Research Article Alkaloidal Extracts from Avicennia africana P. Beauv. (Avicenniaceae) Leaf: An Antiplasmodial, Antioxidant, and Erythrocyte Viable Mustapha A. Ahmed,1,2 Elvis O. Ameyaw ,3 Francis A. Armah ,1 Patrick M. Fynn,4 Isaac Asiamah ,4 George Ghartey-Kwansah,1 Felix K. Zoiku,5 Ebenezer Ofori-Attah,6 and Christian K. Adokoh 7 1Department of Biomedical Sciences, School of Allied Health Sciences, University of Cape Coast, Cape Coast, Ghana 2Small Animal Teaching Hospital, SVM, CBAS, University of Ghana, Legon, Accra, Ghana 3Department of Pharmacotherapeutics and Pharmacy Practice, School of Pharmacy and Pharmaceutical Sciences, University of Cape Coast, Cape Coast, Ghana 4Department of Chemistry, School of Physical Sciences, University of Cape Coast, Cape Coast, Ghana 5Department of Epidemiology, Noguchi Memorial Institute for Medical Research, College of Health Sciences, University of Ghana, Legon, Accra, Ghana 6Department of Clinical Pathology, Noguchi Memorial Institute for Medical Research, College of Health Sciences, University of Ghana, Legon, Accra, Ghana 7Department of Forensic Sciences, School of Biological Science, University of Cape Coast, Cape Coast, Ghana Correspondence should be addressed to Christian K. Adokoh; cadokoh@ucc.edu.gh Received 28 August 2023; Revised 19 December 2023; Accepted 23 December 2023; Published 9 January 2024 Academic Editor: Farshad Mirzavi Copyright © 2024 Mustapha A. Ahmed et al. Tis is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background. Te emergence of drug-resistant parasites impedes disease management and eradication eforts. Hence, a rein- vigorated 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-MTTassay 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 (IC50 = 11.890 µg/mL). Te crude and quaternary alkaloidal extracts demonstrated promising antiplasmodial efects with IC50 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.TeGC-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. Hindawi Advances in Pharmacological and Pharmaceutical Sciences Volume 2024, Article ID 4541581, 17 pages https://doi.org/10.1155/2024/4541581 https://orcid.org/0000-0002-8317-9602 https://orcid.org/0000-0001-6560-1147 https://orcid.org/0000-0003-3451-4379 https://orcid.org/0000-0002-4341-3361 mailto:cadokoh@ucc.edu.gh https://creativecommons.org/licenses/by/4.0/ https://creativecommons.org/licenses/by/4.0/ https://doi.org/10.1155/2024/4541581 1. Introduction Malaria remains a fatal infection with public health sig- nifcance. 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 falci- parum 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 well- known 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, andmalaria in children under fve [11, 12]. Consequently, various alkaloids have been reported to show varying biological activities, including notable an- timalarial efects. Several classes of these alkaloids with an- timalarial activity have been identifed [13]. Bekeo and collaborators reported that antimalarial drugs with an alka- loidal 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 prop- erties, due to the presence of bioactive metabolites [15]. Avicennia africana, which is associated with the West Af- rican mangrove, has been shown to have a high concen- tration 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 quan- titative techniques. Te tetrazolium-based colorimetric (MTT) assay technique was used to test the cytotoxic efects of the alkaloids and total crude extract on human eryth- rocytes. Furthermore, the phytocompounds of the alkaloidal isolates were identifed using GC-MS analysis. 2. Materials and Methods 2.1. 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 Bi- ological Sciences, University of Cape Coast. A voucher number (CC3096) was assigned to the plant specimen for future reference. 2.2. 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 pres- sure 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). 