Scientific African 24 (2024) e02151 Available online 4 March 2024 2468-2276/© 2024 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Phytochemical profiling and bioactivity study of Adenia panduriformis in Zambia using UHPLC-MS/MS-MZmine3, GNPS, and METLIN Gen2 Bitwell Chibuye a,b,c,*, Indra Sen Singh b, Luke Chimuka c, Kakoma Kenneth Maseka b a Department of Chemistry Education, Mukuba University, Itimpi Campus, Kitwe PO Box 20382, Zambia b Department of Chemistry, School of Mathematics and Natural Sciences, The Copperbelt University, Kitwe PO box 21692, Zambia c Molecular Sciences Institute, School of Chemistry, University of Witwatersrand, Johannesburg, South Africa A R T I C L E I N F O Editor: DR B Gyampoh Keywords: Adenia panduriformis (Passifloraceae) Antioxidant activity Phytochemical screening LCMS/MS Molecular networking Total phenolic content Total flavonoid content A B S T R A C T Diseases, especially degenerative illnesses, can be treated and avoided altogether, and good health can be enhanced by including health-promoting foods in the diet without side effects. Adenia panduriformis (Passifloraceae) is a wild vegetable consumed as part of the everyday diet in some communities in Zambia. The plant is also used in traditional medicine to effectively treat and manage many diseases, including degenerative diseases. Because of significant health ben- efits and medicinal attributes, this vegetable has very high merit to be investigated for its phy- tochemicals and bioactivities. Therefore, this study screened phytochemicals using qualitative chemical tests and Liquid chromatography-tandem mass spectrometry (LCMS/MS), identified metabolites using molecular networking tools and evaluated the total polyphenolic content and antioxidant activity of the methanolic leaf extract of the vegetable. Solvent extraction by maceration recovered metabolites for screening by qualitative chemical tests, extraction by QuEChERS (quick, easy, cheap, effective, rugged, and safe) was used to recover metabolites for screening by LCMS/MS, and tentative phytochemical constituents were identified using MZmine3, GNPS (global natural products social molecular networking) and METLIN. Total phenolic content (TPC) determination used the Folin-Ciocalteu (FC) method at 765 nm. Total flavonoid content (TFC) was evaluated using the aluminium chloride colorimetric assay at 510 nm. Antioxidant activity was evaluated using 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging assay at 517 nm. Phytochemical screening by qualitative chemical tests revealed the presence of many classes of phytocompounds, such as alkaloids, polyphenols, terpenoids, and saponins. TPC was determined to be 122.30 ± 2.00 mg GAE/g, and TFC was 60.17 ± 0.25 mg QE/g. Screening by LCMS/MS revealed the presence of many metabolites, of which 23 phyto- compounds were identified using MZmine3, 42 by GNPS, and 21 by METLIN. The crude extract exhibited high DPPH free radical quenching activity with a relatively low IC50 value of 53.48 µg/ mL compared to the reference standard ascorbic acid IC50 of 74.47 µg/mL. The significantly lower IC50 value for the wild vegetable than that of ascorbic acid indicates a considerably high effi- ciency of the metabolites in the vegetable as antioxidants. The presence of many metabolites with health-promoting properties and high antioxidant activity of the leaf extract of A. panduriformis may be the science behind its remarkable health benefits and power to treat and manage a variety of diseases, including degenerative illnesses. This study presents results that indicate the potential * Corresponding author at: Department of Chemistry Education, Mukuba University, Itimpi Campus, Kitwe PO Box 20382, Zambia. E-mail address: bchibuye@mukuba.edu.zm (B. Chibuye). Contents lists available at ScienceDirect Scientific African journal homepage: www.elsevier.com/locate/sciaf https://doi.org/10.1016/j.sciaf.2024.e02151 Received 2 April 2023; Received in revised form 22 February 2024; Accepted 3 March 2024 mailto:bchibuye@mukuba.edu.zm www.sciencedirect.com/science/journal/24682276 https://www.elsevier.com/locate/sciaf https://doi.org/10.1016/j.sciaf.2024.e02151 https://doi.org/10.1016/j.sciaf.2024.e02151 http://crossmark.crossref.org/dialog/?doi=10.1016/j.sciaf.2024.e02151&domain=pdf https://doi.org/10.1016/j.sciaf.2024.e02151 http://creativecommons.org/licenses/by-nc-nd/4.0/ Scientific African 24 (2024) e02151 2 of A. panduriformis as a rich source of metabolites with brilliant health-promoting properties and high antioxidant activity, which may open exciting opportunities for further development in the fields of pharmacology and drug development. Introduction The expression ‘Let food be your medicine and medicine be your food’ was highly embraced by the father of medicine, Hippocrates, 2500 years ago [1]. Indeed, food might provide the human body with therapeutic benefits, and a good diet is essential for preventing diseases [2]. It may suffice to postulate that a better diet might be better than drugs and surgeries when fighting illness and diseases. Food may be defined as anything consumed and converted into a substance in the body. Consequently, a poor diet will harm the health of someone because once consumed, food is converted to numerous parts of the body, such as muscles and nerves [3]. This explains why food may be considered medicine to the human body. Therefore, the kind of food consumed determines their good health or lack thereof. Before resorting to surgeries and drugs, one must monitor one’s diet. Consuming certain foods may prevent any diseases that may require surgery and drugs for treatment [4]. Some diseases, such as degenerative diseases, are caused mainly by free radicals. It may suffice to say that free radicals containing oxygen are termed reactive oxygen species (ROS) and are the most biologically sig- nificant free radicals [5]. Naturally, humankind has been relentlessly trying to discover healthier foods with antioxidants that ameliorate the adverse effects of free radicals and other bioactive phytocompounds with health-promoting properties [6]. As for antioxidants or free radical scavengers, they play several physiological benefits in human biological systems. Antioxidants quench free radicals, thereby preventing cell damage that might arise from chemical reactions involving oxidants. Furthermore, antioxidants stabilize or deactivate free radicals before the latter cause damage to cells and other biological targets. Thus, antioxidants are essential for maintaining optimum healthy cells and their well-being. When human cells utilize oxygen, they spontaneously form free radicals as by-products, which in turn can damage cells. Antioxidants act as “free radical scavengers,” thereby preventing and repairing the damage done or may be done by free radicals. Above