds did show promising IC50 values for compounds 2 (IC50 = 4.60 µM) and 5 (IC50 = 5.11 µM), and less activity for compounds 3 (IC50 = 7.86 µM) and 4 (IC50 = 8.20 µM). The activity was, however, not comparable to that of the reference compounds (artesunate and chloroquine (CQ)) ( Table  3). It was also observed that compounds 1, 6 and 7 displayed no activity against the chloroquine-sensitive strain of the malaria parasite (P. falciparum NF54). These results suggested some interesting structure-activity relationships (Figure 4). Compound 2, with the highest number of methoxy groups, also exhibited the highest activity, suggesting that methoxy groups might play an important role in the molecule's mode of action against the chloroquine-sensitive strain of the malaria parasite (Plasmodium falciparum NF54), while compound 1, which contains no methoxy groups, did not present any activity against P. falciparum NF54 ( Table 3). The influence of a methoxy group in biflavonoid compounds' antiplasmodial activity was not a surprise because a similar trend was observed in our previous report [12]. The presence of methoxy groups with steric hindrances in the molecule does seem to reduce the compound's antiplasmodial activity. This supports the lack of antiplasmodial activity of compound 7 (isoflavonoid). This conclusion has been confirmed by molecular docking.

Antiplasmodial Activity
The antiplasmodial activity of the studied compounds did show promising IC 50 values for compounds 2 (IC 50 = 4.60 µM) and 5 (IC 50 = 5.11 µM), and less activity for compounds 3 (IC 50 = 7.86 µM) and 4 (IC 50 = 8.20 µM). The activity was, however, not comparable to that of the reference compounds (artesunate and chloroquine (CQ)) ( Table 3). It was also observed that compounds 1, 6 and 7 displayed no activity against the chloroquine-sensitive strain of the malaria parasite (P. falciparum NF54). These results suggested some interesting structure-activity relationships (Figure 4). Compound 2, with the highest number of methoxy groups, also exhibited the highest activity, suggesting that methoxy groups might play an important role in the molecule's mode of action against the chloroquine-sensitive strain of the malaria parasite (Plasmodium falciparum NF54), while compound 1, which contains no methoxy groups, did not present any activity against P. falciparum NF54 ( Table 3). The influence of a methoxy group in biflavonoid compounds' antiplasmodial activity was not a surprise because a similar trend was observed in our previous report [12]. The presence of methoxy groups with steric hindrances in the molecule does seem to reduce the compound's antiplasmodial activity. This supports the lack of antiplasmodial activity of compound 7 (isoflavonoid). This conclusion has been confirmed by molecular docking.
From the anti-HIV-1 integrase and antiplasmodial activities exhibited by these isolated compounds, some important structure-activity relationships can be established for all the tested compounds, and the most important are highlighted and summarized in Figure 5.
As shown in Figure 5, the presence and number of free hydroxy groups seem responsible for the inhibitory activity of HIV-1 integrase, while methoxy groups seem to have the opposite effect. Sterically hindered methoxy groups, as illustrated in Figure 5 for compound 7, leads to the absence of activity against Plasmodium falciparum strain NF54.    (7).
From the anti-HIV-1 integrase and antiplasmodial activities exhibited by these isolated compounds, some important structure-activity relationships can be established for all the tested compounds, and the most important are highlighted and summarized in Figure 5.  (1), (2) and (7).
As shown in Figure 5, the presence and number of free hydroxy groups seem responsible for the inhibitory activity of HIV-1 integrase, while methoxy groups seem to have the opposite effect. Sterically hindered methoxy groups, as illustrated in Figure 5 for compound 7, leads to the absence of activity against Plasmodium falciparum strain NF54.

