Alkaloidal Extracts from Avicennia africana P. Beauv. (Avicenniaceae) Leaf: An Antiplasmodial, Antioxidant, and Erythrocyte Viable

Background The emergence of drug-resistant parasites impedes disease management and eradication efforts. Hence, a reinvigorated attempt to search for potent lead compounds in the mangroves is imperative. Aim This study evaluates in vitro antiplasmodial activity, antioxidant properties, and cytotoxicity of A. africana leaf alkaloidal extracts. Methods The A. africana leaves were macerated with 70% ethanol to obtain a total crude extract. Dichloromethane and chloroform-isopropanol (3 : 1, v/v) were used to extract the crude alkaloids and quaternary alkaloids from the total crude. The antiplasmodial activities of the alkaloidal extracts were performed against 3D7 P. falciparum chloroquine-sensitive clone via the SYBR Green I fluorescence assay with artesunate serving as the reference drug. The alkaloidal extracts were further evaluated for antioxidant properties via the total antioxidant capacity (TAC), the total glutathione concentration (GSH), the DPPH (2,2-diphenyl-1-picrylhydrazyl) assay, and the ferric-reducing antioxidant power (FRAP) methods. The cytotoxic activity of the alkaloidal extracts was tested on erythrocytes using a 3-(4,5-dimethylthiazol-2-yl)-5-diphenyltetrazolium bromide-MTT assay with little modification. The phytocompounds in the alkaloidal extracts were identified via gas chromatography-mass spectrometry (GC-MS) techniques. Results The total crude extract showed good antiplasmodial activity (IC50 = 11.890 µg/mL). The crude and quaternary alkaloidal extracts demonstrated promising antiplasmodial effects with IC50 values of 6.217 and 6.285 µg/mL, respectively. The total crude and alkaloidal extracts showed good antioxidant properties with negligible cytotoxicity on erythrocytes with good selectivity indices. The GC-MS spectral analysis of crude alkaloidal extracts gave indole and isoquinoline alkaloids and several other compounds. Dexrazoxane was found to be the main compound predicted, with an 86% peak area in the quaternary alkaloidal extract. Conclusion The crude and quaternary alkaloidal extracts exhibited antiplasmodial activities and ability to inhibit oxidative stress with negligible toxicity on erythrocytes. This may be good characteristics to avoid oxidative stress related to Plasmodium infection in the treatment of malaria.

Background.Te emergence of drug-resistant parasites impedes disease management and eradication eforts.Hence, a reinvigorated attempt to search for potent lead compounds in the mangroves is imperative.Aim.Tis study evaluates in vitro antiplasmodial activity, antioxidant properties, and cytotoxicity of A. africana leaf alkaloidal extracts.Methods.Te A. africana leaves were macerated with 70% ethanol to obtain a total crude extract.Dichloromethane and chloroform-isopropanol (3 : 1, v/v) were used to extract the crude alkaloids and quaternary alkaloids from the total crude.Te antiplasmodial activities of the alkaloidal extracts were performed against 3D7 P. falciparum chloroquine-sensitive clone via the SYBR Green I fuorescence assay with artesunate serving as the reference drug.Te alkaloidal extracts were further evaluated for antioxidant properties via the total antioxidant capacity (TAC), the total glutathione concentration (GSH), the DPPH (2,2-diphenyl-1-picrylhydrazyl) assay, and the ferric-reducing antioxidant power (FRAP) methods.Te cytotoxic activity of the alkaloidal extracts was tested on erythrocytes using a 3-(4,5-dimethylthiazol-2-yl)-5-diphenyltetrazolium bromide-MTT assay with little modifcation.Te phytocompounds in the alkaloidal extracts were identifed via gas chromatography-mass spectrometry (GC-MS) techniques.Results.Te total crude extract showed good antiplasmodial activity (IC 50 = 11.890µg/mL).Te crude and quaternary alkaloidal extracts demonstrated promising antiplasmodial efects with IC 50 values of 6.217 and 6.285 µg/mL, respectively.Te total crude and alkaloidal extracts showed good antioxidant properties with negligible cytotoxicity on erythrocytes with good selectivity indices.Te GC-MS spectral analysis of crude alkaloidal extracts gave indole and isoquinoline alkaloids and several other compounds.Dexrazoxane was found to be the main compound predicted, with an 86% peak area in the quaternary alkaloidal extract.Conclusion.Te crude and quaternary alkaloidal extracts exhibited antiplasmodial activities and ability to inhibit oxidative stress with negligible toxicity on erythrocytes.Tis may be good characteristics to avoid oxidative stress related to Plasmodium infection in the treatment of malaria.