all, and interestingly, antioxidants may enhance the immunity of the body and reduce the risk of many infections and diseases [7–9]. Consequently, it is crucial to consume foods containing antioxidants and other therapeutic metabolites for the human body to ward off infection or treat disease naturally. Such foods may also include functional foods. Functional foods possess some bacterial strains; they may originate from plants or animals and possess physiologically active molecules beneficial to human health and lowering the risk of chronic infections and disease [10]. Functional foods are neither pills nor capsules, but are consumed as part of the everyday diet, offering health benefits and reducing the risk of chronic disease beyond the universally recognized nutritional effects. In the Copperbelt province and elsewhere in Zambia, a medicinal wild vegetable, Adenia panduriformis, belonging to the family Passifloraceae, offers a myriad of health benefits and is consumed as part of the everyday diet in some communities [11,12]. Additionally, traditional healers prescribe the herb to patients with weakened immunity, such as those with HIV/AIDS. It is believed that wild vegetable is an outstanding immune booster. In addition to such health benefits, it is claimed that the leafy vegetable can heal several diseases such as diabetes, hypertension, malaria, and diarrhea. Worldwide, the woody climber plant is only found in Tanzania, Mozambique, Zambia, and Zimbabwe [11–15]. However, the plant should also grow under similar climatic conditions elsewhere globally. Due to the amazing health benefits of this plant, it is prudent to explore the hidden secrets of this wild vegetable. The determination of the phytochemicals present in it and its antioxidation properties would be an exciting study as it will scientifically corroborate the persistent claims about the wild vegetable as a panacea for many diseases. Therefore, the current study determined the phytochemical profile of A. panduriformis leaf extract, its total phenolic and flavonoid content, and its antioxidant potential. Fig. 1 shows a picture of the climber plant captured in a forest in Zambia’s Kalulushi District of the Copperbelt province. Fig. 1. Leaves of Adenia panduriformis. B. Chibuye et al. Scientific African 24 (2024) e02151 3 Materials Materials The following materials were obtained from the chemical stores of the School of Chemistry, University of Witwatersrand, South Africa: 2.0 mL vials, and various glassware such as test tubes and flasks, 50 mL polytetrafluoroethylene (PTFE) centrifuge tubes, 0.45 µm PTFE filters, and 10 mL syringes and needles. Authentication of the plant Fresh plant leaves and stems were collected from the bush in Kalulushi town, Copperbelt Province of the Republic of Zambia. Plant parts were taken to the Herbarium of the Ministry of the Green Economy and Environment of the Republic of Zambia for authenti- cation. The plant was identified and authenticated as A. panduriformis. Reagents The following HPLC grade reagents were obtained from Merck (Johannesburg, South Africa): gallic acid (G7384), quercetin (Q4951), 2,2-diphenyl-1-picrylhydrazyl (DPPH) (D9132), and acetonitrile (HPLC grade, ≥ 99.9%) Folin–Ciocalteau (FC) reagent (1,090,010,100), anhydrous sodium carbonate (≥99.5%), sodium nitrite (≥97.0%), sodium hydroxide (≥ 98.0%), and anhydrous aluminium chloride (≥ 98.0%), and primary secondary amine (PSA) (Supelclean™ PSA SPE). In addition, the following reagents were obtained from chemical stores of the School of Chemistry, University of Witwatersrand, South Africa: chloroform (HPLC grade, ≥ 99.9%), acetic anhydride (≥ 99.5%), ferric chloride (≥ 99.99%), magnesium ribbon (≥ 99.0%,), lead acetate (≥99%), hydrochloric acid (≥37%), methanol (HPLC grade, ≥ 99.9%), glacial acetic acid (≥99%), sulphuric acid (≥ 98%), Hager’s reagent (1.3%, in water, saturated), and ammonia (Reagent, ≥32%). Methods Extraction of phytocompounds for phytochemical screening Preliminary phytochemical screening of A. panduriformis leaf extract was carried out in the Environmental Analytical Chemistry Laboratory, School of Chemistry, University of Witwatersrand, South Africa. The fresh leaves collected were dried in the shade and then reduced to powder using a mortar and pestle. Phytocompounds were extracted in 80% methanol following a method by Vieira (2022) with some modifications [16]. In brief, 5.0 g of the powdered sample was placed in 100 mL flasks, and 50 mL methanol (80%) was added. This solid-solvent ratio of 1:10 (m/v) was chosen based on its effective extraction yield for the maceration extraction of metabolites from other food matrices [17]. The flasks were sealed and subsequently placed in a water shaker with continuous agitation fixed on 150 rpm at 40◦C for the extraction time of 24 hours for maximum phytochemical recovery. The recovered extracts were filtered using 0.45 µm PTFE filters. Finally, the crude extract was subjected to phytochemical screening through qualitative reactions as part of phytochemical studies to identify the various families of secondary bioactive metabolites. QuEChERS Extraction Process for LC-MS QTOF Analysis An adapted QuEChERS extraction procedure by Guo (2018) was used to recover metabolites from powdered leaf material [18]. The modification of the method was in terms of the amount of the sample used. In this study, a 3.0 g sample was used instead of the 5.0 g used in the previous study. This was to attain the solid-to-solvent ratio of 1:10 that assures maximum extraction [19]. Essentially, 3.0 g of plant sample was weighed and placed in 50 mL PTFE centrifuge tubes. To hydrate the plant matrix, 3 mL of double distilled water was added, and the contents were mixed well by vortex for 30 s and left to stand for 30 min. After that, 10 mL of acetonitrile was added, and the contents vortexed for 60 s. Subsequently, 4.0 g of MgSO4, and 1.0 g of NaCl were added and the contents were shaken well by vortex for 60 s and immediately centrifuged at 4000 rpm for 10 min. During the clean-up stage, 5.0 mL of organic supernatant was recovered and transferred to a clean polypropylene tube, and then 150 mg PSA sorbent and 75 mg MgSO4 were added. The contents were then shaken well by vortex for 60 s and promptly centrifuged for 10 min. Eventually, an aliquot of 2.0 mL supernatant was collected and used for LCMS/MS analysis. The adsorbent, PSA, was used to remove unwanted substances such as pigments, proteins, carbohydrates, organic acids, and polar interferences. Further, graphitized carbon black was used to remove chlorophylls. These unwanted substances are removed because they interfere with the LCMS analysis. Extraction of polyphenols for total polyphenol content, and antioxidant activity determination Polyphenols were extracted following a previous method by