2.3. Extraction of Alkaloids from the Total Crude Extract (AAE) of A. africana. Te crude extract (412.18 g) was dis- solved in 30% acetic acid and fltered. Te clear acidic so- lution 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 magne- sium 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 (NH4)2SO4 in 85% H3PO4 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]. 2.4. Parasite Cultivation. Te efcacy of the alkaloidal ex- tracts (AAA and AAQ) and total crude (AAE) was tested against a 3D7 P. falciparum clone (chloroquine sensitive) obtained from the Immunology Department, Noguchi Me- morial Institute for Medical Research, University of Ghana. Te asexual forms of P. falciparum were preserved in con- tinuous cultures by employing techniques suggested by Rapoport and Holden [26], with minor modifcations. Te parasites were cultured in 2% packed cell volume (ORh + noninfected human erythrocytes) and maintained in a complete culturemedium (CPM).Temedium is composed of RPMI-1640 supplemented with 5.94 µg/L HEPES, 5 µg/L AlbuMAX II, 50mg/L hypoxanthines, and 2.1 µg/L sodium carbonate (NaHCO3). All chemicals and reagents utilized in this study were procured from Sigma-Aldrich (Germany) and QualiChem’s Lab Reagents (India).Te incubation conditions were 3% O2, 4% CO2, 93% N2, and 37°C. Te culture media were changed daily to ensure that parasitaemia was greater than 5%. A solution of 5% sorbitol was used to treat cultures and incubated for 48 hours to attain synchrony of ring-stage parasites. Te parasites were subcultured to obtain 1% par- asitaemia before being used in assays. 2.5. Antiplasmodial Activity of Crude andAlkaloidal Extracts. Te stock solutions (1000 μg/mL) of the alkaloidal extracts (AAA and AAQ) and total crude extract (AAE) were flter- sterilized using a 0.2 μmMillipore flter. A working solution (100 μg/mL) was achieved by diluting the stock solution 10- fold. It was further diluted to attain concentrations ranging from 100 to 0.39 μg/mL. An aliquot of 100 µL of each of the nine dilutions was plated in duplicate in each well of a 96- well coastal plate. A standard antimalarial reference drug, artesunate (15 ng/ml working concentration), was serially diluted up to the 9th concentration (15−0.06 ng/mL) and plated alongside. Each well received 100 μL of parasite culture (1% parasitaemia at 2% haematocrit). All the other extracts were taken through the same procedure. Te plates were gassed for 5minutes in a modular incubation chamber with 90% N2, 5% CO2, and 5% O2 before being kept at 37°C for 72 h for incubation. 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, 5mM EDTA, 20mMTris- 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 30–60minutes 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. 2.6. 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)-5- diphenyltetrazolium 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 eryth- rocytes was added to each well and incubated for 3 days at 37°C in a humidifed incubator (5% CO2 and O2). Following that, each well received 20 µL of 7.5mg/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 iso- propanol 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 (CC50 values) were determined using Microsoft Excel 2016 software to create a graph of the extract concentrations versus per- centage mean cell viability with dose-response curves. Te CC50 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 (CC50) to efective bioactive doses (IC50) are used to determine the amount of extract that inhibits or kills the parasites with no toxicity. 2.7. Antioxidant Assays 2.7.1. Evaluation of the Total Antioxidant Capacity (TAC) of Alkaloidal and Crude Extracts. Te total antioxidant ca- pacity of alkaloidal extracts (AAA and AAQ) and total crude Figure 1: Photograph ofAvicennia africanawhole plant and leaves. Source: feldwork, Ahmed et al. [17, 18]. Advances in Pharmacological and Pharmaceutical Sciences 3 extract (AAE) was determined using the phosphomo- lybdenum assay with minor modifcations [29]. In this method, 50 µL of AAA, AAQ, and AAE were mixed with 500 µL of reagent solution (4mM ammonium molybdate, 28mM sodium phosphate, and 0.6M 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 DMSOwith 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. 