Introduction
Malaria remains a fatal infection with public health signifcance.Te infection is caused by plasmodial species such as Plasmodium vivax, P. malariae, P. ovale, P. knowlesi, and P. falciparum.Plasmodium falciparum is predicted to cause 99.7% of all the malaria cases in Africa [1].Te infection is spread by the bites of infected female Anopheles mosquitoes [2].Te World Health Organization (WHO) projected 241 million malaria cases in 2020, with 627,000 mortalities, the vast majority of which were children under the age of fve [2].Africa continues to be the infection hotspot, accounting for 94% of the global disease and mortality burden [2].
In several countries, including Ghana, artemisinin-based combination drugs are still the frst-line treatment regime for uncomplicated malaria [3].However, concerns about emerging and widespread antimalarial drug resistance pose a serious drawback for malaria control.Plasmodium falciparum has been reported to resist quinoline-based drugs such as chloroquine [4] and artemisinin [5,6].Resistance to these and other antimalarial drugs, as well as the lack of an efective vaccine [7], necessitates a renewed efort to fnd new, efective, and afordable antimalarial agents from a variety of natural resources available.
Natural compounds found in plants have been suggested to possess infnite therapeutic potential [8], with many of these agents having promising antimalarial properties.Both quinines, isolated from Cinchona bark [9], and artemisinin, developed from the Artemisia annua plant, are two wellknown antimalarial lead compounds [10].But quinine is often used to treat uncomplicated malaria in pregnancy, severe malaria, treatment failure in artemisinin-based combination treatment (ACT) therapies, and malaria in children under fve [11,12].Consequently, various alkaloids have been reported to show varying biological activities, including notable antimalarial efects.Several classes of these alkaloids with antimalarial activity have been identifed [13].Bekeo and collaborators reported that antimalarial drugs with an alkaloidal base could be a good alternative to ACTs in Ghana [9].
Several mangrove plants used in folk medicine have been proposed to have tremendous therapeutic potential [14].Preliminary research revealed that their extracts exhibit a diverse array of biological activities, including antifungal, antibacterial, anticancer, antidiabetic, and antiviral properties, due to the presence of bioactive metabolites [15].Avicennia africana, which is associated with the West African mangrove, has been shown to have a high concentration of alkaloids and saponins [16,17] and a variety of pharmacological properties, including antimalarial efects, in both in vitro and in vivo assays [18].
In malarial pathogenesis, haemoglobin degradation by malarial parasites generates redox-active by-products such as free haem [19], hydrogen peroxide, and hydroxyl radicals in P. falciparum-infected RBCs [20], which cause oxidative insult to host cells.Tis could suggest an association between parasite pathophysiology and free radical generation, as well as a drop in antioxidant levels in the host system [21].Te oxidative stress caused by malaria infections may cause signifcant pathological damage to important organs in humans, such as the liver and spleen, as well as cognitive impairment.It has been revealed that the use of antimalarial drugs frequently leaves residues of this damage following therapy, as evidenced by memory impairment after cerebral malaria [22].Hence, plants or compounds with antimalarial efects and antioxidant properties could help in malaria management and possibly prevent infection afterefects.In view of this, alkaloidal extracts from A. africana leaves found in mangroves may have the potential to revolutionise the battle against malaria.
Tis study investigated the antiplasmodial activity of the 70% ethanol total crude extract and alkaloidal extracts of A. africana leaf against the chloroquine-sensitive clones of 3D7 parasites using the SYBR Green I fuorescent assay.In addition, the antioxidant activities of the extracts as well as their alkaloids were determined by utilising various quantitative techniques.Te tetrazolium-based colorimetric (MTT) assay technique was used to test the cytotoxic efects of the alkaloids and total crude extract on human erythrocytes.Furthermore, the phytocompounds of the alkaloidal isolates were identifed using GC-MS analysis.

Collection and Authentication of the Plant.
Te leaves of the A. africana plant, also locally called "Dwira Akyinim," in Akan were sampled from a mangrove forest area called "Iture," a coastal community near Elmina, Cape Coast, in the Central Region of Ghana (Figure 1).Te plant sample was identifed and validated by a botanist at the Department of Environmental Studies' herbarium at the School of Biological Sciences, University of Cape Coast.A voucher number (CC3096) was assigned to the plant specimen for future reference.

Plant Extraction.
Te A. africana leaves were cleaned with tap water, shade-dried, and ground into a fne powder.Te dried and powdered leaves (6.5 kg) were extracted by cold maceration in 70% ethanol (3 x 1.5 L) for 72 h [23].Te combined extracts were concentrated under reduced pressure in a Rotary thin-flm evaporator (R-114 SABITA) to aford a greenish-gummy crude extract (AAE, 412.18 g, 6.34% w/w).All solvents and reagents used in the total crude and alkaloidal extractions were of analytical-grade quality and obtained from Merck Chemical Supplies (Darmstadt, Germany) and Sigma-Aldrich (Germany).