Ralepele, Chimuka, Nupia, and Risenga (2021) with modifications [20]. In this study, the sample–solvent ratio used was 1: 10 instead of using the 1:6 sample-solvent, due to the high phytochemical recovery for a 1:10 sample-solvent ratio [21]. Briefly, 5.0 g of powdered leaf sample were placed in a 50 mL PTFE centrifuge tube containing 45 mL of 80% methanol for 36 hours with intermittent agitation for 150 rpm in a temperature-controlled water shaker at 40◦C. After spontaneous cooling, the resultant extract was centrifuged at 4000 rpm for 10 min to separate solids from the solution. The procedure was repeated and the recovered supernatants combined. A 0.45 µm PTFE filter was used to remove the solid particles. The filtered extracts were used for total polyphenolic (phenolic and flavonoids) content determination and evaluation of the antioxidant B. Chibuye et al. Scientific African 24 (2024) e02151 4 activity. Phytochemical screening using chemical tests Preliminary phytochemical screening was carried out using extracts described in the earlier procedure (3.1) to identify broad categories of bioactive metabolites present in the sample using standard qualitative tests reported in previous studies, that is, Alkaloids (Hager’s Test), Tannins (Braymer’s Test), Saponins (Foam Test), Steroids (Salkowski Test), phenolics (Libermann’s test), flavonoids (Shinoda Test), Carbohydrates (Molisch’s Test), Glycosides (Liebermann’s Test), and Anthraquinones (Borntrager’s Test) [21–25]. LC-MS/QTOF analysis UHPLC-MS2 screening was achieved by following a previous method by Ralepele, Chimuka, Nupia, and Risenga (2021) with modifications [20]. Essentially, 10 µL of the crude extract recovered through QuEChERS extraction was injected into and chro- matographically separated on a Phenomenex C18 column. The UHPLC system was connected to a quadrupole time-of-flight (Bru- kinjection ver Daltonik GmbH, Bremen, Germany). Chromatographic separation was attained using a binary LC solvent system. Mobile phase A consisted of HPLC-grade formic acid and mobile phase B contained 100% acetonitrile, which was pumped at a flow rate, of 0.50 mL/min. The gradient was scheduled in the following order, that is, B increment: fixed at 5% from 0 to 10 min, raised from 5 to 100% from 10 to 25 min, and kept at 100% until 35 min. MS2 analysis was achieved through Electrospray Ionization in the negative ion mode with the following optimized operating conditions: collision energy, 20–50 eV; capillary voltage, + 4000 V; dry gas tem- perature, 210◦C; nebulizer pressure, 2.0 bar; flow rate, 8.0 L/min; spectra rate, 1 s− 1; and scan mass range, 50 – 1300 m/z. Measurement of total phenolic content (TPC) TPC was measured using a UV–Vis spectrophotometer (Varian, Cary 50 Conc, Darmstadt, Germany) following a previously re- ported method by Aryal et al (2019) with some modifications [26]. Briefly, 0.2 mL of extract was added to 0.75 mL of Folin-Ciocalteau reagent (1:10 dilution with distilled deionized water (DDW)). After 3 min, 2 mL 7.5% (w/v) Na2CO3 solution was added, and then DDW was also added to dilute the mixture to a final volume of 10.0 mL. The contents were mixed well and left to stand in the dark at ambient conditions for 2 h. Absorbance was measured after 30 min at 765 nm using a UV–Vis spectrophotometer, and 80% MeOH was used as the blank. Measurements were carried out with gallic acid standard concentrations of 25, 50, 150, 250, 350, and 450 µg/mL prepared in 80% MeOH. Absorbance was transformed to phenolic content in mg of gallic acid equivalent (GAE) per g. Determination of total flavonoid content (TFC) Total flavonoids were determined following an adapted previous method by Pakade, Cukrowska, and Chimuka (2013) [27]. In essence, 0.3 mL extract was placed in a 10-mL volumetric flask containing 4.0 mL DDW. Afterward, 0.3 mL NaNO2 (5 %) was added, and the constituents were shaken well by Voltex. After 5 minutes, 3.0 mL of AlCl3 (1.0%) solution was added. Further, 2.0 mL of NaOH (1.0 M) was added after allowing the contents to stand for 6 min. Finally, 0.4 mL of DDW was added, bringing the total volume to 10.0 mL. The contents were mixed well by vortex, and absorbance was measured at 510 nm (Varian, Cary 50 Conc, Darmstadt, Germany). Quercetin was used to prepare the standard curve and TFC expressed as quercetin equivalent (QE) in mg per g. Determination of DPPH antioxidant activity A previously reported radical-scavenging assay by Aryal et al (2019) was used with modifications to determine the capacity of leaf extracts to scavenge DPPH [26]. In brief, 50.0 mg DPPH was dissolved in 100 mL of MeOH (80%) to make a stock solution, from which a working solution was made through dilution (1:5) with MeOH (80%). There were six quantities of the extract, that is, 20, 40, 60, 80, 100, and 120 µg/mL. The DPPH radical scavenging assay consisted of 0.2 – 1.2 mL extract and 1.4 mL working DPPH solution. The contents were added with MeOH (80%) to make up 2.0 mL. The mixture underwent incubation for 45 minutes in the dark before measuring the absorbance at 517 nm. Similarly, the reference, standard ascorbic acid, was prepared as follows. 2.0 mg ascorbic acid was dissolved in 2.5 mL of DDW, thereby making the concentration of stock reference solution 0.8 mg/mL. Subsequently, serial di- lutions of 20, 40, 60, 80, 100, and 120 µg/mL were made. Subsequently, 1.4 mL of DPPH was added, and the contents were added with methanol to make up to 2.0 mL. The mixture underwent incubation for 45 minutes in the dark before measuring the absorbance at 517 nm. Inhibition (%) was determined using the following equation. % Scavenging / Inihibition = ( ABS sample − ABS blank ABS control ) × 100 Where ABS sample = sample absorbance or reference standard absorbance, ABS blank = absorbance of MeOH, and ABS control = absorbance of DPPH working solution. B. Chibuye et al. Scientific African 24 (2024) e02151 5 Statistical Analysis All measurements were done in triplicate and recorded as mean ± SD. Numerical data were analyzed using Microsoft Excel 2016 and calibration curves were constructed using Origin 2018. Results and data analysis Phytochemical screening using qualitative chemical tests Phytochemical screening using standardized qualitative tests of the crude methanolic leaf extract revealed the presence of many groups of metabolites, as depicted in Table 1. LCMS/MS screening Phytochemical screening of A. panduriformis leaf using UHPLC− ESI-MS/MS revealed the presence of exciting phytocompounds, that may cause important pharmacological effects on humans when the wild vegetable is consumed in the diet. The phytochemical LCMS/MS screening was carried out in the negative ion mode, where many metabolites were found with higher intensity. Molecular identification The study used tandem mass spectrometry-based tools to identify