2.7.2. Scavenging Activity of Alkaloidal and Crude Extracts on DPPH Radical. Te antioxidant activities of alkaloidal extracts were assessed using a slightly modifed DPPH (2,2- diphenyl-1-picrylhydrazyl) assay [30]. Ascorbic acid (0.5mg/mL in methanol) was diluted 2-fold, which served as the positive control. Te total crude (AAE), crude alkaloids (AAA), and quaternary alkaloids (AAQ) (2.5mg/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 0.5mM 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 20minutes. 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 cal- culated as follows: (Ao − Ae) Ao 􏼢 􏼣 × 100, (1) 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 scav- enging activity (EC50) was found by plotting a graph of the percentage of free radical scavenging activity vs. the con- centration of the sample. 2.7.3. 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 (5mg/mL in DMSO). In addition, 180 µL of GSH bufer (100mM NaH2PO4, 1N NaOH, 5mM EDTA, and pH 8.0) and 10 µL of O- phthaldehyde (0.75mM) were used for the reaction. Te mixture, together with the GSH standard solution (0.0001563–0.1mg/mL in DMSO (2-fold serial dilution)), was kept at room temperature for 15minutes. Te Pulverized leaves of A. africana (6500 g) Alkaloids AAA (5.23 g, 1.27% w/w) Partitioned with CHCl3: Isopropanol (3:1, v/v) Quaternary alkaloids AAQ (6.13 g, 1.49% w/w) Organic layer Total crude AAE (412.18 g, 6.34% w/w) Organic layer Organic layer Aqueous layer Aqueous layer Aqueous layer Cold maceration (70% ethanol for 72 h) i. Acidifed (dissolved in 30% Acetic acid) ii. Washed 3x with chloroform i. Basifed with 25% NH3(eq) ii. Extracted with DCM, 3x i. Dried with anhydrous MgSO4 ii. Concentrated to dryness iii. Tested with Mayer’s and Dragendof iv. TLC i. Dried with anhydrous MgSO4 ii. Concentrated to dryness iii. Tested with Mayer’s and Dragendof iv. TLC Figure 2: Schematic diagram for the extraction of alkaloids from A. africana. 4 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, R2 � 0.998) was made to fgure out howmuch glutathione was present in the extracts. Te total glutathione in the extracts was expressed as glutathione equivalent (GSH). 2.7.4. 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, 2.5mL of 20mmol/L FeCl3 solutions, 2.5mL of 10mmol/L TPTZ solution, and 25mL of 300.0mmol/L acetate bufer were mixed to make the FRAP reagent. Te AAE, AAA, and AAQ (20 μL) were mixed vi- olently together with 180 μL of the FRAP reagent. In the presence of antioxidants, the complex compound ferric tri- pyridyltriazine (Fe3+-TPTZ) is reduced to its ferrous tripyr- idyltriazine (Fe2+-TPTZ) form, resulting in an intense blue colour that can absorb maximally at a wavelength of 593 nm. 2.8. Profling ofA. africanaExtractsUsingGasChromatography- Mass Spectrometry (GC-MS). 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 30m+10m EZ Guard× 0.25mm internal diameter-fused silica capillary coated with VF-5ms (0.25mm 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 25min. Te system temper- ature was increased up to 150°C (3°C/min), 200°C (8°C/min), and 280°C (2.133minutes). Helium was the carrier gas with a constant fow rate of 2.25mL/minute, with nitrogen serving as the collision gas with a constant fow rate of 1.5mL/minute. Te septum purge was performed at a rate of 30mL/minute for 0.75minutes at a pressure of 27.5 psi. Te mass detector could scan at m/z values ranging from 50 to 550. To identify the phytocomponents in the extracts, an injection volume of 2µL (10mg/mL in acetonitrile) of the samples was used for analysis. Te compounds detected were identifed by correlating the various peaks produced with the mass spectral library NIST 2014 (National Institute of Standards and Technology, Mass Spectral Library) [33].Te confrmation and characterisation of the alkaloidal metabolites were done using isotopic ft ratios (iFit) and mass accuracy. Tis fragmentation pattern analysis provides important information regarding the number of isotopes in the molecule to facilitate molecular formula de- termination which ensured the dependability and correctness of the phytocompounds identifed. 