Extraction of Alkaloids from the Total Crude Extract (AAE) of A. africana.
Te crude extract (412.18g) was dissolved in 30% acetic acid and fltered.Te clear acidic solution was extracted with chloroform (3x500 mL).Te chloroform layer was discarded and the aqueous layer was basifed to a pH of 10.5 using 25% aqueous ammonia and extracted with dichloromethane (3x150 mL).Te dichloromethane layer was dried using anhydrous magnesium sulphate and evaporated under reduced pressure to 2 Advances in Pharmacological and Pharmaceutical Sciences dryness to obtain a light brownish crude (AAA, 5.23 g, 1.26% w/w,) [24].Te screening of this extract using Dragendof reagent, Mayer's reagent, and 3% Ce (NH 4 ) 2 SO 4 in 85% H 3 PO 4 revealed the presence of alkaloids.Once again, the aqueous layer was extracted with chloroform-isopropanol mixture (3 :1 v/v, 3x250 mL).Te chloroform-isopropanol layer was concentrated to give a light brownish solid (AAQ, 6.13 g, 1.49%) [24,25].Tis light brownish solid gave a positive test with the Mayer's reagent [25,26].After 72 h of incubation, the cultures were treated with 100 µL of lysing bufer (SYBR Green I fuorescent), which is composed of 0.08% Triton-X 100, 5 mM EDTA, 20 mM Tris-Cl (pH 7.5), and 0.008% saponin as suggested by Johnson et al. [27], with slight modifcations.Te lysing bufer was treated carefully to avoid creating bubbles in the wells.Before reading the plates, they were left at room temperature for 30-60 minutes in the dark.Te plates were read at 470 and 520 nm using the FLUOstar OPTIMA Fluorometer plate reader with Control Software version 2.20.

Screening of Alkaloidal and Crude Extracts for Cytotoxic
Efect.Te cytotoxic properties of the alkaloidal extracts and total crude extracts were tested on erythrocytes using a slightly modifed version of the 3-(4,5-dimethylthiazol-2-yl)-5diphenyltetrazolium bromide-MTT assay as described by Ayisi et al. [28].A total of 100 µL of extracts (twofold serial dilution) ranging in concentration from 6.25 to 100 µg/mL were dispensed (in duplicate) into separate wells of a 96-well microtiter plate.A volume of 100 µL of noninfected erythrocytes was added to each well and incubated for 3 days at 37 °C in a humidifed incubator (5% CO 2 and O 2 ).Following that, each well received 20 µL of 7.5 mg/mL MTT in PBS and was kept for 2 h.After that, an aliquot (150 µL) of culture media was removed from each well and discarded.Te plates were treated with 200 µL of Triton X-100 in acidifed isopropanol to dissolve any formazan that had formed.Te plates were maintained in the dark at room temperature for 24 hours before being read at 570 nm with a plate reader.Te concentrations at which the extracts kill 50% of the cells (CC 50 values) were determined using Microsoft Excel 2016 software to create a graph of the extract concentrations versus percentage mean cell viability with dose-response curves.Te CC 50 values were compared to standard values to fnd out if the total crude and the alkaloidal extracts were harmful to cells.In addition, to fnd the selectivity indices (SI), the ratios of toxic concentrations of extracts (CC 50 ) to efective bioactive doses (IC 50 ) are used to determine the amount of extract that inhibits or kills the parasites with no toxicity.extract (AAE) was determined using the phosphomolybdenum assay with minor modifcations [29].In this method, 50 µL of AAA, AAQ, and AAE were mixed with 500 µL of reagent solution (4 mM ammonium molybdate, 28 mM sodium phosphate, and 0.6 M sulfuric acid).Te compositions were kept for one hour at 95 °C before being cooled and read at 695 nm with a FLUOstar Optima (BMG Labtech) against a blank (50 µL of DMSO).Ascorbic acid (a standard antioxidant) in DMSO with varying concentrations (1, 0.5, 0.250, 0.125, 0.0625, 0.0312, and 0.0156 μg/mL) was used to create the calibration curve.Te total antioxidant activity was expressed as mg/g of ascorbic acid.

Scavenging Activity of Alkaloidal and Crude Extracts on DPPH Radical.
Te antioxidant activities of alkaloidal extracts were assessed using a slightly modifed DPPH (2,2diphenyl-1-picrylhydrazyl) assay [30].Ascorbic acid (0.5 mg/mL in methanol) was diluted 2-fold, which served as the positive control.Te total crude (AAE), crude alkaloids (AAA), and quaternary alkaloids (AAQ) (2.5 mg/mL in methanol) were individually constituted to generate seven distinct concentrations.Te reaction began with the transfer of 100 µL of each total crude or alkaloidal extract or ascorbic acid into a 96-well plate, followed by the addition of 100 µL of a 0.5 mM DPPH solution into the wells.Te absorbances at 517 nm were measured with a plate reader (Tecan Infnite M200 Pro, Austria) after the mixture was kept for 20 minutes.Methanol was used as a negative control.Te experiments were conducted in triplicates.Te antioxidant activities of the extracts were expressed as a percentage of free-radical scavenging activity (%FRSA), which was calculated as follows: where Ao � absorbance of the blank solution and Ae � absorbance of the test (extract) solution or the standard (ascorbic acid).
Te efective concentration at 50% free radical scavenging activity (EC 50 ) was found by plotting a graph of the percentage of free radical scavenging activity vs. the concentration of the sample.