phytochemicals. Four tools used in this study included MZmine 3, GNPS library match, GNPS feature networking advance analysis, and METLIN GEen2. MZmine3 molecular identification. Twenty-six (26) phytochemicals were identified using MZmine 3.2.8 software, forty-two (42) were identified using library matching, whereas fourteen (14) were identified by advanced feature-based analysis of GNPS [28,29]. The Bruker data were initially converted to an “mzML” file compatible with MZmine and GNPS. The file conversion was attained using the MSConvert package (Version 3.0.19330, Proteowizard Software Foundation, USA). The converted “mzML” file was initially imported to the MZmine3.2.8 platform and processed following standard MZmine 3 workflow enabling the exportation of processed data as MGF and quantification table files for further use by the GNPS platform. The mzML data file was imported to MZmine 3.2.8. MS level (1) mass detection settings were performed by setting the noise level at 25.0 and m/z tolerance at 5.0 ppm. Mass detection level (2) was set at a noise level of 20.0 and m/z tolerance at 5.0 ppm. Chro- matogram builder settings included a minimum group size in the number of scans to be 5, group intensity threshold of 35.0, a min- imum highest intensity of 45.0, and scan-to-scan accuracy (m/z) at 30.0 ppm. Resolving was achieved using a retention time tolerance of 0.2 minutes, MS1 to MS2 precursor tolerance (m/z) at 10.0 ppm, the minimum merged intensity at 250, chromatographic threshold at 0.8, minimum absolute height at 4000.0, Minimum ratio of peak top/edge at1.2, and Min number of data points of 5. 13C isotope filtering parameters included m/z tolerance of 20 ppm, retention time tolerance of 0.1 minutes, and the most intense representative isotope to be considered as representative isotope. Aligning of feature list step set parameters as m/z tolerance at 20.0 ppm, retention time tolerance at 0.2 minutes, weight for RT at 2.0, and mobility weight at 2.0. Feature list filtering settings included minimum features in a row (1.0), minimum features in an isotope pattern (2), and m/z tolerance at 5.00 ppm. The parameters set in Gap filling step included intensity tolerance at 0.2, m/z tolerance at 5.0 ppm, retention time tolerance at 0.6 minutes, and minimum data points at 3. For MZmine 3 molecular networking, spectral library match used MoNA (MoNA-export-Experimental Spectra.json) library (1,545,052 spectra) using only MS level 2 scans, precursor m/z tolerance at 20.0 ppm, CCS tolerance [%] at 0.05 and minimum ion density was 1.0. Molecular networking identified twenty-six (26) phytochemicals, as depicted in Table 2. Table 1 Results of phytochemical screening of the leaf extract of A. panduriformis. # Metabolites Methanolic extract 1 Phenolics + 2 Saponins + 3 Anthraquinones + 4 Steroids + 6 Terpenoids + 7 Cardiac glycosides + 8 Proteins + 9 Anthocyanins + 10 Carbohydrates + 11 Tannins + 12 Flavonoids + 13 Alkaloids + 15 Volatile oils - - = absent, + = present B. Chibuye et al. Scientific African 24 (2024) e02151 6 Global natural product social molecular networking (GNPS) library matching The UHPLC-MS/MS data for the A. panduriformis obtained from its QuEChERS leaf extract were subjected to GNPS library match analysis available online at: http://gnps.ucsd.edu/ProteoSAFe/status.jsp?task=160cbd2a9f5e43edb471190f4622c1c3. The Bruker data converted “mzML” files were uploaded and submitted to the GNPS platform using Filezilla Client 3.62.0 [30,31]. Table 3 shows forty-two (42) GNPS-identified phytocompounds by UHPLC− ESI-MS/MS in the negative ion mode. Base peaks are shown in bold numbers. GNPS advanced analysis The UHPLC-MS/MS data of the A. panduriformis obtained from its QuEChERS leaf extract was subjected to GNPS feature networking advanced analysis tool. The GNPS analysis used mzML, MS2 MGF, and quantification table files. The Bruker data converted “mzML” files were uploaded and submitted to the GNPS platform using Filezilla Client 3.62.0 [30,31]. MGF and quantification table files derived from MZmine 3.3 were also uploaded. The results are available online at https://gnps.ucsd.edu/ProteoSAFe/status.jsp? task=dc4f0f62cfd343029e412b55fd60cf88. Table 4 shows identified phytocompounds by UHPLC− ESI-MS/MS in the negative ion mode and subsequently using feature networking advanced analysis of GNPS. It is interesting to note from Tables 2, 3, and 4 that there are some distinct compounds, such as Bergenin, Suberic acid, Azelaic acid, and (Z)-5,8,11-trihydroxyoctadec-9-enoic acid, were identified by both mzmine3 and GNPS. The GNPS analyses afforded 37 metab- olites more than mzmine3. MZmine 3 identified a smaller number of molecules because of access to only one database-MoNA used in this study. Analysis of the GNPS molecular network that was earlier obtained from the arrangement of the LC-MS/MS data of the crude leaf extract and fractions of A. panduriformis leaf extract revealed the presence of a total of 404 nodes equivalent to precursor ions visu- alized in Cytoscape 3.9.1, partially depicted in Fig. 2 [32,33]. Fig. 2 is available online at: https://gnps-cytoscape.ucsd.edu/process? task=dc4f0f62cfd343029e412b55fd60cf88%26override_path=gnps_molecular_network_iin_collapse_graphml%2F Table 2 MZmine 3.2.8 based tentative identification of phytocompounds from crude extract of A. panduriformis leaves by UHPLC− ESI-MS/MS in the negative ion mode. ID RT [M-H]− Exact Mass MS2 Tentative Compound Formula Cosine similarity 1 4.17 145.0484 146.0734 83, 99, 127 (100) 2-methylglutaric acid C6H10O4 1.000 2 4.70 327.0664 326.0722 207 (100), 234 Bergenin C14H16O9 0.840 3 4.72 327.0662 326.0722 192, 207 (100), 234 Bergenin isomer C14H16O9 0.840 4 5.59 173.0795 174.0671 111 (100), 129, 84, 83 Suberic acid C8H14O4 1.000 5 5.68 343.2122 344.4480 127, 171, 229, 325 FA 18:2+4O C18H32O6 0.845 6 5.93 [M+H]+ 187.0965 186.0892 97, 112, 116, 125 (100), 143, 169 Gabapentin Related Compound E, 1-(carboxymethyl) cyclohexane carboxylic acid) C9H14O4 1.000 7 5.93 187.0946 188.0440 125 (100), 169 Azelaic acid C9H16O4 1.000 8 6.17 305.1190 306.0370 75, 105 (100), 129, 143, 205, 243 8,8-dimethyl-2-phenylpyrano[2,3-f]chromen-4-one C20H16O3 0.982 9 6.23 [M+H]+ 199.0978 198.0905 130, 155 (100), 2-amino-3-methylimidazol [4,5-f] quinoline C11H10N4 0.998 10 6.36 [M+H]+ 327.2122 326.2067 327 (100), 171, 211 Hydroquinidine C20H26N2O2 0.994 11 6.47 [M+H]+ 325.1956 324.1838 97, 127, 171, 211, 229, 251 (R)-[(2S,4S,5R)-5-ethenyl-1-azabicyclo[2.2.2] octan-2-yl]- (6-methoxyquinolin-4-yl) methanol (Quinine) C20H24N2O2 1.000 12 6.47 325.1956 326.1834 171, 211, 229, 251 Quinidine C20H24N2O2 1.000 13 6.59 [M+H]+ 329.2275 328.2334 99, 211 (Z)-5,8,11-trihydroxyoctadec-9-enoic acid C18H34O5 0.952 14 6.72 137.0223 138.0253 75, 93 (100), 108 4-Hydroxybenzoic acid C7H6O3 15 6.72 137.0223 138.0226 75, 93 (100), 108 