2.9. Analysis of Data. Te tests were conducted in triplicate. Te data were shown as the mean± standard deviation (SD). Te IC50 values were obtained from graphs of dose-response curves through the application of GraphPad Prism 5.0 version software (GraphPad Software Inc., San Diego, CA). Te CC50 and EC50 values were also derived from a dose- response curve using Microsoft Excel 2016. Te student t- test was employed for analysis, and statistical signifcance was set at p< 0.05. 3. 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 alka- loids (AAQ: 6.13 g, 1.49%), as shown in Figure 2 [25, 26].Te basifed aqueous extract with DCM was preliminary con- frmed 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]. 3.2. 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 IC50 values for the extracts (AAE, AAA, and AAQ) were 11.890 µg/mL, 6.217 µg/ mL, and 6.285 µg/mL, respectively. Te IC50 value for the control drug, artesunate, was 0.9×10−3 µg/mL. Previous re- search suggests that extracts with IC50s 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 IC50s of less than 10 µg/ mL are classifed as having “promising” antiplasmodial ac- tivity. Tey also said that IC50s between 10 and 20 µg/mL, 20 and 40 µg/mL, 40 and 70 µg/mL, andmore than 70 µg/mL had “moderate,” “good,” “marginally potent,” and “poor” anti- plasmodial activity, respectively. Te latter antiplasmodial activity score categorization was used in this study. Based on the IC50 values obtained for AAE, AAA, and AAQ, the ex- tracts demonstrated moderate to promising activity against 3D7 P. falciparum parasite clones. 3.3. 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 5 3.4. Antioxidant Activity of Total Crude and Alkaloidal Extracts. Te alkaloidal extracts and the total crude of A. africana yielded an appreciable amount of overall anti- oxidant activity. Te AAE and AAQ had total antioxidant capacities (TAC) of 375.506± 0.047 and 373.638± 0.040mg/ g, respectively (Figure 4). Te AAA had the highest TAC value, at 494.39± 0.058mg/g ascorbic acid equivalent. In this test, both the total crude and the alkaloidal extracts con- tained 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 pre- sented in Figure 5.Te various efective concentrations (EC50) of the extracts were found to be 0.929±0.008mg/mL, 0.287 ± 0.044mg/mL, 0.245±0.040mg/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 scav- enging 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± 0.0002mg/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. Simi- larly, the ferric-reducing antioxidant power (FRAP) of AAE, AAA, and AAQ extracts showed a considerable variance in EC50s (AAE, 1.722± 0.268mg/mL; AAA, 3.568± 0.759mg/ mL, and AAQ, 3.386± 0.015mg/mL) compared to ascorbic acid (0.077± 0.005mg/mL) (Figure 7). Te EC50s of the an- tioxidant activities of the extracts were concentration de- pendent. AAE exhibited the highest power-reducing activity in comparison to ascorbic acid, followed by AAQ and AAA. 3.5. GC-MS Analysis of Compounds from the Alkaloidal Ex- tracts 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 16.683minutes was assigned to gramine. Gramine is an aminoalkylindole alkaloid (C11H14N2; MW-174.24 gmol−1) (M+) and had the highest peak area (31.97%) of all the compounds found in the AAA extract. On the other hand, dexrazoxane (C11H16N4O4; MW-268.27 gmol−1) with Table 1: Antiplasmodial, cytotoxic activities, and therapeutic indices of the AAE, AAA, and AAQ extracts of A. africana. Extracts Antiplasmodial efcacy against 3D7 P. falciparum IC50± SD (µg/mL) Cytotoxicity