Assessment of Glutathione GSH Concentration.
Te approach proposed by Cereser et al. [31], with minor modifcations, was used in the assessment of the glutathione (GSH) concentration inherent in the AAE, AAA, and AAQ.Te reaction solutions were made up of 10 µL of the crude extract and alkaloidal extracts (5 mg/mL in DMSO).In addition, 180 µL of GSH bufer (100 mM NaH 2 PO 4 , 1N NaOH, 5 mM EDTA, and pH 8.0) and 10 µL of Ophthaldehyde (0.75 mM) were used for the reaction.Te mixture, together with the GSH standard solution (0.0001563-0.1 mg/mL in DMSO (2-fold serial dilution)), was kept at room temperature for 15 minutes.Te   Advances in Pharmacological and Pharmaceutical Sciences fuorescence was read at 350 nm (the excitation wavelength) and 420 nm (the emission wavelength).Te tests were run in triplicate.A calibration curve for the GSH standard solution, with a regression equation (y � 651473x + 103.69,R 2 � 0.998) was made to fgure out how much glutathione was present in the extracts.Te total glutathione in the extracts was expressed as glutathione equivalent (GSH).

Ferric-Reducing Antioxidant Power (FRAP) Assay.
Te FRAP test, as proposed by Benzie and Strain [32], was used in this study with a minor modifcation using a 96-well microplate.In a 10 : 1:1 ratio, 2.5 mL of 20 mmol/L FeCl 3 solutions, 2.5 mL of 10 mmol/L TPTZ solution, and 25 mL of 300.0 mmol/L acetate bufer were mixed to make the FRAP reagent.Te AAE, AAA, and AAQ (20 μL) were mixed violently together with 180 μL of the FRAP reagent.In the presence of antioxidants, the complex compound ferric tripyridyltriazine (Fe 3+ -TPTZ) is reduced to its ferrous tripyridyltriazine (Fe 2+ -TPTZ) form, resulting in an intense blue colour that can absorb maximally at a wavelength of 593 nm.

Results
3.1.Total Crude and Alkaloidal Extracts Yield.Te 70% v/v ethanol cold maceration of 6.5 kg of pulverised leaf material yielded 412.18 g (6.34% w/w) of the total crude extract (AAE).To allow the extraction of the alkaloidal components, the crude extract was treated with aqueous acid and washed with chloroform.Te aqueous layer was made basic to convert the alkaloids back into their neutral forms and subsequently extracted with DCM to aford the alkaloidal extract (AAA: 5.23 g, 1.26% w/w).Te aqueous layer was further partitioned into chloroform-isopropanol (3 : 1 v/v) to give a light brownish solid believed to be quaternary alkaloids (AAQ: 6.13 g, 1.49%), as shown in Figure 2 [25,26].Te basifed aqueous extract with DCM was preliminary confrmed by positive Mayer's and Dragendof tests.After chloroform-isopropanol extraction, the basifed aqueous extract was tested for quaternary alkaloids and found to be positive for Mayer's test [25,26].

Antiplasmodial Efects of A. africana Total Crude and
Alkaloidal Extracts.Te antiplasmodial efects of the total crude and crude alkaloidal extracts from the leaves of A. africana were tested against 3D7 P. falciparum strains, and the results are shown in Table 1.Te IC 50 values for the extracts (AAE, AAA, and AAQ) were 11.890 µg/mL, 6.217 µg/ mL, and 6.285 µg/mL, respectively.Te IC 50 value for the control drug, artesunate, was 0.9 × 10 −3 µg/mL.Previous research suggests that extracts with IC 50 s below 5 µg/mL have "very active" antiplasmodial action, while those between 5 and 50 are "active," 50 and 100 are "weakly active," and those above 100 are "inactive."[34].Similarly, Kamaraj et al. [35] also suggested that plant extracts with IC 50 s of less than 10 µg/ mL are classifed as having "promising" antiplasmodial activity.Tey also said that IC 50 s between 10 and 20 µg/mL, 20 and 40 µg/mL, 40 and 70 µg/mL, and more than 70 µg/mL had "moderate," "good," "marginally potent," and "poor" antiplasmodial activity, respectively.Te latter antiplasmodial activity score categorization was used in this study.Based on the IC 50 values obtained for AAE, AAA, and AAQ, the extracts demonstrated moderate to promising activity against 3D7 P. falciparum parasite clones.