Salicylic acid C7H6O3 1.000 16 6.76 [M+H]+ 327.2117 326.2112 80, 129, 187 (100), 209 Ajmaline C20H26N2O2 0.983 17 6.76 112.9846 113.9834 69.9, 70.7 Acetylenedicarboxylic acid C4H2O4 1.000 18 6.98 209.1145 210.1256 137, 165 (100) Jasmonic acid C12H18O3 1.000 19 6.98 329.2272 328.2334 99, 199, 329 (100) (Z)-5,8,11-trihydroxyoctadec-9-enoic acid C18H34O5 0.952 20 7.80 299.0506 300.0901 176, 192, 207 (100), 284 3′,5,7-Trihydroxy-4′-methoxy-flavone (Diosmetin) C16H12O6 0.905 21 7.86 269.1698 270.0530 148, 205, 221 (100), 236 Genistein C15H10O5 1.000 22 8.76 265.1422 266.1552 96, 165, 265 (100) dodecyl hydrogen sulfate C12H26O4S 0.999 23 8.78 [M+H]+ 293.2060 292.2110 179, 195, 223, 256,275, 293(100) 8-[3-oxo-2-[(E)-pent-2-enyl]cyclopenten-1-yl]octanoic acid C18H28O3 0.869 24 8.82 293.2122 294.2195 179,195, 223, 256, 275 HOTrE C18H30O3 0.907 25 9.10 265.1426 266.1552 96, 265 (100) Dodecyl sulfate isomer C12H26O4S 1.000 26 9.84 [M+H]+ 384.1728 383.1594 164, 177, 194, 237, 384 (100) Prazosin C19H21N5O4 1.000 B. Chibuye et al. http://gnps.ucsd.edu/ProteoSAFe/status.jsp?task=160cbd2a9f5e43edb471190f4622c1c3 https://gnps.ucsd.edu/ProteoSAFe/status.jsp?task=dc4f0f62cfd343029e412b55fd60cf88 https://gnps.ucsd.edu/ProteoSAFe/status.jsp?task=dc4f0f62cfd343029e412b55fd60cf88 https://gnps-cytoscape.ucsd.edu/process?task=dc4f0f62cfd343029e412b55fd60cf88%26override_path=gnps_molecular_network_iin_collapse_graphml%2F https://gnps-cytoscape.ucsd.edu/process?task=dc4f0f62cfd343029e412b55fd60cf88%26override_path=gnps_molecular_network_iin_collapse_graphml%2F Scientific African 24 (2024) e02151 7 Table 3 GNPS identification of phytocompounds from the crude extract of A. panduriformis leaves by UHPLC− ESI-MS/MS in the negative ion mode. No. RT (min) Precursor MS2 Fragments Tentative Compound Formula Cosine Library class library 1 4.64 327.072 192.01, 193.01, 207.03, 234.02, 237.04, 249.04, 312.05, 327.07 Bergenin C14H16O9 0.84 Bronze Trent Northen 2 4.65 327.072 192.01, 193.01, 207.03, 234.02, 237.04, 249.04, 312.05, 327.07 Bergenin C14H16O9 0.84 Bronze Trent Northen 3 4.69 327.072 164.01, 166.03, 177.02, 192.00, 193.01, 194.02, 207.03, 234.01,249.04, 312.05, 327.07 Bergenin C14H16O9 0.94 Bronze Massbank 4 4.69 327.072 164.01, 166.02, 177.02, 190.03, 192.01, 193.01, 194.02, 207.03, 234.02, 249.04, 312.05, 327.02 Bergenin C14H16O9 0.83 Bronze Massbank 5 5.13 429.191 91.02, 135.12, 137.10, 161.10, 179.11, 205.09, 223.10 Anziaic acid C24H30O7 0.71 Bronze Jean-Luc WOLFENDER Pierre-Marie ALLARD 6 5.23 431.098 283.06, 311.06, 312.06, 323.06, 341.06, 431.10 Isovitexin(4) C21H20O10 0.78 Bronze Massbank 7 5.24 431.098 268.04, 283.06, 311.06, 323.03, 341.07, 431.10 Isovitexine C21H20O10 0.81 Bronze Stephane GREFF 8 5.57 173.0 57.30, 66.80, 81.40, 83.30, 109.20, 111.20, 129.40, 131.00 Suberate|1,8-Octanedioic acid| Cork acid|Suberic acid| Octanedioic acid|1,6- Hexanedicarboxylic acid C8H14O4 0.86 Bronze Massbank 9 5.90 187.098 57.03, 97.07, 123.08, 123.10, 143.11, 169.09, 187.10 Azelaic acid (isomer) C9H16O4 0.88 Bronze Trent Northen 10 5.91 187.097 97.07, 123.08, 125.10, 126.10, 143.11, 187.10 Massbank:PR309132 Azelaic acid (Not validated) C9H16O4 0.91 Bronze Massbank 11 5.92 187.098 97.06, 125.10, 126.10, 187.10, Azelaic acid C9H16O4 0.88 Bronze MoNA 12 5.93 243.16 181.16, 225.15, 143.16 Brassylic acid C13H24O2 0.80 Gold PI 13 6.34 327.218 85.03, 97.07, 171.10, 183.14, 211.13, 221.12, 229.14, 327.22 (10E,15E)-9,12,13- trihydroxyoctadeca-10,15-dienoic acid C18H32O5 0.89 Gold Wolfender 14 6.36 657.437 329.22, 330.23 h_163_prostanozol_3′OH C20H28N2O2 0.91 Gold Mehdi Beniddir 15 6.54 659.14 217.08, 241.08, 285.07, 329.06, 330.07 Nornotatic acid C17H14O7 0.72 Bronze Joel BOUSTIE Pierre LE POGAM 16 6.56 329.231 171.10, 183.14, 211.13, 229.14, 329.23 Massbank:PR309108 FA 18:1+3O C18H34O5 0.94 Bronze Massbank 17 6.80 227.129 92.99, 142.99, 165.13, 183.14, 184.14, 209.98, 227.13, 228.99 Spectral Match to Butanedioic acid, 2-(4,4-dimethyl-2- methylenepentyl)- from NIST14 C12H20O4 0.90 Bronze Data from Wiemann 18 6.81 227.129 111.08, 165.13, 83.14, 209.12, 227.13 Traumatic Acid - 40.0 eV C12H20O4 0.88 Gold PI 19 6.82 227.129 165.13, 183.14, 09.12, 227.13 Traumatic Acid - 30.0 eV C12H20O4 0.83 Gold PI 20 6.97 329.233 99.08, 169.12, 199.13, 211.13, 329.23 Massbank:PR309110 FA 18:1+3O C18H34O5 0.79 Bronze Massbank 21 7.11 309.206 137.10, 141.08, 154.08, 171.10, 172.10, 185.11, 223.13, 247.20, 251.16, 291.20, 309.20 Massbank:PR309084 FA 18:3+2O C18H30O4 0.70 Bronze Massbank 22 7.14 329.22 127.02, 167.05, 71.08, 195.11, 199.10, 01.09, 211.08, 227.10, 75.16, 293.17 (Z)-5,8,11-trihydroxyoctadec-9- enoic acid C18H34O5 0.71 Bronze Mona 23 7.24 307.19 71.05, 97.06, 121.07, 125.09, 167.14, 185.12, 209.11, 211.13, 235.13, 289.18, 307.19 Massbank:PR309076 FA 18:4+2O C18H28O4 0.83 Bronze Massbank 24 7.33 881.273 325.05, 395.13, 441.14, 881.27 ginkgolide C C20H24O11 0.82 Gold Sang Hee SHIM Kyo Bin 25 7.83 294.146 71.01, 96.96, 220.15, 221.15, 236.10, 293.18 1-(5,10-dioxo-2,3,5a,6,7,8- hexahydro-1H-dipyrrolo[1,2- C14H21N3O4 0.78 Bronze MoNA (continued on next page) B. Chibuye et al. Scientific African 24 (2024) e02151 8 Notably, most of the 404 nodes in the molecular network show ions for metabolites. For example, depicted in Fig. 3 is a nine (9) node cluster extracted from Fig. 2 showing four (4) isomeric molecular ions of one metabolite, bergenin, and five (5) unknown me- tabolites. Indeed, the information extractable from feature-based mass spectrometry for metabolites seems to be fingerprints of me- tabolites, analogous to information from DNA for living organisms. Phytochemical identification using METLIN Gen2 The MGF file used in GNPS previously described was submitted to METLIN Gen2 with a tolerance set at 30 ppm [34]. Table 5 shows a list of 21 compounds generated using METLIN The tentative structures of the metabolites from Table 5 are presented in Fig. 4. Table 3 (continued ) No. RT (min) Precursor MS2 Fragments Tentative Compound Formula Cosine Library class library d:1′,2′-f]pyrazin-10a-yl)propan-2- yl carbamate 26 7.83 293.212 148.00, 164.08, 177.08, 192.11, 205.08, 220.10, 221.12 13-S-hydroxy-6Z,9Z,11E- octadecatrienoic acid C18H30O3 0.77 Bronze Massbank 27 7.85 293.212 195.23, 221.20, 236.15, 275.26, 293.15 (9Z,11E,15Z)-13-Hydroxy- 9,11,15-octadecatrienoic acid C18H30O3 0.73 Bronze Massbank 28 7.85 293.212 97.04, 148.06, 164.03, 177.04, 205.08, 220.09, 221.14 (9Z,11E,15Z)-13-Hydroxy- 9,11,15-octadecatrienoic acid C18H30O3 0.82 Bronze Massbank 29 7.86 293.176 57.03, 71.01, 177.09, 192.12, 205.12, 220.15, 221.16, 236.11, 274.88 6-Gingerol C17H26O4 0.85 Bronze Trent Northen 30 8.20 264.124 61.99, 79.96, 96.96 Anisomycin C14H19NO4 0.91 Bronze MoNA 31 8.22 313.237 99.08, 129.09, 183.13, 195.13, 246.55, 277.22, 313.23 Massbank:PR309105 FA 18:1+2O C18H34O4 0.70 Bronze Massbank 32 8.25 313.239 99.08, 