against RBCs CC50 (µg/mL) Selectivity indices CC50/IC50 Crude extract (AAE) 11.890± 0.011∗∗ >100 >8.410∗∗ Crude alkaloids (AAA) 6.217± 0.012∗∗ >100 >16.085∗∗ Quaternary alkaloids (AAQ) 6.285± 0.456∗∗ >100 >15.910∗∗ Artesunate (control)∗ 0.09± 0.03 (×10−3) >100 >10000 ∗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 (∗∗). AAE AAQ ART AAA 6.25 12.5 25 50 1000 Conc (μg/mL) 0 20 40 60 80 100 120 % M ea n C el l V ia bi lit y 6.25 12.5 25 50 1000 Conc (μg/mL) 0 20 40 60 80 100 120 % M ea n C el l V ia bi lit y 6.25 12.5 25 50 1000 Conc (μg/mL) 0 20 40 60 80 100 120 % M ea n C el l V ia bi lit y 20 40 60 80 1000 Conc (ng/mL) 0 50 100 150 % M ea n C el l V ia bi lit y Figure 3: Erythrocytes’ survival following the subjection of A. africana total crude (AAE), crude alkaloids (AAA), quaternary alkaloids (AAQ), and artesunate (ART) to uninfected red blood cells (RBCs). Te experiments were triplicated (n� 3), with cytotoxic efect (CC50) values greater than 100 for all the extracts as well as the control drug (ART). 6 Advances in Pharmacological and Pharmaceutical Sciences a retention time of 5.252minutes 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. 3.5.1. 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 y = 0.1071x-0.0051 R2=0.9977 0 0.4 0.8 1.2 A bs or ba nc e 2 4 6 8 10 120 Conc (mg/mL) (a) 0 200 400 600 A sc or bi c a ci d eq ui va le nt s (m g/ g) AAA AAQAAE Sample (b) 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). AAQ AAA Ascorbic acid EC50= 0.9291± 0.0081 EC50= 0.245± 0.040 EC50= 0.065± 0.006 EC50= 0.287± 0.044 AAE 0.3125 0.625 1.25 2.5 50 Conc (mg/mL) 0 20 40 60 80 100 % M EA N A N TI O XI D A N T A CT IV IT Y 0 20 40 60 80 100 % M EA N A N TI O XI D A N T A CT IV IT Y 0.50 1.00 1.500.00 CONC. (mg/mL) 0 20 40 60 80 100 % M EA N A N TI O XI D A N T A CT IV IT Y 0.5 1 1.50 CONC. (mg/mL) 0 20 40 60 80 100 % M EA N A N TI O XI D A N T A CT IV IT Y 0.05 0.1 0.15 0.2 0.250 CONC. (mg/mL) Figure 5: Te percentage mean antioxidants activities (DPPH free-radical scavenging) of A. africana total crude (AAE), crude alkaloids (AAA), quaternary alkaloids (AAQ), and ascorbic acid. Data show the mean± standard deviation SD for three repeated runs (n� 3) (p< 0.0001). y = 651473x + 103.69 R2 = 0.998 0.00 10000.00 20000.00 30000.00 40000.00 50000.00 60000.00 70000.00 M ea n Fl uo re sc en ce 0.02 0.04 0.06 0.08 0.1 0.120 [GSH] (mg/mL) (a) 0 0.4 0.8 1.2 1.6 2 G SH E qu iv al en ts (m g/ g) AAQ AAAAAE Sample (b) Figure 6: (a) GSH standard curve and (b) reduced GSH concentrations in AAE, AAQ, and AAA of theA. africana plant.Te data are shown as the mean± standard deviation (SD) (n� 3). Te experiments were triplicated. Advances in Pharmacological and Pharmaceutical Sciences 7 overall MS accuracy was within 5mDa, the molecular for- mulae 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 5mDa 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 alka- loid with a molecular weight and formulae of MW- 174.24gmol−1 [M+] and C11H14N2, respectively. Tis com- pound was fragmented by the loss of CH3· to produce a pre- cursor 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 (−26Da), amine (−18Da), and methyl groups (−15Da) 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 re- tention times of 5.578 and 14.744min, 1,2,3,4-tetrahy- droisoquinoline 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 C9H11N and C10H10N2O, respectively (Table 2). Te fragmentation of 1,2,3,4-tetrahydroisoquinoline led to the abstraction of two hydrogens to a more stable 1,4- dihydroisoquinoline (m/z 131.5), confrming the structure of the compound (Figure 11) [37]. In the case of 1H indole-oxime, the loss of –OHgave a precursor ion atm/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 5.252min (Figure 9) with a mo- lecular ion at m/z 267.27 [M+]- (C11H16N4O4) and m/z 267.27 [M+]- (C11H16N4O4), respectively (Figure 13). Sur- prisingly, the molecular masses of these two compounds were the same, with a similar fragmentation pattern. Tese compounds