Cytotoxicity of Alkaloidal Extracts of A. africana.
Te outcome of the erythrocytes' survival is shown in Figure 3 after RBCs were subjected to diferent concentrations of the alkaloidal extracts.Te cell survival rate of the alkaloidal extracts was similar to that of the artesunate reference drug.Also, both the crude and alkaloidal extracts showed good selectivity for 3D7 parasites, as indicated by their selectivity indices of >2 (Table 1).
Advances in Pharmacological and Pharmaceutical Sciences

Antioxidant Activity of Total Crude and Alkaloidal
Extracts.Te alkaloidal extracts and the total crude of A. africana yielded an appreciable amount of overall antioxidant activity.Te AAE and AAQ had total antioxidant capacities (TAC) of 375.506 ± 0.047 and 373.638 ± 0.040 mg/ g, respectively (Figure 4).Te AAA had the highest TAC value, at 494.39 ± 0.058 mg/g ascorbic acid equivalent.In this test, both the total crude and the alkaloidal extracts contained adequate quantities of antioxidants required to neutralise free radicals at varying concentrations.Te scavenging abilities of AAE, AAA, and AAQ crude extracts compared to that of ascorbic acid (control) are presented in Figure 5. Te various efective concentrations (EC 50 ) of the extracts were found to be 0.929 ± 0.008 mg/mL, 0.287 ± 0.044 mg/mL, 0.245 ± 0.040 mg/mL, and 0.065 ± 0.006 mg/mL, respectively.Compared to ascorbic acid (0.065 ± 0.006), the alkaloidal extracts (AAA and AAQ) had the strongest scavenging activities (p < 0.0001), with the total crude (AAE) extract having the least.Tis suggests that the alkaloidal components of the plant are mainly responsible for its radical scavenging activity.
Te total glutathione (GSH) content inherent in AAE, AAA, and AAQ extracts yielded varying concentrations.Te total GSH levels in the total crude and the alkaloidal extracts were AAE: 0.269 ± 0.0001, AAQ: 1.764 ± 0.0001, and AAA: 1.495 ± 0.0002 mg/g GSH equivalent, as shown in Figure 6.Te AAQ and AAA extracts were found to have higher levels of glutathione concentrations (p < 0.0001) than the AAE.Similarly, the ferric-reducing antioxidant power (FRAP) of AAE, AAA, and AAQ extracts showed a considerable variance in EC 50 s (AAE, 1.722 ± 0.268 mg/mL; AAA, 3.568 ± 0.759 mg/ mL, and AAQ, 3.386 ± 0.015 mg/mL) compared to ascorbic acid (0.077 ± 0.005 mg/mL) (Figure 7).Te EC 50 s of the antioxidant activities of the extracts were concentration dependent.AAE exhibited the highest power-reducing activity in comparison to ascorbic acid, followed by AAQ and AAA.

GC-MS Analysis of Compounds from the Alkaloidal Extracts of A. africana.
GC-MS analysis of the extract showed that AAA had 19 peaks and AAQ had 7 peaks, showing the presence of diferent phytocompounds.As presented in Figure 8 and Table 2, the peak with a retention time of 16.683 minutes was assigned to gramine.Gramine is an aminoalkylindole alkaloid (C 11 H 14 N 2 ; MW-174.24gmol −1 ) (M + ) and had the highest peak area (31.97%) of all the compounds found in the AAA extract.On the other hand, dexrazoxane (C 11 H 16 N 4 O 4 ; MW-268.27gmol −1 ) with    Advances in Pharmacological and Pharmaceutical Sciences a retention time of 5.252 minutes was the major compound with the highest percentage composition (90.7%) in the AAQ extract, with traces of other six compounds as shown in Figure 9 and Table 3.

Identifcation and Characterization of Alkaloidal
Metabolites.Te respective MS spectrum of each alkaloidal metabolite was generated, and on the basis of isotopic ft ratios (iFit) close to zero and, more importantly, that the  Advances in Pharmacological and Pharmaceutical Sciences overall MS accuracy was within 5 mDa, the molecular formulae were computed [36].Te Dictionary of Natural Products online database (https://dnp.chemnetbase.com)was used to identify compounds.Te molecular formulae of these respective alkaloids were carefully chosen on the 5 mDa mass accuracy range scale.
Te MS fragmentation pattern analysis of the alkaloidal extract (AAA) identifed several alkaloidal derivatives (Figure 8).For instance, the most abundant peak (Figure 10, Table 2) was cautiously identifed to be gramine, an aminoalkylindole alkaloid with a molecular weight and formulae of MW-174.24gmol −1 [M + ] and C 11 H 14 N 2 , respectively.Tis compound was fragmented by the loss of CH 3 • to produce a precursor ion at m/z 155.6.Further fragmentation gave a more stable molecule (Figure 10) to generate precursor ions at m/z 129.5 and 96.4 from the loss of amide (−26 Da), amine (−18 Da), and methyl groups (−15 Da) side chains after a possible 1,3 methyl McLaferty rearrangement of the dimethyl derivative of gramine to a more stable (E)-N-((3H-indol-3-ylidene)methyl) methanimine derivative (Figure 10(a)) [36].Similarly, at retention times of 5.578 and 14.744 min, 1,2,3,4-tetrahydroisoquinoline and 1H-indol oxime derivatives were tentatively identifed with molecular weights of m/z 131.5 and 173.7, respectively (Figures 11 and 12).Te molecular formulae for these compounds are C 9 H 11 N and C 10 H 10 N 2 O, respectively (Table 2).Te fragmentation of 1,2,3,4-tetrahydroisoquinoline led to the abstraction of two hydrogens to a more stable 1,4dihydroisoquinoline (m/z 131.5), confrming the structure of the compound (Figure 11) [37].In the case of 1H indole-oxime, the loss of -OH gave a precursor ion at m/z 159, followed by the loss of an amide and propyl groups to a molecular ion of m/z 129 and 96, respectively (Figure 12(a)).
Te GC-MS profles of phytocompounds from the quaternary alkaloidal extract (AAQ) of the A. africana plant also presented seven compounds.Te major molecules were identifed at RT 5.111 and 5.252 min (Figure 9) with a molecular ion at m/z 267.Advances in Pharmacological and Pharmaceutical Sciences has been reported by Rapoport and Holden [26].Te fragmentation pattern of this molecule at a higher CE level produced product ions at m/z 140.14 and 112.50, resulting from the cleavage of the tertiary carbon (Figure 13(a)).Te rest of the compounds were in trace amounts (Table 3 and Figure 9).