129.09, 183.14, 184.14, 195.14, 77.22, 295.23, 313.24, 314.24 Spectral Match to 12,13-DiHOME from NIST14 C18H34O4 0.82 Bronze Data from Wiemann 33 8.50 341.138 107.05, 123.01, 147.04, 165.02, 179.03, 183.03 8-Prenylnaringenin|(2S)-5,7- dihydroxy-2-(4-hydroxyphenyl)-8- (3-methylbut-2-enyl)-2,3- dihydrochromen-4-one C20H20O5 0.90 Bronze Massbank 34 8.53 341.139 107.05, 123.01, 137.02, 147.02, 165.02, 183.03, 285.07 MLS001360619–01 C20H20O5 0.87 Gold Dorrestein 35 8.55 341.138 107.05, 123.01, 147.04, 165.02, 179.03, 183.03 8-Prenylnaringenin|(2S)-5,7- dihydroxy-2-(4-hydroxyphenyl)-8- (3-methylbut-2-enyl)-2,3- dihydrochromen-4-one (isomer) C20H20O5 0.89 Bronze Massbank 36 8.59 341.139 99.01, 123.01, 137.02, 165.02, 183.03, 285.07 5,7-dihydroxy-2-(4- hydroxyphenyl)-6-(3-methylbut-2- enyl)-2,3-dihydrochromen-4-one 0.89 Gold Dorrestein 37 8.64 434.931 170.01, 183.02, 217.99, 243.99, 249.96, 277.95, 317.97, 329.96, 365.94, 398.95 Fipronil|5-amino-1-[2,6-dichloro- 4-(trifluoromethyl)phenyl]-4- (trifluoromethylsulfinyl)pyrazole- 3-carbonitrile C12H4Cl2F6N4OS 0.78 Bronze Massbank EU 38 8.65 434.931 249.96, 251.95, 277.95, 317.97, 319.97, 329.96, 331.96, 365.94 Fipronil|5-amino-1-[2,6-dichloro- 4-(trifluoromethyl)phenyl]-4- (trifluoromethylsulfinyl)pyrazole- 3-carbonitrile C12H4Cl2F6N4OS 0.82 Bronze Massbank 39 8.67 434.931 249.96, 277.95, 317.97, 329.96, 331.96, 365.94, 398.95 Fipronil|5-amino-1-[2,6-dichloro- 4-(trifluoromethyl)phenyl]-4- (trifluoromethylsulfinyl)pyrazole- 3-carbonitrile C12H4Cl2F6N4OS 0.83 Bronze Massbank 40 8.71 293.212 121.11, 183.14, 235.17, 275.20, 293.21 Massbank:PR309079 FA 18:3+1O C18H30O3 0.76 Bronze Massbank 41 8.80 734.599 294.29, 296.30, 30.43, 448.33, 580.57, 91.62, 734.60 Spectral Match to 1-(1Z- Octadecenyl)-2-(5Z,8Z,11Z,14Z- eicosatetraenoyl)-sn-glycero-3- phosphoethanolamine from NIST14 C43H78NO7P 0.84 Bronze Katrina Waters; Yoshihiro Kawaoka;Richard Smith;Thomas Metz 42 8.82 293.213 97.13, 171.21, 179.19, 195.19, 196.20, 205.17, 224.18, 249.33, 275.33 Spectral Match to 13S-Hydroxy- 9Z,11E,15Z-octadecatrienoic acid from NIST14 C18H30O3 0.77 Bronze Data from Albert Rivas Ubach B. Chibuye et al. Scientific African 24 (2024) e02151 9 Table 4 Phytocompounds identified by UHPLC− ESI-MS/MS in the negative ion mode and subsequent use of feature based advanced analysis of GNPS. No. Precursor MS2 Fragments Tentative Compound Formula Cosine Library class library 1 329.231 99.08, 139.11, 171.10, 183.14, 211.13, 229.14 Massbank:PR309108 FA 18:1+3O C18H34O5 0.93 Bronze Massbank 2 327.072 164.01, 192.00, 194.02, 207.03, 234.01, 249.04, 312.05 Massbank:PR307403 Bergenin C14H16O9 0.91 Bronze Massbank 3 327.218 85.03, 97.07, 171.10, 211.13, 229.14, 327.22, (10E,15E)-9,12,13-trihydroxyoctadeca-10,15- dienoic acid C18H32O5 0.89 Gold Wolfender 4 327.072 192.01, 193.01, 207.03, 234.02, 249.04, 312.05, 327.07 Massbank:PR307395 Bergenin C14H16O9 0.87 Bronze Massbank 5 293.176 71.01, 177.09, 192.12, 205.12, 220.15, 221.16, 236.11, 274.88, 293.18 6-Gingerol C17H26O4 0.84 Bronze Trent Northen 6 363.049 192.01, 193.01, 207.03, 234.02, 327.07 bergenin CollisionEnergy:205,060 C14H16O9 0.83 Bronze Trent Northen 7 363.049 192.01, 207.03, 234.02, 249.04, 312.05, 327.07 bergenin CollisionEnergy:102,040 C14H16O9 0.83 Bronze Trent Northen 8 434.931 183.02, 249.96, 277.95, 317.97, 329.96, 398.95 Fipronil/ 5-amino-1-[2,6-dichloro-4- (trifluoromethyl)phenyl]-4- (trifluoromethylsulfinyl)pyrazole-3-carbonitrile C12H4Cl2F6N4OS 0.83 Bronze Massbank 9 311.092 79.96, 119.05, 170, 183.01, 184.01, 197.03 5,6,2′-Trimethoxyflavone C18H16O5 0.77 Bronze MoNA 10 329.233 99.08, 169.12, 199.13, 211.13, 329.23 Massbank:PR309110 FA 18:1+3O C18H34O5 0.76 Bronze Massbank 11 307.19 121.07, 185.12, 209.11, 211.13, 235.13, 307.19 Massbank:PR309076 FA 18:4+2O C18H28O4 0.75 Bronze Massbank 12 265.146 97.08, 265.48 Spectral Match to Dodecyl sulfate from NIST14 C12H26O4S 0.75 Bronze Data from Luan 13 329.288 127.02, 171.08, 199.10, 201.09, 211.08, 293.17 (Z)-5,8,11-trihydroxyoctadec-9-enoic acid C18H34O5 0.75 Bronze MoNA 14 313.239 99.08, 129.09, 183.14, 184.14, 195.14, 77.22, 295.23, 313.24, 314.24 Spectral Match to 12,13-DiHOME from NIST14 C18H34O4 0.71 Bronze Data from Wiemann Fig. 2. Molecular network (showing clusters of metabolites of interest) based on LCMS/MS data in the negative ionization mode of A. panduriformis leaf extract. The network is shown as a pie chart to display the relative abundance of each ion in the analyzed sample. B. Chibuye et al. Scientific African 24 (2024) e02151 10 Total polyphenolic content Total phenolic content (TPC) TPC in methanolic leaf extract of A. panduriformis was determined using the FC assay with gallic acid as standard. TPC was evaluated using the calibration curve of the regression equation, y = 0.00111x + 0.11836, R2 = 0.997, and expressed as mg gallic acid equivalent per gram (mg GAE/g), as described in Table 6. Total flavonoid content (TFC) TFC was expressed as quercetin equivalent (QE) in mg/g of extract. The leaf extract contained 60.17 ± 0.25 mg QE/g total fla- vonoids, as depicted in Table 6. Table 6 shows that the total phenolic content (122.30 ± 2.00 mg GAE/g) was twice more than the Fig. 3. Network showing a pie chart displaying nodes of molecular ions (red dots) of bergenin based on collision energies. Table 5 Phytocompounds identified by UHPLC− ESI-MS/MS- METLIN Gen2 in the negative ion mode. # METLIN MID PubChem Name Mass Formula 1 1,936,754 1,398,069 5-chloro-N-[(3-methoxyphenyl)methyl]-1H-indole-2-carboxamide 580.9340 C18H16Br2F3N5O2S 2 1,794,738 3,120,608 2,5-diiodo-n-(2-methyl-5-{[1,3]-oxazolo[4,5-b]pyridine-2-yl}phenyl)benzamide 580.9100 C20H13I2N3O2 3 1,814,226 1,247,375 N-(4-bromo-3-methylphenyl)-3,5-bis(methylsulfanyl)-1,2-thiazole-4-carboxamide 387.9373 C13H13BrN2OS3 4 1,794,988 2,819,020 N-(5-bromo-4-methoxythiophene-3-carbonyl)-2-chlorobenzohydrazide 387.9280 C13H10BrClN2O3S 5 1,788,386 1,037,008 Methyl 2-(5-bromo-2-chlorobenzamido)-4-methyl-1,3-thiazole-5-carboxylate 387.9280 C13H10BrClN2O3S 6 1,965,269 5,348,344 (2,2-diethoxyethyl)-5-methyl-8-thia-4,6-diazatricyclo[7.4.0.0^(2,7)]trideca-1 (9),2,4,6-tetraen-3-amine 351.9610 C14H10BrClN2O2 7 1,965,197 5,348,748 3,5-dimethyl-4-(4-hydroxy-3,5-dimethoxyphenyl)-1-[(4-methyoxyphenyl)methyl]- 4H-pyridine-3,5-dicarboxylate 351.9520 C13H9BrN2O3S 8 1,675,950 1,673,703 2-{[(2-chloro-6-fluorophenyl)methyl]sulfanyl)-5-(2,4-dichlorophenyl)-1,3,4- oxadiazole 387.9410 C15H8Cl3FN2OS 9 1,775,422 1,034,227 3-bromo-2-chloro-5-(4-methylpiperidin-1-ylsulfonyl)pyridine 351.9650 C11H14BrClN2O2S 10 1,773,801 135,460,546 2-bromo-N’-[(E)-(5-chloro-2hydroxyphenyl)methylidene)benzohydrazide 351.9610 C14H10BrClN2O2 11 1,678,386 22,588,545 2-(4-bromophenyl)-4H-1lambda6,2,4-benzothiadiazine-1,1,3-trione 351.9520 C13H9BrN2O3S 12 1,609,292 2,739,683 5-(4-{[(2,6-dichlorophenyl)sulfanyl]methyl}phenyl)-1,2,4-thiadiazole 