were identifed as dexrazoxane (85.59%) (C11H16N4O4), m/z 268.27, and its isomer razoxane (5.14%) (C11H16N4O4), m/z 268.27, a synthesized bisdioxopiperazine compound. It was determined that these two compounds were isomers of quaternary alkaloids in the form of QA1 and its zwitter-ionic form, QA2 (Figure 13(a)), similar to what 0 0.4 0.8 1.2 M ea n Ab so rb an ce AAE 12102 4 60 8 Conc (mg/mL) ASCORBIC ACID 0 0.2 0.4 0.6 0.8 M ea n Ab so rb an ce 0.2 0.4 0.8 1 1.20.60 Conc (mg/mL) AAQ 5 10 150 Conc (mg/mL) 0 0.2 0.4 0.6 0.8 M ea n Ab so rb an ce 100 82 1264 Conc (mg/mL) AAA 0 0.2 0.4 0.6 0.8 1 M ea n Ab so rb an ce Figure 7: Ferric-reducing antioxidant power (FRAP) of the total crude AAE (1.722± 0.268mg/mL) and alkaloidal extracts AAA (3.568± 0.759mg/mL), AAQ (3.386± 0.015mg/mL), and ascorbic acid (0.077± 0.005mg/mL). 1 0.8 0.6 0.4 0.2 0 ×109 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 Counts vs. Acquisition Time (min) +EI TIC Scan 663-pes2-22.D Figure 8: GC profle of the crude alkaloidal extracts (AAA) 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. 8 Advances in Pharmacological and Pharmaceutical Sciences https://dnp.chemnetbase.com Ta bl e 2: G C -M S pr of le s of ph yt oc om po un ds of th e cr ud e al ka lo id al ex tr ac t( A A A ) of 70 % et ha no ll ea fe xt ra ct of th e A .a fri ca na pl an t. S. no s. RT (m in ) N am e of co m po un d M F M W (g /m ol ) % Pe ak ar ea 1 5. 57 8 1, 2, 3, 4- Te tr ah yd ro iso qu in ol in e C 9H 11 N 13 3. 19 2. 28 2 5. 85 0 N -[ 3- [N -A zi ri dy l] pr op yl id en e] te tr ah yd ro fu rf ur yl am in e C 10 H 18 N 2O 18 2. 26 0. 61 3 6. 05 8 (+ )- D ib en zo yl -L -t ar ta ri c ac id an hy dr id e C 18 H 12 O 7 34 0. 30 2. 47 4 6. 14 0 3- Ph en yl -3 -p en ta no l C 11 H 16 O 16 4. 24 1. 05 5 6. 64 3 7- M et hy l-2 -o xa -7 -a za tr ic yc lo [4 .4 .0 .0 (3 ,8 )] de ca ne C 9H 15 N O 15 3. 22 2. 47 6 6. 81 0 H yd ra zi ne ,1 -( 2- et hy l-6 -m et hy lp he ny l)- C 9H 14 N 2 15 0. 22 2. 92 7 7. 62 6 2- Ph en yl -l- p- to lu en e es ul fo ny la zi ri -d in e C 15 H 15 N O 2S 27 3. 40 1. 15 8 7. 95 2 Be nz en ep ro pa no ic ac id ,o ct yl es te r C 17 H 26 O 2 26 2. 40 2. 57 9 8. 02 0 2, 5- O ct ad ec ad iy no ic ac id ,m et hy le st er C 19 H 30 O 2 29 0. 40 1. 75 10 9. 99 9 2- Pr op en oi c ac id ,3 -p he ny l- C 9H 8O 2 14 8. 16 4. 95 11 10 .1 63 Et ha no ne ,1 -h yd ro xy -2 ,6 ,6 -t ri m et hy l-2 ,4 -c yc lo he xa di en -1 -y l- C 11 H 16 O 2 18 0. 24 3. 19 12 12 .8 08 5, 5, 8a -T ri m et hy l-3 ,5 ,7 ,8 ,8 a- he xa hy dr o- 2H -c hr om en e C 12 H 20 O 18 0. 29 4. 83 13 13 .2 21 2H -O xe ci n- 2- on e, 3, 4, 7, 8, 10 -h ex ah yd ro -4 -h yd ro xy l-1 0- m et hy l-, [4 s- (4 R∗ ,5 E, 10 S∗ )] - C 10 H 16 O 3 18 4. 23 13 .1 1 14 14 .1 27 (1 H -in do l-3 -y l) ac et al de hy de ox im e C 10 H 10 N 2O 17 4. 20 8. 10 15 14 .7 44 (1 H -in do l-3 -y l) ac et al de hy de ox im e C 10 H 10 N 2O 17 4. 20 7. 04 16 16 .6 83 G ra m in e C 11 H 14 N 2 17 4. 24 31 .9 7 17 22 .1 39 7- N on en am id e C 9H 17 N O 15 5. 24 3. 43 18 26 .1 39 8, 11 ,1 4- Ei co sa tr ie no ic ac id ,( Z, Z, Z) - C 20 H 34 O 2 30 6. 50 2. 39 19 28 .7 21 4- N itr o- be nz oi c ac id ,1 -m et hy l-h ep ty le st er C 15 H 21 O 4 27 9. 33 3. 38 G C -M S� ga s ch ro m at og ra ph y- m as s sp ec tr om et ry ,R T � re te nt io n tim e (m in s) ,M F � m ol ec ul ar fo rm ul a, M W � m ol ec ul ar w ei gh t, an d A A A � cr ud e al ka lo id s of A .a fri ca na le af . Advances in Pharmacological and Pharmaceutical Sciences 9 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). 4. 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, antioxi- dant, 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 me- tabolites 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 high- throughput 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 IC50 values that ranged from 6.217 to 11.890 µg/mL in the order AAA100 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 parasite- mediated 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 prop- erties 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 [65–68]. Te arte- sunate reference drug used in this study had been reported to have antioxidant properties [69–72]. 