Discussion
Plant medicine is largely prepared locally by decoction [38] or soaking in locally brewed alcohol for maximum infusion of phytoconstituents and A. africana is no exception.Tis plant has been an important folk remedy for people who have lived in mangrove-covered areas for a very long time.
Te paucity of data regarding the plant's antimalarial efects reinforces the need to investigate its antimalarial, antioxidant, and cytotoxic activities.Consequently, the extract yield in this study was within the predicted range of 1%-10% or more [39].It is noteworthy that in an extraction process, the extract yield may be afected by several factors, including the type of solvent used, the extraction method employed, and the duration of the extraction [39].In this study, the cold maceration extraction process was used for the extraction of the total crude (AAE).It was the most convenient and suitable method for the supposed thermolabile alkaloidal extracts inherent in the plant [39].Several secondary metabolites have been identifed in this plant in previous studies [17,18], and such bioactive compounds have served as the foundation for the advancement and production of novel conventional drugs [8,40].
In this study, crude alkaloids (AAA and AAQ) were extracted from the total crude extract (AAE) obtained from the leaves of A. africana.Te extracts were evaluated for antiplasmodial activity using the SYBR Green I fuorescence assay.Tis method has been found not only to be fast, reliable, and relatively inexpensive but also to provide highthroughput screening of antimalarial drugs [4].Te SYBR Green I dye binds to the parasite's DNA in infected RBCs, resulting in high fuorescence that is detectable by fow cytometry.
Te antiplasmodial properties of the alkaloidal extracts and the total crude extract yielded IC 50 values that ranged from 6.217 to 11.890 µg/mL in the order AAA < AAQ < AAE of activity.Te results of this research suggest that the alkaloidal extracts (AAA � 6.217 µg/mL and AAQ � 6.287 µg/ mL) showed better antiplasmodial efects than the total crude extract (AAE � 11.890 µg/mL) as opposed to the reference drug (artesunate).Although the hydroethanolic extract of A. africana (AAE) showed good activity, as was similarly reported by Okokon et al. [41] for other plants, the alkaloidal extracts in our current study performed better and looked more promising.It further suggests that the antiplasmodial efects can be attributed mainly to the alkaloids present or may also be due to the synergy efect of the various compounds in this plant.Our fndings are consistent with those of Erhunse et al. regarding the antimalarial efect of isoquinoline alkaloids such as berberine and palmatine [42].Kyei et al. also demonstrated the potential antimalarial activity of cryptolepine and isocryptolepine [43].Te display of the potent antiplasmodial activity of the alkaloidal extracts in the present study confrmed earlier reports regarding the diversity of bioactive metabolites and, importantly, the antiplasmodial properties of several classes of alkaloids, such as the indole and isoquinoline classes [13,44,45].
Te GC-MS profle of AAA revealed about nineteen phytocompounds (Table 2).A good number of compounds were predicted to be present in the extract used in this study.Notable among them are indole and isoquinoline alkaloids.Of the various compounds identifed in the alkaloidal extract (AAA), gramine, a simple indole alkaloid, was found to have the highest percentage peak area of all the phytocompounds.Gramine has recently gained prominence due to its diverse biological activities, which include insecticidal, antibacterial, antiviral, antitumor, and anti-infammatory properties [46].It may be suggested that the antiplasmodial efects of the AAA extract in this study are largely due to the intrinsic indole alkaloids and isoquinoline alkaloids in the extract.Several indole alkaloids, including vinblastine and vincristine, have shown promising antimalarial efects.Tey are thought to disrupt the parasite's microtubule assembly, afecting its growth [47].Tryptanthrin kills the parasite by interfering with the parasite's DNA synthesis [48].Importantly, indole alkaloids are basic compounds that ionise in the acidic environments of the parasite's food vacuoles, contributing to their antimalarial activity.Te quinoline ring was reported to have the potential to interfere with parasites' production of hemozoin, resulting in a buildup of toxic haem species that are harmful to the parasites.Also, some indole alkaloids have lipophilicity, which is a vital pharmacokinetic property that allows these drugs to penetrate the membrane of the parasite and interact with target sites [49].
Our current study supports previous research regarding the antimalarial properties of indole alkaloids isolated from natural products [50,51].It is important to note that indole alkaloids have long been suggested to possess a signifcant number of potent pharmacological properties, such as antiinfammatory, cytotoxic, antiparasitic, antiviral, antagonistic, and serotonin-related activities [52].Omar and 10 Advances in Pharmacological and Pharmaceutical Sciences colleagues reported on the antiplasmodial activities of two indole alkaloidal compounds, ellipticine and olivacine, that were isolated from plants of the genus Aspidosperma [49].
In addition, the GC-MS spectral analysis of the AAQ extracts revealed several compounds (Table 3).Among them were two enantiomorphic-pair compounds, dexrazoxane and razoxane (5.14%), with dexrazoxane having the highest (86%) peak area and the other fve compounds having peak areas ranging from 0.93 to 3.09%.Te antiplasmodial activity of the extract in this study may be due to the synergy of the various compounds or the single efect of dexrazoxane identifed in AAQ.Dexrazoxane has been shown to have cardioprotective efcacy against doxorubicin-induced cardiotoxicity in breast cancer therapy [55].Research has shown that the hydrolyzed product of dexrazoxane chelates with both bound and free iron [56], and the iron-chelator's strong iron binding properties may inhibit the intraerythrocytic development of plasmodium parasites [57].
Plasmodium metabolism is iron dependent.Te ironcontaining enzymes such as delta-aminolevulinate synthase and ribonucleotide reductase, which are needed for DNA synthesis, de novo haem production in the parasite, electron transport, and mitochondrial activity [58,59], may have been deprived of iron by dexrazoxane in AAQ.It may be possible that the antiplasmodial efects of the AAQ extract are due to dexrazoxane.Dexrazoxane may have chelated with the irons required for the optimum function of these enzymes, which interferes with the parasite's metabolic activities and inhibits the intraerythrocytic growth process.
In natural product-based pharmacotherapy research for infectious diseases, the plant extract may possess high therapeutic efcacy but could cause potential harm to cells or organs; hence, screening for toxicity of promising or lead compounds is crucial.Te current study evaluated the    Advances in Pharmacological and Pharmaceutical Sciences AAE extracts were determined to be >100 µg/mL in this study, and they have negligible cytotoxic efects on red blood cells (RBCs) [60].A similar outcome was obtained for artesunate (the positive control drug).Te high cell survival percentages recorded for alkaloidal extracts, total crude, and artesunate support the low toxicity or weak cytotoxicity to erythrocytes.Te inability of plant extracts to cause the lysis of red blood cells in vitro may be highly connected to the inherent biological constituents in the plant, which ensure the protection of the erythrocytes against malaria parasitemediated cellular damage [61].Te alkaloidal extracts (AAA and AAQ) and the total crude extract (AAE) gave good selectivity indices (greater than 2), which suggests that the extracts possess curative properties against P. falciparum parasites [62].Te determination of selectivity indices is key for evaluating the therapeutic potential of extracts in natural product drug discovery; it seeks to assess the relative safety as well as the efcacy of the plant extracts and their ability to target specifc pathogens or cells, while at the same time, minimising any detrimental efects on normal cells.
Te antioxidant properties of alkaloidal extracts and the total crude extract of A. africana were tested using various procedures based on assay principles and assay conditions [63].Te following assays were used in this study to assess antioxidant activity: total antioxidant capacity, DPPH radical scavenging activity, total glutathione, and ferric-reducing antioxidant power.Te alkaloidal extracts and total crude extract demonstrated good antioxidant and reducing properties in a concentration-dependent manner.Te display of scavenging abilities of the extracts for free radicals may be, to a greater extent, associated with the extracts' intrinsic properties [64].Te fndings of this study on the antioxidant and antiplasmodial properties of isoquinoline and indole alkaloids, as well as several other compounds in the plant, agree with previous studies on alkaloids [65][66][67][68].Te artesunate reference drug used in this study had been reported to have antioxidant properties [69][70][71][72].
Te reactive oxygen species (ROS) produced by oxidative stress-mediated damage to host RBCs and other organs during blood-stage schizogony may aggravate the infection.