351.9660 C15H10Cl2N2S2 13 1,541,241 5,818,377 (2E)-N-(4-bromophenyl)-3-(5-ntrothiophen-2-yl)prop-2-enamide 351.9517 C13H9BrN2O3S 14 1,541,238 6,018,482 (2E)-N-(3-bromophenyl)-3-(5-nitrothiophen-2-yl)prop-2-enamide 351.9517 C13H9BrN2O3S 15 1,465,742 2,917,869 2-chloro-4-nitro-N-[5-(trifluoromethyl)-1,3,4-thiadiazol-2-yl]benzamide 351.9640 C13H9BrN2O3S 16 1,459,445 2,800,574 4-(3,4-dichlorophenyl)-3-(phenylamino)-1,3-thiazole-2-thione 351.9660 C15H10Cl2N2S2 17 1,456,140 1,294,178 5-bromo-N-(2-carbamoylphenyl)-2-chlorobenzamide 351.9610 C14H10BrClN2O2 18 1,431,466 2,765,805 4-[(2,4-dichlorophenyl)sulfanyl]-2-(methylsulfanyl)quinazoline 351.9660 C15H10Cl2N2S2 19 1,430,006 3,739,319 5-(benzylsulfinyl)-4-(2,4-dichlorophenyl)1,2,3-thiadiazole 351.9660 C15H10Cl2N2S2 20 1,426,795 2,765,804 4-[(2,5-dichlorophenyl)sulfanyl]-2-(methylsulfanyl)quinazoline 351.9660 C15H10Cl2N2S2 21 1,240,614 3,492,160 5-{[(2,4-dichlorophenyl)methyl]sulfanyl}-4-phenyl-1,2,3-thiadiazole 351.9660 C15H10Cl2N2S2 B. Chibuye et al. Scientific African 24 (2024) e02151 11 Fig. 4. The structures of metabolites tentatively identified in the leaf extract of A. panduriformis by LC-MS/MS- METLIN Gen 2. B. Chibuye et al. Scientific African 24 (2024) e02151 12 Fig. 4. (continued). B. Chibuye et al. Scientific African 24 (2024) e02151 13 amount of flavonoids (60.17 ± 0.25 mg QE/g). The higher phenolic content than flavonoids might be because flavonoids are a subset of polyphenolic compounds. Antioxidant activity The radical scavenging activity of the A. panduriformis leaf extract was evaluated using the DPPH assay, and the results are pre- sented in Table 7. The free radical scavenging activity of A. panduriformis leaf extract ranged from 26.10 % to 97.16 % relative to ascorbic acid (25.81 % to 78.92 %) at respective concentrations between 20 and 120 μg/ml. Similarly, the sample extract IC50 value of 53.48 µg/mL was low compared to that of ascorbic acid of 74.47 µg/mL. DPPH assay results generally indicated that A. panduriformis leaf extract is a potential source of antioxidants of natural origin. Discussions, conclusion, and Outlook Discussion Preliminary phytochemical screening results of the study revealed the presence of alkaloids, glycosides, tannins, saponins, steroids, polyphenols, terpenoids, and volatile oils. Interestingly, these phytocompounds provide strong scientific support for consuming A. panduriformis, a wild vegetable for medicinal purposes in the Zambian traditional healthcare system. For example, alkaloids might account for the analgesic properties of this wild vegetable. In this regard, codeine and morphine alkaloids can be cited to have analgesic properties [35]. Similarly, anthraquinones are present, which are reportedly anti-cancerous, laxative, and anti-arthritic [36]. Furthermore, the presence of polyphenols is remarkable, and these compounds have been associated with antioxidation properties [37]. Flavonoids present in the vegetable can offer antibacterial, antidiabetic, antifungal, and antiviral potential, as reported earlier [38,39]. The presence of such a diversity of molecules in one vegetable is significant. It may well account for its use in the traditional management of many diseases, including degenerative diseases such as cancer and diabetes mellitus in Zambia. The presence of cardiac glycosides may be behind the effectiveness of the consumption of A. panduriformis in controlling hyper- tension in traditional healthcare. Cardiac glycosides are effective diuretics and affect the heart positively thereby aiding the heart’s sturdiness and rate of shortening during contractions [21,40]. Therefore, this wild vegetable might be a possible alternative for controlling and managing cardiac diseases. Furthermore, the wild vegetable was found to possess saponins. These compounds essentially reduce the amount of cholesterol in the blood and further assist the body in absorbing calcium [41]. Thus, A. panduriformis is a potential food for treating and preventing atherosclerosis, a health problem usually caused by significantly higher amounts of cholesterol. In addition, the presence of tannins may account for the plants’ several medicinal properties [42]. For example, tannins are used in treating diarrheal, wounds, and catarrh and protecting the inflamed outer layer of the mouth [43,44]. Finally, the presence of steroids further validates the consumption of this vegetable for treating diarrheal in traditional herbal medicine [45]. LCMS- molecular networking tools, mzmine3 (table 2), GNPS (Tables 3 and 4), and METLIN (Table 6 and Fig. 4) results showed the presence of metabolites known to have interesting medicinal properties and nutritional value. The study, therefore, corroborates the reason for the prevalent use of the traditional wild vegetable in treating diseases in the traditional healthcare system. Consumption of this vegetable is pharmacologically significant when it comes to consuming food for medicine. For instance, quinine and its derivatives in this vegetable effectively treat malaria and illnesses such as nocturnal leg cramps, inflammation, and relieving pain [46]. In addition, Salicylic acid is an effective component in fighting acne owing to its oil-fighting capacities [47]. Further, ajmaline, an indole alkaloid, is an effective antiarrhythmic agent. Its presence in the wild vegetable renders it effective at preventing and treating a heart rhythm that may be too fast or irregular [48]. Jasmonates such as jasmonic acid are hormones in plants but exhibit anticancer and anti-inflammatory properties in humans [39]. Its presence, therefore, is pharmacologically significant. The other metabolite present is prazosin. It is an alpha-blocker that treats high blood pressure by relaxing and widening blood vessels, thereby enabling blood to flow more easily [49]. This effect helps prevent heart attacks, kidney problems, and strokes. Further, diosmetin, a flavonoid, reduces swelling and inflammation and restores normal vein function [50]. As for bergenin, it stimulates both the anti-inflammatory messengers and protective prostaglandin secretions. Breaking down lipids is an essential function of bergenin, making it a popular constituent of thermos genic fat-burning dietary supplements. Bergenin has no adverse effects, even at large dosages [51]. Dietary suberic acid has been reported as an effective agent for the treatment of skin photoaging [50]. Many more metabolites present in this wild vegetable possess medicinal effects. They include anziaic acid that works as an anti- bacterial compound; isovitexin, a flavonoid, possesses multifaceted pharmacological effects, such as being anticancer, antioxidant, antidiabetic, anti-inflammatory, neuroprotective, and antinociceptive [51,39]. As for 6-gingerol, it possesses an assortment of phar- macological