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. O O O O O O O O O OO OO O O N HN O HN N N NH N N N N N N N HN OH Cl Cl QA1 QA2 QA3 H3C H3C H3C H m/z 268 m/z 112.5 N H N H m/z 140.6 (a) MS Spectrum ×10 8 1 0.8 0.6 0.4 0.2 0 50 100 150 200 250 300 350 400 450 500 550 Counts vs. Mass-to-Charge (m/z) Cpd 1: Dexrazoxane: +EI Scan (rt: 5.202-5.270 min, 16 scans) 666-pes2-22.D (b) Figure 13: (a) Proposed fragmentation pathway and (b) mass spectrum of dexrazoxane. Advances in Pharmacological and Pharmaceutical Sciences 13 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 in- fected red blood cells after the endoperoxide bridge is ac- tivated [73]. In this regard, it has been suggested that any potential antimalarial drugs should have scavenging prop- erties to get rid of the extra free radicals generated by parasite metabolism and other exogenous factors [67, 74–76]. 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. Fur- thermore, the indole and isoquinoline alkaloids identifed in the AAA extract potentially mediated the antiplasmodial and antioxidant activities in the current study. Te phar- macodynamic 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 advan- tages 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 ex- tracts to obtain pure phytocompounds for structure eluci- dation and optimisation studies. Tis will potentially introduce novel lead compounds from this plant into the antimalarial drug discovery pipeline. 5. Conclusion Te fndings of this study suggest that the alkaloidal extracts of A. africana leaves possess promising antiplasmodial ef- fects against 3D7 P. falciparum chloroquine-sensitive par- asites. 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 studymay not translate to clinical application as the protocol used has some limitations though the outcome of the study supports folklore application for malaria infection treat- ment. 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. Abbreviations ART: Artesunate ACT: Artemisinin-based combination treatment AAA: Crude alkaloids CPM: Complete culture medium CC50: Cytotoxic concentration DCM: Dichloromethane DPPH: 2,2-diphenyl-1-picrylhydrazyl assay EDTA: Ethylenediaminetetraacetic acid EC50: Efective concentration at 50% Fe2+-TPTZ: Ferrous tripyridyltriazine Fe3+-TPTZ: Ferric tripyridyltriazine FRAP: Ferric-reducing antioxidant power GC-MS: Gas chromatography-mass spectrometry GC: Gas chromatograph GSH: Glutathione IC50: Inhibition concentration MF: Molecular formula MW: Molecular weight NIST: National Institute of Standards and Technology AAQ: Quaternary alkaloids RBCs: Red blood cells RT: Retention time ROS: Reactive oxygen species SD: Standard deviation TAC: Total antioxidant capacity GSH: Total glutathione concentration AAE: Total crude extract WHO: Te World Health Organization MTT: Te tetrazolium-based colorimetric TLC: Tin layer chromatography. Data Availability Te data used to support the fndings of this study are in- cluded within the article. Ethical Approval Te University of Cape Coast’s Institutional Review Board examined and approved the study protocol (ID: UCCIRB/ CHAS/2016/13). Conflicts of Interest Te authors declare that they have no conficts of interest. Acknowledgments Te authors wish to extend their appreciation to the Uni- versity of Cape Coast and the University of Cape Coast’s Directorate of Research, Innovation, and Consultancy (DRIC) for fnancial assistance. 14 Advances in Pharmacological and Pharmaceutical Sciences References [1] A. T. Tsegaye, A. Ayele, and S. Birhanu, “Prevalence and associated factors of malaria in children under the age of fve years in Wogera district, northwest Ethiopia: a cross-sectional study,” PLoS One, vol. 16, no. 10, 2021. 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