Advances in Pharmacological and Pharmaceutical Sciences
In addition, haemolysis produced by oxidative stress is a typical clinical occurrence after a few days of antimalarial therapy with artemisinin drugs and derivatives.Te drug kills parasites by inducing ROS to be produced within infected red blood cells after the endoperoxide bridge is activated [73].In this regard, it has been suggested that any potential antimalarial drugs should have scavenging properties to get rid of the extra free radicals generated by parasite metabolism and other exogenous factors [67,[74][75][76].
Generally, all the AAE, AAA, and AAQ extracts exhibited antioxidant properties in this study, and this may have contributed to the antiplasmodial activities recorded.Particularly, the dexrazoxane identifed in AAQ, which is a known iron chelator, may be responsible for both the antiplasmodial and anticytotoxic efects on RBCs.Furthermore, the indole and isoquinoline alkaloids identifed in the AAA extract potentially mediated the antiplasmodial and antioxidant activities in the current study.Te pharmacodynamic profle of indole alkaloids and isoquinoline in malaria infection has been established [44].Te mechanism of dexrazoxane's antiplasmodial activity has been linked to iron but its anticardiotoxicity remains unknown.
Te anticytotoxic and antioxidant properties of the extracts in this study may have contributed to protecting against the development of complications related to malarial infections.Tis may have also increased the efcacy of the extracts to promote rapid recovery.Te identifcation of indole and isoquinoline alkaloids, as well as dexrazoxane compounds, in A. africana for the frst time ofers signifcant promise for advancing drug discovery and contributing to the development of novel therapies with potential advantages to human health.Tis study used the 3D7 P. falciparum strain for the antimalarial activity assay.Hence, other strains of the parasites are recommended to ascertain the efect of the alkaloids on them.Te current study confrmed the antimalarial efects of these known indole and isoquinoline alkaloidal compounds.However, this is the frst time these alkaloids, including dexrazoxane, have been identifed in A. africana leaves.
We recommend in vivo studies of the alkaloidal extracts.Furthermore, we suggest that more studies be conducted by fractionating, isolating, and purifying these alkaloidal extracts to obtain pure phytocompounds for structure elucidation and optimisation studies.Tis will potentially introduce novel lead compounds from this plant into the antimalarial drug discovery pipeline.