Molecular Docking
In the next experiments, in silico analysis with the isolates was conducted using the Molegro software. The best poses were selected from each docking analysis and pictures saved, as shown in Figures 6 and 7. Moreover, different interactions of the ligands with amino acids, including hydrogen bonds, van der walls interactions and total binding energies, were calculated and represented in Tables 4 and 5 below. The software has also identified all the amino acids involved in the binding and their respective positions, as shown in Tables 6 and 7, respectively, for integrase and Plasmodium receptors.  Chicoric acid-Int  The aim of docking analysis was to predict the binding capacities of isolated compounds on integrase and Plasmodium receptors; as well as the amino acids involved in the binding pockets. Therefore, compounds 1 to 7 were docked against integrase and the Plasmodium-6 cysteine s48/45 domain, two membrane proteins necessary for viral and parasite invasion into human cells. As far as integrase enzyme is concerned, compounds 3, 4, 5 and 6 showed very low binding scores with the enzyme (data not shown); suggesting no interaction of these compounds with the binding site of the enzyme. This result corroborates the observations obtained in vitro with no inhibition detected with these compounds. However, both compounds 1 and 2 were found bound to the active cavity of integrase receptor protein and showed binding with amino acids of the enzyme's active site. The binding energy of compound 1 with amino acids (Tyr83, Ala86, Arg107, Asn184, Tyr83, Glu85, Trp108, Glu177, Val180, Asn184) was compared with the binding energy of Dolutegravir with amino acids (Asp116, Asn144, Gln62, Glu152, Ser153, Met154, Asn155Arg657, Asp655) and of chicoric acid with amino acids (Asp116, Asn144, Gln62) ( Table 5). The results showed that compounds 1 and 2 bind to integrase with the highest score and strongest energy (MolDock score: −121.35 and −131 Kcal/mol, respectively) in comparison with chicoric acid and Dolutegravir (MolDock score: −116 and −100 Kcal/mol respectively). As observed in vitro, compound 1 seems to be a promising inhibitor of integrase with an IC50 close to that of chicoric acid. Binding poses showed that compound 1 establishes five hydrogen bonds with the active site's amino acids while chicoric acid has six and Dolutegravir only three, suggesting that in silico and in vitro observations are quite similar. Contrary to what was observed in vitro, compound 2 exhibited an interesting binding score in docking, suggesting that other factors may influence its activity in vitro but deserves further investigations to confirm its antiviral activity. Docking of compound 7 also showed a lower binding score in comparison to compounds 1, 2 and the reference compounds, confirming the result obtained in vitro ( Table 2).
As described above, docking analyses were also performed with the Plasmodium-6cysteine s48/45 domain, which is a membrane protein responsible for parasite entry into human cells. An analysis of binding interactions revealed that compounds 1 and 2 displayed high affinity for Plasmodium protein PDB ID: 2LOE with three and two hydrogen bonds with seven amino acids, in comparison to one hydrogen bond with chloroquine. The same observation was obtained for the binding score with −125 Kcal/mol for compounds 1 and 2 and −84 Kcal/mol for chloroquine. As it was observed in vitro,  The aim of docking analysis was to predict the binding capacities of isolated compounds on integrase and Plasmodium receptors; as well as the amino acids involved in the binding pockets. Therefore, compounds 1 to 7 were docked against integrase and the Plasmodium-6 cysteine s48/45 domain, two membrane proteins necessary for viral and parasite invasion into human cells. As far as integrase enzyme is concerned, compounds 3, 4, 5 and 6 showed very low binding scores with the enzyme (data not shown); suggesting no interaction of these compounds with the binding site of the enzyme. This result corroborates the observations obtained in vitro with no inhibition detected with these compounds. However, both compounds 1 and 2 were found bound to the active cavity of integrase receptor protein and showed binding with amino acids of the enzyme's active site. The binding energy of compound 1 with amino acids (Tyr83, Ala86, Arg107, Asn184, Tyr83, Glu85, Trp108, Glu177, Val180, Asn184) was compared with the binding energy of Dolutegravir with amino acids (Asp116, Asn144, Gln62, Glu152, Ser153, Met154, Asn155Arg657, Asp655) and of chicoric acid with amino acids (Asp116, Asn144, Gln62) ( Table 5). The results showed that compounds 1 and 2 bind to integrase with the highest score and strongest energy (MolDock score: −121.35 and −131 Kcal/mol, respectively) in comparison with chicoric acid and Dolutegravir (MolDock score: −116 and −100 Kcal/mol respectively). As observed in vitro, compound 1 seems to be a promising inhibitor of integrase with an IC 50 close to that of chicoric acid. Binding poses showed that compound 1 establishes five hydrogen bonds with the active site's amino acids while chicoric acid has six and Dolutegravir only three, suggesting that in silico and in vitro observations are quite similar. Contrary to what was observed in vitro, compound 2 exhibited an interesting binding score in docking, suggesting that other factors may influence its activity in vitro but deserves further investigations to confirm its antiviral activity. Docking of compound 7 also showed a lower binding score in comparison to compounds 1, 2 and the reference compounds, confirming the result obtained in vitro ( Table 2).

Chloroquine-Plasm
As described above, docking analyses were also performed with the Plasmodium-6-cysteine s48/45 domain, which is a membrane protein responsible for parasite entry into human cells. An analysis of binding interactions revealed that compounds 1 and 2 displayed high affinity for Plasmodium protein PDB ID: 2LOE with three and two hydrogen bonds with seven amino acids, in comparison to one hydrogen bond with chloroquine. The same observation was obtained for the binding score with −125 Kcal/mol for compounds 1 and 2 and −84 Kcal/mol for chloroquine. As it was observed in vitro, compound 2 seems to be a promising antiplasmodial compound. Compounds 3 and 4 showed no binding with the enzyme (data not shown). Contrary to in vitro results, compound 1 showed a high binding score with the enzyme in comparison to the reference drug chloroquine, suggesting that further in vitro and in vivo experiments should be performed in the future to confirm its antiplasmodial effect. Several plant compounds isolated from Dioscorea bulbifera showed antiplasmodial effects in the docking analysis, thereby supporting this strategy of in silico analysis as a platform of discovering new lead compounds against malaria [30].
These results confirm the potential inhibitory effect of compounds 1 and 2 for HIV and malaria treatment.

Conclusions and Recommendations
The phytochemical investigation of the root barks of Ochna rhizomatosa led to the isolation and structure elucidation of six biflavonoids (1-6) and one isoflavonoid (7). The anti-HIV-1 replication and antiplasmodial activities were assessed during this study. Compound 1 exhibited a noteworthy inhibition of HIV-1 integrase (IC 50 = 0.047 µM), whereas compound 2 displayed the highest antiplasmodial activity (IC 50 = 4.60 µM). Simultaneously, a structure-activity relationship was established. The presence and number of free hydroxy groups seem responsible for the inhibitory activity of HIV-1 integrase, while methoxy groups seem to have the opposite effect. Sterically hindered methoxy groups lead to the absence of activity against Plasmodium falciparum strain NF54. Docking studies showed that compounds 1 and 2 are good inhibitors for both integrase and Plasmodium 6-cysteine s48/45 domain receptor proteins with a binding score higher than control drugs and well-known inhibitors, indicating their potential effect as lead compounds against HIV and malaria. These results indicate the necessity to confirm our observations in vivo, which will be the direction of our future research.