effects, including anti-inflammation, anticancer, and antioxidation [52]. Furthermore, anisomycin is an effective Table 6 TPC and TFC of A. panduriformis leaves (n = 3). Leaves TPC (mg GAE/g) 122.30 ± 2.00 TFC (mg QE/g) 60.17 ± 0.25 B. Chibuye et al. Scientific African 24 (2024) e02151 14 antibiotic molecule [53]. The preceding are some of the health benefits of some of the metabolites found in A. panduriformis. Of interest is to note that the individual metabolites can act singly or in concert. However, the synergistic action of therapeutic metabolites produces a more sig- nificant effect when combined than the sum of their effects if not combined [54]. The health benefits of polyphenolic compounds including their antioxidant effects are well alluded to and are well documented in the literature [55]. The results of polyphenolic content are presented in Table 4. The content of phenolic compounds and flavonoids is commensurate with the LCMS/MS screening results, which have shown several polyphenols. Further, the enhanced TPC and TFC are in tandem with the enhanced antioxidative potential of the extract, as revealed by DPPH radical quenching results. The sample IC50 value of 53.48 µg/mL compared to that of ascorbic acid of 74.47 µg/mL shows higher radical quenching ability and consequently desirable antioxidant potential. In recent decades, flavonoid and phenolic-rich natural diets with antioxidant activity have encouraged interest in food science and nutrition [55]. Many literature reports suggest that polyphenolic compounds affect peroxide decomposition, oxygen scavenging, free radical inhibition, or metal inactivation in biological systems and assist in averting oxidative stress disease burden [26]. It is undeniable that natural antioxidants from leafy vegetables have an important role in safeguarding from the undesirable effects of free radicals [56]. Epidemiological studies have shown that the consumption of leafy vegetables possessing polyphenolic compounds with strong anti- oxidant capacity is mainly associated with reduced chances of diabetes, cardiovascular diseases, neurodegenerative diseases, and cancer [27,55,56]. The results suggest that the consumption of A. panduriformis wild vegetable as part of the diet can help enhance human health and significantly lower the damaging effects of free radicals that foster the progression of oxidative stress-related diseases. Conclusion This phytochemical study and identification of metabolites using molecular networking platforms have provided good visibility, not only on the phytochemical profile of A. panduriformis leaf but has also revealed its importance in nutrition, traditional healthcare, and in the search for therapeutic metabolites from plant sources. Phytochemical studies revealed the presence of natural products with interesting medicinal properties. These were polyphenols (flavonoids, tannins, phenolic compounds), alkaloids, steroids, terpenoids, Glycosides, saponins, and volatile oils. Total polyphenolic content was significant (TPC = 122.30 ± 2.00 mg GAE/g, and TFC = 60.17 ± 0.25 mg QE/g), and the antioxidant potential was high with the extract’s IC50 value of 53.48 µg/mL being lower in comparison to that of reference ascorbic acid of 74.47 µg/mL. This indicated that the wild vegetable is a potential source of antioxidants. In total, 26 metabolites were identified by mzmine3.2.8, 42 through GNPS, 21 metabolites were identified by METLIN Gen2 and previous studies confirmed the nutritional and pharmacological importance of these metabolites. Indeed, A. panduriformis wild vegetable, if included as part of the everyday diet, would boost health and fight and prevent infection and disease. The findings of the study will profoundly help drug developers. Outlook Although this study is the first investigation of the phytochemistry of A. panduriformis wild vegetable, the findings are very encouraging and lead to further studies, and possibly the isolation and characterization of compounds not revealed by LCMS2, which may also be responsible for the nutritional benefits as well as the use of the plant in traditional healthcare. Notes This work is part of the doctoral dissertation by Chibuye Bitwell and supervised by Professor Indra Sen Singh, Professor Maseka Kenneth Kakoma, and Professor Chimuka Luke. The study focussed on phytochemical studies and metal analysis of some medicinal herbs used in the traditional healthcare system in Zambia and elsewhere. CRediT authorship contribution statement Bitwell Chibuye: Conceptualization, Methodology, Validation, Software, Formal analysis, Investigation, Data curation, Resources, Writing – original draft, Writing – review & editing, Visualization, Project administration. Indra Sen Singh: Conceptualization, Table 7 DPPH antioxidant activity results. [SAA](µg/mL) [SE](µg/mL) Abcont AbSAA AbSE %RSA of SAA %RSA SE IC50 of SAA µg/mL IC50 of SE µg/mL 20 20 0,74 0,612 0,549 17,30 25,81 74.47 53.48 40 40 0,74 0,539 0,419 27,16 43,38 60 60 0,74 0,428 0,347 42,16 53,11 80 80 0,74 0,356 0,238 51,89 67,83 100 100 0,74 0,252 0,137 65,94 81,48 120 120 0,74 0,156 0,021 78,92 97,16 Note: [] = concentration, SAA = standard ascorbic acid, SE = sample extract, Ab cont= control absorbance, Ab SAA = absorbance of standard ascorbic acid, Ab SE = absorbance of sample extract, RSA = radical scavenging activity B. Chibuye et al. Scientific African 24 (2024) e02151 15 Resources, Funding acquisition, Methodology, Validation, Writing – review & editing, Supervision. Luke Chimuka: Conceptualiza- tion, Methodology, Validation, Investigation, Writing – review & editing, Funding acquisition, Supervision. Kakoma Kenneth Maseka: Conceptualization, Methodology, Supervision. 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Introduction Materials Materials Authentication of the plant Reagents Methods Extraction of phytocompounds for phytochemical screening QuEChERS Extraction Process for LC-MS QTOF Analysis Extraction of polyphenols for total polyphenol content, and antioxidant activity determination Phytochemical screening using chemical tests LC-MS/QTOF analysis Measurement of total phenolic content (TPC) Determination of total flavonoid content (TFC) Determination of DPPH antioxidant activity Statistical Analysis Results and data analysis Phytochemical screening using qualitative chemical tests LCMS/MS screening Molecular identification MZmine3 molecular identification Global natural product social molecular networking (GNPS) library matching GNPS advanced analysis Phytochemical identification using METLIN Gen2 Total polyphenolic content Total phenolic content (TPC) Total flavonoid content (TFC) Antioxidant activity Discussions, conclusion, and Outlook Discussion Conclusion Outlook Notes CRediT authorship contribution statement Declaration of competing interest Acknowledgments References