Conclusion
Te fndings of this study suggest that the alkaloidal extracts of A. africana leaves possess promising antiplasmodial effects against 3D7 P. falciparum chloroquine-sensitive parasites.Te results also showed that the A. africana leaves had antioxidant properties with negligible cytotoxic efects on erythrocytes.We acknowledge that the in vitro antimalarial study may not translate to clinical application as the protocol used has some limitations though the outcome of the study supports folklore application for malaria infection treatment.So far, this is the frst time that isoquinoline alkaloids, indole alkaloids, as well as razoxane and dexrazoxane have been identifed in A. africana leaves to the best of our knowledge.

Figure 2 :
Figure 2: Schematic diagram for the extraction of alkaloids from A. africana.

Figure 4 :Figure 5 :
Figure 4: (a) TAC standard calibration curve and (b) total antioxidant capacity of A. africana total crude and alkaloidal extracts AAE, AAA, and AAQ.Data are presented as the mean value ± standard deviation SD (n � 3).

Figure 6 :
Figure 6: (a) GSH standard curve and (b) reduced GSH concentrations in AAE, AAQ, and AAA of the A. africana plant.Te data are shown as the mean ± standard deviation (SD) (n � 3).Te experiments were triplicated.

Figure 9 :
Figure 9: GC-MS profle of the quaternary alkaloidal extract (AAQ) of A. africana shows the retention time (min) of the compounds on the X-axis, and the Y-axis represents the percentage (%) of peak area.
the application of GraphPad Prism 5.0 version software (GraphPad Software Inc., San Diego, CA).Te CC 50 and EC 50 values were also derived from a doseresponse curve using Microsoft Excel 2016.Te student ttest was employed for analysis, and statistical signifcance was set at p < 0.05.
Te profles of alkaloidal extracts (AAA and AAQ) from A. africana leaf (70% ethanol extract, AAE) were analysed on an Agilent 7890 B GC with an Agilent Technologies GC sampler 80 (Agilent Technologies, CA, USA).Te device was equipped with an MS Agilent 7000 °C triple quadrupole with a column size of 30 m + 10 m EZ Guard × 0.25 mm internal diameter-fused silica capillary coated with VF-5 ms (0.25 mm flm) from Agilent or equivalent.Te temperatures of the injector (in splitless mode) and the MSD transfer line were set to 280 °C and 325 °C, respectively.Te extract was dissolved in methanol and injected at an initial column temperature of 70 °C for 25 min.Te system temperature was increased up to 150 °C (3 °C/min), 200 °C (8 °C/min), and 280 °C (2.133 minutes).Helium was the carrier gas with a constant fow rate of 2.25 mL/minute, with nitrogen serving as the collision gas with a constant fow rate of 1.5 mL/minute.2.9.Analysis of Data.Te tests were conducted in triplicate.Te data were shown as the mean ± standard deviation (SD).Te IC 50 values were obtained from graphs of dose-response curves through

Table 1 :
Antiplasmodial, cytotoxic activities, and therapeutic indices of the AAE, AAA, and AAQ extracts of A. africana.Artesunate was utilized as the reference drug.Te data show averages for duplicate runs ± SD (standard deviation).Diferences in mean values that are statistically signifcant (p < 0.01) were shown using the symbols ( * * ).

Table 2 :
GC-MS profles of phytocompounds of the crude alkaloidal extract (AAA) of 70% ethanol leaf extract of the A. africana plant.